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S Majumder

Publications and source records attributed to S Majumder.

At least 73 records · Page 4Linked to original sources
PubMed1994

Requirements for DNA transcription and replication at the beginning of mouse development.

In mice, the first round of DNA replication occurs in fertilized eggs (1-cell embryos), while the onset of zygotic gene transcription begins approximately 20 hours after fertilization, a time that normally coincides with formation of a 2-cell embryo. One approach to investigating the mechanisms that control these developmentally regulated events has been to microinject plasmid DNA into the nuclei of mouse oocytes and embryos in order to determine the requirements for unique DNA sequences that regulate transcription and replication. The results from these and other studies have revealed two important mechanisms that regulate the beginning of animal development. The first is a time dependent "zygotic clock" of unknown detail that delays the onset of transcription, regardless of whether or not a 2-cell embryo is formed. The second is a mechanism that represses the activity of promoters and origins of replication specifically in maternal pronuclei of oocytes and 1-cell embryos, and in all nuclei of 2-cell embryos, regardless of their parental origin or ploidy. This repression is linked to chromatin, but the striking ability to relieve this repression with specific embryo-responsive enhancers first appears with formation of a 2-cell embryo. The need for a TATA-box to mediate enhancer stimulation of promoter activity appears even later when cell differentiation becomes evident. Thus, a biological clock delays transcription until both paternal and maternal genomes are replicated and remodeled from a post-meiotic state to one in which transcription is repressed by chromatin structure in a manner that can be relieved by cell-specific enhancers at appropriate times during development.

Animals↗
PubMed1994

Changes in Trypanosoma cruzi infectivity by treatments that affect calcium ion levels.

The possible role of the intracellular Ca2+ level in the regulation of Trypanosoma cruzi infectivity was explored by measuring the capacity of trypomastigote forms of this organism to invade mammalian host cells after treatments which decrease or elevate cytoplasmic Ca2+. Parasites loaded with either bis-(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid (BAPTA) or 2-([2-bis(carboxymethyl)-amino-5-methylphenoxy]methyl)-6-nethoxy-8 - bis(carboxymethyl)aminoquinoline (Quin-2) to chelate Ca2+ displayed significantly decreased infectivity. This effect was denoted by reductions in both the proportion of rat heart myoblasts invaded by the parasite in vitro and the number of trypanosomes penetrating these host cells, the extents of which were BAPTA or Quin-2 concentration dependent. Consistent with these observations, inhibitory effects were also recorded when the parasite was pretreated with the calmodulin-binding phenothiazines trifluoperazine and chlorpromazine or with felodipine, a chemically different type of calmodulin antagonist, for as little as 5 min. In contrast, pretreatment with the Ca2+ ionophore ionomycin, which elevated Ca2+ levels in T. cruzi, significantly enhanced the infective capacity of the parasite. These results point to the existence of a Ca(2+)-dependent mechanism that regulates the invasive capacity of T. cruzi.

Aminoquinolines↗
PubMed1994

TATA-dependent enhancer stimulation of promoter activity in mice is developmentally acquired.

Herpes simplex virus (HSV) thymidine kinase (tk) promoter activity depends on four transcription factor binding sites, one of which is a TATA box sequence, and the presence of either a cis-acting enhancer sequence or a transactivator protein. Studies presented here show that this TATA box was required for promoter activity only after cells began to differentiate and then only when promoter activity was stimulated by either an enhancer or a transactivator. When the HSV tk promoter was utilized by mouse embryos from the one-cell to eight-cell stage of development or by undifferentiated mouse embryonic stem cells, disruption of the HSV tk TATA box by site-specific mutations did not reduce promoter activity. This was true even when HSV tk promoter activity was stimulated strongly by either the embryo-responsive polyomavirus F101 enhancer or its natural transactivator, the HSV ICP4 gene product. However, stimulated expression was dependent on a distal Sp1 DNA binding site. Similarly, disruption of the TATA box did not reduce tk promoter activity in primary mouse embryonic fibroblasts or in immortalized 3T3 mouse fibroblasts; in fact, promoter activity was increased up to 2.6-fold. However, in these differentiated cells, stimulation of the HSV tk promoter by either the F101 enhancer or ICP4 protein required the TATA box. HSV tk promoter activity also was dependent on its TATA box in the mouse oocyte, a terminally differentiated cell with an endogenous transactivating activity. These results reveal that the need for a TATA box is developmentally acquired and depends on at least two parameters: the differentiated state of the cell and stimulation of the promoter by either an enhancer or a transactivator.

