Ethanol is a potent inhibitor of canine cerebrospinal fluid production: an acute and reversible effect

Poly(ethylene glycol) and dextran aqueous two-phase systemis (ATPS) were developed to facilitate the separation of components of the proteose peptone fraction of bovine milk, which are mostly large casein derived peptides or glycoproteins. These have proved difficult to purify using conventional chromatographic procedures. ATPS exploit differences in hydrophobicity, size and ionic properties of the proteose peptones with a view to developing methods for future large scale preparations of the individual components of this whey protein fraction.     

Characterization of protein-tyrosine phosphatases that dephosphorylate the high affinity IgE receptor

An early event that follows aggregation of the high affinity receptor for IgE (FcepsilonRI) is the phosphorylation of protein tyrosines, especially those on the beta- and gamma-subunits of the receptor. Disaggregation of the receptors leads to their rapid dephosphorylation, but even stably aggregated receptors undergo continual rounds of phosphorylation and dephosphorylation. We developed assays to study dephosphorylation of the receptors and other cellular proteins. Whole cell extracts dephosphorylated both subunits of the receptors rapidly and were as active against aggregated as against disaggregated FcepsilonRI. Upon disaggregation, the in vivo dephosphorylation of the FcepsilonRI and several other proteins followed first-order kinetics with closely similar rate constants despite substantial differences in the extent of phosphorylation. These results suggest that the level of phosphorylation of FcepsilonRI is largely controlled by the aggregation-induced action of kinase(s) and not from changes in susceptibility to or activity of the phosphatases. Much of the total phosphatase is lost when the cells are permeabilized, but the rate of dephosphorylation of disaggregated FcepsilonRI was comparable in intact and permeabilized cells. Thus, much of the activity utilized by the cell to dephosphorylate the FcepsilonRI is likely to be associated with the plasma membrane.     

Haloenol lactone is a new isozyme-selective and active site-directed inactivator of glutathione S-transferase

A haloenol lactone derivative has been synthesized and found to be an isozyme-selective and active site-directed inactivator of glutathione S-transferase (GST). Preincubation of the haloenol lactone (100 microM) with murine Alpha, Mu, or Pi GST isozyme (1.0 microM) at pH 6.5, 37 degrees C resulted in time-dependent loss of enzyme activity with highly selective inhibition of the Pi isozyme (t1/2, approximately 2 min). In a separate experiment, a 10-fold excess of the lactone was incubated with GST-Pi isozyme at 37 degrees C for 3 h, followed by dialysis against Nanopure water. GST activity lost upon incubation with the lactone could not be restored by exhaustive dialysis, and only 8% of enzyme activity for the modified GST remained relative to the control that was treated identically except the lactone was omitted from the incubation. Both control and modified GST were characterized using electrospray ionization mass spectrometry. No native GST (23,478 Da) was observed in the spectrum of modified GST. Instead, protein incubated with the lactone exhibited an increase in molecular mass of 230 Da relative to control GST. The lactone (100 microM) was incubated with GST Pi isozyme (1.0 microM) in the presence of the competitive inhibitor S-hexylglutathione (10 microM), which suppressed time-dependent inhibition of GST by the lactone. The results suggest that this haloenol lactone is an irreversible and active site-directed inhibitor of GST that appears to inhibit the enzyme through two consecutive steps of nucleophilic attack.     

The inhibition of human salivary alpha-amylase by type II alpha-amylase inhibitor from Triticum aestivum is competitive, slow and tight-binding

The release of newer quinolones (such as pefloxacin, ofloxacin, and ciprofloxacin) from biodegradable poly(D, L lactic acid) has been investigated. The in vitro study showed that drug delivery takes place for about two months and a maximum in concentration was recorded after fifteen days. The release from poly(lactic acid) slabs seemed to give high drug doses that are adequate for the treatment of infections caused by common pathogens.     

