Structural equivalents of latency for lysosome hydrolases.
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1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: beta-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl beta-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of beta-glucuronidase and beta-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the beta-glycerophosphate concentration. The fractional free activity of beta-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free beta-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with beta-glycerophosphate or p-nitrophenyl beta-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of "intact" lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl beta-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane. (+info)
Identification of sulfated oligosaccharide-based inhibitors of tumor growth and metastasis using novel in vitro assays for angiogenesis and heparanase activity.
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Inhibitors of tumor angiogenesis and metastasis are rapidly emerging as important new drug candidates for cancer therapy. To facilitate the identification of such drugs, we recently developed novel and rapid in vitro assays for human angiogenesis and for the extracellular matrix-degrading enzyme heparanase, which has been implicated in tumor metastasis. In this study, sulfated oligosaccharides, which are structural mimics of heparan sulfate, were investigated as drug candidates because these compounds may interfere with heparan sulfate recognition by many angiogenic growth factors and may inhibit cleavage of heparan sulfate by heparanase. In the preliminary screening studies, it was found that inhibitory activity in both assay systems was critically dependent on chain length and degree of sulfation, highly sulfated linear oligosaccharides of five or more monosaccharides in length being the most active. However, two sulfated oligosaccharides stood out as potential antitumor drugs, phosphomannopentaose sulfate (PI-88) and maltohexaose sulfate, both of these compounds having the important property of simultaneously being potent inhibitors of in vitro angiogenesis and heparanase activity. Due to the ease of manufacture of the starting material, phosphomannopentaose, PI-88 was studied in more detail. PI-88 was shown to inhibit the primary tumor growth of the highly invasive rat mammary adenocarcinoma 13762 MAT by approximately 50%, inhibit metastasis to the draining popliteal lymph node by approximately 40%, and reduce the vascularity of tumors by approximately 30%, all of these effects being highly significant. Acute hematogenous metastasis assays also demonstrated that PI-88 was a potent (>90%) inhibitor of blood-borne metastasis. Thus, by the use of novel in vitro screening procedures, we have identified a promising antitumor agent. (+info)
Metabolism and excretion of atorvastatin in rats and dogs.
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Atorvastatin (AT) is a second-generation potent inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, clinically approved for lowering plasma cholesterol. Using a mixture of [D(5)/D(0)] AT and/or [(14)C]AT, the metabolic fate and excretion of AT were examined in rats and dogs following single and multiple oral doses. Limited biliary recycling was examined in one dog after a single dose of AT. AT-derived metabolites in bile samples were identified by metabolite screening of the [D(5)/D(0)] AT molecular clusters using tandem mass spectrometry. Bile was a major route of [(14)C] drug-derived excretion, accounting for 73 and 33% of the oral dose in the rat and dog, respectively. The remaining radioactivity was recovered in the feces; only trace amounts were excreted in urine. Radioactive components identified in rat and dog bile were the para- and ortho-hydroxy metabolites, a glucuronide conjugate of ortho-hydroxy AT, and unchanged AT. Two minor radioactive components were identified as beta-oxidation products of AT with one confirmed as a beta-oxidized AT derivative. The reappearance of AT and major metabolites in bile from a dog administered a sample of its previously excreted bile indicated biliary recycling is an important component in AT metabolism. Multiple dose administration in rats did not alter biliary metabolic profiles. Rat and dog plasma profiles after multiple dose administration were similar and showed no additional metabolites not found in bile. Examination of rat and dog bile and plasma indicates that AT primarily undergoes oxidative metabolism. (+info)
Immunomodulatory action of citrus auraptene on macrophage functions and cytokine production of lymphocytes in female BALB/c mice.
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The modifying effects of auraptene isolated from the peel of citrus fruit (Citrus natsudaidai Hayata) on macrophage and lymphocyte functions were investigated in mice. Female BALB/c mice were gavaged with auraptene at a dose of 100, 200 or 400 mg/kg once a day for 10 consecutive days. Glucose consumption of peritoneal macrophages was significantly higher than that in the control group (P < 0.05-0.001) in auraptene-treated mice at all doses at 24, 48 and 72 h incubation except for mice given 200 mg/kg auraptene at 24 h incubation. Activity of acid phosphatase in peritoneal macrophages was significantly increased in mice treated with auraptene at a dose level of 100 mg/kg (P < 0.001). Activity of beta-glucuronidase in peritoneal macrophages in the auraptene-treated mice at all doses was significantly higher than that in the control group (P < 0.001), but there was no significant difference in lactate dehydrogenase activity of peritoneal macrophages at any dose. Interleukin (IL)-1beta production of peritoneal macrophages in the auraptene-treated mice at all doses was significantly higher than that in the control group (P < 0.05-0.001). Tumor necrosis factor alpha production of peritoneal macrophages in mice gavaged with auraptene at a dose of 200 mg/kg was significantly higher than that in the control group (P < 0.05). Auraptene did not affect proliferation of spontaneous splenic lymphocytes in mice at any dose. Stimulation indices in mice given auraptene at a dose of 200 mg/kg were significantly higher than that in the control group (P < 0.05). When spleenic lymphocytes were cultured without concanavalin A (Con A), IL-2 and interferon (IFN) gamma productions were not detectable in the supernatant. However, IL-2 and IFN production stimulated by Con A were significantly increased in mice gavaged with auraptene at dose levels of 100 and 200 mg/kg (P 0.05-0.001). Auraptene did not enhance spontaneous IL-4 production by splenocytes. There was no significant difference in IL-4 production of splenic lymphocytes stimulated by Con A in all groups. These findings might suggest that oral administration of citrus auraptene effectively enhanced macrophage and lymphocyte functions in mice. (+info)
Development and testing of a microbiological assay to detect residual effects of disinfectant on hard surfaces.
