Senescence, Cancer and 'Endogenous Parasites': A Salutogenic Hypothesis

The integrity and health of an organism can be considered as a state actively imposed by specialised 'salutogenic' mechanisms (immune, endocrine, paracrine etc) on an innate tendency towards internal conflict. One major source of internal conflict arises from the operation of natural selection upon replicating sub-organismal components such as cells, organelles and gene sequences. From this perspective cancer is seen not as a pathological process arising in a healthy organism but as caused by the capacity of replicating cells to evolve 'selfish' adaptations and elude the finite repertoire of integrative mechanisms. Cancer can therefore be regarded as one instance of a more general tendency towards senescence due to the failure of salutogenesis and accumulation of endogenous parasites. This may provide a new and potentially fruitful approach to framing, analysing and understanding the aetiology of the degenerative diseases of senescence.

approach to framing, analysing and understanding the aetiology of the degenerative diseases of senescence.
Cancer research has led to a massive expansion of knowledge at the genetic level. A great deal of detailed information has emerged, but also a few simple principles. The model of neoplasia is roughly as follows [1][2][3]. Cancer is defined as uncontrolled cell replication, and the process that causes it is natural selection.
Cancer arises from a single ancestral cell with enhanced replicadve capacity due to a gene mutation or serial mutations. The mutation may either enhance the growth of the cancer cell itself (eg activation of oncogenes) or remove the inhibitory restraints on cell growth (eg inactivation of tumour suppressor genes). Mutations favourable to the promotion of uncontrolled replication accumulate progressively among progeny of the original ancestral cell, and lineal descendants will undergo adaptive evolution (tumour progression).
Knowledge concerning the genetic lesions which may contribute to the cancer process can be seen as filling in detail within this general framework. To use Mayr's terminology [4], the ultimate causes of neoplasia are clear in outline, while cancer research has been mainly a matter of delineating the proximate causes?those mechanisms that give rise to particular cancer diseases in particular situations. What follows should be seen not as supplying novel theories of cancer, nor as a review of the latest information concerning mechanisms, but as suggesting a broad perspective in which to view neoplasia. Cancer is seen not as a qualitatively distinct entity but as an instance of a more general senescence phenomenon named endogenous parasitism. It is a perspective that embeds the modern, gene-centred, evolutionary view of the nature of the cancer process into a humancentred view which concentrates on the evolution of the organism and the nature of health.

Endogenous parasitism
The evolutionary process by which endogenous cancers are generated can be seen as inevitable where there is replication of genes in a context of selection pressure. Natural selection will act such that gene mutations associated with 'selfish' phenotypic adaptations will differentially be favoured [5]. For instance, mutations coding for adaptations that enhance cell replication will be retained, while mutations coding for phenotypic functions that have a role in maintaining organismal functions (the 'somatic duties' of the cell [6]) will progressively be discarded. This tendency must continually be thwarted by the organism if health is to be maintained.
The process of within-organism natural selection for 'selfish' phenotypes is not, however, limited to cells, and applies equally to any gene-bearing component (or 'vehicle' [5]) that codes for its own reproduction?such as an organelle [7]. For example, it seems plausible to speculate that evolved selfish adaptation in mitochondria might explain the progressive decline in the performance of mitochondrial 'duties' of oxidative phosphorylation, combined with increased replicative efficiency due to chromosomal shortening [8,9], A similar example could be the proliferation of noncoding 'selfish DNA' in the genome when there may be no direct phenotypic consequences, merely a differential survival of DNA on the basis of its capacity to self-replicate [5]. Such changes would not usually be referred to as 'cancer' but would be nonetheless explicable as a result of the same basic process of natural selection.
This tendency for natural selection at suborganismal levels of organisation can be described as parasitic, in that phenotypic evolution is in the direction of exploiting the organismal environment rather than contributing to its functional integrity. Parasites will pursue replicative goals at the expense of organismal fitness. I have therefore suggested the term endogenous parasitism to describe the process of natural selection at the sub-organismal level [9].
There is also a close analogy bettveen the selective process that generates endogenous parasites (eg tumour progression) and the process of within-host BRUCE G CHARLTON, md, Lecturer, Department of Epidemiology and Public Health, University of Newcastle upon Tyne evolution of 'exogenous' parasites such as pathogenic bacteria [10]. Because a rapidly replicating parasite can evolve but the host cannot, parasite variants are selected that have the greatest resistance to the host's immunological defence mechanisms. Although the parasite may initially be held in check by the immune system, the selective accumulation of adaptive mutations within the parasite genome means that host defences are progressively eluded by new generations ?f parasites, and ultimately overwhelmed. This has been suggested as a possible cause of senescence [10].

Possibility of organisms
Organisms arise owing to natural selection acting upon the germ line genome [5][6][7]. The organism is necessary to the germ line because the sex cells are the only route by which the whole set of cooperating genes are able to reach future generations of new organisms. Selection pressure at the level of the organism will therefore favour the evolution of mechanisms that maintain integration and harmonious functioning at least until reproduction has occurred [7]. Multicellular organisms commence life as a somatic clone, but the operation of natural selection upon random mutations in succeeding generations of cells will mean that organisms progressively evolve into chimeras composed of the original zygotic genome plus newly evolved cell lineages [6]. The integrity of a clone is easily explained on the basis of identical cellular genomes having identical replicative interests, but the different genetic compositions of different cells in a chimera are a potential source of intercellular conflict.
The tendency to generate endogenous parasites means that natural selection operates in a sequential fashion, first to create the organism, then to destroy it. Organism-level selection dominates until the usual age of reproduction, after which the balance progressively tips away from the need to maintain organisinal integrity and towards 'allowing' sub-organism selection [11]. This sequence may seem paradoxical, futile, or even tragic, from an organism-centred viewpoint. However, from the gene-centred perspective of natural selection, the organism is 'merely' a vehicle for producing further copies of the germ line genome [5]. Because maintaining organismal integrity is costly, a 'disposable' organism is the most efficient way of ensuring gene survival and replication [9,11].

Salutogenic mechanisms
Traditionally medicine has assumed health and sought to explain disease. The study of disease is pathology, and the processes that cause disease are described as pathological. Cancer has generally been interpreted, from an organism-centred perspective and within the pathological framework, as an 'abnormal' event arislng in the context of an integrated organism and dis-rupting its state of health [12]. In other words the occurrence of cancer is seen as the state that requires explanation.
However, an organism is not intrinsically harmonious and is a site of potential conflict at every level of organisation [13]. Health should be seen as something that cannot be assumed and must itself be explained [14]. To rephrase Dawkins [15], there is an infinite number of ways to be unhealthy, but only a few ways of being healthy. Health, not disease, is the state that requires explanation, and disease may most plausibly be considered a consequence of the failure to maintain health.
The study of health and its determinants has been dubbed salutology, and the processes by which organismal health is created and maintained may be considered salutogenic [14] (the name was coined by Antonovsky [16] and derives from 'salutary', meaning health-promoting or beneficial). Salutology is not merely the reciprocal of pathology [14,17], since the processes that 'cause' health are often different from the processes or events that 'cause' disease.
The ultimate, foundational salutogenic processes that 'cause' (or enable) health will include the evolutionary history, the organism's developmental history from fertilised egg to adult capable of reproduction, and?in humans?a vast range of social processes [6,7,16,17], The proximate salutogenic causes would include the whole spectrum of mechanisms that have evolved to create and sustain organismal integration.
Proximate mechanisms range from organism-wide systems (such as immune, endocrine and nervous systems) to a large and growing number of described intercellular control and signalling mechanisms to regulate cell reproduction, differentiation etc, such as those coded for by the various 'tumour suppressor genes', and the signals to induce apoptosis or programmed cell death [1][2][3]18]. An analogous role may be surmised for many transcription regulating processes, eg the control of intragenomic conflict [19]. Another vital class of salutogenic mechanisms is that which monitors and repairs damaged DNA [7]. The value of a salutogenic perspective is heuristic. I suggest that it is often useful to consider health and disease as alternative outcomes of a dynamic conflict between salutogenic forces (which act to maintain organismal integrity) and pathological forces (which cause disintegration or destruction). This may have implications both as a framework for research and as a background theorem for therapeutic intervention [9,14,17], Senescence and degenerative disease Slow maturation and a lengthy period of development means that transmission of genes in a human depends upon many years of good health leading to reproductive maturity. Following reproduction the need to look after dependent infants likewise places a premium B G Charlton upon an extended lifespan. Such selection pressures have led to the evolution of highly effective salutogenic mechanisms to control the endemic threats to organismal integration, including neoplasia. Human health and longevity [20] are therefore byproducts of the constraints on effective human reproduction. Although multicellular organisms have 'solved' the problem of controlling endemic neoplasia well enough to enable organisms to reproduce and pass on their genes to future generations, this solution is temporary for each organism. Natural selection is constantly operating on dividing cells, and cancer can only be held in check by salutogenic mechanisms which will inevitably develop imperfections.
The production of endogenous parasites is a phenomenon of senescence, the effects of which accumulate with time. This implies that cancer is not a distinct entity. Cancer is a particular instance of the generalised phenomenon of natural selection acting upon organismal components. This is important because the same process that gives rise to cancer cells may be expected to give rise to other deleterious outcomes both at the cellular level (including the various 'pre-cancerous' states) and in other subcellular replications such as organelles and gene sequences.
Cancer is likely to be a major problem the longer an animal lives and the lower its death rate from accident, predation or exogenous parasites. Natural selection of sub-organismal components will eventually lead to variants able to elude any conceivable integrative system. This is consistent with the observation that cancer becomes a relatively more common cause of death among humans as average lifespan increases.
A consideration of natural selection within organisms offers the possibility of extending the reach of Darwinian medicine?an approach that uses insights from evolutionary theory to throw light upon human health [21]. It is a truism to observe that 'degenerative' diseases of senescence are increasingly important in developed countries [22]. Endogenous parasitism opens the door to a different way of approaching and classifying such 'diseases of civilisation'. It seems reasonable to speculate that the aetiology of disease processes such as atherosclerosis and the pathologies associated with Alzheimer-type dementia might be fruitfully analysed in terms of the development of endogenous parasites [9]. The message is bleak. The organism is disintegrating from the moment of its formation, due to the tendency for endogenous parasitism among replicat-ing components. Senescence is apparently inevitable, either from neoplasia or from some other 'selfish' adaptation in some other sub-organismal component. If cancer does not kill us, it is likely that a different kind of endogenous parasite eventually will.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Myristylation of FBR v-fos dictates the differentiation pathways in malignant osteosarcoma.

