Chemical studies on purified exopolysaccharide from a virulent strain, ICMP 9758 of Pseudomonas solanacearum.
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Carbohydrate Sequence↗
Biomedical subjects
S Basu
Publications and source records attributed to S Basu.
Developmental expression of the embryonic chicken brain DNA polymerase alpha and its binding with monoclonal antibodies against human KB cell DNA polymerase alpha.
Changes in DNA polymerase alpha activity accompanying tissue development have been well established in several systems. In most cases, DNA polymerase alpha activity decreases with development. Here, we report observed changes in DNA polymerase alpha activity throughout embryonic chicken brain (ECB) development. The level of DNA polymerase alpha activity was found to gradually decrease by 60% (2.3 to 0.8 nmol of [3H]dCMP incorporated/mg protein/h) between 9- and 19-day-old ECB. An enzyme-linked immunosorbent assay of DNA polymerase alpha utilizing monoclonal antibody SJK 237-71 (human KB cell DNA pol-alpha binder) also demonstrated a gradual decrease (up to 60%) of antigen over this same range of development. Analysis of DNA polymerase alpha from 11- and 19-day-old ECB by a 10 to 30% glycerol density gradient revealed a high molecular weight peak sedimenting near catalase (11.3 S) with activity at the 11th day being approximately 3-fold greater than activity at the 19th day. A Western immunoblot analysis utilizing monoclonal antibody SJK 237-71 (against human KB cell DNA polymerase alpha) showed a decrease in DNA polymerase alpha from 186 kilodaltons in 9- and 11-day ECB cell-free extracts to 120 kilodaltons in extracts from 13- to 19-day ECB. The conversion of DNA polymerase alpha from a higher to a lower molecular weight form may be a regulatory mechanism in eukaryotic DNA replication.
Animals↗
Isolation, purification and preliminary studies on fish eye lens glycoprotein.
Glycoprotein from the eye lens of fish, Mystus cavasius, was isolated by extraction with 1% Triton X-100 in saline. The crude extract which was found to be electrophoretically heterogeneous was fractionated on a DEAE-cellulose column. One of the fractions obtained in major amount was further resolved by column chromatography using Sephadex G-150 into two homogeneous fractions (GP-1 and GP-2]. GP-1 contained carbohydrates (11.2%) and protein (77.5%). The constituent sugars were D-glucose, D-mannose, D-galactosamine and N-acetyl neuraminic acid. The principal amino acids were aspartic acid, serine, glutamic acid, glycine and alanine. The proportions of these residues were determined.
Amino Acids↗
Synthesis of methyl 2-O-alpha-D-galactopyranosyl-3-O-beta-D-glucopyranosyl-alpha-D-glucopyr anoside, a trisaccharide of the R-1 core antigen of Enterobacteriaceae.
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Antigens, Bacterial↗
Site-directed mutagenesis of Moloney murine leukemia virus reverse transcriptase. Demonstration of lysine 103 in the nucleotide binding site.
Lys103 and Lys421 of Moloney murine leukemia virus reverse transcriptase have been implicated in the dNTP binding function as judged by their reactivity to a substrate binding site-directed reagent, pyridoxal 5'-phosphate (Basu, A., Nanduri, V. B., Gerard, G. F., and Modak, M. J. (1988) J. Biol. Chem. 263, 1648-1653). To assess the true catalytic importance of the individual lysine residues in Moloney murine leukemia virus reverse transcriptase, we mutated Lys103 and Lys421 to leucine and alanine, respectively. Analysis of the mutant enzymes revealed that mutation at the 103 position had a drastic effect on the DNA polymerase activity whereas the 421 mutation had no effect. Both mutants exhibited normal RNase H activity as well as the ability to bind to RNA or DNA templates as judged by UV-mediated cross-linking of the enzyme to the template primers. The enzyme with mutation at codon 421 (Lys----Ala) exhibited properties that were indistinguishable from the wild type with respect to its mode of catalysis, i.e. preference of template primer and divalent metal ion, RNA- or DNA-dependent DNA polymerase activity, RNase H activity, and the processive mode of DNA synthesis. These observations suggest that only Lys103 and not Lys421 is the catalytically important residue that is involved in the binding of substrate dNTP in Moloney murine leukemia virus reverse transcriptase.
Base Sequence↗
Influences of laser pulse duration and anastomotic disruption on laser-assisted microvascular anastomoses (LAMA).
