Presence of sauvagine-like epitopes in the interrenal gland of the bullfrog Rana catesbeiana

The objective of this study was to determine if a variety of hepatotoxicants could induce the level of heat shock protein 70I, and whether or not elevated levels of heat shock proteins (hsp's) could provide cytoprotection from those hepatotoxicants. Exposure of HepG2 cells to cytotoxic concentrations of bromobenzene, cadmium, cyclophosphamide, or diethylnitrosamine increased the level of hsp 70I protein and mRNA, while carbon tetrachloride and cocaine had no effect on hsp 70I or mRNA levels. To determine if induction of hsp 70I might afford protection against cytotoxicity, HepG2 cells were given a prior sublethal heat shock (sub-LHS) (43 degrees C for 1 hr) to induce hsp's and then challenged 24 hr later with the hepatotoxicants. Sub-LHS pretreatment diminished toxicity from bromobenzene, cadmium, cyclophosphamide, or diethyl-nitrosamine, but not carbon tetrachloride or cocaine. In cells treated with [14C]carbon tetrachloride or [3H]cocaine, no detectable covalent binding to proteins was observed; whereas, [14C]-bromobenzene treatment resulted in substantial covalent binding to cellular protein. The apparent absence of formation of reactive metabolite adducted proteins from cocaine and carbon tetrachloride may explain why no hsp 70I induction was observed with these agents. The correlation between hepatotoxicant induction of hsp 70I and cytoprotection afforded by sub-LHS pretreatment suggests that hsp 70I induction may represent an important cellular defense mechanism in the liver.     

Immunochemical detection of quinol--thioether-derived protein adducts

Glutathione (GSH) conjugates of hydroquinone (HQ) and 2-bromohydroquinone (2-BrHQ) produce severe renal proximal tubular necrosis in rats. Since the reactivity of quinones lies, in part, in their ability to alkylate proteins, our goal was to develop an immunochemical method with which to investigate the role of protein adduct formation in quinone-thioether-mediated toxicity. An immunogen was synthesized by coupling 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone (2-BrHQ-NAC) to keyhole-limpet hemocyanin (KLH). Anti-2-BrHQ-NAC-KLH antibodies were raised in rabbits and purified by affinity chromatography. Antibody binding to the 2-BrHQ-NAC epitope was confirmed by competitive enzyme-linked immunosorbent assay (ELISA) with a bovine serum albumin conjugate of 2-BrHQ-NAC. Affinity-purified anti-2-BrHQ-NAC-KLH antibodies recognized adducted proteins in the kidneys of rats treated with HQ, 2-BrHQ, 2-bromo-bis(glutathion-S-yl)hydroquinone, 2-(glutathion-S-yl)hydroquinone, 2, 5-bis(glutathion-S-yl)hydroquinone, and 2,3, 5-tris(glutathion-S-yl)hydroquinone. Immunoreactive proteins were found in all renal subcellular fractions of 2-BrHQ-treated rats, and the distribution of adducts was similiar to that obtained by quantifying 2-Br[14C]HQ covalent adducts. Western blot analysis revealed that three proteins, at 42, 46, and 79 kDa, were adducted by all the compounds examined. The identification of these adducted proteins will be required to assess their significance in quinol-thioether-mediated nephrotoxicity.     

Production of antiserum for sensitive enzyme-linked immunosorbent assay of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid by chemiluminescence

To obtain a specific antiserum for use in enzyme-linked immunosorbent assay (ELISA) of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), we prepared a hapten-carrier conjugate in which the CMPF hapten was linked to a carrier protein through the 5-(1-hydrazopropyl) group. The antisera raised against this antigen in guinea pigs had excellent specificity for CMPF, showing little cross-reactivity with closely related compounds and no significant cross-reactivities with other furan compounds. The results indicated that a specific antiserum to CMPF could be produced by an antigen whose CMPF moiety is linked to a carrier protein through a position remote from the inherent functional groups. A standard curve of CMPF by ELISA using a chemiluminescence system showed a high sensitivity and a linearity in the range of 5-100 ng/mL.     

Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein

A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.     

