In Silico Screening of Mutated K-Ras Inhibitors from Malaysian Typhonium flagelliforme for Non-Small Cell Lung Cancer.

K-ras is an oncogenic GTPase responsible for at least 15-25% of all non-small cell lung cancer cases worldwide. Lung cancer of both types is increasing with an alarming rate due to smoking habits in Malaysia among men and women. Natural products always offer alternate treatment therapies that are safe and effective. Typhonium flagelliforme or Keladi Tikus is a local plant known to possess anticancer properties. The whole extract is considered more potent than individual constituents. Since K-ras is the key protein in lung cancer, our aim was to identify the constituents of the plant that could target the mutated K-ras. Using docking strategies, reported potentially active compounds of Typhonium flagelliforme were docked into the allosteric surface pockets and switch regions of the K-ras protein to identify possible inhibitors. The selected ligands were found to have a high binding affinity for the switch II and the interphase region of the ras-SOS binding surface.

A comparison of liver transplantation outcomes in the pre- vs. post-MELD eras.

BACKGROUND: The model for end stage liver disease (MELD)-based organ allocation system is designed to prioritize orthotopic liver transplantation (OLT) for patients with the most severe liver disease. However, there are no published data to confirm whether this goal has been achieved or whether the policy has affected long-term post-OLT survival. AIM: To compare pre-OLT liver disease severity and long-term (1 year) post-OLT survival between the pre- and post-MELD eras. METHODS: Using the United Network of Organ Sharing database, we compared two cohorts of adult patients undergoing cadaveric liver transplant in the pre-MELD (n = 3857) and post-MELD (n = 4245) eras. We created multivariable models to determine differences in: (i) pre-OLT liver disease severity as measured by MELD; and (ii) 1-year post-OLT outcomes. RESULTS: Patients undergoing OLT in the post-MELD era had more severe liver disease at the time of transplantation (mean MELD = 20.5) vs. those in the pre-MELD era (mean MELD = 17.0). There were no differences in the unadjusted patient or graft survival at 1 year post-OLT. This difference remained insignificant after adjusting for a range of prespecified recipient, donor, and transplant centre-related factors in multivariable survival analysis. CONCLUSIONS: Although liver disease severity is higher in the post- vs. pre-MELD era, there has been no change in long-term post-OLT patient or graft survival. These results indicate that the MELD era has achieved its primary goals by allocating cadaveric livers to the sickest patients without compromising post-OLT survival.

Endothelin-1 stimulates human colonic myofibroblast contraction and migration.

BACKGROUND: Although the contractile, migratory, and proliferative responses of subepithelial myofibroblasts to injury have been postulated to be important events in intestinal wound healing, contractile force generation and migration by these cells has not been investigated previously, and the signals that regulate proliferation by these cells are poorly understood. AIMS: The primary aim of this study was to test the hypothesis that the inflammatory mediator endothelin-1 modulates contraction, migration, and proliferation of intestinal myofibroblasts. We also sought to examine the signal transduction pathways which might underlie these putative effects. METHODS: Contraction, migration, proliferation, cytosolic [Ca(2+)], and myosin phosphorylation were measured in human colonic subepithelial myofibroblasts in the absence and presence of endothelin receptor agonists and antagonists. RESULTS: Endothelin-1, but not interleukin 1 alpha, interleukin 6, interleukin 8, interleukin 10, or tumour necrosis factor alpha, induced a rapid and robust generation of contractile force, which was associated with an increase in cytosolic [Ca(2+)] and myosin phosphorylation. Inhibition of rho associated kinase reduced endothelin-1 stimulated myosin phosphorylation and contractile force development. Endothelin-1 stimulated migration with a dose-response relationship similar to that observed for contraction. Endothelin A and B receptors mediated contraction while migration was mediated predominantly through endothelin B receptors. Platelet derived growth factor and serum, but not endothelin-1, induced proliferation. CONCLUSIONS: Endothelin-1 stimulates colonic subepithelial myofibroblast contraction and migration via endothelin receptor mediated myosin phosphorylation. These results support an important role for subepithelial myofibroblasts in the injury response of the gut and consequently intestinal wound repair.

Hepatitis C screening strategies in hemodialysis patients.