The success rate of laser-assisted microvascular anastomosis (LAMA) can be influenced by many factors, including the parameters of the laser technique used. This study examined the effects of laser pulse duration and the occurrence of anastomotic disruption immediately following first repair attempts on the incidence of aneurysm formation and the patency rate in LAMA. Fifty transected rat femoral arteries in 25 rats were anastomosed with three stay sutures and welded with a CO2 laser at a power of 80 mW. In each rat, the laser was applied in the form of 45 pulses of 0.2 sec duration to one femoral artery (group 1) and in the form of 15 pulses of 0.6 sec duration to the contralateral femoral artery (group 2). Comparison of group 1 with group 2 showed that overall rates of aneurysm formation (30% vs. 17%) and patency rates (92% vs. 96%) did not differ significantly (P less than 0.05) at 3 weeks postoperatively. However, disrupted vessels in both groups had a significantly higher incidence of subsequent aneurysm formation than nondisrupted vessels (54% vs. 0% in group 1, P less than 0.05; 43% vs. 6% in group 2, P less than 0.06; groups 1 and 2 pooled, P less than 0.001). These findings indicate that changes in laser pulse duration such as those tested do not affect the aneurysm and patency rates of LAMA. The results demonstrate, however, that disruption after laser welding plays a significant role in subsequent aneurysm formation.
Anastomosis, Surgical↗
Cerebrospinal fluid drainage and steroids provide better spinal cord protection during aortic cross-clamping than does either treatment alone.
We investigated whether intravenous methylprednisolone (30 mg/kg) before 30 minutes of aortic cross-clamping and after 4 hours could enhance the effects of cerebrospinal fluid drainage on spinal cord perfusion pressure and postoperative paraplegia when proximal blood pressure was controlled with sodium nitroprusside and partial exsanguination. Dogs were randomized into three groups: group 1 (n = 6), control; group 2 (n = 7), steroids; and group 3 (n = 6), steroids with cerebrospinal fluid drainage. During aortic cross-clamping, blood pressure proximal to the clamp decreased significantly in each group compared with baseline (p less than 0.05), but did not differ among groups (group 1 = 82.2, group 2 = 82.1, group 3 = 86.6 mm Hg, p greater than 0.05). Mean distal pressure decreased from systemic values to 8.4, 8.5, and 3.7 mm Hg, respectively, after aortic cross-clamping (p less than 0.05); these values did not differ from one another (p greater than 0.05). During aortic cross-clamping, cerebrospinal fluid pressure in groups 1 and 2 did not differ significantly compared with baseline (12.2 versus 8.2, 14.2 versus 10.7 mm Hg, p greater than 0.05), whereas in group 3 the baseline cerebral spinal fluid pressure of 10.7 mm Hg decreased to 0.4 mm Hg (p less than 0.05). Spinal cord perfusion pressure in group 3 was significantly higher than in groups 1 and 2 (3.3 versus -3.9 and -5.7 mm Hg, p less than 0.05), but did not differ between groups 1 and 2 (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites.
The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.
Base Sequence↗
Effect of a fatty acid moiety of phospholipid and ceramide on purified GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) activity from embryonic chicken brain.
Galactosyltransferase, GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) has been characterized and solubilized from 19-day-old embryonic chicken brain, and purified to over 2000-fold using mixed-modal chromatography on a omega-aminohexyl Sepharose column and affinity chromatography on a UDP-hexanolamine Sepharose column. The activity of purified GalT-3 was modulated by phospholipids in vitro with stimulation observed specifically with dipalmitoyl phosphatidylethanolamine (PE). All natural phospholipids tested (PE, PC and PI) inhibited GalT-3 activity. Enzyme activity was affected by the structure of the phospholipid vesicle. It was stabilized by the hexagonal (dipalmitoyl PE) structure and inhibited by the bilayer (dielaidoyl PE) structure. The long-chain fatty acid moiety of the glycosphingolipid substrate, GM2, was found to be necessary for optimum enzyme activity. In the absence of fatty acid, the modified substrates, lyso-GM2 and acetyl-GM2, had a 10-fold increased Km and a 4-8 fold decreased Vmax compared to the normal substrate. We postulate that GalT-3 belongs to a group of glycosyltransferases having recognition for both the carbohydrate as well as the hydrophobic domains (HY-CARS) of their substrates and that the fatty acid moiety of either the substrate (GM2) or a heterotropic effector (phospholipid) plays an important role in regulating the activity of this enzyme.
Animals↗
Carbohydrate and hydrophobic-carbohydrate recognition sites (CARS and HY-CARS) in solubilized glycosyltransferases.