Myristylation of FBR v-fos, a c-fos retroviral homologue that causes osteosarcomas in mice, determines many of its transcriptional properties in vitro. To determine whether myristylation of FBR v-fos contributes to in vivo tumorigenicity, we examined its transforming capability in comparison to a nonmyristylated FBR v-fos (G2A-R). Retroviral infections with FBR v-fos and G2A-R transform BALB/c-3T3 cells. The number, size, and cellular morphology of foci generated by both FBR and G2A-R are indistinguishable. However, marked biological differences were found in transgenic mice expressing either the myristylated FBR v-fos or the nonmyristylated G2A-R. 11 of 26 FBR v-fos transgenic mice died as a result of gross tumor burden. None of the 28 G2A-R transgenic mice died from tumor burden, and only two of the G2A-R mice developed bone tumors. Histologic examination of the tumors reveals that the FBR v-fos bone tumors contain malignant cells with features of four cell lineages (osteocytes, chondrocytes, myocytes, and adipocytes) in an environment rich in extracellular matrix (ECM). However, the G2A-R tumors exist in an environment devoid of ECM and display malignant cells with features of adipocytes. Masson staining reveals that the ECM of the FBR tumors stains strongly for collagen. Immunohistochemical staining with collagen III antibody demonstrates an abundance of collagen III expression in this ECM. While NH2-terminal myristylation is not required for FBR immortalization and transformation, it is essential in determining the degree of differentiation and tumorigenicity of malignant cells.

mice developed bone tumors. Histologic examination of the tumors reveals that the FBR v-fos bone tumors contain malignant cells with features of four cell lineages (osteocytes, chondrocytes, myocytes, and adipocytes) in an environment rich in extracellular matrix (ECM). However, the G2A-R tumors exist in an environment devoid of ECM and display malignant cells with features of adipocytes. Masson staining reveals that the ECM of the FBR tumors stains strongly for collagen. Immunohistochemical staining with collagen III antibody demonstrates an abundance of collagen III expression in this ECM. While NH2-terminal myristylation is not required for FBR immortalization and transformation, it is essential in determining the degree of differentiation and tumorigenicity of malignant cells.
FBR v-los is expressed as a 75-kD nuclear protein that differs from c-Fos by deletion and replacement of both NH2-and COOH-terminal regions with retroviral gag and mouse fox sequences, respectively Van Beveren et al., 1984;Wilson and Treisman, 1988). FBR v-fos represents an important model in the study of carcinogenesis, since its cellular analogue (c-Fos) is a transcriptional regulator whose early expression is implicated in cellular growth and differentiation. Previously we have shown that FBR v-fos demonstrates both losses of function (inability to transactivate from AP-1 sites and inability to repress from the serum response element in the c-los promoter) and gains of function (transcriptional activation of the collagen III promoter) when compared to c-Fos (Kamata et. al., 1991;Kamata and Holt, 1992;Jotte et al., 1994). The expression of collagen III, which is normally tightly controlled during the differentiation of 3T3-L1 adipocytes, is markedly disrupted by the presence of FBR v-los (Jotte et al., 1994). These altered transcriptional properties of FBR v-fos are dependent upon its myristylated NH2-terminal glycine. A nonmyristylated point mutant FBR protein, G2A-R (for glycine to alanine at the second amino acid site), not only regains its ability to transactivate from AP-1 sites and repress at the serum response element, but it also loses its ability to disrupt the differentiation-dependent expression of collagen III. Absence of myristylation therefore restores FBR's molecular mechanisms to that of its normal cellular homologue, c-Fos.
While myristylation is essential for the differentiationdependent transcriptional properties of FBR in vitro, its functions through the process of immortalization and tumorigenesis remain undefined. To address the biological relevance of FBR v-fos myristylation, we examined FBR and G2A-R's ability to transform mouse fibroblasts in tissue culture. Myristylation's contribution to tumorigenicity was further characterized in vivo in transgenic mice expressing either the myristylated or nonmyristylated FBR proteins.

Cell Cultures and Transfections
FBR v-fos 3T3-L1 stable cell lines and BALB/c-3T3 cells were grown in DME supplemented with 10% FCS (Sigma Chemical Co., St. Louis, MO), 2 mM L-glutamine (Sigma Chemical Co.), and 1x antibiotic/antimycotic (Sigma Chemical Co.). Bosc 23 cells were maintained in DME containing 10% dialyzed FCS, 0.25 mg/ml xanthine (Sigma Chemical Co.), 2 i~g/ml aminopterin (Sigma Chemical Co.), 6 i~g/ml thymidine (Sigma Chemical Co.), 25 I~g/ml mycophenolic acid (GIBCO BRL, Gaithersburg, MD), 2 mM L-glutamine, and lx antibiotic/antimycotic. 3T3-L1 cells were transfected as proliferating fibroblasts at 30% confluency. Cells were transfected with 10 I~g of MuSV-FBR expression vector and I Ixg of RSV-neomycin resistance gene by the calcium phosphate-DNA coprecipitation with glycerol shock (Graham and van der Eb, 1973). After glycerol shock, cells were refed in DME with 10% FCS. 24 h later, transfected cells were split 1:10 and replenished in DME with 10% FCS and 100 I.~g/ml G418. Cells were maintained in tissue culture in the presence of G418 for 3 wk at which time colonies were isolated and screened for FBR v-fos expression by Western blot.

Generation of FBR and G2A-R Transient Supernatants
G2A-R viral vector was generated by inserting the NcoI fragment of MuSV LTR-driven G2A-R (Kamata and Holt, 1992) into FBR v-fos. 20 I~g of FBR or G2A-R was transiently transfected into Bosc 23 cells (plated at a density of 5 × 105 cells the day before) by the calcium phosphate coprecipitation method. 12 h after transfection, media (DME with 10% FCS) was replaced. 48 h after transfection, viral supernatant was harvested and spun at 3,000 rpm to remove cellular debris. Viral supernatants were stored at -70°C.

Titering of FBR and G2A-R Viral Supernatants
For titering by viral RNA hybridization, 20 ml of media in which the viralproducing Bosc 23 cells were growing was centrifuged at 4°C at 10,000 rpm to pellet any producer cells. Supernatants were transferred to a SW27 polyallomer tube and spun for 2 h at 27,000 rpm at 4°C. Viral particles were resuspended in 0.5 ml of 10 mM Tris-HCl, pH 7.5. Viral suspensions were layered onto a 4-ml step gradient in SW50.1 tubes consisting of 2 ml 25% sucrose in 100 mM Tris-HC1, pH 7.5, 1 ml 40% sucrose in 100 mM Tris-HC1, pH 7.5, and I m150% sucrose in 100 mM Tris-HC1, pH 7.5. Viral suspensions were centrifuged 3 h at 35,000 rpm at 4°C. Viruses resting at the 40% sucrose cushion were phenol-chloroform extracted twice and once with chloroform. RNA was precipitated with an equal volume of isopropanol. Precipitated RNA was resuspended in 250 Ixl of 15% formaldehyde and 250 td of 20x SSC. Suspended RNA was heated at 50°C for 15 min and transferred to an ice bath. Serial dilutions in 10x SSC of each sample were made and adsorbed onto a Gene Screen Plus membrane (Dupont/NEN, Wilmington, DE) through a dot blot minifold apparatus. Blots were rinsed in 2x SSC and allowed to air dry. Blots were then baked at 80°C for 2 h to reverse the formaldehyde reaction. RNA transcripts were hybridized with T7 RNA polymerase generated antisense FBR v-fos coding sequence at 60°C and washed according to the protocol provided by the Gene Screen Plus manufacturers.
FBR and G2A-R Transformation Assays in BALB/c-3T3 Cells 2 X l0 s BALB/c-3T3 cells were plated per 10-cm dish the day before infection. Serially diluted viral supernatants (10 1, 10-2, 10-3, 10-4, and 10 -5) were added to BALB/c-3T3 cells in the presence of 6 Ixg/ml polybrene. The following day, plates were split 1 to 10 and allowed to grow in DME, 10% FCS with a media change every 3 d. At 3 wk, cells were stained with Giemsa stain and scored for loci formation. Foci >5 mm in diameter were scored as positive.

Establishment of Transgenic Mouse Lines
The DNAs injected were the BamHI-XhoI fragment of either the FBR or G2A-R expression vectors that encompassed the Moloney Leukemia virus LTR and the oncogenic region extending through the polyadenylation site (Van Beveren, 1984). Transgenic mice were established through the Vanderbilt University Cancer Center Transgenic Mouse Core. C57BL6 x DBA F1 fertilized eggs were injected with either MuSV-LTR driven FBR or G2A-R encoding DNA (Hogan et al., 1986). At 2 wk of age, mice had 0.5 in of their tails resected and placed into 300 ILl of buffer containing 50 mM Tris, pH 8.0, 100 mM EDTA, 100 mM NaC1, 1% SDS, and 175 g proteinase K (Boehringer Mannheim Corp., Indianapolis, IN). Tails were incubated for 12 h at 55°C and then extracted with an equal volume of phenol and chloroform. Precipitated DNA was spooled and resuspended into sterile distilled H20. Transmission of the FBR and G2A-R genes in founder and progeny mice was confirmed by PCR analysis of mouse tail DNA using 5'-CAGAGCGGGAATGGTGAAGA-3' and 5'-TGATGC-CGGAAACAAGAAGT-3' oligonucleotides as PCR primers. Founder mice were crossed with C57BL6 nontransgenic mice to determine germline transmission, and progeny mice were screened by PCR as described above.