3T3 Cells↗
PubMed1994

Subacute toxicity of fenvalerate in broiler chicks: concentration, cytotoxicity and biochemical profiles.

Subacute toxicity study of fenvalerate was carried out in broiler chicks after oral administration @ 525.6 mg/kg once daily for 28 days. The blood concentration of fenvalerate following 1 day post-administration (pd) was 39.65 +/- 2.67 micrograms/ml and maintained plateau thereafter up to day 21 pd, and then declined (18.46 +/- 1.47 micrograms/ml) on day 28 pd. Intestine contained maximum residue (7.46 +/- 1.96 micrograms/g) followed by fat (5.95 +/- 1.16 micrograms/g), brain (5.06 +/- 0.96 micrograms/g), liver (3.93 +/- 0.51 micrograms/g), kidney (3.79 +/- 0.72 micrograms/g) and heart (1.72 +/- 0.35 micrograms/g). Histopathological examinations showed focal areas of necrosis in liver, proliferation and fibrosis of bile duct, larger size of glomeruli, glomerular and tubular necrosis in treated birds. Fenvalerate significantly increased the cholesterol level in brain, GPT activity in liver and heart, GOT activity in heart, and alkaline phosphatase activity in heart and brain tissue. It significantly decreased the glycogen content in liver and heart, GOT activity in brain and acid phosphatase activity in all the tissues analyzed. It appears that comparatively fowl is resistant to fenvalerate toxicity.

Animals↗
PubMed1993

Analysis of gene expression in mouse preimplantation embryos demonstrates that the primary role of enhancers is to relieve repression of promoters.

Enhancers are generally viewed simply as extensions of promoters, lacking a function of their own. However, previous studies of mouse preimplantation embryos revealed that 1-cell embryos can utilize enhancer-responsive promoters efficiently without an enhancer, whereas 2-cell embryos require an enhancer to achieve the same levels of expression. This suggested that enhancers relieved a repression in 2-cell embryos that is absent in 1-cell embryos. Results presented here demonstrate first that the ability of 1-cell embryos to dispense with enhancers does not result from the absence of specific activation proteins. Under conditions where GAL4-VP16 activated a GAL4-dependent promoter in both embryos, GAL4-VP16 activated a GAL4-dependent enhancer only in 2-cell embryos. Moreover, the role of an enhancer is not to compensate for either changes in promoter requirements, or for reduced levels of promoter-specific transcription factors. Linker-scanning mutations in a natural promoter revealed that both embryos utilized the same promoter elements, and comparison of different promoters revealed that these embryos have equivalent transcriptional capacities. In addition, titration experiments revealed less Sp1 activity in 1-cell embryos where enhancers are dispensable than in 2-cell embryos where enhancers are required. Therefore, we propose that the primary function of enhancers, first evident with formation of a mouse 2-cell embryo, is to prevent repression of weak promoters, probably by altering chromatin structure. Consistent with this hypothesis is the fact that butyrate, an agent that alters chromatin structure, stimulated promoters in 2-cell embryos, but not in 1-cell embryos.

Animals↗
PubMed1993

N,N'-thiophene-substituted polyamine analogs inhibit mammalian host cell invasion and intracellular multiplication of Trypanosoma cruzi.

We studied the effects of two N,N'-thiophene-substituted polyamine analogs (MDL 28302 and MDL 29431) on the capacities of Trypanosoma cruzi, the etiologic agent of Chagas' disease, to invade and multiply within a mammalian host cell. Both compounds inhibited infectivity significantly in a time- and concentration-dependent manner. This inhibition resulted from a selective effect on the parasite, because pretreatment of T. cruzi but not host cell cultures with either MDL 28302 or MDL 29431 reduced infectivity. The parasite gradually recovered its infective capacity after removal of unincorporated polyamine analog, denoting the reversible nature of the inhibitory effect. Some biochemical modification of MDL 28302 and MDL 29431 appeared to be required for their inhibitory activities to be exerted, since the effects of these drugs on T. cruzi infectivity were abrogated by MDL 72527, a drug known to inhibit polyamine oxidase (PAO) activity specifically. Supporting the notion of that products of MDL 28302 and MDL 29431 oxidation by PAO were involved in the activity of these compounds was the finding that PAO competitive substrates (N1-acetylspermine and N1-acetylspermidine) also abolished the inhibition of T. cruzi infectivity mediated by MDL 28302 or MDL 29431. However, we can not rule out that MDL 72527 and the PAO competitive substrates might have altered an alternative mechanism because no significant polyamine oxidase activity could be demonstrated in preparations of lysed or intact T. cruzi in assays monitoring conversion of [14C]spermine to [14C]spermidine. When either MDL 28302 or MDL 29431 was added to infected cell cultures, a marked reduction in the rate of intracellular parasite growth ensued.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Inhibition of host cell invasion and intracellular replication of Trypanosoma cruzi by N,N'-bis(benzyl)-substituted polyamine analogs.