Covalent modification and site-directed mutagenesis of an active site tryptophan of human prostatic acid phosphatase

Because tryptophans are found as part of the phosphate binding sites in a number of proteins, human prostatic acid phosphatase (hPAP) was examined for the presence and the role of essential tryptophan residues. The pH dependence of the intrinsic fluorescence of hPAP resembled the kinetic pH dependence. Chemical modification by N-bromosuccinimide (NBS) resulted in an inactivation of the enzyme and produced a characteristic reduction of the protein absorbance at 280 nm. Two tryptophans per subunit were modified, and this was accompanied by an apparently complete loss of enzymatic activity. In the presence of the competitive inhibitor L-(+)-tartrate, the loss of enzyme activity was significantly reduced as compared to the rate of tryptophan modification. After labeling the protein with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), two tryptic peptides containing DNPS-labeled tryptophans were isolated and the sequences were identified by amino acid sequence analysis and mass spectroscopy. One peptide corresponded to residues 172-176, and included Trp174. The other corresponded to the C-terminal sequence, including Trp336. It was concluded that Trp174 was at the active site of the human enzyme because it was protected by the competitive inhibitor tartrate in the DNPS-Cl modification studies. This is also consistent with the location of a homologous residue in the structure of the rat enzyme. Using site-directed mutagenesis, Trp174 was replaced by Phe or Leu. Both mutants showed altered kinetic properties, including lower Km values with several aromatic substrates, and also exhibited reduced stability towards urea denaturation.     

The active site specificity of the Yersinia protein-tyrosine phosphatase

Previous studies have shown that B700, an albumin-like murine melanoma antigen, has a human homologue termed H700. Polyclonal antibodies to B700 also bind to all cultured human, swine and hamster melanoma cells, suggesting that B700 is a "pan-melanoma" antigen. The objects of this investigation were: (a) to determine if 2-3-3, a monoclonal antibody to B700, can be used to identify human melanomas in formalin-fixed, paraffin-embedded tissues, and (b) to determine the specificity and potential diagnostic value of 2-3-3. Forty-eight of the 49 human melanomas, including spindle melanoma cells, stained positively, as did five of the eight pigmented naevi including cellular spindle naevi. Twenty-six of the 32 human non-melanomatous lesions were negative for 2-3-3 staining (weakly positive on one breast carcinoma and positive on five neural tumours). These results indicate that 2-3-3, a monoclonal antibody to the mouse melanoma antigen B700, can be used to identify H700 in archival specimens. 2-3-3 may have an advantage over HMB45, which is the most commonly used antibody for melanoma diagnosis, because of its immunoreactivity with spindle melanocytic lesions. Antibodies to B700 may prove to be a useful adjunct in the diagnosis of human melanoma and related lesions.     

Genetic complexity, structure, and characterization of highly active bovine intestinal alkaline phosphatases

Mammalian alkaline phosphatases (APs) display 10-100-fold higher kcat values than do bacterial APs. To begin uncovering the critical residues that determine the catalytic efficiency of mammalian APs, we have compared the sequence of two bovine intestinal APs, i.e. a moderately active isozyme (bovine intestinal alkaline phosphatase, bIAP I, approximately 3,000 units/mg) previously cloned in our laboratory, and a highly active isozyme (bIAP II, approximately 8, 000 units/mg) of hitherto unknown sequence. An unprecedented level of complexity was revealed for the bovine AP family of genes during our attempts to clone the bIAP II cDNA from cow intestinal RNAs. We cloned and characterized two novel full-length IAP cDNAs (bIAP III and bIAP IV) and obtained partial sequences for three other IAP cDNAs (bIAP V, VI, and VII). Moreover, we identified and partially cloned a gene coding for a second tissue nonspecific AP (TNAP-2). However, the cDNA for bIAP II, appeared unclonable. The sequence of the entire bIAP II isozyme was determined instead by a classical protein sequencing strategy using trypsin, carboxypeptidase, and endoproteinase Lys-C, Asp-N, and Glu-C digestions, as well as cyanogen bromide cleavage and NH2-terminal sequencing. A chimeric bIAP II cDNA was then constructed by ligating wild-type and mutagenized fragments of bIAP I, III, and IV to build a cDNA encoding the identified bIAP II sequence. Expression and enzymatic characterization of the recombinant bIAP I, II, III, and IV isozymes revealed average kcat values of 1800, 5900, 4200, and 6100 s-1, respectively. Comparison of the bIAP I and bIAP II sequences identified 24 amino acid positions as likely candidates to explain differences in kcat. Site-directed mutagenesis and kinetic studies revealed that a G322D mutation in bIAP II reduced its kcat to 1300 s-1, while the converse mutation, i.e. D322G, in bIAP I increased its kcat to 5800 s-1. Other mutations in bIAP II had no effect on its kinetic properties. Our data clearly indicate that residue 322 is the major determinant of the high catalytic turnover in bovine IAPs. This residue is not directly involved in the mechanism of catalysis but is spatially sufficiently close to the active site to influence substrate positioning and hydrolysis of the phosphoenzyme complex.     