Tumor Detection, Histology, and Immunohistochemistry
Tumors were initially detected roentgenographically on a General Electric MSI.1250 IV Radiographic Imager (Milwaukee, WI) at settings of 1.5 kV and 1.5 mA. Tumors were resected from the transgenic mice and immersed in 3.7% formaldehyde. Tumors were then embedded in low melting point Ameraffin (Baxter, Marietta, GA) and sectioned at 4-5 i~m on a Reichert-Jung microtome. Processing was conducted according to standard procedure by the Vanderbilt University Medical Center Research Management Service. Standard Mayers hematoxylin (Fisher Scientific, Pittsburgh, PA) and pholxine B/eosin y (Fisher Scientific), Masson trichrome (Fisher Scientific), and Sudan black B (Chroma-Gesellschaft, Germany) stains were conducted on paraffin-embedded sections. Immunohistochemistry was conducted on an automated stainer (model Techmate; Biotech Res. Labs, Inc., Rockville, MD) using a universal ABC detection kit with DAB chromagen using the following antibodies: actin prediluted monoclonal antibody (Enzo Biochem Inc., New York), cytokeratin cam 5.2 monoclonal antibody (Becton-Dickinson Immunocytometry Sys., Mountain View, CA) diluted 1:20, desmin monoclonal antibody (Dako Corp., Corpinteria, CA) diluted 1:500, and a rabbit anti-rat collagen type III antibody (Pasteur Institute, Lyon, France) diluted 1:20.
Tumor RNA Isolation and Reverse Transcriptase PCR RNA was purified from resected tumor tissue as previously described (Chirgwin et al., 1979). Tumors were resected and quick-frozen in liquid Nz. 10 ml of 4 M guanidinium isothiocyanate, 50 mM Tris, 10 mM EDTA solution was added per gram of tissue, and tissue was ground with a tissuemizer. Suspensions were centrifuged 10 min at 10,000 rpm at 12°C, and supernatants were heated to 65°C for 2 min in the presence of 2% Sarkosyl. CsCI was then added at a concentration of 0.1 g/ml solution and layered over 5 ml of 5.7 M CsCI in a SW-28 polyallomer tube. Samples were spun overnight at 25,000 rpm at 22°C. RNA pellets were resuspended in 5 mM EDTA, 0.5% Sarkosyl, and 5% 2-mercaptoethanol. Samples were extracted with equal volumes of phenol/chloroformhsoamyl alcohol and precipitated. Quantity and quality of RNA was determined by spectrophotometric ultraviolet light absorbance at 260 nm and by ethidium bromide staining for ribosomal RNA within the total RNA samples. 5 ~g total RNA from each sample was treated with 5 U RNAse-free DNAse I (Promega Corp., Madison, WI) at 37°C for 30 min. Samples were extracted with an equal volume of phenol and chloroform and ethanol precipitated. RT-PCR was performed by our published methods (Thompson et al., 1995) using the following basic method: RNA samples were reverse transcribed for 1 h at 37°C using 2 ixg of total RNA, 1 p.g random hexamers (Boehringer Mannheim Corp.), 1 x first strand buffer (GIBCO BRL), 0.01 M DTT, 0.5 mM each dATP, dCTP, dGTP, and dTTP and 200 U Superscript II RNaseH-reverse transcriptase (GIBCO BRL). The RNA/ DNA duplexes were used as templates for 20-cycle PCR reactions using FBR and G2A-R retroviruses were generated as described in Materials and Methods. Dilutions were made from the retroviral stocks, l0 6 logarithmically growing BALB/c-3T3 cells were infected with 1 ml of the respective dilutions in the presence of polybrene. 12 h after infection, plates were passaged l:10 and allowed to grow for 3 wk. Colony foci were scored as positive if colony diameter was >/2 ram. Numbers indicate the number of viral particles per ml of initial infecting dilution and represent the average of three separate infections. for the viral oncogene, 2534 to 2719 from the published sequence (Van Beveren et al., 1984). 20% of PCR products were run on a 2% agarose gel and visualized with ethidium bromide intercalation and UV lighting.

Transformation by FBR is Myristylation Independent
To address the biological relevance of FBR v-los myristylation, retroviral vectors encoding myristylated FBR v-los and non-myristylated G2A-R ( Fig. 1) were generated as described in Materials and Methods. The FBR v-los and G2A-R retroviral vectors were transiently transfected into Bosc 23 cells (which harbor the Moloney-murine leukemia helper virus) by the calcium phosphate coprecipitation method (Graham and van der Eb, 1973). Viral supernatants were harvested and stored at -70°C. Viral RNA was   v-los and 28 G2A-R transgenic mice were generated as described in Materials and Methods. Over the course of 20 mo, 11 of the 26 FBR transgenic mice succumbed to tumor burden and died (a 58% tumor-dependent mortality). Of the 28 G2A-R transgenic mice, only two developed tumors. These mice were sacrificed at 9 and 20 mo for tumor histology studies. The first G2A-R mouse death was secondary to pneumonia at 4 mo of age. This death and the two mice sacrificed for tissue examination were the only G2A-R transgenic deaths (a 4% study mortality). Dotted line, the average age and standard error of 7.3 --_ 1.2 mo at which FBR transgenic mice succumbed to tumor burden.
purified from these viral supernatants as described in Materials and Methods, applied to nylon membranes, and subjected to hybridization with an antisense F B R radiolabeled probe capable of hybridizing with R N A generated from both the F B R v-los and G 2 A -R viral vectors (data not shown). A n equal number of either F B R v-fos or G 2 A -R serially diluted virus particles (as determined by the viral R N A assay) were used to infect 5 x 105 BALB/c-3T3 cells. Cells were infected with 104, 103, or 102 virus particles of either F B R v-fos or G 2 A -R in the presence of polybrene to enhance viral adherence to the plasma membrane. 12 h after infection, BALB/c-3T3 cells were passaged at a 1:10 dilution and maintained in tissue culture for up to 3 wk. A t 3 wk, media was aspirated and the plates were stained with Giemsa. Infection with myristylated F B R v-fos as well as nonmyristylated G 2 A -R retrovirus causes transformation of BALB/c-3T3 cells as determined by foci formation (Fig. 2, A and B). The growth and size of transformed colonies between the two retroviruses are indistinct, and the cellular morphology of the transformed BALB/c-3T3 cells generated by both retroviruses are also very similar (Fig. 2, C and D). A n average of three separate loci transformation assays reveals that the number and size of BALB/c-3T3 loci generated by both F B R and G 2 A -R are indistinguishable (Table I). These experiments demonstrate that F B R and G 2 A -R retroviruses have functionally indistinct qualitative and quantitative transforming characteristics, indicating that myristylation of F B R v-fos does not affect either its ability or efficiency to cause transformation of fibroblasts. Although the gross morphology of the cells transformed by either the myristylated or nonmyristylated forms appears similar, it is conceivable that a more detailed structural analysis might reveal differences which would correlate with the observed in vivo effects. Prior studies have shown no differences in the actin cytoskeleton of these cells using immunohistochemistry, but more subtle structural effects may be present.

