Regulation of tyrosine aminotrasferase activity in two liver-derived permanent cell lines.

The regulation of tyrosine aminotransferase (TAT) activity has been examined in two liver-derived heteroploid cell lines. One (hepatoma tissue culture cells [HTC]) was derived from a hepatoma, the other (rat liver culture cells [RLC]) was derived from normal liver. The two cell lines show the following striking similarities in the control of this specific protein: (a) The kinetics of TAT induction by dexamethasone phosphate (DxP) are similar in randomly growing cells of both lines; (b) During mitosis and early G(1) phase of the cell cycle TAT activity cannot be induced by DxP in either cell line; (c) 2-3 h into G(1), when both lines become sensitive to inducer, basal enzyme activity declines to a new steady-state level; (d) Preinduced cells collected in mitosis show approximately twice the level of TAT activity as fully induced, randomly growing cultures and this activity is maintained in early G(1) with or without the inducer; and (e) Inhibition of RNA synthesis by 5 microg/ml of actinomycin D in preinduced, synchronized cells allows TAT activity to remain at constitutive levels throughout G(1), even in the absence of inducer. These results are presented in support of a previously described model which states that glucocorticoid hormones exert posttranscriptional control of the synthesis of specific proteins in mammalian cells.

Receptor isoforms mediate opposing proliferative effects through gbetagamma-activated p38 or Akt pathways.

The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst(2) receptor isoforms, which couple to identical Galpha subunit pools (Galpha(i3) > Galpha(i2) >> Galpha(0)), were both inhibited following betagamma sequestration. The sst(2(a)) receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70(rsk) induced by sst(2(b)) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst(2(a)) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21(cip1) was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst(2(a)) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst(2(a)) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst(2) receptor isoforms may determine the selection of appropriate betagamma-pairings necessary for interaction with distinct kinase cascades.

On-target sorafenib toxicity predicts improved survival in hepatocellular carcinoma: a multi-centre, prospective study.

BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and has high mortality despite treatment. While sorafenib has a survival benefit for patients with advanced HCC, clinical response is highly variable. AIM: To determine whether development of sorafenib toxicity is a prognostic marker of survival in HCC. METHODS: In this prospective multicentre cohort study, patients with advanced-stage HCC receiving sorafenib were recruited from five international specialist centres. Demographic and clinical data including development and grade of sorafenib toxicity during treatment, radiological response to sorafenib and survival time (months) were recorded prospectively. RESULTS: A total of 634 patients with advanced-stage HCC receiving sorafenib were recruited to the study, with a median follow-up of 6692.3 person-months at risk. The majority of patients were male (81%) with Child-Pugh A stage liver disease (74%) and Barcelona Clinic Liver Cancer stage C HCC (64%). Median survival time was 8.1 months (IQR 3.8-18.6 months). 94% experienced at least one sorafenib-related toxicity: 34% diarrhoea, 16% hypertension and 37% hand-foot syndrome (HFS). Twenty-one per cent ceased sorafenib due to toxicity and 59% ceased treatment due to progressive disease or death. On multivariate analysis, sorafenib-related diarrhoea (HR 0.76, 95% CI 0.61-0.95, P = 0.017), hypertension (HR 0.531, 95% CI 0.37-0.76, P < 0.0001) and HFS (HR 0.65, 95% CI 0.51-0.81, P < 0.0001) were all significant independent predictors of overall survival after adjusting for age, severity of liver disease, tumour stage and sorafenib dose. CONCLUSION: Development of sorafenib-related toxicity including diarrhoea, hypertension and hand-foot syndrome is associated with prolonged overall survival in patients with advanced-stage HCC on sorafenib.

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.

Purification of a 20 kDa phosphoprotein from epithelial cells and identification as a myosin light chain. Phosphorylation induced by enteropathogenic Escherichia coli and phorbol ester.

