Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases. It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini. The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5. The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs. Neither RNA nor RNA-DNA hybrid are degraded by the enzyme. The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence. Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase. We suggest that this enzyme be designated as endonuclease V of E. coli.
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PMID:Endonuclease V of Escherichia coli. 1 59

Peripheral PMNs were collected from blood, and crevicular PMNs separated by filtration from gingival washings in 13 patients, aged 22-75 y, who had varying degrees of gingivitis and periodontitis. After pre-incubation with cytochalasin B, the same number of crevicular and peripheral cells were incubated either in PBS (with Ca2+ and Mg2+) (spontaneous release) or in the same buffer containing increasing concentrations of FMLP (stimulated release); elastase activity was measured in the supernatant by a fluorometric technique. There was a higher spontaneous release of enzyme from crevicular than from peripheral neutrophils. The average elastase activity in the supernatant of 1 x 10(4) crevicular cells was more than five times higher than that obtained from the same number of peripheral cells. However, stimulated crevicular PMNs liberated smaller amounts of enzyme than did stimulated peripheral PMNs. These results suggest that crevicular PMNs are already releasing elastase, and are consistent with the possibility that lysosomal enzymes contribute to tissue damage during gingivitis and periodontitis.
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PMID:In vitro release of elastase from human blood and gingival crevicular neutrophils. 177 24

A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg2+. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. The maximal difference in fluorescence between untreated and HN2-treated cells was observed after heating at 100 degrees C for 5 min in PBS containing 1.25 mM MgCl2. Higher concentrations of MgCl2 inhibited MOAB binding to HN2-treated cells and heating at lower concentrations induced binding to control cells. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl2. Thus, the presence of phosphates and MgCl2 during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S1 nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. Specific reactivity of MOAB F7-26 with deoxycytidine was established by avidin-biotin ELISA. Single-stranded conformation was necessary for the binding of MOAB to deoxycytidine on the DNA molecule. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.
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PMID:Decreased stability of DNA in cells treated with alkylating agents. 225 76

The DNA repair enzyme uracil-DNA glycosylase from Mycoplasma lactucae (831-C4) was purified 1,657-fold by using affinity chromatography and chromatofocusing techniques. The only substrate for the enzyme was DNA that contained uracil residues, and the Km of the enzyme was 1.05 +/- 0.12 microM for dUMP containing DNA. The product of the reaction was uracil, and it acted as a noncompetitive inhibitor of the uracil-DNA glycosylase with a Ki of 5.2 mM. The activity of the enzyme was insensitive to Mg2+, Mn2+, Zn2+, Ca2+, and Co2+ over the concentration range tested, and the activity was not inhibited by EDTA. The enzyme activity exhibited a biphasic response to monovalent cations and to polyamines. The enzyme had a pI of 6.4 and existed as a nonspherical monomeric protein with a molecular weight of 28,500 +/- 1,200. The uracil-DNA glycosylase from M. lactucae was inhibited by the uracil-DNA glycosylase inhibitor from bacteriophage PBS-2, but the amount of inhibitor required for 50% inhibition of the mycoplasmal enzyme was 2.2 and 8 times greater than that required to cause 50% inhibition of the uracil-DNA glycosylases from Escherichia coli and Bacillus subtilis, respectively. Previous studies have reported that some mollicutes lack uracil-DNA glycosylase activity, and the results of this study demonstrate that the uracil-DNA glycosylase from M. lactucae has a higher Km for uracil-containing DNA than those of the glycosylases of other procaryotic organisms. Thus, the low G + C content of the DNA from some mollicutes and the A.T-biased mutation pressure observed in these organisms may be related to their decreased capacity to remove uracil residues from DNA.
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PMID:A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase. 234 31

In order to establish metastatic lesions, 2.5 x 10(6) AH100B cells were injected into the left carotid artery of male Donryu rats. Each metastatic nodule in the liver or kidney, 1 mm or less in diameter, thus obtained was then injected into the peritoneal cavity in which these metastatic cells come to free. About 3 weeks later, each ascites was collected from the rats, while not bloody. Then, cancer cells obtained from each ascites were suspended in Dulbecco's PBS without Ca2+ and Mg2+ (pH 7.2) after washing. Then, 10(6) metastasized or control cancer cells were incubated in 0.1 ml of PBS mentioned above together with 0.1 microCi of (1-14C)-AA at 24 degrees C for 3 min, respectively. After the extraction procedure, AA metabolites formed were separated by means of TLC, and each TLC plate was subjected to autoradiography. In the metastasized cells, PG production ability was generally accelerated and especially in that of PGF2 alpha as compared with that of the control.
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PMID:Some features of the metastatic cancer cells in prostaglandin production. 251 Mar 66

Chemiluminescence (CL) production by phagocytosing polymorphonuclear leukocytes (PMNLs) was measured by an automatic photoluminometer with built-in mixing and temperature controls. Agitation of the vials with PMNLs and opsonized zymosan particles influenced both the lag time and the CL production. Maximal production was obtained by continuous mixing of the samples, the reaction peak occurring within 6 min. Increasing the temperature from 20 to 40 degrees C also increased the CL production, and in further experiments 37 degrees C was used. Aggregation of the PMNLs was avoided by washing the cells in PBS containing gelatin 1 g/l. Glucose, Ca2+ and Mg2+ in the final reaction mixture were necessary for maximal CL responses. The measurements of CL per s up to 4 min, the peak CL value, or the integral below the CL curve up to 6 min were all linearly proportional to the number of PMNLs in the reaction mixture. Since the lag time and the time before reaching peak CL may vary, the integral below the curve up to 6 min was chosen as the mode of CL measurement. On repeated measurements the coefficient of variation was 6.3%. The mean CL integral value for PMNLs from 14 healthy individuals was 205 +/- 19 mVs, indicating a good reproducibility of the standardized assay.
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PMID:Factors important for the measurement of chemiluminescence production by polymorphonuclear leukocytes. 300 25