Nonmyristylated FBR (G2A-R) Does Not Cause Malignant Tumor Growth
To further address myristylation's possible role in F B R ' s tumorigenic properties, transgenic mice carrying either the myristylated FBR or the non-myristylated G2A-R genes were generated as described in Material and Methods. Carriers of the transgene were confirmed by PCR analysis of D N A isolated from a resected portion of each mouse's tail. PCR analysis was designed to detect both the endogenous c-los gene as well as the exogenous transgene (data not shown). Two FBR and three G2A-R founders were generated from the initial transgenic injections. However, only one FBR and two G2A-R mice from these founders displayed germ-line transmission of the respective transgenes. These founders were mated with nontransgenic B6D2 mice, and F2 progeny were screened for the presence of the transgene by PCR analysis (data not shown). 26 mice harboring the myristylated FBR gene and 28 mice carrying the nonmyristylated G2A-R gene were sired from the corresponding founders and compared for tumor growth and mouse survival. Marked differences were observed between the FBR and G2A-R transgenics. Of 26 FBR transgenic mice, 11 developed tumors and died over the course of 20 mo. Each of these deaths were secondary to tumor burden. However, of 28 G2A-R transgenic mice, only three died over the same time period. The first G2A-R transgenic mouse death occurred at 3 mo of age and was due to pneumonia; no tumors were detected at autopsy. The two remaining deaths were sacrifices at 9 and 20 mo to examine tumor histology. More importantly, the two G2A-R mice sacrificed for tumor histology were the only two G2A-R transgenic mice that developed tumors. While 11% of the G2A-R mice died during this study, there was only a 4% mortality rate which is attributed to a pneumonia. 0% of the G2A-R transgenic mouse deaths were due to tumor burden. However, 58% of the FBR transgenics died, all of which were deaths caused by tumor burden (Fig. 3). Although the data presented was obtained with a single line of FBR transgenic mice, a second founder (female) developed a large pelvic osteosarcoma which may have contributed to her lack of offspring and proved ultimately fatal. This second founder confirms that FBR v-los transgenics develop lethal osteosarcomas.
FBR-and G2A-R-induced tumors were initially detected roentgenographically as early as 2 mo of age. Fig. 4 A demonstrates a normal roentgenogram from a control nontransgenic mouse. In comparing the FBR and G2A-R tumors, the malignant nature of the FBR tumors compared to that of the G2A-R tumors is readily visible (compare Fig. 4, B and C). Tumors became grossly detectable as early as 3 mo of age with an average survival of 7.3 ___ 1.2 mo in the FBR transgenic mouse group.
To ensure that the tumors isolated from the FBR and G2A-R transgenics did express the respective transgenes, reverse transcriptase polymerase chain reactions specific for FBR and G2A-R sequences were conducted. Total RNA was isolated from the different tumors by cesium chloride purification as described in Materials and Methods and digested with RNAse-free DNAse to ensure samples were not contaminated with DNA. Primers were designed to encompass FBR and G2A-R specific sequences which flank the fos/fox junction in FBR and G2A-R. Because the amplified region lies near the 3' end of FBR, only completely transcribed message is detected. Fig. 5 Figure 5. Reverse transcriptase PCR of FBR and G2A-R transcripts isolated from tumors. Reverse transcriptase PCR was performed on total RNA isolated from FBR and G2A-R resected tumors. Equal levels of RNAs were confirmed by spectrophotometric ultraviolet light absorption at 260 nm and by ribosomal staining with ethidium bromide. Samples without reverse transcriptase served as a negative control to ensure that amplified sequences were driven from an RNA template and not DNA. Total RNA isolated from FBR and G2A-R stably transfected 3T3-L1 cell lines served as positive controls. Amplified PCR products were resolved on a 2% agarose gel and visualized by ethidium bromide staining. (Lane 1) 100-bp molecular weight standards demonstrates that both the FBR-induced tumors and the G2A-R induced tumors express message for their respective genes (Fig. 5, lanes 7 and 9). Total RNA isolated from 3T3-L1 cell lines stably transfected with the FBR or G2A-R genes served as positive controls (Fig. 5, lanes 3 and 5). Omission of the reverse transcriptase cDNA step served as a negative control to ensure that amplified fragments were not generated from contaminating D N A (Fig. 5,  lanes 2, 4, 6, and 8). This study confirms that the FBR and G2A-R tumors express messages encoded by the respective transgenes. mors were excised and compared with tumors isolated from the sacrificed G 2 A -R transgenics. Tumors and normal long bone were fixed in 3.7% formaldehyde, paraffin embedded, and sectioned for histologic examination. Fig.  6, A and E, is haematoxylin and eosin stains of normal long bone for comparison. H & E staining of F B R tumor sections reveals that these tumors are osteosarcomas (Fig.  6 B) with additional features of chondrosarcoma, rhabdomyosarcoma, and liposarcoma. F B R tumors also possess a peripheral band of fibrous tissue which encapsulates the tumor (Fig. 6 C). High power views reveal the multiple differentiated features of the F B R osteosarcomas throughout the tumor (Fig. 6, F and G). G 2 A -R tumors, however, are liposarcomas and lack features of multiple differentiated cell lineages other than the adipocyte (Fig. 6  D). Although the G2A-R liposarcomas possess a large proportion of poorly differentiated mesenchymal cells, there are adipocyte-like cells with lipid-laden vesicles (Fig.  6 H). Sudan black staining for lipid and phospholipid reveals that FBR tumors express low levels of lipid, and G2A-R tumors express significant levels (Fig. 7, A and D). Immunohistologic stains for actin reveals that FBR tumors stain positive in regions consistent with rhabdomyosarcomatous features (compare Fig. 7, B and C). However, G 2 A -R tumors do not stain for actin, confirming that G 2 A -R tumors lack cells with myocyte-type features (compare Fig, 7, E and F). Additional stains for cytokeratin were conducted to determine if either the FBR or G 2 A -R tumors contain cells of epithelial origin. Neither FBR or G 2 A -R tumors stain positively for cytokeratin (data not shown). Thus, FBR tumors display characteristics with four differentiating features (osteosarcoma, chondrosarcoma, rhabdomyosarcoma, and liposarcoma). However, G 2 A -R tumors only display features of the differentiating liposarcoma.

Myristylation of FBR Defines Tumor Histology
Closer examination of the FBR osteosarcomas reveals an abundance of extracellular matrix (ECM) throughout the tumor (Fig. 8 A). G 2 A -R liposarcomas display a largely cellular component that is devoid of the E C M seen in FBR tumors (Fig. 8 D). Masson staining of the tumor sections reveals that the high levels of ECM seen in FBR tumors stain strongly for collagen (Fig. 8 B). FBR osteosarcomas also contain a fibrous capsule that encompasses the tumor and is rich in collagen (Fig. 8 C, arrows). G2A-R tumors not only lack this fibrous capsule, but they also fail to stain for collagen throughout the tumor (Fig. 8 E). These studies demonstrate that the FBR tumors that are rich in ECM express high levels of collagen protein, whereas the G2A-R tumors lack ECM and therefore fail to stain for collagen.
Because FBR is known to transcriptionally activate the collagen III promoter (Setoyama et al., 1986a;Jotte et al., 1994), specific immunoperoxidase staining for collagen III was performed on the FBR and G2A-R tumors as described in Materials and Methods. Immunoperoxidase positive staining was greatest at the periphery of the FBR tumors within the fibrous capsule (compare Fig. 9, A and  B). This suggests that the fibrous capsule surrounding the FBR osteosarcomas consists largely of collagen III protein. Higher magnification reveals the extent of ECM-rich FBR osteosarcoma which stains for collagen III protein (compare Fig. 9, C and D). G2A-R liposarcomas demonstrate a markedly lower level of collagen III protein expression throughout the tumor (compare Fig. 9, G and H). There is also a lack of enhanced collagen III expression at the tumor periphery, as the G2A-R liposarcomas lack a fibrous capsule (compare Fig. 9, E and F).

Discussion
These data demonstrate that while myristylation of a viral oncogene is independent of its ability to transform and immortalize cells in tissue culture, its role in vivo is crucial for tumorigenesis. Myristylated FBR-induced tumors are osteosarcomas with features of chondrosarcoma, rhabdomyosarcoma, and fibrosarcoma. The nonmyristylated mutant G2A-R induced tumors are liposarcomas. The presence of the myristoyl moiety on FBR protein allows malignant mesenchymal cells to enter and disrupt multiple differentiation pathways leading to tumors with features of osteosarcomas, chondrosarcomas, rhabdomyosarcomas, and liposarcomas. But nonmyristylated FBR disrupts the differentiation of a single cell lineage leading to liposarcomas. The differentiation pattern of osteocytes, chondrocytes, and myocytes are not disrupted and therefore appear normal in the G2A-R transgenic mice. Although we do not detect the level of lipid-accumulating adipocytes in the FBR osteosarcomas as in the G2A-R liposarcomas, there are a high number of fibroblasts and primitive mes- enchymal cells (Fig. 6 G), which have also been seen in FBR retrovirus-induced tumors (Price et al., 1972;Finkel et al., 1975;Ward and Young, 1976). These primitive mesenchymal cells may represent preadipocytes which are blocked from entering the adipocyte differentiation pathway (Fig. 10). FBR v-los transformed tumor cells also exist in an extracellular matrix rich in collagen III that is transcriptionally activated by FBR v-los (Setoyama et al., 1986a, b;Jotte et al., 1994). Such an environment may alter programmed cellular differentiation and allow FBR transformed ceils to disrupt multiple differentiation pathways (Liau et al., 1985;Yoakum et al., 1985;Coppola and Cole, 1986).
The indistinguishable efficacy by which FBR and G2A-R cause immortalization indicates that myristylation is not necessary for cellular transformation to occur. Other regions within the FBR protein must be responsible for this initial step toward tumorigenicity. Fos sequences within FBR are a likely candidate for its immortalizing features, as unregulated expression of Fos has been shown to overcome the limited proliferative capacity of different cell lineages causing transformation (Jenuwein and Mtiller, 1987;Lassar et al., 1989). Although Fos sequences may initiate tumorigenesis by causing cellular transformation, these se-quences are not the sole determinant of the differentiation pathways followed by the transformed cells. This is evidenced by the different predominant lineages observed in the FBR and G2A-R tumors. Myristylation of FBR protein defines which differentiation pathways will be disrupted. Thus, it would appear that the parameters which dictate cellular growth and immortalization are dependent upon Fos sequences, and the differentiation patterns of FBR transformed cells are dependent upon myristylation.
The mechanisms by which FBR and nonmyristylated G2A-R transformed cells disrupt differentiation remains unknown. Perhaps myristylation of FBR endows it with a novel transcriptional activity or function which disrupts the differentiation pathway of various cell lineages. Previous work in our lab and other's has examined FBR's myristylation-dependent transcriptional activity of the collagen III gene (Setoyama et al., 1986a, b;Jotte et al., 1994). Collagen III expression is markedly down-regulated during the differentiation of adipocytes (Weiner et al., 1989;Jotte et al., 1994). Collagen III's expression is shut off by day two of 3T3-L1 adipocyte differentiation (Jotte et al., 1994). 3T3-L1 cells stably transfected with FBR demonstrate a disruption of this expression pattern, whereas G2A-R stably transfected 3T3-L1 cells demonstrate a normal col- Figure 9. Collagen III expression in FBR and G2A-R tumors. (A) FBR control collagen III stain at tumor periphery. FBR tumors were fixed, sectioned, and stained as described in Materials and Methods. Collagen III positive staining is represented by an immunoperoxidase generated brown color. Section represents the background staining pattern for the collagen III antibody observed in FBR tumor sections at the fibrous capsule (FC). (B) Collagen III staining seen in the periphery of FBR tumors. Arrows demarcate the border of collagen Ill expression within the fibrous capsule (FC). Also note the overall increase in generalized staining for collagen III throughout the tumor section. (C) FBR control collagen III stain at higher magnification. Section represents the background immunoperoxidase staining pattern for the collagen III antibody observed within the FBR tumor. (D) FBR collagen III staining pattern at higher magnification. Note the marked staining for collagen III within the ECM of the FBR tumor. (E) G2A-R control collagen III stain at the tumor periphery. Section represents the background immunoperoxidase staining pattern for the collagen III antibody observed near the periphery of the G2A-R tumor. (F) Collagen III staining seen in the periphery of G2A-R tumors. Note the lack of enhanced collagen III staining at the periphery of G2A-R tumors compared to FBR tumors in B. Also note the overall decreased expression of collagen III throughout the G2A-R tumor. (G) G2A-R control collagen III stain at higher magnification. Section represents the background immunoperoxidase staining pattern for the collagen III antibody observed within the G2A-R tumor. (H) G2A-R collagen III staining pattern at higher magnification. Note the lack of staining for collagen III in the G2A-R liposarcoma. Compare this section with that of the FBR tumor in Fig lagen III expression pattern (Abbott, D., and J.T. Holt, unpublished data). Novel FBR transcriptional properties such as this may activate genes crucial to the disruption of specific cell lineage differentiation pathways. Such a phenomenon is mimicked by fibroblasts treated with the D N A methylation inhibitor, 5-azacytidine (Chiang, 1981), which enables them to differentiate into chondrocytes, myocytes, and adipocytes (Sager and Kovac, 1982). Absence of myristylation may eliminate these novel FBR properties, restoring FBR's activity to that of its cellular homologue, c-Fos.
Earlier data has demonstrated that the known molecular mechanisms of c-Fos are restored in FBR v-los when its myristylation site is disrupted (Kamata and Holt, 1992;Kamata et al., 1992;Jotte et al., 1994). A restoration of transcriptional properties in G2A-R (so that it behaves as the normal cellular Fos protein) may allow the transformed cells to proceed down the differentiation pathway of the adipocyte lineage and leave other differentiation pathways intact. Many studies implicate Fos protein as playing a role in modulating adipocyte differentiation-specific gene expression (Distel et al., 1987;Rauscher et al., 1988;Herrera et al., 1989;Barcellini-Couget et al., 1993). Fos sequences within FBR may allow transformed cells to proceed down the adipocyte lineage, and lack of myristylation prevents FBR from disrupting multiple differentiation pathways.
Another striking myristylation-dependent feature is the malignant nature of the FBR tumors compared to that of the G2A-R tumors. Not only do a greater number of FBR transgenic mice develop tumors, but they also die from their tumor burden. This does not occur in the G2A-R transgenic mice. Myristylation endows FBR with a greater tumorigenic and malignant potential compared to nonmyristylated G2A-R. This correlates with data observed with c-Fos transgenic mice which present with benign tu- Figure 10. Schematic model depicting sites of F B R and G2A-R disruption of cell-specific differentiation lineages. Diagram represents a mechanism by which F B R may disrupt the differentiation pattern of multiple cell lineages. Broken lines represent the points at which either F B R or G2A-R disrupt normal differentiation. Myristylated FBR disrupts the differentiation pathways of four cell lineages (adipocytes, chondrocytes, osteocytes, and myocytes) early, leading to malignant tumors. When F B R is not myristylated the differentiation pathways of chondrocytes, osteocytes, and myocytes proceed normally.
Nonmyristylated G2A-R still disrupts the differentiation pathway of the adipocyte, causing liposarcomas. However, liposarcomas are a tumor of lesser malignant potential (compared to osteosarcomas, chondrosarcomas, and rhabdomyosarcomas).


