Inhibitory properties of the regulatory domains of human protein kinase Calpha and mouse protein kinase Cepsilon

Two fusion proteins in which the regulatory domains of human protein kinase Calpha (Ralpha; amino acids 1-270) or mouse protein kinase Cepsilon (Repsilon; amino acids 1-385) were linked in frame with glutathione S-transferase (GST) were examined for their abilities to inhibit the catalytic activities of protein kinase Calpha (PKCalpha) and other protein kinases in vitro. Both GST-Ralpha and GST-Repsilon but not GST itself potently inhibited the activities of lipid-activated rat brain PKCalpha. In contrast, the fusion proteins had little or no inhibitory effect on the activities of the Ser/Thr protein kinases cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, myosin light chain kinase, and mitogen activated protein kinase or on the src Tyr kinase. GST-Ralpha and GST-Repsilon, on a molar basis, were 100-200-fold more potent inhibitors of PKCalpha activity than was the pseudosubstrate peptide PKC19-36. In addition, a GST-Ralpha fusion protein in which the first 32 amino acids of Ralpha were deleted (including the pseudosubstrate sequence from amino acids 19-31) was an effective competitive inhibitor of PKCalpha activity. The three GST-R fusion proteins also inhibited protamine-activated PKCalpha and proteolytically activated PKCalpha (PKM), two lipid-independent forms of PKCalpha; however, the IC50 values for inhibition were 1 order of magnitude greater than the IC50 values obtained in the presence of lipid. These results suggest that part of the inhibitory effect of the GST-R fusion proteins on lipid-activated PKCalpha may have resulted from sequestration of lipid activators. Nonetheless, as evidenced by their abilities to inhibit the lipid-independent forms of the enzyme, the GST-R fusion proteins also inhibited PKCalpha catalytic activity through direct interactions. These data indicate that the R domains of PKCalpha and PKCepsilon are specific inhibitors of protein kinase Calpha activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.     

Regional epidemiological features of lip, oral cavity, and oropharyngeal cancer

Lip, oral cavity, and oropharyngeal cancer are among the most common forms of the disease in the world. These types of cancer display significant geographic, ethnic, and socioeconomic variations. We examined the cases of cancer of the lip, oral cavity, and oropharynx diagnosed in the Department of Otolaryngology at the University of Uluda? School of Medicine during the last 5 years, July 1990 to June 1995, and recorded the epidemiological features of these tumors. The Department of Otolaryngology treated a total of 26,225 in- and outpatients during the 5-year period. 320 of these patients (1.2%) were seen for head and neck cancer. 42 of the 320 patients (13.1%) were diagnosed with cancer of the lip, oral cavity, and oropharrynx. After the larynx, this was the second most frequent location of malignant head and neck tumors. We discovered the following epidemiological and pathological features: (1) The incident rate was highest in patients between 41 and 60 years of age. (2) 70% of the patients were male, and 76% of them had a history of tobacco/alcohol use. (3) Occupation had no apparent relevance (four of the patients were farmers). (4) Approximately one third of the patients had undergone medical therapy prior to diagnosis. (5) One third of the patients had initially seen a dentist for treatment, and approximately half had poor dental and oral hygiene. (6) The most frequent symptom was ulceration. (7) Histopathological examination revealed squamous cell carcinoma in 88% of the cases. (8) The cancer was localized to the lip in 31% of cases, oral cavity, 50%, and oropharynx, 19%. (9) Almost half of the cancer cases were diagnosed in advanced stage (stages III and IV).     

Absence of PTEN/MMAC1 germ-line mutations in sporadic Bannayan-Riley-Ruvalcaba syndrome

Bannayan-Riley-Ruvalcaba syndrome (BRRS) is a rare hamartomatous polyposis condition with features of macrocephaly, intestinal juvenile polyposis, developmental delay, lipomas, and pigmentation spots of the male genitalia. An autosomal dominant pattern of inheritance exists in some families, but others appear as sporadic cases. Germ-line mutations in PTEN, a tyrosine phosphatase and putative tumor suppressor gene, have been demonstrated in two families with BRRS, and chromatin loss at the PTEN gene locus on chromosome 10q23 has been demonstrated in two BRRS patients. Germ-line mutations in PTEN have also been described in Cowden disease and in a small number of patients with juvenile polyposis syndrome. In an attempt to assess the nature of PTEN mutations in BRRS, we analyzed three sporadic BRRS patients for chromosome 10q23 deletion or PTEN germ-line mutations. All 3 patients demonstrated no loss of parental alleles at 15 chromosome 10q23 markers that encompassed the region of PTEN. In addition, analysis of mRNA and genomic DNA revealed no nonsense, missense, or insertion/deletion mutations of PTEN. Thus, other mechanisms besides mutation of PTEN must have occurred to cause BRRS in these patients. We speculate that BRRS and juvenile polyposis syndrome may have a heterogeneous etiology to cause their syndromes.     

The human gallbladder increases cholesterol solubility in bile by differential lipid absorption: a study using a new in vitro model of isolated intra-arterially perfused gallbladder

In this study, we first developed and validated a new in vitro isolated, intra-arterially perfused, gallbladder model and then applied the method to investigate the absorption of biliary lipids by the gallbladder wall and the effect of this process on the composition of human bile. Oxygenated and glucose-added buffer was perfused through the cystic artery to maintain organ viability. A standard pooled natural bile, radiolabeled with H3-cholesterol and C14-palmitoyl-linoleoyl-phosphatidylcholine, was instilled in the lumen via a cystic duct catheter. Changes in bile volume and lipid concentrations were monitored at time intervals to evaluate the disappearance of lipids from bile caused by gallbladder absorptive function. Organ viability was demonstrated by stable lactate dehydrogenase (LDH) organ release and oxygen consumption throughout the experiments. In the pig, disappearance rates of lipids from bile were similar in vitro and in vivo, demonstrating the validity of the isolated in vitro model for functional studies. By applying our in vitro isolated preparation to the human gallbladder, we found that 23% of cholesterol and 32% of phosphatidylcholine, but only 9% of bile salts, disappeared from bile in 5 hours. As a consequence, at the end of the experiments, cholesterol (P < .05) and phospholipid (P < .05) molar percentages were significantly reduced, while the bile salt (P < .05) molar percentage was significantly increased with respect to values at the beginning of the studies. Our findings are of pathophysiological relevance and support the concept that the human gallbladder modifies the relative composition of biliary lipids in such a way as to increase cholesterol solubility in bile.     

A comparison of morphine, pethidine and fentanyl in the postsurgical patient-controlled analgesia environment

Oncogene and tumor suppressor gene mutations are candidate biomarkers for cancer risk assessment and lesion detection. The K-ras oncogene has previously been associated with non-small cell lung cancer (NSCLC), particularly adenocarcinomas in which reported rates of mutation have approached 30-40%. We have analyzed non-malignant lung tissue from patients with lung cancer and primary lung cancers for K-ras gene mutations. Mutations were detected in 32% cancers and 29% non-malignant lung tissue from patients with cancer. The majority of tumors testing positive were adenocarcinoma of the lung. Normal DNA controls, including peripheral blood lymphocytes and normal lung from non-smokers, were negative. The ability to detect genetic alterations in non-malignant lung tissues is consistent with the concept that genetic alterations are involved in field cancerization of the aerodigestive tract.     

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.     

Caspase-dependent ceramide production in Fas- and HLA class I-mediated peripheral T cell apoptosis

We recently demonstrated that the engagement of HLA class I alpha1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.