Previous studies on the mechanism of enteropathogenic Escherichia coli (EPEC) infection have revealed an increase in the phosphorylation state of a number of proteins in human laryngeal HEp-2 cells. The most prominent was an acidic phosphoprotein(s) of Mr 20-21 kDa. The present study reports: (a) a simple method for purification of phosphorylated 20 kDa protein; (b) identification of the 20 kDa phosphoprotein as myosin light chain; and (c) that the phorbol ester, TPA, also increased the phosphorylation of the 20 kDa myosin light chain. In contrast to the effects of EPEC, TPA stimulation resulted in the dissociation of myosin from the cytoskeleton to the cytosol.

Immunohistochemical localization of the somatostatin SST2(A) receptor in the rat brain and spinal cord.

The neuropeptide somatostatin is widely distributed in the CNS and is believed to play a role as a neurotransmitter or a neuromodulator. Somatostatin mediates its actions by the binding of the peptide to high affinity membrane receptors. The genes for five somatostatin receptor types have been cloned recently and Northern blotting and in situ hybridization studies have shown that the transcripts of all five types are expressed in the CNS. Here we report the cellular distribution of somatostatin sst2(a) receptor protein in the adult rat CNS, using a polyclonal anti-peptide antibody directed against a portion of the C-terminal domain of the receptor. The specificity of the affinity-purified antibody was demonstrated by Western blotting and immunolabelling of cells transfected with a hemagglutinin epitope-tagged version of the sst2(a) receptor. Immunohistochemistry showed a distinct distribution of the receptor protein in the rat brain. Cells and processes were labelled in a number of areas, including the basolateral amygdala, the locus coeruleus, the endopiriform nucleus, the deep layers of the cerebral cortex, the subiculum, the claustrum, the habenula, the interpenduncular nucleus, the hippocampus and the central grey. In the spinal cord, the substantia gelatinosa showed strongly-labelled cell bodies and their processes. This study provides an improved understanding of the distribution of the sst2(a) receptor in rat brain.

Somatostatin sst5 inhibition of receptor mediated regeneration of rat aortic vascular smooth muscle cells.

1. The aim of the present study was to determine the effect of somatostatin (SRIF) on mitogen-induced regeneration of rat aortic vascular smooth muscle cells (VSMC) and for comparison Chinese hamster ovary (CHO)-K1 cells expressing human recombinant sst5 receptors (CHOsst5), following partial denudation of a confluent cell monolayer. Regeneration was assessed by measuring areas of recovery into the denuded area and by counting total cell numbers. 2. In VSMC, SRIF (0.1 nM - 1 microM) had no effect on the basal levels of regeneration but caused a concentration-dependent inhibition (pIC50 8.0-8.6) of the stimulated regeneration induced by submaximal concentrations of basic fibroblast growth factor (bFGF, 10 ng ml[-1]), platelet-derived growth factor-BB (PDGF, 5 ng ml[-1]) or endothelin-1 (ET-1, 100 nM). SRIF (pIC50 8.8) also inhibited bFGF-induced regeneration of CHOsst5 cells. 3. In VSMC, the inhibitory action of SRIF on the regeneration induced by bFGF (10 ng ml[-1]) was due to an anti-proliferative effect, rather than an effect on cell migration, as SRIF (0.1 nM - 1 microM) abolished bFGF-induced increases in total cell numbers. The bFGF-induced increase in cell numbers was also abolished by actinomycin D (0.1 microg ml[-1]). 4. The sst5 receptor-selective agonist, L-362,855 (pIC50 10.5), was about 100 times more potent than SRIF at inhibiting bFGF-induced regeneration of both VSMC and CHOsst5 cells whilst the sst2 receptor-selective agonist, BIM-23027 (pIC50 6.8), was approximately 20 times weaker than SRIF. 5. The sst5 receptor antagonist, BIM-23056 (100 nM), antagonized SRIF-induced inhibition of bFGF-induced regeneration in both VSMC and CHOsst5 cells (estimated pKB values 8.8 and 8.3, respectively). 6. SRIF-induced inhibition of bFGF-induced regeneration of VSMC and CHOsst5 cells was abolished by pretreating cells with pertussis toxin (100 ng ml[-1]) for 20 h. 7. These findings suggest that SRIF-induced inhibition of the proliferation of rat aortic VSMC is mediated via activation of receptors which are similar to human sst5 receptors. Furthermore this inhibitory effect is transduced via pertussis toxin-sensitive Gi/Go proteins.