As an outgrowth of in-vitro fertilization and embryo transfer, detection of genetic and metabolic defects prior to implantation might be possible in the future. The objective for preimplantation diagnosis would be to sample a minimum of cell material of the conceptus for diagnosis prior to transfer. Different protocols for isolating individual blastomeres out of 2-cell mouse embryos were evaluated. 2-cell mouse embryos (from F1 hybrids C57B1 females x CBA males) were collected and the zona pellucida was removed by enzyme treatment (pronase), by exposure to Ca2+-Mg2+-free acid Tyrode (pH = 2.5) or by mechanical force. Individual blastomeres were obtained by exposure to an enzyme (pronase), to a chelating agent (EDTA-glycine mixture), to Ca2+-Mg2+-free PBS or after isolation by mechanical force. The biopsied blastomeres were then cultured in vitro as such or first replaced into a host zona pellucida. Evaluation was performed by culture in vitro up to the blastocyst stage and by transfer of embryos appearing morphologically normal into pseudopregnant foster mothers. A chromosomal study of the second mitotic division of the isolated blastomere was also performed. All isolation procedures had a negative impact on the in-vitro and in-vivo growth patterns of the isolated blastomeres. After culture in vitro to the blastocyst stage, different abnormalities could be observed: embryos lacking compaction, embryos with double blastocoelic cavities and embryos with no inner cell mass (trophoblastic vesicle). After replacement of the isolated blastomeres into a host zona pellucida, similar observations could be made. Chromosomal analysis did not reveal a clear influence of the different biopsy methods on the mitosis of the isolated blastomeres.
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PMID:Evaluation of different biopsy methods of blastomeres from 2-cell mouse embryos. 320 55

Eight day (8-d CEF) and 16 day old chick embryo fibroblasts (16-d CEF) obtained after a mild trypsin treatment (50 micrograms/ml in Ca2+ and Mg2+-free PBS, plus 10 mM EDTA) for 10 min at 37 degrees C present the same number of fibronectin (FN) binding sites at their surface (approximately 550,000 sites per cell) with a Kd approximately equal to 1.40 microM in both cases. Furthermore, FN interacted with high molecular weight plasma membrane proteins (150,000 and 125,000) insensitive to trypsin treatment. Both 8-d and 16-d CEF adhered and spread to the same extent on a fibronectin coated substratum (80% of the CEF adhered in 60 min). In contrast, 8-d and 16-d CEF behaved differently towards laminin (LM). 8-d CEF exhibited approximately 5500 binding sites per cell with a Kd of 1.5 nM (Codogno P., Doyennette, M.-A. and Aubery M., 1987, Experimental Cell Research, 169, 478-489.) and were highly sensitive to trypsin treatment, whereas 16-d CEF do not express cell surface binding sites for laminin. Differences were also observed in the adhesive capacities of 8-d and 16-d CEF on LM substrata: 8-d CEF adhered and spread on LM in a very specific manner (60% of the cells adhere in 60 min) and 16-d CEF did not adhere to LM even after long periods of incubation exceeding 360 min.
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PMID:Alterations of fibroblast interactions with fibronectin and laminin during chick embryo development. 344 Feb 93

As an extension of in-vitro fertilization and embryo transfer, detection of genetic and metabolic defects prior to implantation might be possible in the future. The objective of pre-implantation diagnosis would be to sample a minimal amount of cellular material of the conceptus for diagnosis prior to transfer. Different protocols for isolating individual blastomeres from two-cell mouse embryos were evaluated. Two-cell mouse embryos were collected and the zona pellucida was removed by enzyme treatment (pronase), by exposure to acid Tyrode (pH = 2.5) or by mechanical force (suction into a small pipette, removal with a microblade). Individual blastomeres were obtained by exposure to a chelating agent (EDTA-glycine mixture), to Ca2+-Mg2+-free PBS or after isolation by mechanical force (bisection with a microblade or suction in a small pipette). The isolated blastomeres were then cultured in vitro without zonae pellucidae. All isolation procedures had a negative impact on the growth patterns of the isolated blastomeres. Different abnormalities could be observed at the blastocyst stage including embryos lacking visible compaction features, embryos with double blastocoelic cavities and embryos with no inner cell mass (trophoblastic vesicles).
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PMID:Assessment of different isolation procedures for blastomeres from two-cell mouse embryos. 365 31

Rabbit red blood cells (RaRBC, 3 x 10(7)/ml PBS) were incubated with different amounts of purified human C3 at 37 degrees for 30 min and washed twice in PBS. Different amounts of normal human serum containing 2 mM Mg2+ and 5 mM EGTA were added to the C3-treated and control RaRBC. The extent of lysis was measured after a further incubation at 37 degrees for 40 min. Enhanced lysis was observed with C3-treated RaRBC as compared to control cells. The enhancing effect was dependent on the dose of C3 used for the treatment of RaRBC. Investigation the kinetics of lysis, the lag phase was observed to be significantly shorter with the C3-treated than with the control RaRBC. No enhancement was found when RaRBC were pretreated with preformed C3b fragment. KSCN-treated C3 (C3b-like C3), however, had a lysis-enhancing effect. These results suggest that noncovalently bound C3 molecules may have a role in the initiation and/or maintenance of the alternative pathway activation on activator cells.
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PMID:Non-covalently bound C3 enhances lysis of rabbit erythrocytes through the alternative pathway. 393 72


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