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Regional Conference in Sheffield

Professor P Baylis (Royal Victoria Infirmary, Newcastle upon Tyne) summarised the factors influencing fluid balance. The pituitary secretion of vasopressin is controlled by both osmoreceptor and baroreceptor input integrated in anterior hypothalamic cells, although baroreceptor input usually plays little part due to minimal fluctuations in blood pressure. Osmoreceptors in the anterior hypothalamus are sensitive to serum sodium and are of two types, leading to an increase or decrease in vasopressin secretion with hyperor hypo-osmolality respectively. Vasopressin secretion produces a maximum antidiuresis at a concentration of 4 pmol/1; any further increase in plasma osmolality then causes thirst and thus regulates fluid intake, but there is no further increase in the degree of antidiuresis. Drinking stops before osmoreceptors

Hospital, Sheffield in September 1995. The conference was designed to update physicians on a wide range of medical topics, and to discuss controversies that were pertinent at the time. The presentations were given by local and national experts, and were well received by the large audience.

Endocrinology
The management of thyrotoxicosis Professor A P Weetman (Northern General Hospital, Sheffield) gave a resume of the current management of thyrotoxic patients. It depends above all on establishing the correct diagnosis, whether primary or secondary. Graves disease should be treated, in all patients under 50 years, with a six-month 'block and replace' regime of carbimazole, followed by thyroxine once euthyroid status is reached. During pregnancy antithyroid drugs alone should be used, propylthiouracil being the drug least likely to cause adverse fetal side-effects. When the mother has thyroid stimulating receptor antibodies, the infant may become thyrotoxic one week after birth when levels of trans-placental^ transferred antithyroid drugs have diminished. Patients treated with antithyroid drugs should be given a written warning of their possible side effects. If agranulocytosis occurs, treatment with these drugs must be permanently discontinued; transient granulocytopenia, however, resolves spontaneously. Radioactive iodine is used in thyrotoxicosis relapses during treatment with antithyroid drugs, unless the patient is pregnant or breast feeding. The risk of genetic abnormalities is minute (0.002%), thus conception is safe from six months after radioiodine treatment. Although a small but significant increase in the incidence of stomach cancer is noted, the overall cancer rate is not increased. Thyroidectomy provides a rapid cure and is the treatment of choice in large nodular goitres before planned pregnancy, and for resistant thyrotoxicosis in pregnancy and children. Osmoreceptors in the anterior hypothalamus are sensitive to serum sodium and are of two types, leading to an increase or decrease in vasopressin secretion with hyper-or hypo-osmolality respectively. Vasopressin secretion produces a maximum antidiuresis at a concentration of 4 pmol/1; any further increase in plasma osmolality then causes thirst and thus regulates fluid intake, but there is no further increase in the degree of antidiuresis. Drinking stops before osmoreceptors can adjust to lowered plasma osmolality because the mechanism of swallowing appears directly to inhibit vasopressin secretion. Polyuria may be due to a greater fluid intake, a small vasopressin release or a decreased renal response to vasopressin. Diagnosis may be made either by water deprivation or by measuring plasma vasopressin following an osmotic stimulus. Chronic hypernatraemia may be caused by an isolated osmoreceptor defect where both thirst sensation and vasopressin response to hyperosmolality are suppressed yet baroreceptor stimuli continue to elicit vasopressin secretion, or may be a result of resetting the osmoreceptors to a higher setpoint yet with otherwise normal regulation. In the former case patients are at risk from the effects of hypo-or hyper-natraemia, while in the latter there is no increased risk.
Growth hormone treatment in the adult Dr R Ross (Northern General Hospital, Sheffield) reviewed the effects of growth hormone deficiency and growth hormone therapy. Growth hormone deficiency in adults increases the amount of subcutaneous fat and is associated with decreased fat metabolism, poor muscular development, and impaired psychological well being [1], and a higher risk of cardiovascular deat^i. The most common cause of growth hormone deficiency is pituitary tumour or its treatment; it is diagnosed by measuring growth hormone, insulin like growth factor-1 (IGF-1) and IGF-BP3 secretion but these rates are influenced by age, gender and nutrition. Thus to confirm the diagnosis of growth hormone deficiency an insulin stress test should be performed, or in the presence of known pituitary disease a deficit of one other pituitary hormone should be confirmed. Treatment with growth hormone lowers body fat and raises fat-free mass, but total bone mass increases only in subjects with childbirth-onset growth hormone deficiency; it improves the quality of life in perhaps 50% of patients. It is very expensive (?5,000 a year) and only 50% of patients Rapporteurs: A M ROBINSON, mrcp, Senior Registrar to Sheffield Hospitals continue with long-term treatment due to the side effects. These considerations must be borne in mind when deciding whom to treat, at what dose and for how long, and how to monitor treatment.
Another use for growth hormone may be for protein-malnourished people whose nutritional state is an important predictor of survival. These people are growth hormone resistant and have high levels of growth hormone and low levels of IGF-1, yet some patients do respond to growth hormone treatment with an increase in IGF-1, a cumulative positive nitrogen balance [2]  Patients on long-term parenteral nutrition in the first year of life may develop cholestatic jaundice and will then require combined liver and small intestine transplantation. Professor R F M Wood (Northern General Hospital, Sheffield) described some of the challenges ?f small bowel transplantation. They arise because near perfect function must be obtained and complex immunological problems occur because the large amount of lymphoid tissue provides the potential for graft-versus-host-disease (GVHD), as well as rejection. Extensive migration of host T lymphocytes five days after transplantation leads to an early rejection reaction, and the migration of graft T lymphocytes to the host's Peyer's patches within 24 hours triggers GVHD. Cyclosporin A can prevent early rejection in the presence of continuing T lymphocyte migration, leading to stable chimerism. Before Cyclosporin A was available, mortality was mainly due to sepsis because of mipairment of the intestinal barrier function, but now death after transplantation is caused by lympho-Proliferative disease associated with cytomegalovirus infection and OKT3 usage. Of the seven small bowel transplant recipients in Britain, three are still alive.
Further developments in immobilisation of both antigen recognition systems and cytokines must occur before small intestinal transplantation can become commonplace. Stents cannot be used in small distal vessels because of a high reocclusion rate but in proximal vessels they remain patent longer and have a lesser residual gradient than vessels treated by angioplasty. Thrombolysis can be used for critical limb ischaemia provided irreversible damage has not occurred. Lysis is successful in 40-78% of patients, however 51% have haemorrhagic complications which are mostly minor at the puncture site but severe in 5%. Death occurs in 1-2%, mostly due to cerebral haemorrhage. Carotid angioplasty is currently being studied at the Royal Hallamshire Hospital as part of the Cavitas Trial. Early results indicate that carotid angioplasty is relatively safe, can reduce stenosis to less than 50% and is clinically effective. Endovascular management of aortic aneurysms can be achieved by placing a covered stent within the body of an aneurysm. Although patients treated by the above procedures often have other health problems or limited life expectancy, the procedures are relatively non-invasive.
Adult respiratory distress syndrome Dr T W Evans (Royal Brompton Hospital, London) discussed recent advances in the treatment of adult respiratory distress syndrome (ARDS). It is associated with a range of medical and surgical conditions, both pulmonary and extrapulmonary, and may vary from mild pulmonary oedema to dense inflammatory infiltrate, hypoxaemia and death. Pulmonary endothelial cells may be the target of inflammatory mediators, and subclinical endothelial damage also occurs in other organs. The aims of treatment are adequately to oxygenate the tissues and to provide multisystem support until recovery occurs. Continuous positive airways pressure (CPAP) is often adequate for respiratory support in mild cases although in more severe cases inverse ratio ventilation (IRV) or pressure controlled IRV causes less barotrauma. Extracorporeal membrane oxygenation (ECMO), intravascular oxygenation (IVOX), and the use of pulmonary surfactant to lower the pulmonary transfer coefficient, require further research before introduction into clinical use. Pulmonary hypertension is a significant cause of mortality in ARDS. In 50% of patients inhalation of nitric oxide has been found to lower local vascular resistance whereas intravenous vasodilators, including salbutamol, may adversely affect ventilation/perfusion. On withdrawal there may be a marked increase in vascular resistance and thus the side effects need to be further elucidated. Patients with ARDS secondary to trauma do well, but when it is associated with sepsis, mortality has remained high.
The Linacre Lecture The role of matrix degradation in liver fibrosis Professor M J P Arthur (University of Southampton) discussed the role of matrix degradation in liver fibrosis.
In cirrhosis, lipocytes proliferate. They are the predominant matrix-producing cells, forming collagen type I and III, with resulting fibrosis irrespective of the initial liver injury. Proliferation of lipocytes is mediated by platelet-derived growth factors produced by Kupffer cells, and by factors produced by hepatocytes undergoing apoptosis. The activated lipocytes produce monocyte chemotactic factor which is profibrogenic and increases mast cell response. As liver fibrosis progresses, interstitial collagenase activity diminishes so that there is less degradation of collagen I and III. Collagenase activation is also inhibited by the plasminogen activating system (PAS), components of which are expressed in lipocytes from injured but not from normal liver. Activated lipocytes also produce tissue inhibitors of metalloproteinase (TIMP) such as TIMP-1. TIMP is increased 4 to 5 fold in the sinusoidal lipocytes from fibrotic explants of human liver but there is no change in interstitial collagenase level. Thus liver fibrosis is a result of lipocyte activation and proliferation mediated by fibrogenic cytokines and other factors, with consequent alteration in matrix synthesis and degeneration. Current treatment of liver fibrosis includes removal of the stimulus, and anti-inflammatory drugs. Future therapeutic possibilities include inhibition of lipocyte activation, decreasing matrix synthesis, and enhancing matrix degradation; the last is Professor Arthur's preferred option.