Six different glycosyltransferases that are active with glycosphingolipid substrates have been purified from Golgi-membranes after solubilization with detergents. It appears that GalT-4(UDP-Gal:GlcNAc-R1 beta 1-4GalT), GalNAcT-2(UDP-Gal:Gal alpha-R2 beta 1-3GalNAcT) and FucT-2(GDP-Fuc:Gal beta GlcNAc-R3 alpha 1-2FucT) are specific for oligosaccharides bound to ceramide or to a protein moiety. These are called CARS (carbohydrate recognition sites) glycosyltransferases (GLTs). On the other hand, GalT-3(UDP-Gal:GM2 beta 1-3GalT), GalNAcT-1(UDP-GalNAc:GM3 beta 1-4GalNAcT) and FucT-3 (GDP-Fuc:LM1 alpha 1-3FucT) recognize both hydrophobic moieties (fatty acid of ceramide) as well as the oligosaccharide chains of the substrates. These GLTs are called HY-CARS (hydrophobic and carbohydrate recognition sites). D-Erythro-sphingosine (100-500 microM) modulates the in vitro activities of these GLTs. Modulation depends on the binding of D-sphingosine to a protein backbone, perhaps on more than one site and beyond transmembrane hydrophobic domains. Control of GLTs by free D-sphingosine was suggested with the concomitant discovery of ceramide glycanase in rabbit mammary tissues. The role of free sphingosine as an in vivo homotropic modulator of glycosyltransferases is becoming apparent.
Animals↗
Characterization of solubilized GlcAT-1 (UDP-GlcA: nLcOse4Cer beta 1-3 glucuronyltransferase) activity from embryonic chicken brain and its inhibition by D-erythro-sphingosine.
Glycolipid glucuronyltransferase activity (GlcAT-1) has been solubilized and characterized from 19-day-old embryonic chicken brain Golgi-rich membranes. The enzyme catalyzes the biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer glycolipid using neolactetraosylceramide (nLcOse4Cer, Gal beta 1-4GlcNAc beta 1-3Gal beta-1-4Glc-Cer) as the substrate. The membrane-bound enzyme shows optimum activity in the presence of neutral detergents such as Triton CF-54, Triton DF-12, and Nonidet P-40. Approximately 60% of the enzyme activity can be solubilized from the Golgi membrane by Nonidet P-40. The solubilized GlcAT-1 activity is inhibited by different salts such as NaCl, NaBr, NaI, and NaOAc, but not by sodium fluoride (up to 0.4 M concentration). Desialyzed alpha 1 acid glycoprotein (SA alpha 1AGP) can be used as a substrate for glucuronyltransferase. Competition studies between glycolipid (nLcOse4Cer) and glycoprotein SA alpha 1AGP) substrates show a mixed type of inhibition. Phospholipids, in particular phosphatidylglycerol, stimulate solubilized GlcAT-1 activity, while D-erythro-sphingosine, a metabolite of glycosphingolipids, is inhibitory (50% inhibition at 0.8 mM D-erythro-sph). These results demonstrate that both phospholipid as well as sphingosine might be involved in modulating glucuronyltransferase activity.
Animals↗
Physical demonstration of a high molecular weight bacteriocin plasmid in Vibrio cholerae by genetic transformation process.
Bacteriocinogeny was transferred at high frequencies from bacteriocinogenic (Bac+) V. cholerae strains to non-bacteriocinogenic (Bac-) recipients in the in situ genetic transformation system on agar surface. DNA extracted from samples of growth of bacteria transformed to Bac+ were obtained at 2 h intervals following contact with the sterile agar surface where the donor had grown previously. This showed acquisition of a high molecular weight plasmid which could be physically demonstrated best in the 6 h sample of the Bac+ transformants; their 4 h samples failed to show this specific plasmid, while it was demonstrable only as a faint band in the 8 h samples.
Bacteriocins↗
Physiochemical studies on achatininH, a novel sialic acid-binding lectin.
