Regulation of NAD(P)H oxidase by associated protein disulfide isomerase in vascular smooth muscle cells.
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NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22(phox), Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22(phox), Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling. (+info)
1H NMR study on the conformation of bacitracin A in aqueous solution.
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The conformation of bacitracin A, a widely used cyclic dodecapeptide antibiotic in aqueous solution, has been investigated using 500 MHz 1H NMR and molecular modeling. Findings revealed that a region (residues 1-6) is folded over the cyclic ring, resulting in metal coordination sites, a thiazoline ring, and Glu4 and His10 being proximate to each other. (+info)
Pharmacological characterization of angiotensin-induced depolarizations of rat superior cervical ganglion in vitro.
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1. The depolarizing responses to angiotensin II and angiotensin III of the rat superior cervical ganglion have been characterized in vitro, by the use of peptidase inhibitors, peptide and non-peptide antagonists and dithiothreitol (DTT). 2. Angiotensin II and III depolarized the ganglion in a concentration-related manner. Angiotensin II was approximately 30 fold more potent than angiotensin III. 3. The endopeptidase inhibitor, bacitracin, increased the potency of angiotensin II and III by approximately 4 and 20 fold respectively. The aminopeptidase inhibitor, amastatin, further increased the potency of angiotensin III (but not angiotensin II) by approximately 4 fold. In the presence of bacitracin and amastatin, angiotensin II and III were equipotent. 4. The peptide antagonist [Ile7]angiotensin III (0.01-0.3 microM) produced a non-parallel rightward displacement of the angiotensin II concentration-response curve, with a suppression of the maximum response. The potency of [Ile7]angiotensin III was increased by bacitracin and amastatin. 5. The AT1-selective non-peptide antagonist losartan (DuP 753; 0.03 and 0.1 microM) produced a parallel rightward displacement of the angiotensin II concentration-response curve, with an apparent pKB of 8.3 +/- 0.1. A higher concentration of losartan (0.3 microM) depressed the maximum agonist response by 32 +/- 6.5%, possibly reflecting non-competitive behaviour of the antagonist. The potency of losartan was not influenced by bacitracin. 6. The AT2-selective non-peptide antagonist, PD123177 (3 microM) failed to antagonize the angiotensin II-induced depolarizations. 7. DTT (1 mM) produced a 22% reduction of the maximum response to angiotensin II.8. We conclude that the angiotensin II-induced depolarizations of the rat superior cervical ganglion are mediated by angiotensin II receptors of the AT1 subclass. The ability of peptidase inhibitors to modify the potency of peptide agonists and antagonists highlights the difficulties associated with the use of peptide agents to characterize angiotensin II receptors in this preparation. (+info)
Nasal recombinant hirudin-2 delivery: absorption and its mechanism in vivo and in vitro studies.
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The objective of this study was to investigate the feasibility of systemic absorption of recombinant hirudin-2 (rHV2) by nasal delivery, and its possible absorption mechanism. The degradation of rHV2 in the nasal tissue homogenate and extracts of mucosae of rabbit, as well as the degradation inhibition of enzyme inhibitor (bacitracin) was evaluated. The bioavailability of rHV2 and the improvement with enhancers, after nasal administration in rats was investigated. For further understanding of the transport and uptake characteristics of rHV2, in vitro transport experiment under various conditions using diffusion chamber technique in excised rabbit nasal epithelium was performed. It was found that rHV2 underwent rapid degradation in rabbit nasal homogenate, but it was more stable in the extracts of nasal mucosae surface. Bacitracin was able to inhibit the degradation of rHV2 to certain extent. rHV2 was detected in the rat plasma by chromogenic substrate assay after nasal administration and some enhancers also significantly increased the nasal absorption of rHV2. The transport and uptake of rHV2 across nasal epithelium was concentration-dependent and unsaturated, and was significantly inhibited by low temperature, NaN(3), DNP and colchicines, while was less affected by alteration of transport direction. These results demonstrate that the possible absorption mechanism of rHV2 by nasal mucosa appears to be associated with the endocytosis as well as passive diffusion process. (+info)
Comparison of culture media and chairside assays for enumerating mutans streptococci.
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AIM: This study compared several traditional culture-based media and chairside cultural assays for ability to recover mutans streptococci (MS) from pure cultures and from saliva samples. METHODS AND RESULTS: When pure cultures were used with traditional culture-based media, mitis-salivarius bacitracin (MSB) agar demonstrated less support for bacterial recovery than trypticase-yeast extract-cysteine sucrose-bacitracin (TYCSB) agar and the modified medium of Ritz (HLR-S). One species of MS, Streptococcus ferus (c), was not recovered on MSB medium. Chairside cultural tests displayed considerable disparity between tests in recovering bacteria from pure cultures. On the glass adherence assay (Mucount), S. ferus was not detected and Streptococcus criceti was not detected on the dipslide assay (Cariescreen SM) or on the plastic adherence assay (Dentocult SM Strip mutans). The frequency of isolation of pure strains of bacteria other than MS was common. From saliva samples, the frequency of isolation of MS on HLR-S and TYCSB media and the glass adherence assay was 91-97%. The frequency of isolation on MSB medium and on the dip-slide and plastic adherence assays was significantly decreased (37, 47 and 69%, respectively). Recovery scores varied considerably among the culture methods studied and tended to be highest on the HLR-S medium and on the glass adherence assay. CONCLUSIONS: Growth and recovery profiles of pure bacterial cultures and of saliva samples for the MS varied according to different media. SIGNIFICANCE AND IMPACT OF THE STUDY: Caution should be exercised in comparing results between studies that employ different cultural methods for MS enumeration. (+info)
A role for sperm surface protein disulfide isomerase activity in gamete fusion: evidence for the participation of ERp57.