Movement disorders
Professor H Sagar (Royal Hallamshire Hospital, Sheffield) reviewed the recent changes in emphasis that have occurred in the treatment of Parkinson's disease. It is now thought that the cause is an environmental insult in patients with a genetic predisposition which leads to cell death in the substantia nigra. He dismissed the role of selegeline in the early treatment of the disease. He discussed the evidence that treatment with levodopa given as pulsatile treatment rather than as controlled release preparation is more likely to lead to motor complications. He suggested therefore that low-dose controlled release formulations should be used in the early part of treatment, followed by dopamine agonists as there is now some evidence that they might be neuroprotective. If fluctuations occur, the dose of levodopa should be increased until the patient is 'on'. To improve levodopa transport, selegeline and a high protein diet can be added. However, once complications have developed the mainstay of treatment is dopamine agonists. There is evidence that if one fails to produce desired effects, another should be tried; pergoline appears to be the most useful to date. The role of apomorphine was discussed, and although this drug still has to be given parenterally, it remains very useful for treating the on/off phenomenon; its therapeutic effect showed no fall-off after a five year follow-up study; it can also be used if there is uncertainty about the diagnosis of Parkinson's disease. Future therapeutic possibilities include a role for surgery: pallidotomy may be useful for the treatment of akinesia and rigidity; thalamic stimulation may be as effective and is reversible.

Seizure disorders
Dr P Baxter (Sheffield Children's Hosptial) discussed the problems of epilepsy from the patient's perspective. Patients with uncomplicated epilepsy have a poorer quality of life due to problems in gaining employment, restriction on driving and drug side effects. When the social progress of two children with similar IQs is followed, the one with epilepsy will fare the worse; this relates to prejudice at school, in particular lower expectations from teachers, who restrict patients' activities; also drugs such as vigrabatin and phenobarbitone may cause behavioural problems. Dr Baxter suggested that there should be no restrictions on childhood activity, drugs with adverse effects should be avoided and patients should be empowered to be more in control of their situation.
He also emphasised the need for specialist care, as it is important to make the correct diagnosis; to illustrate this point, he described Rolandic epilepsy which occurs in the morning in young teenagers but disappears by 16 years of age and does not require treatment, while Janz epilepsy is clinically similar but never remits and is made worse if treated with carbamazepine. Improvements in management may be expected from the newer drugs with fewer side effects and absence of drug interactions and the availability of controlled release preparations. Despite this improvement in the therapeutic options, he suggested that there was still a role for surgery in patients with intractable epilepsy.

Neuromuscular disorders
Dr R Petty (Royal Hallamshire Hospital, Sheffield) used myasthenia gravis as an illustration of autoimmune neuromuscular disorder, and showed how this model can be used to unravel other apparently autoimmune neurological disorders. Antibodies are pathogenic in the disease and reducing the antibody load should be the aim of treatment. Treatment would be aimed more specifically at antibody elimination, and animal models are being developed to produce antibodygenerated antagonists against the autoantibody. In Guillain-Barre syndrome (GBS) patients with mainly demyelination usually recover well while those with axonal degeneration tend to do badly. In the Miller-Fisher variant of GBS, an antibody against Gql6 is universally found in neuronal tissues. Its site of action is close to that of botulin toxin, which suggests that GBS has an autoimmune basis. This is further supported by a recent study in which treatment with gangliosides inadvertently caused severe GBS in 7 out ?f 15 patients; these patients developed an IgG antibody to the ganglioside GM1 which has also been found in patients with previous Campylobacter infection and axonal GBS.
The chronic inflammatory demyelinating polyneuropathies may also have an autoimmune basis, especially those 35% who have a monoclonal band in their blood; 60% of them have a severe sensory ataxia and an IgM band which binds to myelin associated glycoprotein (MAG), a component of myelin sheath. This ataxia responds poorly to steroids but can be treated with cyclophosphamide or chlorambucil.
Multifocal motor neuropathy with conduction block is a purely motor syndrome which mimics motor neuron disease but does not affect speech or swallowing and the EMG shows a patchy slowing of conduction. It is treatable with immunoglobulins, and probably has an autoimmune basis.

Cerebrovascular disorders
Dr G Venables (Royal Hallamshire Hospital, Sheffield) suggested that treatments aimed at primary prevention in high risk patients prevent only 5% of strokes. It is therefore important to develop therapy to reduce the immediate morbidity and mortality. At the onset of a stroke, a cascade of metabolic disturbances occurs which leads to cell death. It should be possible to interrupt this cascade at several steps and so reduce cell death. Lifarizine, an ion channel blocker, is presently being evaluated. So far, however, the only study to have shown an improved patient outcome was in patients treated in stroke units. Dr Venables felt it was beneficial to maintain the diastolic blood pressure below 110 nimHg, but due to impaired autoregulation of blood Pressure following a stroke it might be more prudent to treat persistent mild hypertension later rather than during the acute event. Dr Venables said that there is no place for acute carotid endarterectomy, and aspirin does not improve outcome. The role of heparin is being evaluated at the present time in the International Stroke Trial. Studies on the use of thrombolysis in acute stroke have been stopped prematurely, but there may be a subgroup of patients who would benefit from this treatment. Haemodilution, calcium antagonists, steroids, praxilene, naloxone and glycerol have no Place in the treatment of acute stroke.

Rheumatology
Early rheumatoid arthritis Dr J Winfield (Nether Edge Hospital, Sheffield) described the present nation-wide study to determine the features of early rheumatoid arthritis of less than two years' duration which will be used to develop a rationale of when and whom to treat. Early results show the expected female predominance (2:1), with an average age of 55. Nearly half the patients presented with an acute onset, but only a third initially had small joint involvement, while 17% (mainly elderly) presented with only large joint disease. Three years after the first patients were recruited, 47% have persistent disease, 14% have remitted, while the rest follow a relapsing/remitting course. There appear to be no predictive factors to date, the best indicator being the quality of life score. Most patients show an improvement in the first six months which is maintained for two years, but the disease then progresses.
After the first year 95% of patients were on disease modifying treatment: sulphasalazine was the drug most often used, methotrexate was under-used at that stage of the study because its rise to prominence occurred later. Serious adverse drug events were few and all were reversible. However, overall mortality was higher than expected, mainly due to ischaemic heart disease. Although there were no deaths directly related to rheumatoid arthritis, nearly all the patients had lost at least four weeks' work due to the disease and a quarter had retired on medical grounds.