Leukotriene C4 is a tight-binding inhibitor of microsomal glutathione transferase-1. Effects of leukotriene pathway modifiers

Microsomal glutathione transferase-1 (MGST-1) is an abundant protein that catalyzes the conjugation of electrophilic compounds with glutathione, as well as the reduction of lipid hydroperoxides. Here we report that leukotriene C4 is a potent inhibitor of MGST-1. Leukotriene C4 was found to be a tight-binding inhibitor, with a Ki of 5.4 nM for the unactivated enzyme, and 9.2 nM for the N-ethylmaleimide activated enzyme. This is the first tight-binding inhibitor characterized for this enzyme. Leukotriene C4 was competitive with respect to glutathione and non-competitive toward the second substrate, CDNB. Analysis of stoichiometry supports binding of one molecule of inhibitor per homotrimer. Leukotrienes A4, D4, and E4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C4 analogues, which have been developed as antagonists of leukotriene receptors, were found to display varying degrees of inhibition of MGST-1. In particular, the cysteinyl-leukotriene analogues SKF 104,353, ONO-1078, and BAYu9773 were strong inhibitors (IC50 values: 0.13, 3. 7, and 7.6 microM, respectively). In view of the partial structural similarity between MGST-1, leukotriene C4 synthase, and 5-lipoxygenase activating protein (FLAP), it was of interest that leukotriene C4 synthesis inhibitors (which antagonize FLAP) also displayed significant inhibition (e.g. IC50 for BAYx1005 was 58 microM). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 microM). Our discovery that leukotriene C4 and drugs developed based on its structure are potent inhibitors of MGST-1 raises the possibility that MGST-1 influences the cellular processing of leukotrienes. These findings may also have implications for the effects and side-effects of drugs developed to manipulate leukotrienes.     

Saquinavir, an HIV protease inhibitor, is transported by P-glycoprotein

Saquinavir, a peptidomimetic HIV protease inhibitor, has been shown to be effective in reducing patient viral load and reducing mortality. In this report we investigated whether saquinavir is a substrate for the multidrug resistance transporter P-glycoprotein (P-gp), which may reduce the effective intracellular concentration of the drug. G185 cells, which highly express P-gp, are resistant to saquinavir-mediated cytotoxicity, and co-administration of cyclosporine reversed this resistance. Saquinavir and saquinavir mesylate inhibited basolateral to apical transport of the fluorescent dye rhodamine 123 in a polarized epithelial transport assay, a result that suggests competition of these drugs for the P-gp transporter. Finally, we measured specific, directional transport of saquinavir and saquinavir mesylate in an epithelial monolayer model. Transport in the basolateral to apical direction was 3-fold greater than apical to basolateral flux for both saquinavir and saquinavir mesylate and was blocked by co-incubation with the established P-gp reversal agents cyclosporine and verapamil. These data provide evidence that saquinavir is a substrate for the P-gp transporter and suggest that this protein may affect intracellular accumulation of the drug and contribute to its poor oral bioavailability.     