We have purified a sialic acid-binding lectin, achatininH, in a single step by affinity chromatography, having high affinity for 9-O-acetylneuraminic acid. The physicochemical characterization of the interaction of achatininH with bivalent metal ions and sialic acid derivatives by the use of spectrofluorimetry, spectropolarimetry and precipitin reaction is reported. From fluorescence quenching studies the binding of Ca2+ (Ka = 251 +/- 9 M-1) and of Mn2+ (Ka = 86 +/- 5 M-1) was found to be weak, but their presence is absolutely necessary for sugar binding as well as biological activity. The nature and position of the substituent group play a very important role in the binding affinity. AchatininH shows a high affinity for 9-O-acetylneuraminic acid (Ka = 1.20 x 10(3) +/- 0.07 x 10(3) M-1) compared with that for the 4-O-acetyl derivative. In oligomers the binding strength increases in the order monosaccharide less than disaccharide less than trisaccharide. The binding affinity of achatininH for the disaccharide was found to reach a peak around pH 8. From c.d. spectral studies achatininH was found to have a high beta-sheet content (46%) and a low alpha-helix content (24%). From precipitin analysis at least one sugar-binding site on each of the 16 monomer subunits of the protein is indicated.
Animals↗
Endogenous inhibition of arachidonic acid metabolism in the endometrium of the sheep.
Arachidonic acid is metabolised via the cyclo-oxygenase pathway to several biologically active metabolites. These metabolites control important reproductive functions like luteolysis of the corpus luteum. The metabolism of arachidonic acid was studied by the enzymatic conversion of [1-14C]-labelled arachidonic acid in sheep endometrial tissue. The inhibitory capacity of sheep endometrial tissue was measured by the enzymatic conversion of [1-14C]-arachidonic acid by sheep seminal vesicular gland microsomes. Endometrial microsomes converted arachidonic acid into different prostaglandins and monohydroxy acids but at a low rate. A factor(s) inhibiting both prostaglandin and monohydroxy acid synthesis was found in both the microsomal and cytosolic fractions of endometrial tissue. A very high inhibitory potency of prostaglandin and monohydroxy acid synthesis, calculated as IC50 values, was found in cytosolic fractions. For comparison IC50 values of indomethacin, mefenamic acid, carprofen and acetylsalicylic acid were also calculated in this in vitro system. These data indicate that both prostaglandin and monohydroxy acid synthesizing capacities and an inhibitory factor(s) are present in sheep endometrium and possibly regulate arachidonic acid metabolism in this tissue.
Animals↗
Characterization of versilin-sensitive sites in self-sensitive producer and sensitive non-producer or unrelated organism.
Studies on self-sensitivity of producer mutant vs. sensitivity of non-producer parent and unrelated organism showed that versilin inhibited spore germination and sporulation in the self-sensitive producer mutant, non-producer parent Aspergillus versicolor N5 and the unrelated sensitive Trichophyton rubrum. Sporulation appeared to be more sensitive than spore germination. The inhibition of in vivo synthesis of protein was very marked, but inhibition of RNA and DNA was slight and moderate, respectively. Thus versilin was not specific in its action, but the principal sensitive site was protein synthesis, as further suggested by inhibition of polyU-directed in vitro synthesis of polyphenylalanine. The activation of leucine was unaffected, but the formation of leucyl-tRNA was severely inhibited in all three strains. The differences in sensitivities between the strains were the same, whether as whole cells or as cell-free extracts. Thus the nature of the sensitive site appeared to be identical in the self-sensitive producer and sensitive non-producer or unrelated organism.
Anti-Bacterial Agents↗
Effect of flunixin meglumine on prostaglandin F2 alpha synthesis and metabolism in the pig.
The effect of flunixin meglumine on prostaglandin synthesis and metabolism was evaluated in the pig in vivo. It was found that the prostaglandin metabolite, 15-ketodihydro-PGF2 alpha, was decreased in the peripheral circulation within 20 min of injection of the drug. In therapeutic doses in the pig the drug had no effect on the metabolism of PGF2 alpha. Flunixin was compared with some other non-steroidal anti-inflammatory drugs in an in vitro test system utilizing sheep vesicular gland microsomes. It was concluded that this drug is a potent inhibitor of prostaglandin synthesis.
Animals↗
Fertility and mortality in tribal populations of Bastar District, Madhya Pradesh, India.
Demographic analysis of genealogical data, collected in 1983-1984 on 792 households of the 4 tribal populations, namely Muria, Madia, Bhattra and Halba, from Jagdalpur and Konta tehsils of Gastar district of Madhya Pradesh (India) is conducted employing indirect estimation techniques. The results of the study indicate that the total fertility rate of these 4 groups is higher than the Madhya Pradesh rural non-tribal population groups and Indian national population which is in accordance with high tribal fertility levels as reported in some studies from India. The study groups show mortality differentials, but feature distressing mortality conditions as compared to the Indian national population. There are also sex-specific mortality differentials in the present study, with males experiencing higher mortality than females. The trends are discussed in the light of prevailing socioeconomic, cultural, and health care practices among these tribal groups.
Age Factors↗