Why, when and how in the treatment of arthritis
Dr R Amos (Nether Edge Hospital, Sheffield) questioned previously published statements that rheumatoid arthritis is a mild disease. He suggested that such evidence had been based on population studies where few of the patients met all the necessary criteria for the diagnosis of rheumatoid arthritis. Most patients who do meet them will develop permanent joint damage and there will be an excess of deaths compared with the normal population. Patients with a normal acute phase response develop fewer erosions than those with a high response. Therefore the rationale for treatment must be to reduce the acute phase response. In this respect NSAIDs have no role, but can be used for symptomatic relief. The treatment of choice is the early use of disease modifying drugs that reduce the acute phase response as they are then better tolerated and more likely to affect outcome. Methotrexate is probably the most effective drug. If he himself had rheumatoid arthritis, Dr Amos would start sulphasalazine on day one, and add methotrexate after three months. There is no evidence to suggest that combining these drugs increases side effects and the drugs may work better together; finally, after five months he would add prednisolone at low dose. If the disease remits, the drugs should not be stopped as this would double the flare-up rate.

Nephrology
Drug induced renal disease Dr A El Nahas (Northern General Hospital) reviewed the role of NSAIDs in renal disease by illustrating three cases, described as the good, the bad and the ugly. These cases were based on the inhibiting effect of NSAIDs on prostaglandin activity; this prevents vasodilatation of the afferent arteriole and vasoconstriction of the capillary bed. The effects on the afferent arteriole can be beneficial in a young patient with chronic mesangiocapillary glomerulonephritis and heavy proteinuria. NSAIDs by their action on the arteriole can reduce proteinuria and slow the rate of decline of the glomerular filtration rate. However, NSAIDs given to a patient with chronic analgesic abuse and impaired renal function may precipitate acute or chronic renal failure because the kidney depends on the afferent vasodilatation to maintain glomerular filtration. If this is inhibited the glomeruli are under-perfused, and both prerenal failure and tubular necrosis can develop. Azapropazone appears to be the worst culprit. Improvement is likely in 90% of patients if the drug is withdrawn, but often four to six weeks of dialysis are required; full recovery occurs in only 50-70%. The third case illustrates the ugly side of the NSAIDs. The patient had congestive cardiac failure, intermittent claudication and was being treated with a diuretic and an ACE inhibitor. The patient was given a NSAID for arthritic pain and, due to unrecognised bilateral renal artery stenosis, developed acute renal failure secondary to afferent arteriolar hypotension. This is caused by the combined effect of the NSAID and ACE inhibitor. If the drugs are stopped, patients may improve, but often require dialysis for life. Such patients should avoid this combination of drugs if possible, but if that is not possible, renal function should be checked before and soon after starting the drug.

Update on lipid management
Professor L Ramsay (Royal Hallamshire Hospital, Sheffield) said that the Scandinavian Simvastatin Survival study [6] has convincingly shown that it is possible to identify patients who would benefit from having their total blood cholesterol level reduced by 25% from the starting point and so reduce their coronary heart disease risk. Because the benefit is apparent within two years, there should be no age barrier. Patients with other forms of macrovascular diseases are also at risk of coronary heart disease and should therefore also be aggressively treated if the total cholesterol is above 5.5 mmol/1. In this first of a series of debates concerning present controversies, the opponent was Professor Anne Tattersfield (City Hospital, Nottingham). She opened by saying that there was no doubt that the right betaagonist, given at the right time and in the right dose, could be life saving. However, the mortality peak in the 1960s coincided with the availability of isoprenaline over the counter, and since no other cause has been found it is presumed that this drug was responsible.
The same case has been made for fenoterol which was associated with a mortality peak in New Zealand in the 1980s. This drug was also sold as a high dose formulation and again no other explanation has been found.
There is some evidence that taking beta-agonists on a regular basis is less effective than taking them as required. When patients were changed from high dose beta-agonists to placebo and vice versa, more patients felt better on placebo than on treatment [3]. It has also been suggested that when beta-agonists are taken regularly their protective effect wears off and bronchial reactivity may increase. If the patient has poor lung function and little insight, this could prove to be a significant problem. In the case of long acting beta-agonists, Professor Tattersfield felt there was still some concern, although this was becoming less as experience grew. The early post-marketing surveillance of salmeterol has shown a non-significant threefold increase in mortality. This needs to be followed-up as pharmacological studies with high doses of the drug have shown an increase in heart rate and a reduction in potassium levels. Dr A Morice (Royal Hallamshire Hospital) supported the idea that taking beta-agonists regularly is safe and that despite an increase in the sale of these drugs over the past few years, mortality from asthma has remained unchanged. He felt that if GPs educated their patients to use steroid inhalers more regularly and appropriately, then it was illogical but not dangerous to use regular beta-agonists. The problem in New Zealand could be due to the ethnic mix of patients living in a very rural setting who were less likely to seek help at the appropriate time. He quoted a study [4] which showed that regular salbutamol was better than taking it as required, and a meta-analysis of nebuliser studies suggested that this was a better form of treatment and was not associated with a higher mortality rate . However, in the subsequent debate Dr Morice said if a patient was in extremis and was given a nebuliser without oxygen, the patient's oxygen saturation could be further compromised and this would hasten his death. Should  Furthermore, if patients can complete the exercise protocol on the treadmill they have a better prognosis than those who only manage stage 1, especially if they have to stop because of chest pain. A group of patients can then be identified who require referral for angiography. On these criteria and with patients who have failed on medical treatment, it is possible to select a group of patients who will have a 90% intervention rate following angiography. Dr Channer then discussed the role of the ETT following myocardial infarction. He felt this was safe, prognostically useful and helped rehabilitation. Dr G Oakely (Northern General Hospital, Sheffield) opposed these views, pointing out that the question was whether everyone should have an exercise test. Many patients are physically incapable of performing an ETT adequately. A 60-year old man who smokes, has a positive family history and a non-specific ECG and is on quadruple therapy, should have an angiogram rather than await the results of an ETT. A negative ETT does not exclude ischaemic heart disease and correlates poorly with the pathology. In a recent study of patients after Myocardial infarction [5] only 60% were able to Undergo an ETT. Although the mortality was greater !n patients with a positive test, the mortality in those who could not perform the test at all was four times higher again. Furthermore, most of the mortality occurred in the first 12 months which, in several hospitals, is the duration of waiting list time for angiography and surgery, and an ETT may delay this even more. However, the consensus was that an ETT appears to help the rehabilitation prospects of post-infarct Patients, and should be part of their workup, if appropriate.
Endoscopy: too many, too few? Dr S Riley (Northern General Hospital, Sheffield) reviewed the use of endoscopy over the previous years. The number of endoscopies has risen 8-fold since the 1970s, but the number of barium meals has dropped by only 40%. Endoscopy has several advantages because the patient's history is not very useful in making a diagnosis. The incidence of cancer is agerelated and open access endoscopy for the over forties has increased the number of operable lesions. It was suggested that treating younger patients empirically would reduce the total number of endoscopies, but in response Dr D Dawson (Northern General Hospital, Sheffield) said that those patients that did have endoscopy, even if it was normal, were strongly reassured. Furthermore, early endoscopy leads to less sick leave and reduces drug treatment. Present figures show that there would not have to be a great increase in hospital resources to endoscope all patients referred by GPs, but this needs to be fully costed; and it may be possible, as already being tried in some centres, to employ nurse endoscopists.


extfieldsofstudy: Chemistry Biology Medicine

text:
Endothelial Nitric Oxide Synthase

Localization The endothelial nitric oxide (NO) synthase (eNOS) is a constitutively expressed 135-kD protein predominantly associated with the particulate subcellular fraction, suggesting that the native enzyme is a membrane-bound protein [1]. Membrane association is achieved by attachment of myristic acid to the amino terminal end of the enzyme and as such is consistent with reports that eNOS cDNA contains a consensus sequence for co-translational modification of the enzyme by N-terminal myristoylation. Preventin of myristic acid inorporation in site-directed mutagenesis experiments converse the membrane-associated eNOS to a cytosolic form but has little effect on enzyme activity. Myristoylation alone, however, provides barely enough energy to anchor a protein to a lipid bilayer, therefore other factors are important in determining the association of the enzyme with the plasma membrane, e.g., additonal hydrophobic interactions owing to reversible palmitoylation of a nearby cysteine residue. A recent detailed analysis of the membrane association of eNOS has demonstrated that this enzyme is localized to specific structures in the plasmalemmal membrane identified as caveolae and that this membrane compartmentalization requires both myristoylation and palmitoylation. In cases of such a dual protein acylation the combined hydrophobic interactions of the two covalently attached lipid moieties anchor the protein firmly to the plasma membrane and attachment could be regulated by palmitate turnover. Although eNOS is reported to be reversibly depalmitoylated in response to certain stimuli, the role of actively regulated eNOS palmitoylation in determining its cellular localization remains controversial. The association of eNOS with a subcompartment of the plasma membrane in which several key signal-transducing complexes are concentrated (e.g., G proteins, src-family tyrosine kinases) is likely to have profound repercussions on enzyme activity as well as on its sensitivity to activation by ignal transduction cascades other than those resulting in an increase in intracellular Ca 2+ ([Ca]i). Indeed, the association of eNOS with the caveolar scaffolding and marker protein caveolin-1 attenuates enzyme activity [2]. Moreover, the variety of additional signal molecules detected within the caveolae render these structures ideal sites for cross-talk between receptor-dependent and independent signal-transduction pathways.