Differential expression of urinary inter-alpha-trypsin inhibitor trimers and dimers in normal compared to active calcium oxalate stone forming men

PURPOSE: We determine if the immunoreactive profile of urinary inter-alpha-trypsin inhibitor can be used to distinguish between normal individuals and individuals with calcium oxalate stone disease. MATERIALS AND METHODS: Urinary proteins were dialyzed against water (15 kDa. molecular weight cutoff), lyophilized and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6% acrylamide, reducing conditions) followed by Western blot. Inter-alpha-trypsin immunoreactive proteins were detected by enhanced chemiluminescence. Stone formation was confirmed to be active radiologically or passed as stone or gravel within 12 months of the sample. Stone composition was confirmed crystallographically. Normal individuals had no personal or familial history of urolithiasis and matched stone forming patients regarding race (white) and age (23 to 71 years old). Urine from a total of 101 individuals was analyzed. RESULTS: The intact inter-alpha-trypsin trimer (approximately 220 to 240 kDa.) and heavy chain (HC) 2-bikunin/HC1-bikunin dimers (approximately 115 to 130 kDa.) were detected more often in stone forming men (23 of 26 [89%] and 26 of 26 [100%], respectively) than in normal individuals (6 of 26 [23%] and 5 of 26 [19%], respectively, p < 0.0001). In those normal individuals who expressed inter-alpha-trypsin trimer and HC-bikunins the relative intensities were 5.3+/-1.4% and 16.3+/-17.1% of the stone forming controls, respectively. The identity of high molecular weight-inter-alpha-trypsin immunoreactive bands was confirmed using antibodies against the individual subunits (HC1, HC2, HC3, bikunin). In contrast to men high molecular weight-inter-alpha-trypsin's were readily detected in normal and stone forming women with equal frequency (inter-alpha-trypsin-trimer p=0.1337, HC-bikunins p=0.2836): inter-alpha-trypsin-trimer 17 of 18 [94%] and 9 of 13 [77%]; HC-bikunins 17 of 18 [94%] and 10 of 13 [85%]). Inter-alpha-trypsin-trimer and HC-bikunins, respectively, were detected in 2 and 5 of 10 patients with chronic renal disease. Expression was not related to hematuria or proteinuria. CONCLUSIONS: Immunoreactive profiles of urinary proteins may be able to be developed into a useful diagnostic tool to identify active stone formation, although a separate panel may be required for men and women. It is possible that these differences may provide clues as to why the incidence of stone disease is higher in men than women.     

Distribution of casein-like proteins in various organs of rat

Casein-like proteins were detected in various organs of rat by use of a specific antiserum raised against rat milk caseins. The antiserum specifically recognized alpha 1-, alpha 2-, beta-, and gamma-caseins in rat milk by Western blot analysis, whereas no immunoreactive band was observed in sera of rat and fetal bovine and in bovine caseins. Immunohistochemical studies of this antiserum on formalin-fixed mammary glands showed that immunoreactive caseins were localized to the apical portion of the cytoplasm in lactating mammary epithelial cells and in the luminal secretion, which indicates a directional secretion of caseins to the lumen by the mammary epithelial cells. With this antiserum, immunoreactive substances were detected in various organs, including the pancreatic ducts and islets of Langerhans, the secretory ducts of salivary glands, zona fasciculata cells and ganglion cells of adrenal gland, distal tubules and convoluted collecting tubules of kidney, epithelial cells of bronchioles and large pneumocytes of the lung, hair follicles, sebaceous glands, and the prickle cell layer of skin, uterine glands and epithelium of the endometrium, hepatic bile ducts, and brain. In Western blot analysis, major immunoreactive substances in the above organ extracts showed a similarity in molecular weight to alpha 2-casein of rat milk. Skin was the only tissue that expressed both alpha 2- and beta-caseins. There were no other immunoreactive bands with similarity to beta- and gamma-caseins in the other organ extracts, but higher molecular weight immunoreactive bands (> 100 kD) were detected in some organ extracts, such as salivary gland, kidney, liver, lung, and uterus. These findings suggest that the alpha 2-casein-like substance is localized not only in the mammary gland but also in a variety of organs and may play an important role as a functional molecule in those organs.     

Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus

The biochemical properties of a second protein (CM2) encoded by RNA segment 6 of influenza C virus were investigated. Three forms of CM2 with different electrophoretic mobilities (CM2(0), CM2a, and CM2b) were detected in infected cells by immunoprecipitation with antiserum to the glutathione S-transferase (GST)-CM2 fusion protein. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with endoglycosidase H or peptide-N-glycosidase F suggested that a mannose-rich oligosaccharide core is added to unglycosylated CM2(0) (Mr, approximately 16,000) to form CM2a (Mr, approximately 18,000) and that the processing of the carbohydrate chain from the high-mannose type to the complex type converts CM2a into CM2b, which is heterogeneous in electrophoretic mobility (Mr, approximately 22,000 to 30,000). Labeling of infected cells with [3H]palmitic acid showed that CM2 is fatty acylated. The fatty acid bond was sensitive to treatment with hydroxylamine and mercaptoethanol, which indicates a labile thioester-type linkage. The CM2 protein was also found to form disulfide-linked dimers and tetramers on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Trypsin treatment of infected cell surfaces as well as of microsome vesicles from infected cells followed by immunoprecipitation with antiserum to the GST fusion protein containing the 56 C-terminal amino acid residues of CM2 suggested that this C-terminal domain is intracellular and exposed to the cytoplasms of microsomes. Furthermore, evidence that a small amount of CM2 is incorporated into progeny virus particles was obtained by Western blot analysis. These results, altogether, suggest that CM2 is an integral membrane protein with biochemical properties similar to those of influenza A virus M2 and influenza B virus NB proteins.     

Association between HLA class II genotype and spontaneous clearance of hepatitis C viraemia

The efficiency and reliability of radioactive fucose as a specific label for newly synthesized glycoproteins were investigated. Young adult male rabbits were injected intravitreally with [3H]-fucose, [3H]-galactose. [3H]-mannose, N-acetyl-[3H]-glucosamine or N-acetyl-[3H]-mannosamine, and killed 40 h after injection. In another series of experiments rabbits were injected with either [3H]-fucose or several tritiated amino acids and the specific activity of the vitreous proteins was determined. Vitreous samples were also processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and histological sections of retina, ciliary body and lens (the eye components around the vitreous body) were processed for radioautography. The specific activity (counts per minute per microgram of protein) of the glycoproteins labeled with [3H]-fucose was always much higher than that of the proteins labeled with any of the other monosaccharides or any of the amino acids. There was a good correlation between the specific activity of the proteins labeled by any of the above precursors and the density of the vitreous protein bands detected by fluorography. This was also true for the silver grain density on the radioautographs of the histological sections of retina, ciliary body and lens. The contribution of radioautography (after [3H]-fucose administration) to the elucidation of the biogenesis of lysosomal and membrane glycoproteins and to the determination of the intracellular process of protein secretion was reviewed. Radioactive fucose is the precursor of choice for studying glycoprotein secretion because it is specific, efficient and practical for this purpose.     

GFP:HIV-1 protease production and packaging with a T4 phage expression-packaging processing system

A bacteriophage T4-derived protein expression, packaging and processing system was used to create recombinant phage that encode, produce and package a protein composed of human HIV-1 protease fused to green fluorescent protein (GFP). The fusion protein is targeted within the phage capsid by an N-terminal capsid targeting sequence (CTS), which is cleaved through proteolysis by the viral scaffold protease P21. The fusion protein is designated CTS [symbol see text] GFP:PR. The [symbol see text] symbol indicates the linkage peptide sequence leu(ile)-N-glu that is cleaved by the T4 head morphogenetic proteinase gp21 during head maturation. The fusion protein is fluorescent and has protease activity as detected by the appearance of the expected substrate cleavage product on a Western blot. CTS [symbol see text] GFP:PR packaging occurs at about 200 molecules per phage particle. The CTS [symbol see text] GFP:PR fusion protein, when protected within the phage capsid, has been maintained stably for over 16 months at 4 degrees C. Production and storage of fusion protein within the phage circumvents problems of toxicity and solubility encountered with E. coli expression systems. Because recombinant phage inhibit host proteolytic enzymes, foreign proteins are stabilized. This phage system packages and processes the fusion protein by means of the CTS. Proteins can be purified from the phage to give high yields of soluble, proteolytically processed protein. The T4 phage packaging system provides a novel means of identification, purification and long-term storage of toxic proteins whose folding and DNA-directed activities can be studied readily in vivo.     