We studied the effects of two N,N'-bis(benzyl)-substituted polyamine analogs on the capacities of Trypanosoma cruzi to invade and multiply within a mammalian host cell. At concentrations as low as 1 microM, these compounds reduced significantly the infectivity of the parasite for rat heart myoblasts in a time-dependent manner. Pretreatment of virulent T. cruzi trypomastigotes, but not myoblast pretreatment, reduced the level of infectivity. The inhibitory effects started to subside 3 h after removal of the drugs and were no longer detectable after 4 h. A significant decrease in the rate of intracellular amastigote multiplication was also seen when the drugs were added to myoblast cultures which had been previously infected with untreated T. cruzi. These results show that N,N'-bis(benzyl)-substituted polyamine analogs meet the two most important criteria for potential chemotherapeutic agents against T. cruzi infection, namely, inhibition of both host cell invasion and intracellular replication by this parasite.

Animals↗
PubMed1993

Inhibition of S-adenosyl-L-methionine (AdoMet) decarboxylase by the decarboxylated AdoMet analog 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811) decreases the capacities of Trypanosoma cruzi to infect and multiply within a mammalian host cell.

A decarboxylated S-adenosyl-L-methionine (AdoMet) analog, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811), that specifically and irreversibly inhibits AdoMet decarboxylase (DC) was used to investigate the role of AdoMetDC in the regulation of host cell invasion and intracellular replication by Trypanosoma cruzi. The presence of MDL 73811 in cocultures of T. cruzi and rat heart myoblasts (RHM) significantly inhibited host cell infection in a dose-dependent manner. This effect, evidenced by reductions in the proportion of infected RHM and the number of parasites per 100 RHM, was due to MDL 73811 action on T. cruzi as it was reproduced when the parasites, but not the RHM, were pretreated with the inhibitor. Significant inhibition of infectivity required a 10-min treatment with MDL 73811 although greater effects ensued with additional incubation time. We could not detect signs of recovered infectivity up to 6 hr after the removal of nonincorporated MDL 73811, suggesting that either AdoMetDC turnover in T. cruzi is a relatively slow process or the amounts of MDL 73811 taken up could not be blocked or eliminated during this time period. MDL 73811-mediated inhibition of infectivity was not bypassed by the addition of exogenous spermidine or spermine, suggesting that the mechanism of action does not involve reduced production of these polyamines due to AdoMetDC inhibition. Intracellular accumulation of AdoMet appeared to be a more plausible explanation in view of the fact that exogenous AdoMet significantly inhibited infectivity. When added to infected RHM cultures, MDL 73811 or AdoMet inhibited also intracellular T. cruzi growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosylmethionine Decarboxylase↗
PubMed1992

Alterations in sarcolemmal adrenergic receptors due to intracellular Ca2+ overload in the myocardium.

To investigate the effect of an overload of intracellular Ca2+ on the adrenergic receptors, rat hearts perfused with Ca(2+)-free medium for 5 or 10 minutes were reperfused (Ca(2+)-paradox) with a medium containing 1.25 mM Ca2+. The status of beta-adrenergic and alpha-adrenergic receptors in a purified sarcolemmal preparation was studied by measuring the specific binding of [3H]dihydroalprenolol and [3H]prazosin, respectively. Reperfusion with Ca(2+)-containing medium increased the density of both beta- and alpha-adrenoceptors. While the hearts perfused with Ca(2+)-free medium for 5 minutes did not show any change in the Kd values upon reperfusion, the hearts perfused with Ca(2+)-free medium for 10 minutes showed an increase in the Kd value for beta-receptors and a decrease in the Kd value for alpha-receptors. The alterations in adrenergic receptors upon reperfusion in the hearts perfused with Ca(2+)-free medium for 5 minutes were not evident if the medium contained a low concentration of Na+ (35 mM) or was maintained at a low temperature (21 degrees C) to prevent the occurrence of an overload of intracellular Ca2+. These results suggest that an overload of intracellular Ca2+ might play an important role in changing the characteristics of adrenergic receptors in diseased hearts.