Localization
The endothelial nitric oxide (NO) synthase (eNOS) is a constitutively expressed 135-kD protein predominantly associated with the particulate subcellular fraction, suggesting that the native enzyme is a membrane-bound protein [1]. Membrane association is achieved by attachment of myristic acid to the amino terminal end of the enzyme and as such is consistent with reports that eNOS cDNA contains a consensus sequence for co-translational modification of the enzyme by N-terminal myristoylation. Preventin of myristic acid inorporation in site-directed mutagenesis experiments converse the membrane-associated eNOS to a cytosolic form but has little effect on enzyme activity. Myristoylation alone, however, provides barely enough energy to anchor a protein to a lipid bilayer, therefore other factors are important in determining the association of the enzyme with the plasma membrane, e.g., additonal hydrophobic interactions owing to reversible palmitoylation of a nearby cysteine residue. A recent detailed analysis of the membrane association of eNOS has demonstrated that this enzyme is localized to specific structures in the plasmalemmal membrane identified as caveolae and that this membrane compartmentalization requires both myristoylation and palmitoylation. In cases of such a dual protein acylation the combined hydrophobic interactions of the two covalently attached lipid moieties anchor the protein firmly to the plasma membrane and attachment could be regulated by palmitate turnover. Although eNOS is reported to be reversibly depalmitoylated in response to certain stimuli, the role of actively regulated eNOS palmitoylation in determining its cellular localization remains controversial.
The association of eNOS with a subcompartment of the plasma membrane in which several key signal-transducing complexes are concentrated (e.g., G proteins, src-family tyrosine kinases) is likely to have profound repercussions on enzyme activity as well as on its sensitivity to activation by signal transduction cascades other than those resulting in an increase in intracellular Ca 2+ ([Ca 2+ ] i ). Indeed, the association of eNOS with the caveolar scaffolding and marker protein caveolin-1 attenuates enzyme activity [2]. Moreover, the variety of additional signal molecules detected within the caveolae render these structures ideal sites for cross-talk between receptor-dependent and independent signal-transduction pathways.
Functional Properties eNOS is classified as a Ca 2+ /calmodulin-dependent enzyme and exhibits low NO output at resting levels of [Ca 2+ ] i . Ca 2+ concentration-dependently increases activity of the purified eNOS and an increase in [Ca 2+ ] i , such as that observed following agonist stimulation, enhances endothelial NO production while both the agonist-induced NO formation and the subsequent vasodilatation are abolished in the absence of extracellular Ca 2+ [3]. A basal enzyme activity, which is sensitive to the NO synthase inhibitor, N G -nitro-L-arginine, is however evident event at Ca 2+ concentrations as low as 10 nM indicating that approximately 75% of basally produced NO may be formed via a Ca 2+ -independent pathway. Little physiological relevance was attributed to this phenomenon and the identification of a calmodulinbinding domain in the primary structure of the eNOS together with the finding that calmodulin-binding proteins inhibited enzyme activity strengthened the hypothesis that the binding of a Ca 2+ /calmodulin complex is essential to acti-

Key Words
Nitric oxide synthase · Endothelial cells · Shear stress vate the constitutive enzyme. However, recent evidence indicates that mechanical stimulation of native and cultured endothelial cells by fluid shear stress results in the formation of NO via a pathway which comprises Ca 2+ -dependent as well as Ca 2+ -independent components [4,5]. In native endothelial cells, the shear stress-induced NO production consists of an initial peak followed by a sustained plateau phase which is maintained as long as shear stress is applied. The initial component of this response can be abolished by removal of Ca 2+ from the extracellular medium and coincides with an increase in [Ca 2+ ] i observed in cultured endothelial cells exposed to shear stress in a parallel plate flow chamber. Moreover, the sustained phase of NO production is insensitive to the chelation of extracellular Ca 2+ as well as to calmodulin antagonists [4,5]. Thus a signalling pathway distinct from the classically accepted Ca 2+dependent pathway regulates the activity of eNOS in response to shear stress. While eNOS is highly sensitive to variations in pH i at basal levels of [Ca 2+ ] i , it is likely that additional signalling pathways, protein-protein interactions and/or post-translational modification of the eNOS protein are involved in the regulation of endothelial NO production. Other possibilities include the activation of various protein kinase since several consensus sequence sites for phosphorylation by protein kinase A, B (Akt), C and calmodulin kinase II are found in all the cloned NOS isoforms. However, the functional relevance of these sites has not been fully elucidated. Recently interest has concentrated upon the role of other proteins in the regulation of endothelial NO formation. Indeed, eNOS appears to exist as part of a multimolecular complex in unstimulated endothelial cells and immunoprecipitation of the enzyme results in the selective co-precipitation of a number of other endothelial proteins including 2 as yet unidentified proteins of 2200-210 and 69-72 kD as well as calmodulin, caveolin and Hsp90 [6]. Hsp90 appears to directly activate eNOS as the formation of an eNOS/Hsp90 complex results in an increase in NO production from both cultured and native endothelial cells [6].
Cellular levels of phosphotyrosine appear to have a marked impact on cellular signalling in endothelial cells and a link between enhanced tyrosine phosphorylation and activation of eNOS has been proposed independent of the effects of these inhibitors on Ca 2+ signalling. There are, for example, fundamental difference in the role played by phosphotyrosine in mediating endothelial responsiveness to agonist-induced stimulation and mechanical stimulation as the tyrosine kinase inhibitor, erbstatin A, only slightly attenuates the vasodilator response to acetylcholine in perfused carotid arteries, while it abolishes the Ca 2+ -independent, shear stress-induced NO production [5]. Enhancing cellular levels of phosphotyrosine by inhibiting protein tyrosine phosphatases also elicited the Ca 2+ /calmodulin antiagonist-independent, erbstatin A-sensitive formation of NO [7]. Thus the endothelial response to the application of tyrosine phosphatase inhibitors exhibits characteristics identical to those of the shear stress-induced activation of eNOS. The sensitivity of the shear stress-induced activation of eNOS to tyrosine kinase inhibitors led to the suggestion that eNOS activity may be regulated by tyrosine phosphorylation. Although the tyrosine (and serine) phosphorylation of eNOS does increase transiently following the application of fluid sher stress, this appears to be a Ca 2+ -dependent phenomenon and phosphorylation levels return rapidly to basal levels although the Ca 2+ -independent output of NO is maintained [8]. Examination of the effects of shear stress and tyrosine phosphatase inhibitors on eNOS revealed that both stimuli result in the time-dependent decrease in the recovery of the enzyme from the Triton X-100-soluble cell fraction and a concomitant increase in its recovery in the insoluble fraction [7]. A similar change in detergent solubility was observed with three tyrosine-phosphorylated proteins, i.e., the focal adhesion kinase, the cytoskeletal-associated protein paxillin, and the caveolae marker protein caveolin. The shear stress-induced tyrosine phosphorylation and redistribution of these proteins to the detergent-insoluble fraction was attributed to activation of an integrin-coupled signal transduction pathway since both effects were abolished by interfering with the association of integrins with the extracellular matrix. Given that a change in detergent solubility is frequently indicative of the formation of a protein:protein or a protein:lipid complex, it is conceivable that shear stress elicits the formation of such a multi-molecular complex comprised of eNOS, tyrosine kinases and phosphatases as well as cytoskeletal and caveolar proteins. This complex, which could regulate the maintained Ca 2+ -independent activation of eNOS, may also include Hsp90 as geldanamycin, which prevents the association of Hsp90 and eNOS [6], abrogates the shear stress-induced Ca 2+ -independent activation of eNOS and attenuates the alteration in eNOS detergent solubility [7].

Transcriptional Regulation of eNOS
Although generally referred to as a constitutive enzyme eNOS can be induced or upregulated in response to a number of stimuli. Indeed, the promoter of the eNOS gene contains a number of recognition sites for transcription factors, including SP1, AP-1, AP-2 CF-1 and NF-1 as well as a putative acute phase response/shear stress, and sterol-regulatory cis elements.

NO Synthesis in Endothelial Cells
Kidney Blood Press Res 1998;21:264-266 tracellular pathway resulting in this increase in eNOS expression and increased basal NO production differs from that of insulin as eNOS mRNA levels remained elevated in VEGF-treated cells in the presence of actinomycin D. Thus it is likely that the VEGF-induced upregulation of eNOS expression is the consequence of a post-transcriptional effect on eNOS mRNA stability [10]. Cytokines also effect the expression of eNOS. TNF-α, for example, decreases NOS mRNA levels by increasing the rate of mRNA degradation via a process that involves de novo protein synthesis [11]. The combination of IFN-γ plus TNF-α or IL-1 has, on the other hand, been shown to paradoxically enhance eNOS activity, despite a concurrent decrease in NOS mRNA, an effect which may be accounted for by increased endogenous BH 4 levels [12].
Fluid shear stress plays a role in the chronic regulation of eNOS, an effect which may account for the fact that the basal release of NO is significantly greater from native endothelial cells, continuously exposed to shear, than that from confluent cultured endothelial cells maintained under static conditions. A similar effect in endothelial cells in situ is thought to underlie the beneficial effects of exercise of eNOS levels. Apart from shear stress, eNOS expression can be enhanced by basic fibroblast growth factor and appears to be negatively regulated by protein kinase C. Insulin also enhances the expression of eNOS mRNA and protein in native endothelial cells via a signalling cascade involving the activation of Erk1/2, Jak2, and STAT1 as well as increased DNA-binding activity of the transcription factors AP-1 and SP1 [9]. Vascular endothelial growth factor (VEGF 165 ) also increases eNOS mRNA and protein levels. However, the in-