Betulinans A and B, two benzoquinone compounds from Lenzites betulina

Two lipid peroxidation inhibitors, designated as betulinans A (1) and B (2), were isolated from the MeOH extract of Lenzites betulina. The structures of these compounds have been determined to be 2,5-diphenyl-3,6-dimethoxy-p-benzoquinone and 2-phenyl-3-methoxy-[1H-2-benzopyran][4,3-e][p]benzoquinone, respectively, on the basis of various spectral data. Betulinans A and B inhibited lipid peroxidation with IC50 values of 0.46 and 2.88 micrograms/mL, respectively.     

Analysis of proteins synthesized in mitochondria of cultured mammalian cells. An assessment of current approaches and problems in interpretation

1. The conditions which enable highly efficient utilization of [35S]methionine by cultured mammalian cells and the resolution of selectively labelled mitochondrial products are described. 2. Analysis of mitochondria purified from cells labelled in the presence or absence of inhibitors of cytoplasmic (or mitochondrial) protein synthesis indicated that about 5% of the [35S]methionine incorporated into mitochondrial proteins results from synthesis on mitoribosomes. 3. The electrophoretic profile of the detergent-solubilized proteins of mitochondrial isolated from cells which were labelled in the presence of 50 mug/ml emetine was similar to those obtained with extracts prepared by direct solbuilization of the intact cells after incorporation of label. 4. Pulse-labelling studies suggested that the components resolved by electrophoresis and autoradiography under the conditions described, apparently represent discrete and stable end products radiography under the conditions described, apparently represent discrete and stable end products of mitochondrial protein synthesis. No post-synthetic modification or degradation of these products was detected. 5. Erythromycin was found to suppress the synthesis of additional labelled products which were detected in extracts of one cell line, when analysed by procedures which normally detected only mitochondrially synthesized proteins. These additional bands were attributed to the synthetic activity of Mycoplasma.     

Rapid fluorescent monitoring of total protein patterns on sodium dodecyl sulfate-polyacrylamide gels and western blots before immunodetection and sequencing

The fluorogenic dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) has been used for the detection of total protein patterns on polyvinylidene difluoride (PVDF) membranes. Fluorescent staining of protein bands on membranes with this covalent dye is completed in 20 min. Wet membranes are translucent, allowing protein visualization by transillumination with ultraviolet light. The resulting images can be recorded using Polaroid film or a charge-coupled device camera. Electrophoretic bands containing 5-10 ng of protein can be detected on the MDPF-stained Western blot. When proteins are directly transferred to the membrane using a slot blotting device, as little as 0.5 ng of protein can be detected. Previous visualization of protein bands on sodium dodecyl sulfate-polyacrylamide gels with the noncovalent fluorescent dye Nile red (Alba et al., BioTechniques, 1996, 21, 625-626) does not interfere with further MDPF staining and fluorescent detection of these bands transferred to PVDF membranes. Thus, Nile red and MDPF staining can be performed sequentially, allowing the rapid monitoring of total protein patterns on both the electrophoretic gel and Western blot. Using the conditions described in this study, MDPF staining does not preclude further N-terminal microsequencing and immunodetection of specific bands with polyclonal antibodies.     

Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT)

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.1 glucuronidation activity was 91 +/- 18 pmol/mg x min for atRA and 113 +/- 19 pmol/mg x min for 5,6-epoxy-atRA. The apparent K(M) and V(max) of atRA acid glucuronidation by UGT1.1 were 59.1 +/- 5.4 microM and 158 +/- 43 pmol/mg x min, respectively. SDS-PAGE and Western blot analysis of UGT1.1-transfected HK293 membrane proteins photolabeled with [11,12-3H]atRA revealed a protein of approximately 56 kDa that was labeled by [3H]atRA, detected by anti-pNP UGT antibody and not present in membranes from nontransfected HK293 cells. Liver microsomes from Gunn rats, which lack UGT1.1, had significant activity toward atRA (111 +/- 28 pmol/mg x min).     