Animals↗
PubMed1992

Biochemical evidence for the presence of arginine decarboxylase activity in Trypanosoma cruzi.

Trypanosoma cruzi was found to release 14CO2 from radiolabeled arginine, and this effect was inhibited by either DL-alpha-difluoromethylarginine or monofluoromethylagmatine, both specific inhibitors of arginine decarboxylase (ADC). Furthermore, agmatine, which can be derived metabolically only by ADC-mediated arginine decarboxylation, was produced when T. cruzi was incubated with radiolabeled arginine, and agmatine production was inhibited in the presence of DL-alpha-difluoromethylarginine. These results constitute direct biochemical evidence for the presence in T. cruzi of ADC, an enzyme that does not occur in mammalian cells.

Agmatine↗
PubMed1990

Inhibition of cardiac phosphatidylethanolamine N-methylation by oxygen free radicals.

This study was undertaken to examine the effects of oxygen free radicals on phosphatidylethanolamine (PE) N-methylation in rat heart sarcolemmal (SL) and sarcoplasmic reticular (SR) membranes. Three catalytic sites involved in the sequential methyl transfer reaction were studied by assaying the incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine (0.055, 10, and 150 microM) into SL or SR PE molecules under optimal conditions. In the presence of xanthine + xanthine oxidase (superoxide anion radicals generating system), PE N-methylation was inhibited at site I and III in the heavy SL fraction isolated by the hypotonic shock-LiBr treatment method. In the light SL fraction isolated by sucrose-density gradient, a significant inhibition of PE N-methylation was seen at all three sites. These inhibitory effects of xanthine + xanthine oxidase on PE N-methylation were prevented by the addition of superoxide dismutase. Hydrogen peroxide showed a significant inhibition of PE N-methylation at site I in the heavy SL fraction, and at site I and II in the light SL fraction. Catalase blocked the inhibitory effects of hydrogen peroxide. The effects of both xanthine + xanthine oxidase and hydrogen peroxide on the SR membranes were similar to those seen for the heavy SL fraction. These results suggest that, in addition to lipid peroxidation, the oxygen free radicals may affect the function of cardiac membranes by decreasing the phospholipid N-methylation activity.

Animals↗
PubMed1990

The mouse W/c-kit locus.

The mature cells in the haemopoietic system arise as the result of the extensive developmental and proliferative capacity of pluripotential stem cells. In order to understand the molecular basis for these developmental processes, it will be necessary to identify and characterize the cellular genes that control early steps in haemopoiesis. Mutations at the mouse W locus on chromosome 5 lead to pleiotropic developmental defects, including sterility, coat colour abnormalities, severe macrocytic anaemia and mast cell deficiency. The defects in all these lineages are cell autonomous and intrinsic, suggesting that the W locus encodes a gene product required directly for cellular differentiation. In an attempt to understand this classical mouse developmental mutation, we have demonstrated that the c-kit proto-oncogene, which encodes a transmembrane receptor tyrosine kinase, is very closely linked to W. Several further observations are consistent with the idea that W and c-kit are allelic: first, c-kit is expressed in those cell populations affected by W mutations; second, the expression of c-kit transcripts can be affected by mutations at the W locus; third, the tyrosine kinase activity associated with the protein encoded by c-kit is functionally impaired in mast cells derived from mutant W/Wv mice; and fourth, rearrangements within the c-kit gene have been reported in two W mutant alleles. These observations suggest that the dominant phenotype associated with W mutations results from loss-of-function alterations that affect the receptor tyrosine kinase encoded by c-kit. The demonstration that the W locus encodes a transmembrane growth factor receptor provides a molecular basis for understanding the intrinsic haemopoietic defect in W mutant mice and the role that this cellular proto-oncogene plays in haemopoiesis and other developmental processes.

Animals↗
PubMed1990

Tyrosine protein kinase activity of the HZ4-feline sarcoma virus P80gag-kit-transforming protein.

