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Biomedical subjects

A B Mukherjee

Publications and source records attributed to A B Mukherjee.

At least 19 recordsLinked to original sources
PubMed1992

Surfactant-producing rabbit pulmonary alveolar type II cells synthesize and secrete an antiinflammatory protein, uteroglobin.

Neonatal respiratory distress syndrome (RDS) caused by surfactant deficiency is a common disorder in premature infants. Exogenous surfactant therapy improves survival in infants with RDS. However, the phospholipid component of the surfactant has been suggested to be inactivated by a phospholipid hydrolyzing enzyme, phospholipase A2 (PLA2). Although alveolar type II cells produce the surfactant, it is not known whether these cells have any mechanism to protect surfactant from PLA2 hydrolysis. Since alveolar Clara cells express uteroglobin (UG), a PLA2 inhibitory and antiinflammatory protein, and since it has been suggested that alveolar type II cells are derived from Clara cells, we sought to elucidate whether type II cells are also capable of expressing UG gene. By using radioimmunoassay, immunoprecipitation and Western blotting techniques we demonstrate for the first time that type II cells, isolated from mature rabbit lungs, synthesize and secrete UG. The transcription of the UG gene was detected by in situ hybridization using rabbit UG cDNA probe. These results imply that UG, synthesized by type II cells, may protect both endogenous and exogenous surfactant from PLA2 hydrolysis. Moreover, the antiinflammatory properties of UG may prevent the development of chronic inflammatory lung disease, a frequent complication of RDS.

Animals↗
PubMed1992

Differential processing of osteopontin transcripts in rat kidney- and osteoblast-derived cell lines.

Using immunoprecipitation and tryptic peptide microsequencing we confirmed the identity of normal rat kidney (NRK) cell-secreted 69-kDa major phosphoprotein as osteopontin (OP). We then immunoselected a 1.4-kilobase pair (kb) OP cDNA from a lambda gt11 library prepared from Kirsten sarcoma virus-transformed NRK (KNRK) cellular mRNA, using rabbit anti-69-kDa OP serum. Sequence analysis of this cDNA revealed the presence of a 52-nucleotide-long insert in the 5'-noncoding region, which was absent in OP cDNA cloned from the cDNA library of ROS 17/2.8 rat osteosarcoma cells. The insert sequence is flanked by putative intron splice junctions and is located 15-nucleotide upstream of the translational initiation site. An insert-specific 30-mer oligonucleotide probe hybridized to a single 1.5-kb RNA species from both NRK and KNRK cells, but not from ROS 17/2.8 cells. However, Southern analysis showed the presence of this insert sequence in the genomic DNA of both NRK and ROS 17/2.8 cells. Furthermore, PCR amplification of the insert-containing region using genomic DNAs from both NRK and ROS 17/2.8 cells gave products of identical size and sequence. Since OP is a single copy gene, these data provide strong evidence for differential cell type-specific processing of OP transcripts. In addition, we demonstrate that, in contrast to most transformed cells, levels of OP expression are significantly reduced in KNRK cells as compared to NRK cells.

Amino Acid Sequence↗
PubMed1992

A novel transglutaminase-catalyzed posttranslational modification of HIV-1 aspartyl protease.

We demonstrate that HIV-1 aspartyl protease (AP), the enzyme essential for the maturation of the AIDS virus, covalently incorporates spermidine catalyzed by guinea pig liver transglutaminase (TGase) and human coagulation factor XIIIa. Preliminary evidence indicates that there are at least three reactive glutamyl and lysyl residues in AP which act as acyl donor and acceptor respectively in a TGase reaction. SDS-PAGE and chromatographic analyses indicate that the two TGases tested catalyze the incorporation of radioactive spermidine into pure HIV-1 AP. The chemical identification and quantitation of (gamma-glutamyl) spermidine isopeptide provide conclusive evidence that the formation of this derivative is catalyzed by TGase. These results imply that TGase-catalyzed post-translational modification of HIV-1 AP may take place in a manner similar to the ones demonstrated in porcine pancreatic phospholipase A2.

Animals↗
PubMed1992

Analysis of chromosome 12 aneuploidy in interphase cells from human male germ cell tumors by fluorescence in situ hybridization.