Implication of Ca(2+)-dependent protein tyrosine phosphorylation in carbachol-induced phospholipase D activation in rat pheochromocytoma PC12 cells

The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca2+. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands (111, 91, 84, 74, 65-70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111-, 91-, 84-, and 65-70-kDa proteins peaked within 1 min, and their time-dependent changes seemingly correlated with that of PLD activation. Others (74, 44MAPK, and 42MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65-70 kDa) in a preincubation time- and concentration-dependent fashion. In Ca(2+)-free buffer, CCh-induced [3H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca2+ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65-70 kDa only in the presence of extracellular Ca2+. Extracellular Ca2+ dependency for CCh-induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111-kDa protein. These results suggest that Ca(2+)-dependent protein tyrosine phosphorylation is closely implicated in CCh-induced PLD activation in PC12 cells.     

Structure and glycosylation patterns of surface proteins from woodchuck hepatitis virus

Woodchucks chronically infected with woodchuck hepatitis virus (WHV) are a valuable model for human hepatitis B virus (HBV) in studies of pathogenesis, immunity, and antiviral therapy. For this reason, substantial efforts to characterize both the similarities and the differences between HBV and WHV are being made. The structure of the WHV surface proteins (WHs proteins) has not yet been adequately elucidated. The bands that would be expected for glycosylated and nonglycosylated small (S) WHs protein are found by sodium dodecyl sulfate gel electrophoresis of purified WHs protein, but the bands corresponding to the middle (M) and large (L) WHs proteins of HBV are not seen at the expected sizes, even though the sequences of the WHV and HBV surface protein genes are 60% homologous. By amino-terminal sequencing we have identified two bands at 41 and 45 kDa as the MWHs proteins, 8 kDa larger than expected. We have also confirmed that two bands at 24 and 27 kDa are SWHs proteins. A protein of 49 kDa was blocked at the N terminus, which using immunoblotting with an antiserum against WHV pre-S1 (positions 126 to 146) was identified, together with a part of the 45-kDa protein, as glycosylated and nonglycosylated LWHs protein of the expected size. Sialidase and O-glycosidase digestion showed that the larger size of MWHs protein results from the presence of O glycoside groups which are probably in the pre-S2 domain of MWHs protein. Since the pre-S2 domains of HBV and WHV have similar numbers of potential O glycosylation sites, it appears to be likely that the glycosyltransferases act differently on the viral proteins in woodchucks and humans.     

The cytoprotective effect of leminoprazole on indomethacin-induced damage to rabbit gastric mucosal cells

Leminoprazole (an acid pump inhibitor) has a mucosal protective effect against various experimental gastric lesions, but the underlying mechanism remains unknown. We examined whether leminoprazole prevents indomethacin-induced damage to cultured gastric mucosal cells. The viability of rabbit gastric mucosal cells was assessed by the 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide and dye exclusion methods. [35S]Methionine-labeled proteins were detected by autoradiography after sodium dodecylsulfate-polyacrylamide gel electrophoresis. Western blot analysis was carried out using anti-heat shock protein (HSP)-70 and anti-HSP-72 antibodies. Exposure of gastric mucosal cells to indomethacin for 4 hr apparently reduced their viability in a dose-related manner. Pretreatment with leminoprazole for 4 hr significantly prevented the reduction in cell viability caused by 50 microM indomethacin, although omeprazole was not effective. However, such pretreatment did not prevent the severe damage induced by 500 microM indomethacin. 16,16-Dimethyl prostaglandin E2 significantly prevented the cell damage induced by indomethacin at both 50 and 500 microM. Leminoprazole alone did not affect cell viability. The cytoprotection by leminoprazole was expressed after a 2-hr lag period. Leminoprazole did not promote prostaglandin E2 synthesis by cells, but it apparently induced the synthesis of 83-kDa, 72-kDa, 52-kDa and 35-kDa proteins. Both the cytoprotection and the induction of such protein synthesis were abolished by cycloheximide and actinomycin D. The leminoprazole-induced 72-kDa protein did not react with the antibodies against HSP-70 and HSP-72. These results indicate that leminoprazole directly protects gastric mucosal cells against mild damage caused by indomethacin and that its cytoprotective effect might be mediated through de novo synthesized proteins. In addition, it is suggested that the leminoprazole-induced proteins might be unknown proteins related to cytoprotection, although the exact characters of the proteins are unclear.     