Potent antagonism by BIM-23056 at the human recombinant somatostatin sst5 receptor.

We have investigated the effects of somatostatin (SRIF) and the linear octapeptide BIM-23056 on changes in intracellular calcium ion concentration ([Ca2+]i) and on the formation of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) in CHO-K1 cells transfected with the human recombinant SRIF sst5 receptor. SRIF elicited concentration-dependent increases in [Ca2+]i, with a pEC50 of 7.02 +/- 0.06, while BIM-23056 (1 x 10(-7) M) behaved not as an agonist but as a potent, surmountable antagonist of these increases in [Ca2+]i. The SRIF concentration-effect curve for increases in [Ca2+]i was shifted rightward producing an estimated pKB for the antagonist of 8.0. BIM-23056 (1 x 10(-7) M) also significantly attenuated Ins(1,4,5)P3 increases due to SRIF, but had no effect on either basal or uridine 5'-triphosphate (UTP) (1 x 10(-4) M) stimulated increases in the levels of [Ca2+]i or Ins(1,4,5)P3.

Molecular cloning and functional characterization of a rat somatostatin sst2(b) receptor splice variant.

1. The mouse somatostatin (SRIF) sst2 receptor exists in two splice variants, sst2(a) and sst2(b), which differ in their intracellular carboxy-termini only. The murine sst2(b) receptor was reported to be less prone to agonist-induced desensitization as compared with the sst2(a) receptor. To determine whether a sst2(b) splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst2(b) receptor and expressed the full-length protein in CHO-K1 cells for functional characterization. 2. This study provides the first evidence for the occurrence in the rat of the sst2(b) receptor, which has a 15 amino acid carboxy-terminus differing in composition to the 38 amino acid C-terminus of the rat sst2(a) receptor. 3. In CHO-K1 cells expressing rat recombinant sst2(a) or sst2(b) receptors, SRIF caused concentration-dependent increases in extracellular acidification rates (EAR) with pEC50 values of 9.0 and 9.9, respectively. Pre-treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration-effect curves with pEC50 values of 8.3 (sst2(a) and 8.4 (sst2(b)). 4. SRIF (3 pM-3 nM) also caused concentration-dependent inhibition of forskolin-stimulated cyclic AMP formation in CHO-sst2(a) cells (pIC50 10.5) and CHO-sst2(b) cells (pIC50 10.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell-shaped concentration-effect curves. Following pre-treatment with Ptx, the inhibitory effect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation. 5. Both the SRIF-induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist-induced desensitization, but this was less apparent following pre-treatment with Ptx. 6. This demonstrates that the operational characteristics of the recombinant rat sst2(a) and sst2(b) receptors are broadly similar. Both isoforms couple to Ptx-sensitive as well as -insensitive G proteins and are equally prone to agonist-induced desensitization.

Cloning and characterization of the epsilon and zeta isoforms of the 14-3-3 proteins.

Two prominent proteins (30 and 33 kD) in a purified preparation of the sheep pineal gland were studied. Amino acid analysis of tryptic peptides indicated that the 33-kD protein was the epsilon isoform of the 14-3-3 family of proteins, and that the 30-kD protein was the zeta isoform. The sheep pineal gland was found to have six other 14-3-3 isoforms in addition to the epsilon and zeta, suggesting that copurification of the epsilon and zeta forms may reflect the existence of homo- or heterodimers comprised of these isoforms. To characterize 14-3-3 proteins further in the pineal gland, the full sequence of the epsilon isoform and a partial sequence of the zeta isoform were cloned from a rat pineal cDNA library and are reported here. Tissue distribution studies using Western blot analysis revealed that rat pineal and retina have levels of 14-3-3 protein similar to those found in brain, and that relatively low levels occur in other tissues. This investigation also revealed the epsilon isoform was present at high levels in the rat pineal gland early in development and decreased steadily thereafter and that 30-kD isoforms exhibited the inverse developmental pattern.