The Hardy-Zuckerman 4 feline sarcoma virus (HZ4-FeSV), isolated from a feline fibrosarcoma, is a replication defective acute transforming feline retrovirus that originated by transduction of feline c-kit sequences with feline leukemia virus (FeLV). The v-kit sequences of the HZ4-FeSV, a segment of 1106 nucleotides, correspond to sequences of the cytoplasmic domain of the c-kit receptor kinase. The HZ4-FeSV is known to encode an 80-kilodalton protein with FeLV gag and kit determinants. The P80gag-kit protein and its associated activities from HZ4-FeSV-transformed mink cells were characterized. The P80gag-kit protein was found to be myristoylated, suggesting a membrane association for this protein. In agreement with the predicted relationship with tyrosine kinases, by using the in vitro immune complex-kinase procedure, the P80gag-kit protein was shown to display a tyrosine-specific autophosphorylation activity. In vivo, the P80 protein was found to be phosphorylated on serine and threonine and to a lesser degree on tyrosine. In addition, potential in vivo protein substrates for tyrosine-specific phosphorylation mediated by the P80gag-kit kinase were identified in HZ4-FeSV-transformed cells.

Animals↗
PubMed1989

Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice--evidence for an impaired c-kit kinase in mutant mice.

The proto-oncogene c-kit, a transmembrane tyrosine protein kinase receptor for an unknown ligand, was shown recently to map to the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis, as well as the proliferation and/or migration of primordial germ cells and melanoblasts during development. Here, we show that c-kit is expressed in tissues known to be affected by W mutations in fetal and adult erythropoietic tissues, mast cells, and neural-crest-derived melanocytes. We demonstrate that the c-kit associated tyrosine-specific protein kinase is functionally impaired in W/WV mast cells, thus providing a molecular basis for understanding the developmental defects that result from these mutations.

Alleles↗
PubMed1988

Intermediates in plasmid pT181 DNA replication.

Staphylococcus aureus plasmid pT181 is thought to replicate via an asymmetric rolling-circle mechanism. By studying pulse labeled replicative intermediates, here we report that pT181 replication involves: (1) a post-replicative hypersupercoiled monomer and (2) a partially replicated intermediate which lacks superhelicity but is unlike a typical rolling-circle intermediate in that only nascent strands of less than unit length are released by alkali denaturation. A model for pT181 replication is proposed to accommodate this apparent discrepancy.

Anti-Bacterial Agents↗
PubMed1988

Genetic analysis fo the mycobacillin biosynthetic pathway in Bacillus subtilis B3.

Twelve mycobacillin-negative (My-) mutants of Bacillus subtilis B3 were isolated from an auxotrophically tagged mycobacillin producer organism. In whole-cell fermentations of some of these My- mutants a penta- and a nonapeptide accumulated; these peptides were also obtained in a cell-free system in which a new tripeptide was also detected. The amino acid composition, N- and C-terminal residues and amino acid sequence of these peptides agreed with those of equivalent segments of the mycobacillin molecule. The mycobacillin-synthesizing enzyme can be divided into three fractions that catalyse different steps in biosynthesis, and the defective enzyme fractions in the various mutant strains were identified by reconstitution experiments in vitro. The defects were further pin-pointed in mutant enzyme fractions by an ATP in equilibrium Pi exchange reaction and also by cell-free synthesis involving the use of membrane-bound enzyme. The defects so identified indicated the formation of tri-, penta- and nonapeptides as intermediates in the mycobacillin biosynthetic pathway.

Amino Acids↗
PubMed1988

c-kit protein, a transmembrane kinase: identification in tissues and characterization.

The proto-oncogene c-kit encodes a transmembrane kinase which is related to the receptors for colony-stimulating factor type 1 and platelet-derived growth factor, as well as to the immunoglobulin superfamily. Antibodies specific for the kinase domain of the P80 gag-kit protein of the Hardy-Zuckerman 4 feline sarcoma virus were prepared. These kit-specific antibodies were used to identify and characterize the c-kit protein in cat brain tissue. The c-kit protein product displays an autophosphorylating activity in immune complex kinase assays, and, in turn, this activity was used to identify the c-kit protein in different tissues. In cat brain, a single 145-kilodalton (kDa) glycoprotein was detected. Its N-linked carbohydrates were found to be sensitive to digestion with the endoglycosidases (neuraminidase, endoglycosidase F, and endoglycosidase H), indicating hybrid and/or complex and high-mannose structures. A partial purification of the c-kit protein was achieved by wheat germ agglutinin affinity chromatography, and the autophosphorylating activity of the partially purified c-kit protein was characterized and found to be specific for tyrosine. The kit antibodies cross-react with the murine c-kit protein product, and variant c-kit proteins in different mouse tissues were identified, with sizes of about 145 kDa (brain), 160 kDa (spleen), and 150 kDa (testis).

Animals↗