The i(12p) marker chromosome has been found to be a highly nonrandom chromosome abnormality associated with germ cell tumors (GCTs). We have previously shown that a chromosome 12 centromere specific alpha-satellite DNA probe detects the i(12p) by virtue of differences in the size of the signal originating from the i(12p) and normal chromosome 12 centromeres after fluorescence in situ hybridization (FISH) in metaphase and interphase cells of cultured GCT cell lines. We have now extended this analysis to 72 fresh GCT tumor biopsy specimens. Banded cytogenetic analysis was attempted on each of these tumors, 45 of which were found to be clonally abnormal. Data on i(12p) and chromosome 12 copy number obtained by FISH agreed well with those obtained by cytogenetic analysis. In addition, the FISH method made possible the detection and determination of i(12p) and the chromosome 12 copy number in cases in which conventional cytogenetic analysis was unsuccessful. We found the incidence of i(12p) in seminomas to be low (7%) compared to that in nonseminomas (75%) when tumor biopsy specimens were studied by FISH. Our results show that the FISH technique can be used reliably for detection of the diagnostically and prognostically useful i(12p) marker in GCT tumor biopsy specimens.

Aneuploidy↗
PubMed1992

Regulation of extracellular phospholipase A2 activity: implications for inflammatory diseases.

Phospholipases A2 (PLA2s; E.C.3.1.1.4) are a family of esterases that are involved in myriads of physiological and pathological processes. The involvement of these enzymes, especially the extracellular PLA2s, in the generation of proinflammatory lipid mediators makes them a very important target for investigation. These PLA2s have been suggested to be involved in the pathogenesis of several human inflammatory diseases. Thus, delineating the mechanism(s) of regulation of the activity of these enzymes may provide a better understanding of the pathophysiology of these diseases and allow the rational design and development of novel therapeutic agents. In this article, we provide a brief description of PLA2s in general with a special emphasis on extracellular enzymes, their mechanism(s) of action, and possible role in the pathogenesis of inflammatory diseases. Additionally, we describe: (i) a novel mechanism of activation of extracellular PLA2s by transglutaminases and (ii) the development of one class of antiinflammatory agents, antiflammins, derived from the active site structure of endogenous PLA2-inhibitory proteins.

Amino Acid Sequence↗
PubMed1992

Detection of cell-cycle stage by fluorescence in situ hybridization: its application in human interphase cytogenetics.

Distinct cell-cycle-dependent changes in the conformation of centromeric chromatin in a specific human chromosome containing alpha-satellite DNA have been demonstrated by fluorescence in situ hybridization (FISH). This method, based upon specific FISH signal morphology, allows simultaneous analysis of chromosomal aneuploidy and detection of specific cell-cycle stage(s) of human tumor and/or normal cell populations in a single preparation of interphase cells. This interphase cytogenetic procedure might prove useful for both basic and clinical research involving human cells.

Cell Cycle↗
PubMed1991

Effects of antiflammins on endotoxin-induced uveitis in rats.

Antiflammins are phospholipase A2-inhibitory, anti-inflammatory, synthetic oligopeptides derived from the region of the highest amino-acid sequence similarity between uteroglobin and lipocortin I. Endotoxin-induced uveitis is a model for anterior uveitis of the eye, which has been suggested to be induced through phospholipase A2 activation. In a preliminary report we demonstrated that topical administration of antiflammins could inhibit endotoxin-induced uveitis in rats. In this study, the anti-inflammatory effects of antiflammins were compared with those of corticosteroids on endotoxin-induced uveitis as measured by phospholipase A2 enzyme activity, inflammatory cell counts in the aqueous humor, and histopathologic features. Antiflammins are as effective as corticosteroids in their ability to suppress endotoxin-induced uveitis.

Administration, Topical↗
PubMed1991

Detection and analysis of origin of i(12p), a diagnostic marker of human male germ cell tumors, by fluorescence in situ hybridization.

The i(12p) chromosome marker has been shown to be a diagnostic and prognostic marker of human male germ cell tumors (GCTs). An analysis of the i(12p) and chromosome 12 aneuploidy was performed in five primary cell cultures and three established cell lines derived from human male GCTs by fluorescence in situ hybridization (FISH) with a chromosome 12 centromere-specific alpha-satellite DNA probe. Distinct differences in the centromeric signals originating from the i(12p) and normal chromosome 12 were detected, which were found to be useful for unambiguous distinction between the i(12p) and normal chromosomes 12 at interphase as well as at metaphase in these cultures. This method can be used for rapid screening of large numbers of interphase cells, eliminating the main limitation of conventional karyotypic analysis, namely, frequent inability to obtain sufficient numbers of dividing cells in direct preparations or in short-term culture of fresh biopsies. Our analysis of chromosome 12 centromeric signal size along with karyotypic data and results of analysis of restriction fragment length polymorphisms (RFLPs) on 12q in four GCTs suggested that the i(12p)s are formed by nonreciprocal centromeric interchanges between nonsister chromatids of homologous chromosomes.