Hepatitis C virus (HCV) infection is common in patients undergoing chronic hemodialysis, with an estimated yearly incidence of 0.2% and prevalence between 8% and 10%. Although a screening strategy based on alanine aminotransferase (ALT) values is currently recommended, this strategy has not been evaluated for cost-effectiveness compared with other potential screening strategies. A comparison therefore was made using a decision-analysis model of a simulated cohort of 5,000 hemodialysis patients followed up for 5 years. Using direct medical costs, three strategies were evaluated, including: (1) ALT values with confirmatory testing (biochemical), (2) serial enzyme-linked immunosorbent and strip immunoblot assay testing (serological), and (3) polymerase chain reaction (viral). Under baseline assumptions, the per-patient cost of screening hemodialysis patients for HCV was $378 for biochemical-based testing, $195 for serological-based testing, and $696 for viral-based testing. Our model was robust when varying the costs of testing, as well as the incidence and prevalence of HCV infection. Results of sensitivity analysis by varying costs, HCV incidence, and HCV prevalence indicated that serological-based screening was less costly than biochemical testing. Biochemical testing was in turn less costly than viral-based screening. Serological-based testing was also more effective in the diagnosis of de novo HCV infection, with a likelihood ratio of 85, in contrast to the likelihood ratio of 44 with biochemical-based testing using viral-based screening as the gold standard. A serological-based screening strategy is less costly and more effective than biochemical-based screening in the diagnosis of de novo HCV infection. Serological-based screening should be considered for HCV screening in hemodialysis populations.

Calyculin-A induces focal adhesion assembly and tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin in Swiss 3T3 cells.

Treatment of intact Swiss 3T3 cells with calyculin-A, an inhibitor of myosin light chain (MLC) phosphatase, induces tyrosine phosphorylation of p125(Fak) in a sharply concentration- and time-dependent manner. Maximal stimulation was 4.2 +/- 2.1-fold (n = 14). The stimulatory effect of calyculin-A was observed at low nanomolar concentrations (<10 nM); at higher concentrations (>10 nM) tyrosine phosphorylation of p125(Fak) was strikingly decreased. Calyculin-A induced tyrosine phosphorylation of p125(Fak) through a protein kinase C- and Ca(2+)-independent pathway. Exposure to either cytochalasin-D or latrunculin-A, which disrupt actin organization by different mechanisms, abolished tyrosine phosphorylation of p125(Fak) in response to calyculin-A. Treatment with high concentrations of platelet-derived growth factor (20 ng/ml) which also disrupt actin stress fibers, completely inhibited tyrosine phosphorylation of p125(Fak) in response to calyculin-A. This agent also induced tyrosine phosphorylation of the focal adhesion-associated proteins p130(Cas) and paxillin. These tyrosine phosphorylation events were associated with a striking increase in the assembly of focal adhesions. The Rho kinase (ROK) inhibitor HA1077 that blocked focal adhesion formation by bombesin, had no effect on the focal adhesion assembly induced by calyculin-A. Thus, calyculin-A induces transient focal adhesion assembly and tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin, acting downstream of ROK.

Serum alanine aminotransferase in hepatitis c screening of patients on hemodialysis.

It is recommended that patients on hemodialysis (HD) therapy undergo regular screening for hepatitis C virus (HCV) infection by using alanine aminotransferase (ALT) values. However, the utility of using ALT values in this setting is unknown. The aim of this prospective study at the University of California Los Angeles Hepatitis Screening Program is to determine the sensitivity, specificity, and predictive values of an elevated ALT level for the diagnosis of HCV infection in HD patients. We screened 2,440 HD patients from 39 dialysis centers for viral infection by using hepatitis antibody serological testing and ALT values. We found the sensitivity and specificity of a newly elevated ALT level for acute HCV infection to be 83% and 90%, respectively. According to Bayes' theorem, the positive predictive value was 4% and the positive likelihood ratio was 8.74. For chronic HCV infection, the sensitivity of a newly elevated aminotransferase level was 21%, and specificity was 91%. The positive predictive value was 16% (according to Bayes' theorem), and the positive likelihood ratio was 2.47. The negative predictive value of a newly elevated aminotransferase value was 99% for acute HCV infection and 94% for chronic HCV infection. Our results indicate that although a newly elevated aminotransferase level is sensitive and specific for acute HCV infection, its positive predictive value is inadequate. A newly elevated aminotransferase level was neither sensitive nor positively predictive of chronic infection. Therefore, an elevated ALT level is an ineffective method for screening for HCV infection in HD patients.

RhoA/rho-associated kinase mediates fibroblast contractile force generation.