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In mammals, sperm-egg interaction is based on molecular events either unique to gametes or also present in somatic cells. In gamete fusion, it is unknown which features are gamete specific and which are shared with other systems. Conformational changes mediated by thiol-disulfide exchange are involved in the activation of some virus membrane fusion proteins. Here we asked whether that mechanism is also operative in sperm-egg fusion. Different inhibitors of protein disulfide isomerase (PDI) activity were able to inhibit sperm-egg fusion in vitro. While pretreatment of oocytes had no effect, pretreatment of sperm reduced their fusion ability. Some members of the PDI family were detected on the sperm head, and use of specific antibodies and substrates suggested that the oxidoreductase ERp57 has a role in gamete fusion. The results support the idea that thiol-disulfide exchange is a mechanism that may act in gamete fusion to produce conformational changes in fusion-active proteins. (+info)
Effect of including distillers dried grains with solubles in the diet, with or without antimicrobial regimen, on the ability of growing pigs to resist a Lawsonia intracellularis challenge.
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A disease challenge experiment was conducted to determine if including 10% dried distillers grains with solubles (DDGS) in the diet, with or without antimicrobial supplementation, reduces the incidence or severity, or both, of intestinal lesions in growing pigs after an Lawsonia intracellularis challenge. One hundred 17-d-old weaned pigs were blocked by sex, ancestry, and BW and randomly allotted to 1 of 5 treatment groups: negative control, unchallenged, corn-soy diet; positive control, challenged, corn-soy diet; 10% DDGS diet, challenged; positive control with antimicrobial regimen, challenged; and 10% DDGS diet with antimicrobial regimen, challenged. For antimicrobial-supplemented treatments, diets contained 33 ppm bacitracin methylene disalicylate throughout the experiment, with chlortetracycline (Aureomycin) pulsed at 550 ppm from d 3 prechallenge to d 11 postchallenge. Challenged pigs were orally inoculated with 8.0 x 10(8) L. intracellularis organisms after a 4-wk prechallenge period. On d 21 postchallenge, pigs were euthanized, lesions of intestinal mucosa were evaluated, and ileal tissue samples were analyzed by immunohistochemistry to determine the presence and proliferation rate of L. intracellularis. Compared with other dietary treatments, feeding a diet containing 10% DDGS reduced ileum and colon lesion length and prevalence (P < 0.05) and reduced severity of lesions in the ileum (P < 0.05) and colon (P < 0.10) in challenged pigs. Compared with other challenged pigs, those fed the diet containing the antimicrobial regimen had a lower prevalence and severity of lesions in the jejunum (P < 0.05) and tended to have reduced total tract lesion length (P = 0.11). Compared with other challenged pigs, pigs on the 10% DDGS diet with antimicrobial regimen exhibited no differences in length, severity, or prevalence of lesions (P > 0.15), but fecal shedding of L. intracellularis was reduced on d 14 postchallenge (P < 0.05). No dietary effects on fecal shedding were observed by d 20 postchallenge (P > 0.10). The proportion of cells infected with L. intracellularis was reduced when DDGS (P = 0.05) or antimicrobial (P = 0.10) diets were fed. Under the conditions of this experiment, dietary inclusion of 10% DDGS appears to provide some benefit to growing pigs subjected to a moderate L. intracellularis challenge, similar to those of a currently approved antimicrobial regimen. (+info)
Colicin M exerts its bacteriolytic effect via enzymatic degradation of undecaprenyl phosphate-linked peptidoglycan precursors.
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Colicin M was earlier demonstrated to provoke Escherichia coli cell lysis via inhibition of cell wall peptidoglycan (murein) biosynthesis. As the formation of the O-antigen moiety of lipopolysaccharides was concomitantly blocked, it was hypothesized that the metabolism of undecaprenyl phosphate, an essential carrier lipid shared by these two pathways, should be the target of this colicin. However, the exact target and mechanism of action of colicin M was unknown. Colicin M was now purified to near homogeneity, and its effects on cell wall peptidoglycan metabolism reinvestigated. It is demonstrated that colicin M exhibits both in vitro and in vivo enzymatic properties of degradation of lipid I and lipid II peptidoglycan intermediates. Free undecaprenol and either 1-pyrophospho-MurNAc-pentapeptide or 1-pyrophospho-MurNAc-(pentapeptide)-Glc-NAc were identified as the lipid I and lipid II degradation products, respectively, showing that the cleavage occurred between the lipid moiety and the pyrophosphoryl group. This is the first time such an activity is described. Neither undecaprenyl pyrophosphate nor the peptidoglycan nucleotide precursors were substrates of colicin M, indicating that both undecaprenyl and sugar moieties were essential for activity. The bacteriolytic effect of colicin M therefore appears to be the consequence of an arrest of peptidoglycan polymerization steps provoked by enzymatic degradation of the undecaprenyl phosphate-linked peptidoglycan precursors. (+info)