Aneuploidy↗
PubMed1991

Inhibition of pancreatic phospholipase A2 activity by uteroglobin and antiflammin peptides: possible mechanism of action.

We investigated the possible mechanism of inhibition of porcine pancreatic phospholipase A2 in vitro by rabbit uteroglobin and by the antiflammin peptides. We optimized the conditions of phospholipase A2 assay using a deoxycholate-phosphatidylcholine mixed micellar substrate and established the activity of these inhibitors under optimized conditions. The results of fluorescence studies and crosslinking experiments indicate that the inhibitors interact with the enzyme in solution and affect the increase in intrinsic fluorescence of phospholipase A2 observed upon interaction with a mixed micellar substrate. In addition, we identified a sequence similarity between the antiflammin peptides, the putative active region of uteroglobin and a region in pancreatic phospholipase A2. This region of phospholipase A2 has been previously identified as being involved in the regulation of dimerization of this enzyme, and is conserved in the pancreatic-type enzymes. Taken together, these observations suggest that uteroglobin and antiflammins interact with porcine pancreatic phospholipase A2 and this may, at least in part, explain the enzyme inhibitory effect of these molecules observed in vitro. One possible mechanism of this effect may be an interference with the dimerization process of phospholipase A2 which is associated with interfacial activation.

Amino Acid Sequence↗
PubMed1990

A novel transglutaminase-mediated post-translational modification of phospholipase A2 dramatically increases its catalytic activity.

Transglutaminases (TG), which include coagulation Factor XIIIa, are calcium-dependent ubiquitous enzymes. TGs catalyze the formation of an isopeptide bond by cross-linking a specific glutamine and a lysine residue between two proteins or within the same protein molecule. Phospholipase A2 (PLA2) is a key enzyme in the regulation of prostaglandin and leukotriene biosynthetic pathways, which catalyzes the release of free fatty acids from the sn-2 position of membrane glycerophospholipids. This enzyme has been suggested to be pathophysiologically related to the initiation and propagation of several inflammatory diseases including juvenile rheumatoid and rheumatoid arthritis. Here, we describe a novel TG-catalyzed post-translational modification of PLA2 which dramatically increases the activity of this enzyme. This increase was dependent upon the time of preincubation, the concentration of TG and the presence of Ca2+. Size exclusion chromatography of TG-treated PLA2 yielded two peaks of PLA2 activity, with apparent molecular masses of 26 and 13 kDa, respectively. The 26-kDa species, a putative PLA2 dimer, contained epsilon-(gamma-glutamyl)-lysine isopeptide in about 1:1 molar ratio to PLA2, suggesting an intramolecular rather than intermolecular cross-linking. This hypothesis was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 26- and 13-kDa species under denaturing conditions. The specific activity of the dimeric peak was 10-fold higher with respect to that of the monomeric enzyme. These data suggest that TG-catalyzed covalent cross-linking of PLA2 is intramolecular and that this may promote a noncovalent dimerization and subsequent activation of this enzyme via a conformational change. To our knowledge, this is the first demonstration that TG-mediated post-translational modification of an enzyme (e.g. PLA2) causes a striking increase in the catalytic activity of the enzyme.

Animals↗
PubMed1990

High level bacterial expression of uteroglobin, a dimeric eukaryotic protein with two interchain disulfide bridges, in its natural quaternary structure.