The intracellular signals governing contractile force generation by non-muscle cells remain uncertain. Our aim was to test the hypothesis that the rhoA/rho-associated kinase signaling pathway is a principal mediator of contractile force generation in non-muscle cells. We measured myosin II regulatory light chain (MLC) phosphorylation and directly quantitated force generation by chicken embryo fibroblasts in the absence and presence of selective inhibitors of rhoA, and its downstream effector, rho-associated kinase. Inactivation of rhoA, with C3 transferase, inhibited serum-stimulated MLC phosphorylation and contractile force generation. Y-27632, an inhibitor of rho-associated kinase, reduced basal contractile tension, and inhibited both serum and endothelin-1 stimulated MLC phosphorylation and contractile force generation. The results of this study provide novel evidence indicating that the rhoA/rho-associated kinase signaling pathway is a principal mediator of MLC phosphorylation and consequent contractile force generation by non-muscle cells.

Wound-induced migration of rat hepatic stellate cells is modulated by endothelin-1 through rho-kinase-mediated alterations in the acto-myosin cytoskeleton.

Although migration of stellate cells during hepatic injury is essential for wound-healing and fibrosis of the liver, the extracellular and intracellular signals that regulate stellate cell migration are incompletely understood. In this study we tested the hypothesis that wound-induced migration of stellate cells is modulated by endothelin-1 (ET-1) through rho-kinase-mediated alterations in the acto-myosin cytoskeleton. To address this hypothesis, a method was established for direct visualization of wound-induced migration of culture-activated stellate cells with subcellular resolution. Migration in response to wounding was characterized by (1) plasma membrane ruffling and protrusion into the wound, (2) lamellipodia formation at the leading edge, (3) focal adhesion and stress fiber assembly, and (4) myosin reorganization. Exogenous ET-1 accelerated wound-induced migration of stellate cells, but did not alter wound-induced proliferation. Experiments using ET-1 antagonists in the absence of exogenous ET-1 showed that wound-induced migration was also stimulated by endogenous ET-1. Selective inhibition of rho-associated kinase decelerated migration in response to wounding. Moreover, inhibition of rho-associated kinase was distinguished by abrogation of focal adhesion formation, stress fiber assembly, and myosin reorganization. This study shows that rho-kinase-dependent alterations in the acto-myosin cytoskeleton contribute to wound-induced stellate cell migration, which is accelerated by both exogenous and endogenous ET-1. Consequently, these results provide important new evidence suggesting that, migration of stellate cells is modulated by paracrine and autocrine ET-1 stimulation via the action of rho-kinase on the acto-myosin cytoskeleton.

Peroxisome proliferator-activated receptors and hepatic stellate cell activation.

The present study examined the roles of peroxisome proliferator-activated receptors (PPAR) in activation of hepatic stellate cells (HSC), a pivotal event in liver fibrogenesis. RNase protection assay detected mRNA for PPARgamma1 but not that for the adipocyte-specific gamma2 isoform in HSC isolated from sham-operated rats, whereas the transcripts for neither isoforms were detectable in HSC from cholestatic liver fibrosis induced by bile duct ligation (BDL). Semi-quantitative reverse transcriptase-polymerase chain reaction confirmed a 70% reduction in PPARgamma mRNA level in HSC from BDL. Nuclear extracts from BDL cells showed an expected diminution of binding to PPAR-responsive element, whereas NF-kappaB and AP-1 binding were increased. Treatment of cultured-activated HSC with ligands for PPARgamma (10 microm 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)); 0.1 approximately 10 microm BRL49653) inhibited DNA and collagen synthesis without affecting the cell viability. Suppression of HSC collagen by 15dPGJ(2) was abrogated 70% by the concomitant treatment with a PPARgamma antagonist (GW9662). HSC DNA and collagen synthesis were inhibited by WY14643 at the concentrations known to activate both PPARalpha and gamma (>100 microm) but not at those that only activate PPARalpha (<10 microm) or by a synthetic PPARalpha-selective agonist (GW9578). 15dPGJ(2) reduced alpha1(I) procollagen, smooth muscle alpha-actin, and monocyte chemotactic protein-1 mRNA levels while inducing matrix metalloproteinase-3 and CD36. 15dPGJ(2) and BRL49653 inhibited alpha1(I) procollagen promoter activity. Tumor necrosis factor alpha (10 ng/ml) reduced PPARgamma mRNA, and this effect was prevented by the treatment with 15dPGJ(2). These results demonstrate that HSC activation is associated with the reductions in PPARgamma expression and PPAR-responsive element binding in vivo and is reversed by the treatment with PPARgamma ligands in vitro. These findings implicate diminished PPARgamma signaling in molecular mechanisms underlying activation of HSC in liver fibrogenesis and the potential therapeutic value of PPARgamma ligands for liver fibrosis.

Tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin does not require extracellular signal-regulated kinase activation in Swiss 3T3 cells stimulated by bombesin or platelet-derived growth factor.

The experiments presented here were designed to examine the contribution of the extracellular signal-regulated mitogen-activated protein kinases (ERKs) to the tyrosine phosphorylation of the focal adhesion proteins p125(Fak), p130(Cas), and paxillin induced by G protein-coupled receptors (GPCRs) and tyrosine kinase receptors in Swiss 3T3 cells. Stimulation of these cells with bombesin, lysophosphatidic acid (LPA), endothelin, and platelet-derived growth factor (PDGF) led to a marked increase in the tyrosine phosphorylation of these focal adhesion proteins and in ERK activation. Exposure of the cells to two structurally unrelated mitogen-activated protein kinase or ERK kinase (MEK) inhibitors, PD98059 and U0126, completely abrogated ERK activation but did not prevent tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin. Furthermore, different dose-response relationships were obtained for tyrosine phosphorylation of focal adhesion proteins and for ERK activation in response to PDGF. Putative upstream events in the activation of focal adhesion proteins including actin cytoskeletal reorganization and myosin light chain (MLC) phosphorylation were also not prevented by inhibition of ERK activation. Thus, our results demonstrate that the activation of the ERK pathway is not necessary for the increase of the tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin induced by either GPCRs or tyrosine kinase receptors in Swiss 3T3 cells.

Quantitation of rat hepatic stellate cell contraction: stellate cells' contribution to sinusoidal resistance.

Although it has been hypothesized that contraction of hepatic stellate cells (HSC) regulates blood flow by modulating sinusoidal resistance, neither HSC contraction nor relaxation has been directly quantitated. To test this hypothesis, a force transducer was employed to directly measure the magnitude and rate of contraction and relaxation by primary rat HSC (4.7 x 10(5) +/- 0.2 x 10(5) cells) cultured within a collagen gel. Serial exposures to 10% fetal bovine serum stimulated 81 +/- 14 and 82 +/- 10 dyn of contractile force, respectively. Subsequent stimulation with 2 nM endothelin-1 (ET-1) resulted in the development of 185 +/- 25 dyn of force. Contractions began within 10 s of ET-1 stimulation, and the half time of maximal force development was <5 min. Removal of agonist resulted in complete or nearly complete relaxation within 45 min. These results suggest that the magnitude and rate of HSC contraction and relaxation are capable of modulating blood flow via sinusoidal constriction.

Ca2+-independent myosin II phosphorylation and contraction in chicken embryo fibroblasts.

1. Non-muscle contraction is widely believed to be mediated through Ca2+-stimulated myosin II regulatory light chain (LC20) phosphorylation, similar to the contractile regulation of smooth muscle. However, this hypothesis lacks conclusive experimental support. 2. By modulating chicken embryo fibroblast cytosolic Ca2+ concentration ([Ca2+]i), we investigated the putative role of [Ca2+]i in fetal bovine serum (FBS)-stimulated LC20 phosphorylation and force development in these cells. 3. Eliminating the FBS-stimulated rise in [Ca2+]i with the Ca2+ chelator BAPTA only partially inhibited FBS-stimulated LC20 phosphorylation and did not significantly alter the magnitude of FBS-stimulated isometric contraction. 4. Ionomycin (1 microM) produced a larger but shorter lasting rise in [Ca2+]i relative to FBS. However, ionomycin only stimulated a small and transient increase in LC20 phosphorylation and did not cause contraction. 5. We conclude that fibroblasts differ from smooth muscle in that LC20 phosphorylation and contraction are predominantly regulated independently of [Ca2+]i.

Rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton.