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We describe a glucuronidase bioassay for detecting residual bactericidal activity from the use of disinfectants on hard surfaces; in this assay we used formaldehyde, ethanol, isopropanol, chlorine, and a commercial preparation containing 2-bromo-2-nitro-1, 3-propanediol. Chlorine and the commercial preparation showed bactericidal activity (53.5% and 98.2%, respectively) for a week after disinfection. (+info)
Independent impairment of osteoblast and osteoclast differentiation in klotho mouse exhibiting low-turnover osteopenia.
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We recently identified a new gene, klotho, which is involved in the suppression of multiple aging phenotypes. The mouse homozygous for a disruption of the klotho locus (kl/kl) exhibited multiple pathological conditions resembling human aging. Histomorphometric analysis revealed low-turnover osteopenia in kl/kl mice. The decrease in bone formation exceeded that of bone resorption, resulting in a net bone loss. The number of osteoblast progenitors determined by ex vivo bone marrow cultures was reduced in kl/kl mice. In addition, cultured osteoblastic cells derived from kl/kl mice showed lower alkaline phosphatase activity and matrix nodule formation than those from wild-type mice. Osteoclastogenesis in the coculture of marrow cells and osteoblastic cells was decreased only when marrow cells originated from kl/kl mice independently of the origin of osteoblastic cells. We also found that the expression of osteoprotegerin, an osteoclastogenesis inhibitor, was significantly upregulated in kl/kl mice. We conclude that a defect in the klotho gene expression causes the independent impairment of both osteoblast and osteoclast differentiation, leading to low-turnover osteopenia. Because this state represents a characteristic feature of senile osteoporosis in humans, kl/kl mice can be regarded as a useful model for investigating cellular and molecular mechanisms of age-related bone loss. (+info)
The Xanthomonas Hrp type III system secretes proteins from plant and mammalian bacterial pathogens.
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Studies of essential pathogenicity determinants in Gram-negative bacteria have revealed the conservation of type III protein secretion systems that allow delivery of virulence factors into host cells from plant and animal pathogens. Ten of 21 Hrp proteins of the plant pathogen Xanthomonas campestris pv. vesicatoria have been suggested to be part of a type III machinery. Here, we report the hrp-dependent secretion of two avirulence proteins, AvrBs3 and AvrRxv, by X. campestris pv. vesicatoria strains that constitutively express hrp genes. Secretion occurred without leakage of a cytoplasmic marker in minimal medium containing BSA, at pH 5.4. Secretion was strictly hrp-dependent because a mutant carrying a deletion in hrcV, a conserved hrp gene, did not secrete AvrBs3 and AvrRxv. Moreover, the Hrp system of X. campestris pv. vesicatoria was able to secrete proteins from two other plant pathogens: PopA, a protein secreted via the Hrp system in Ralstonia solanacearum, and AvrB, an avirulence protein from Pseudomonas syringae pv. glycinea. Interestingly, X. campestris pv. vesicatoria also secreted YopE, a type III-secreted cytotoxin of the mammalian pathogen Yersinia pseudotuberculosis in a hrp-dependent manner. YerA, a YopE-specific chaperone, was required for YopE stability but not for secretion in X. campestris pv. vesicatoria. Our results demonstrate the functional conservation of the type III system of X. campestris for secretion of proteins from both plant and mammalian pathogens and imply recognition of their respective secretion signals. (+info)
Active site residues of human beta-glucuronidase. Evidence for Glu(540) as the nucleophile and Glu(451) as the acid-base residue.
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Human beta-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coli beta-galactosidase active sites led us to propose that residues Glu(451), Glu(540), and Tyr(504) in hGUSB are involved in catalysis, Glu(451) being the acid-base residue and Glu(540) the nucleophile. To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts. The extremely low activity in cells expressing Glu(451), Glu(540), and Tyr(504) hGUSBs supported their roles in catalysis. For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. The k(cat)/K(m) values (mM(-1).s(-1)) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu(451) as the acid-base catalyst. We conclude that, like their homologues in E. coli beta-galactosidase, Glu(540) is the nucleophilic residue, Glu(451) the acid-base catalyst, and Tyr(504) is also important for catalysis, although its role is unclear. All three residues are located in the active site cavity previously determined by structural analysis of hGUSB. (+info)