Bacterial expression of eukaryotic proteins is a tool of ever-increasing importance in biochemistry and molecular biology. However, the majority of the recombinant eukaryotic proteins that have been expressed in bacteria are produced as fusion proteins and not in their native conformation. In particular, correct formation of quaternary structures by recombinant proteins in bacterial hosts has been reported very rarely. To our knowledge, correct intracellular formation of multimeric structures containing more than one interchain disulfide bridge has not been reported so far. We have constructed three plasmids which are able to direct expression of recombinant rabbit uteroglobin, a homodimeric protein with two interchain disulfide bridges, in Escherichia coli. Among these, the plasmid pLE103-1, in which the expression of recombinant uteroglobin is controlled by a bacteriophage T7 late promoter, is by far the most efficient. With pLE103-1, recombinant uteroglobin production reached about 10% of total bacterial soluble proteins. This protein accumulated in bacterial cells in dimeric form, as it is naturally found in the rabbit uterus. Recombinant uteroglobin was purified to near-homogeneity and its NH2-terminal amino acid sequence was confirmed to be identical to that of its natural counterpart, except for 2 Ala residues the codons for which were added during the plasmid construction. This protein was found to be as active a phospholipase A2 inhibitor as natural uteroglobin on a molar basis. To our knowledge, this is the first report of high level bacterial expression of a full length eukaryotic homodimeric protein with two interchain disulfide bridges in its natural, biologically active form. The plasmid pLE103-1 may be useful to explore structure-function relationships of rabbit uteroglobin. In addition, this plasmid may be useful in obtaining high level bacterial expression of other eukaryotic proteins with quaternary structure, as well as for other general applications requiring efficient bacterial expression of cDNAs.

Animals↗
PubMed1990

Changes in frequency and localization of human X- and Y-chromatin bodies at interphase during in vitro cellular aging.

We have investigated the degree of hypodiploidy of human X (inactive) and Y chromosomes and their relative localization in the interphase nuclei during in vitro aging of diploid fibroblasts. It is found that significant proportions of both female and male cells lose the inactive X and Y chromosome, respectively during cellular aging. Our results from fibroblasts are consistent with comparable findings of other investigators utilizing lymphocytes and bone marrow cells. However, we have observed a relatively higher proportion of X and Y chromosomal hypodiploidy in older fibroblasts than the frequencies reported for lymphocytes or bone marrow cells from aged people. Significant changes in the relative localization pattern of the inactive X and Y chromosomes in the nuclei are also noted during in vitro aging of female and male cells, respectively, and these changes in localization pattern are not identical in both sexes. We believe that, during cellular aging, the analysis of sex chromosomal aneuploidy at interphase is highly likely to provide more accurate results as opposed to the analysis of metaphase chromosomes since the latter is dependent upon the divisional capacity of cells which declines with age. Analysis of interphase cells also avoids the artifacts that accompany metaphase chromosome preparations.

Aging↗
PubMed1990

Determination of sex chromosomal constitution and chromosomal origin of drumsticks, drumstick-like structures, and other nuclear bodies in human blood cells at interphase by fluorescence in situ hybridization.

The sex chromosomal constitution has been determined in various types of human leukocytes at interphase by use of fluorescence in situ hybridization with X- and/or Y-specific DNA probes. It is found that during aging and differentiation of myelocytes into polymorphs there is no significant change in the relative frequency of various types of male and female cells with a specific type of sex chromosomal constitution. Non-random variability of the relative proximity between the X chromosomes within the nuclei is also observed in female cells. Moreover, we are the first to determine that sex-specific "drumsticks" and "sessile nodules" in female polymorphs originate from the X chromosomes and that non-sex-specific "drumstick-like" bodies in male polymorphs are of Y chromosomal origin.

Cell Nucleus↗
PubMed1989

Novel peptides derived from a region of local homology between uteroglobin and lipocortin-1 inhibit platelet aggregation and secretion.

The active site for uteroglobin inhibition of phospholipase A2 has been localized to a nonapeptide (P1) which is partially homologous to a nonapeptide (P2) in lipocortin, which also inhibits phospholipase A2. P1 and P2 share an identical tetrapeptide (P4) which is required for inhibition, although P4 alone does not inhibit this enzyme. We found the mechanism of inhibition of platelet aggregation and secretion by the nonapeptides and P4 varied depending on whether platelets were thrombin- or ADP-activated. All three peptides decrease thrombin esterolytic activity and thereby inhibit thrombin-induced platelet activation. P1 decreases ADP-induced aggregation and serotonin secretion by inhibiting phospholipase A2 whereas P4 decreases only aggregation by blocking fibrinogen binding to activated platelets. The P4 sequence in P1 may affect the interaction of P1 with platelets since the presence of P4 potentiates P1 inhibition of platelet activation.

Adenosine Diphosphate↗