Mucus glycoprotein gels. Role of glycoprotein polymeric structure and carbohydrate side-chains in gel-formation.

The structure of mucus glycoprotein gels from the pig gastrointestinal tract was investigated by mechanical spectroscopy. Gastric, duodenal, and colonic mucus had the same mechanical profile, characteristic of a viscoelastic gel. The gel structure collapsed on destruction of the polymeric structure of the component glycoprotein by reduction with 0.2M mercaptoethanol or after proteolysis with papain. The progressive weakening of mechanical properties and the decrease in polymeric glycoprotein content were measured as functions of time of reduction. A linear correlation was obtained between the gel quality [defined by tan delta, the ratio of the loss modulus (G'') to the storage modulus (G')] and the proportion of polymeric to subunit glycoprotein in the mucus. Purified mucus glycoprotein, at the same concentration as that in native mucus, resulted in a gel with mechanical properties no different from those of the respective native secretion, demonstrating that the glycoprotein alone could reproduce the gel-forming properties of mucus. After proteolytic digestion, all native secretions and reconstituted mucus showed an absence of Newtonian behaviour in the frequency dependence of dynamic viscosity at low frequencies. This provided evidence that the noncovalent interactions, characteristic of the native gel matrix, were still present after proteolytic digestion when the nonglycosylated protein core accessible to proteinases had been removed. These results were interpreted to show (a) a common mechanism for gel-formation in gastric, duodenal, and colonic mucus; (b) that the polymeric structure of mucus glycoproteins confers the three-dimensional structure necessary for formation of the gel network; and (c) that noncovalent interactions which arise between the glycoprotein molecules by relatively stable interdigitation of the carbohydrate side-chains are involved in formation of the gel network.

Mechanical characterization and properties of gastrointestinal mucus gel.

Mechanical spectroscopy has been used to study the structure of mucus gel taken from the surface of the pig gastrointestinal tract. Mucus from stomach, duodenum and colon was insoluble and its mechanical properties, characteristic of a weak viscoelastic gel, were unchanged in saline, acid (pH 2) and denaturants. Small intestinal mucus gel which was of poorer quality, was disrupted following exposure to acid and denaturants. Concentration of purified glycoprotein produced gels that had mechanical spectra with the same profiles as the respective native secretion except for reconstituted small intestinal mucus which was of better quality and similar to the other native and reconstituted gels. Reduction of S-S linkages or proteolysis of all mucus gels caused a collapse of structure to give profiles typical of a viscous solution. This collapse of gel structure was shown to result from a breakdown of the covalent polymeric structure of the component glycoproteins. A linear correlation for mucus gels was observed between gel quality (as defined by tan delta) and the ratio of polymeric glycoprotein to its degraded lower molecular weight subunit. Human gastric mucus from a histologically normal stomach also had the characteristics of a weak viscoelastic gel, although that from patients with peptic ulcer disease has a significantly reduced content of polymeric glycoprotein.

The gastric mucosal epithelial barrier: role of mucus and fibrin.

A continuous layer of insoluble mucus gel is adherent to the luminal surface of the gastric epithelium. The true thickness of the gel and its continuity can only be observed on unfixed sections of mucosa since histological fixatives cause dehydration and denaturation of mucus. The mucus:bicarbonate barrier can protect the undamaged epithelium from the endogenous luminal aggressors acid and pepsin but does not appear to offer much protection against exogenous damaging agents such as topical alcohol. Following acute ethanol injury, damaged epithelium is replaced by cells migrating from the gastric pits. In rat gastric mucosa this process of re-epithelialisation is protected by a gelatinous coat ten times thicker than the normal adherent mucus layer. Our studies now show this coat to be a fibrin gel with mucus and necrotic cells. Evidence suggests that the existing mucus layer can act as a template for the fibrinogen--fibrin conversion. These results demonstrate that a fibrin based gelatinous coat, quite distinct from the adherent mucus layer and with considerable protective potential, can be formed over the repairing damaged gastric mucosa.