Evidence of quinone metabolites of naphthalene covalently bound to sulfur nucleophiles of proteins of murine Clara cells after exposure to naphthalene

Naphthalene-induced Clara cell toxicity in the mouse is associated with the covalent binding of electrophilic metabolites to cellular proteins. Epoxide and quinone metabolites of naphthalene are proposed to be the reactive metabolites responsible for covalent binding to proteins. To identify the nature of reactive metabolites bound to proteins (cysteine residues), we alkaline-permethylated proteins obtained from mouse Clara cells incubated with 0.5 mM naphthalene in vitro. Alkaline permethylation of protein adducts produced (methylthio)naphthalene derivatives detected by GC-MS. 3,4-Dimethoxy(methylthio)naphthalene was observed to be a predominant (methylthio)naphthalene derivative formed in the alkaline-permethylated protein sample obtained from Clara cells after exposure to naphthalene. This indicates that 1,2-naphthoquinone is a major metabolite covalently bound to cysteine residues of the cellular proteins. We have developed an immunoblotting approach to detect 1,2-naphthoquinone covalently bound to cysteine residues of proteins [Zheng, J., and Hammock, B. D. (1996) Chem. Res. Toxicol. 9, 904-909]. To identify 1,2-naphthoquinone covalently bound to sulfur nucleophiles of proteins, homogenates obtained from naphthalene-exposed Clara cells were separated by SDS-PAGE followed by Western blotting and immunostaining with the antibodies. Two protein bands with 24 and 25 kDa were detected by the antibodies, further supporting the view that 1,2-naphthoquinone is a reactive metabolite of naphthalene which binds to Clara cell proteins in vitro.     

The additional predictive value contributed by quantitative chromatin pattern description as compared to DNA ploidy level measurement in 257 superficial bladder transitional cell carcinomas

The purpose of the present study was to demonstrate the post-translational modifications of sperm plasma membrane proteins by fatty acid acylation during sperm maturation in the epididymis. Rat epididymal spermatozoa were incubated at 37 degrees C with various concentrations (100 microCi and 1 mCi) of [9-10(n)3H]palmitic acid in a medium containing Tyrode's solution supplemented with sodium bicarbonate, sodium pyruvate and sodium lactate. The incorporation of [3H]palmitate in vitro was determined in epididymal spermatozoa and an attempt was made to identify the lipid-linked proteins of purified plasma membranes of maturing epididymal spermatozoa by autoradiography. The studies demonstrated that [3H]palmitate was covalently linked to a subset of membrane cytoskeleton proteins of maturing rat spermatozoa. The pattern of incorporation of lipid was a maturation-associated phenomenon as caput spermatozoa incorporated more radioactivity than did caudal spermatozoa. The labelled proteins appeared to be membrane-bound since 82% of radioactivity was associated with membrane fractions. Autoradiograms of SDS-PAGE gels of labelled caput sperm extract showed three prominent palmitate-incorporating protein bands of about 70, 56 and 36 kDa and few minor bands. Most of these proteins were present in the membrane fraction of caput spermatozoa. Labelled gels of both the sperm extracts and of purified membranes showed resistance to hydroxylamine treatment, suggesting that there are amide bonds between lipid and proteins. The higher incorporation of labelled palmitate by immature spermatozoa of the caput epididymis compared with mature spermatozoa from the cauda epididymis and the addition of palmitate to plasma membrane proteins of caput epididymal spermatozoa suggest that fatty acylation is a post-translational modification of sperm membrane proteins.     

Missouri Advantage

The OprB porin-mediated glucose transport system was investigated in Pseudomonas chlororaphis, Burkholderia cepacia, and Pseudomonas fluorescens. Kinetic studies of [U-14C]glucose uptake revealed an inducible system of low Km values (0.3-5 microM) and high specificity for glucose. OprB homologs were purified and reconstituted into proteoliposomes. The porin function and channel preference for glucose were demonstrated by liposome swelling assays. Examination of the periplasmic glucose-binding protein (GBP) components by Western immunoblotting using P. aeruginosa GBP-specific antiserum revealed some homology between P. aeruginosa GBP and periplasmic proteins from P. fluorescens and P. chlororaphis but not B. cepacia. Circular dichroism spectropolarimetry of purified OprB-like porins from the three species revealed beta sheet contents of 31-50% in agreement with 40% beta sheet content for the P. aeruginosa OprB porin. These findings suggest that the high-affinity glucose transport system is primarily specific for glucose and well conserved in the genus Pseudomonas although its outer membrane component may differ in channel architecture and specificity for other carbohydrates.