Geminin, an inhibitor of DNA replication, is degraded during mitosis

We describe a novel 25 kDa protein, geminin, which inhibits DNA replication and is degraded during the mitotic phase of the cell cycle. Geminin has a destruction box sequence and is ubiquitinated anaphase-promoting complex (APC) in vitro. In synchronized HeLa cells, geminin is absent during G1 phase, accumulates during S, G2, and M phases, and disappears at the time of the metaphase-anaphase transition. Geminin inhibits DNA replication by preventing the incorporation of MCM complex into prereplication complex (pre-RC). We propose that geminin inhibits DNA replication during S, G2, and M phases and that geminin destruction at the metaphase-anaphase transition permits replication in the succeeding cell cycle.     

Clotrimazole, an imidazole antimycotic, is a potent inhibitor of angiogenesis

PURPOSE: To evaluate the roles of fibroblast proteins in the remodeling of the subconjunctival connective tissue, we immunohistochemically assessed the expression of matrix metalloproteinases (MMP)-1 and -2, and the tissue inhibitors of matrix metalloproteinases (TIMP)-1 and -2 in cultured human subconjunctival fibroblasts and in normal and healing human subconjunctival connective tissue. METHODS: Cultured fibroblasts derived from human subconjunctival connective tissue and surgical specimens of normal and healing conjunctiva were immunostained with monoclonal antibodies directed against human MMPs and TIMPs and examined by light and electron microscopy. RESULTS: In the cultured fibroblasts, MMP-1 and TIMP-1 antibodies stained the cytoplasm in a fine granular pattern, suggesting localization of those proteins in the endoplasmic reticulum (ER) and Golgi apparatus. Antibodies to MMP-2 and TIMP-2 reacted with fibroblast cytoplasm in a granular pattern. Electron microscopy of those fibroblasts revealed MMP-1 and TIMP-1 immunoreactivity in the ER cisternae or on the membrane of the ER. In surgical samples, MMP-1 and TIMP-1 were immunohistochemically detected in healing subconjunctival tissue, but not in conjunctival epithelium or normal subconjunctival tissue. CONCLUSIONS: MMPs and TIMPs may be involved in remodeling of subconjunctival connective tissue and in fibroblast population after surgical interventions. These proteins may play a crucial role in the post-operative fibrotic process occurring during scar formation in subconjunctival tissue.     

Cell density-dependent changes in the insulin action pathway: evidence for involvement of protein-tyrosine phosphatases

Three gas chromatographic procedures for the determination of ethanol in postmortem blood using alternative internal standards to n-propanol are presented: a direct injection procedure using t-butanol, and two headspace methods using t-butanol and methyl ethyl ketone. t-Butanol and methyl ethyl ketone were well resolved from ethanol, acetone, methanol and other commonly observed putrefactive volatiles using direct injection or headspace analysis. CVs for the direct injection method were below 5% for ethanol and below 10% for the other volatiles. The lower limits of detection (LOD) were 25-50 mg/L. The CVs for the headspace methods were below 5% for ethanol and below 6% for the other volatiles. The LODs were 10 mg/L using either t-butanol or methyl ethyl ketone as internal standards. The use of t-butanol or methyl ethyl ketone as alternatives to n-propanol avoids the possibility of error in the quantitation of ethanol due to the presence of n-propanol and allows for the identification of other volatiles that may aid in distinguishing antemortem ingestion from postmortem production of ethanol.     

Trapoxin, an antitumor cyclic tetrapeptide, is an irreversible inhibitor of mammalian histone deacetylase

Trapoxin (cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8- oxo-9,10-epoxy-decanoyl)) is a fungal product that induces morphological reversion from transformed to normal in sis-transformed NIH3T3 fibroblasts. Trapoxin was found to cause accumulation of highly acetylated core histones in a variety of mammalian cell lines. In vitro experiments using partially purified mouse histone deacetylase showed that a low concentration of trapoxin irreversibly inhibited deacetylation of acetylated histone molecules. Chemical reduction of an epoxide group in trapoxin completely abolished the inhibitory activity, suggesting that trapoxin binds covalently to the histone deacetylase via the epoxide. In contrast, inhibition by trichostatin A, a known potent inhibitor of histone deacetylase, was reversible. Despite the different mode of inhibition, trapoxin and trichostatin A induced almost the same biological effects on the cell cycle and differentiation. These results strongly suggest that the in vivo effects commonly induced by these agents can be attributed to histone hyperacetylation resulting from the inhibition of histone deacetylase.     

GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis

Dorsal accumulation of beta-catenin in early Xenopus embryos is required for body axis formation. Recent evidence indicates that beta-catenin is dorsally stabilized by the localized inhibition of the kinase Xgsk-3, utilizing a novel Wnt ligand-independent mechanism. Using a two-hybrid screen, we identified GBP, a maternal Xgsk-3-binding protein that is homologous to a T cell protooncogene in three well-conserved domains. GBP inhibits in vivo phosphorylation by Xgsk-3, and ectopic GBP expression induces an axis by stabilizing beta-catenin within Xenopus embryos. Importantly, antisense oligonucleotide depletion of the maternal GBP mRNA demonstrates that GBP is required for the establishment of the dorsal-ventral axis in Xenopus embryos. Our results define a family of GSK-3-binding proteins with roles in development and cell proliferation.     

Prolyl aminopeptidase is not a sulfhydryl enzyme: identification of the active serine residue by site-directed mutagenesis

Prolyl aminopeptidase (PAP) has been classified as a sulfhydryl enzyme on the basis of its high sensitivity to p-chloromercuribenzoate and heavy metals. Recently, however, the possibility of PAP being instead a serine enzyme has been raised as a result of two observations--the conservation of some residues among the PAPs hitherto sequenced, and a similarity to some serine hydrolases. This is the first report describing an attempt to identify the active residue by site-directed mutagenesis. The pap genes from Bacillus coagulans and Aeromonas sobria, were used for the study. The changes made were Cys62Ser and Ser101Ala for the first enzyme, and Thr92Ala and Ser146Ala for the second. For both enzymes, only the changes made on the serine residues resulted in their complete inactivation, indicating that PAP is a serine peptidase.     

Poisoning with a reversible and selective monoaminooxidase inhibitor

Two cases of poisoning with moclobemide are presented. Moclobemide is a reversible inhibitor of the monoamine oxidase type A (RIMA). Intoxication with moclobemide is according to previously published case stories benign. Both patients described presented severe symptoms, such as convulsions, coma, muscle rigidity and respiratory failure. One of the patients, a 37-year-old woman, also suffered cardiovascular collapse and elevated body temperature (more than 41.9 degrees C), which was treated successfully with dantrolene and norepinephrine. The symptoms match the diagnostic criteria for serotonin syndrome. The possible reasons why the two patients suffered life-threatening complications and the treatment of serotonin syndrome are discussed.     

Cryogenic X-ray crystal structure analysis for the complex of scytalone dehydratase of a rice blast fungus and its tight-binding inhibitor, carpropamid: the structural basis of tight-binding inhibition

Scytalone dehydratase is a member of the group of enzymes involved in fungal melanin biosynthesis in a phytopathogenic fungus, Pyricularia oryzae, which causes rice blast disease. Carpropamid [(1RS,3SR)-2, 2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropa necarboxamide] is a tight-binding inhibitor of the enzyme. To clarify the structural basis for tight-binding inhibition, the crystal structure of the enzyme complexed with carpropamid was analyzed using diffraction data collected at 100 K. The structural model was refined to a crystallographic R-factor of 0.180 against reflections up to a resolution of 2.1 A. Carpropamid was bound in a hydrophobic cavity of the enzyme. Three types of interactions appeared to contribute to the binding. (i) A hydrogen bond was formed between a chloride atom in the dichloromethylethylcyclopropane ring of carpropamid and Asn-131 of the enzyme. (ii) The (chlorophenyl)ethyl group of carpropamid built strong contacts with Val-75, and this group further formed a cluster of aromatic rings together with four aromatic residues in the enzyme (Tyr-50, Phe-53, Phe-158, and Phe-162). (iii) Two hydration water molecules bound to the carboxamide group of carpropamid, and they were further hydrogen-bonded to Tyr-30, Tyr-50, His-85, and His-110. As a result of interactions between carpropamid and the phenylalanine residues (Phe-158 and Phe-162) in the C-terminal region of the enzyme, the C-terminal region completely covered the inhibitor, ensuring its localization in the cavity.