Studies on gastrointestinal mucus.

Mucus is secreted throughout the gastrointestinal tract. The primary secretion is the water insoluble gel adherent to the mucosal surface but substantial amounts of mucus also occur in the lumen. The following are discussed: the functions of mucus; the structure and properties of mucus; study of the adherent mucus gel on the mucosal surface; the effects on mucus properties of proteolysis; thiol agents; bile salts; acid and hyperosmolar solutions. Much of our work on mucus has been with that from the stomach but where possible studies on the intestines and colon are discussed. Studies show that mucous secretions from the different regions of the gastrointestinal tract have similar rheological properties although the component glycoproteins differ in their detailed structure.

The role of mucus in the protection of the gastroduodenal mucosa.

There is good evidence that the adherent mucus plays an important role in the protection of gastroduodenal mucosa from the endogenous aggressors acid and pepsin. Adherent mucus provides a stable unstirred layer which supports surface neutralization of acid by mucosal bicarbonate output and acts as a permeability barrier to luminal pepsin. The adherent mucus layer is continuous. True thickness of the mucus layer and its continuity can only be observed on unfixed sections of mucosa, since histological fixatives and preparation for electron microscopy can cause dehydration and shrinkage of the mucus gel. The structure of adherent gastric mucus is deficient in patients with peptic ulcer disease because of decreased polymerization of the component glycoproteins. This impairment of the mucus barrier is associated with raised amounts of pepsin 1, which digests the mucus layer more aggressively than the major pepsin, pepsin 3, under conditions that pertain both in the stomach (pH 2) and duodenum (pH 4-5). Adherent mucus does not appear to offer much protection against exogenous damaging agents, e.g. alcohol and aspirin. These agents permeate the mucus barrier, damaging the underlying epithelium. The subsequent epithelial repair process is protected by a gelatinous coat over ten times thicker and distinct from the normal adherent mucus layer. Our recent studies show this gelatinous coat to be primarily a fibrin-based gel with mucus and necrotic cells.

Education provided to outgoing UK medical elective students regarding HIV risk and post exposure prophylaxis.

Previous studies suggested medical schools were failing to provide sufficient support for students undertaking electives in areas with high HIV prevalence and despite updated Department of Health (DoH) guidelines, not all were advising post exposure prophylaxis (PEP) starter packs where appropriate. This study assessed whether there has been improvement in risk reduction provided by home institutions. Questionnaires were emailed to all 29 UK medical schools offering an elective. A total of 26 medical schools responded. Only one failed to offer PEP starter packs or advice on where to obtain one. Support and advice provided by the other 25 varied considerably. HIV risk education and provision of PEP to elective students has improved. A discrepancy between advice given, supervision of projects and provision of PEP starter packs across UK medical schools remains. We reiterate recommendations put forward previously that there is a need for regularly updated national guidelines published by experts, issued to all medical schools.

Pre-elective HIV postexposure prophylaxis clinic for medical students: design, protocol, uptake and effectiveness.

We sought to evaluate medical student need for HIV postexposure prophylaxis (PEP) prior to their elective and introduce a 'Pilot PEP Clinic'. We undertook a survey of 388 medical students to assess their elective plans. All were offered an appointment in a clinic, assessed via a protocol and provided a PEP 'starter-pack' prescription if criteria were met. A follow-up questionnaire was sent to assess the acceptability of the clinic. The pre-elective questionnaire response rate was 232/388 (60%); 72/232 (31%) of respondents planned their elective in areas of high HIV prevalence and, of these, 32/72 (45%) attended the clinic. Of 32, 31 (97%) met the clinic protocol criteria and received a prescription for PEP. Of 32, 29 (90%) completed the follow-up questionnaire and every respondent rated the clinic as acceptable. The main concern was the cost of antiretroviral medications. We conclude that a 'Pre-elective HIV PEP Clinic' is an acceptable way to provide students with safe access to PEP prior to their elective.