Hepatic stellate cell activation, thought to play a key role in fibrosis of the liver, is characterized by changes in cellular morphology. The intracellular signals regulating morphological alterations associated with stellate cell activation are uncertain. The ras-like guanosine triphosphate-binding protein, rho, has recently emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. The aim of this study was to test the hypothesis that rho signaling pathways direct activation-associated morphological changes in stellate cells by regulating the actin cytoskeleton. The morphology and actin cytoskeleton of primary rat hepatic stellate cells were studied with phase contrast, differential interference contrast, and epifluorescence microscopy. Immunohistochemistry and immunoblot analysis were used to examine rho expression and activity, respectively. Quiescent and activated stellate cells were investigated in the absence and presence of C3 transferase, a bacterial toxin that specifically inhibits rho. Stellate cell activation was characterized by the development of prominent intracellular fibers, and the loss of dendrite-like processes and perinuclear retinoid droplets. Moreover, activation was accompanied by the formation of prominent actin stress fibers and focal adhesions. Both rho expression and activity were demonstrated in stellate cells. C3 transferase blocked and reversed, both activation-associated morphological alterations and activation-associated changes in the actin cytoskeleton, in quiescent and activated stellate cells, respectively. These results indicate that rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton.

Enterococcal bacteremia after transjugular intrahepatic portosystemic shunts (TIPS).

The objective of this study was to analyze a series of patients with Enterococcus faecium infection following transjugular intrahepatic portosystemic shunts (TIPS) in order to define the risk factors, outcome, and role of treatment including hepatic transplantation. This study is a case series from a tertiary referral center for liver transplantation. The medical records of four patients referred to one teaching hospital in San Francisco between 1990 and 1995 for evaluation or management of Enterococcal infection following TIPS were reviewed. A review of the microbiology records of all 314 patients who underwent TIPS at that institution and a MEDLINE search were performed to assess whether any other cases existed. The effect of therapy on survival was assessed, in particular, the repeated use of TIPS and prolonged courses of antibiotics. All four patients had thrombosis of their TIPS at the time of diagnosis of enterococcal bacteremia. All were treated with prolonged courses of intravenous antibiotics. One patient had echocardiographic evidence of subacute bacterial endocarditis with chronic aortic insufficiency. In all cases, liver transplantation was contraindicated in the acute setting because of uncontrolled endovascular infection. Two of four patients survived; these were the only two patients who had had a successful repeat TIPS. Enterococcal bacteremia is a rare complication following TIPS but carries a high mortality. It usually occurs in the setting of technically difficult TIPS with shunt thrombosis. Management should be focused on long term antibiotics and attempts at reestablishment of portal decompression with another TIPS. Liver transplantation should not be considered until the infection is cleared. Prophylaxis for Enterococcus species should be considered in technically difficult or unsuccessful TIPS.

Visualization of internalization and recycling of the gastrin releasing peptide receptor-green fluorescent protein chimera expressed in epithelial cells.

Gastrin releasing peptide (GRP) regulates critical gastrointestinal functions via the GRP receptor (GRPR). GRPR internalization and recycling have been proposed to play an important role in the cellular response to GRP. Our aim was to develop a direct method for investigating GRPR trafficking in living cells. A chimeric protein, consisting of GRPR fused to green fluorescent protein (GFP), was expressed in epithelial cells. Ligand and receptor interactions were examined with radiolabeled agonist and fluorescent imaging. In comparison with epithelial cells expressing wild-type GRPR, the GRPR-GFP expressing cells showed similar ligand binding affinity, GRP-stimulated Ca2+ signaling, and GRP-initiated internalization. In GRPR-GFP expressing cells treated with fluorescently labeled ligand, receptor and ligand trafficking was directly visualized. Upon ligand binding, the receptor-ligand complex coalesced into vesicles prior to internalization and migration to the perinuclear space. Whereas a portion of the receptors were observed to return to the plasma membrane, the ligand remained in the perinuclear space. Hyperosmolar solution prevented ligand and receptor internalization, and bafilomycin inhibited receptor recycling. We demonstrate that GRPR-GFP is physiologically similar to wild-type GRPR, and permits direct visualization of intracellular trafficking processes in individual living cells with minimal toxicity over several hours.

Fatal Clostridium difficile enteritis after total abdominal colectomy.

A 71-year-old man who had undergone an ileorectal anastomosis some years earlier, developed fulminant fatal Clostridium difficile pseudomembranous enteritis and proctitis after a prostatectomy. This case and three reports of C. difficile involvement of the small bowel in adults emphasize that the small intestine can be affected. No case like ours, of enteritis after colectomy from C. difficile, has hitherto been reported.