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Biomedical subjects

S Sarkar

Publications and source records attributed to S Sarkar.

At least 343 records · Page 19Linked to original sources
PubMed1984

Murine monoclonal antibodies that identify antigenically distinct subpopulations of human sperm.

Murine monoclonal antibodies (MoAbs) were isolated to characterize antigenically distinct subpopulations of human sperm. Spleen cells from Balb/c mice immunized with freshly prepared human sperm, were fused with murine P3-NS1-Ag4-1 myeloma cells by somatic cell hybridization, and supernatants from the IgG-secreting hybridomas were screened by an enzyme immunoassay (EIA) for reactivity against fresh human sperm and a panel of human somatic cells. Two MoAbs, SP1D1 and SP7A7, reacted specifically with human sperm, whereas three others, SP2A9, SP3B3, and SP4F5, cross-reacted with a variety of human somatic cells. The binding of MoAbs were characterized by immunofluorescence, agglutination, and Staphylococcus aureus binding assays. We found that certain MoAbs bound to common antigens of the head and tail, or to tail alone, and had agglutinating activity. However, not all sperm were reactive to antibody, and the binding activity could only be demonstrated in subpopulations of sperm ranging from 5 to 50% of the total number.

Agglutination Tests↗
PubMed1984

Swimming behavior of X and Y human sperm.

A laminar-flow fractionation method, developed primarily for removing dead sperm from human semen, was successfully modified to enrich X and Y sperm to 80% purity, and to characterize each enriched fraction for individual swimming behavior. Y-sperm fractions were rapidly detected by fluorescent cytogenetic staining. Subsequently, the degree of enrichment was quantitated with DNA extracted from each sperm fraction probed with a human male-specific recombinant DNA clone. In stationary fluid, X and Y sperm swam in circles with the same average speed. However, in a flowstream, X sperm shifted to a nearly straight path of movement in a significantly decreased angular velocity. This shift was four times more pronounced in X sperm than in Y sperm, especially after the initial transition from stationary fluid to flow. The velocity gradient across the flow axis was essential for separating X and Y sperm; uniform flow velocity did not separate them effectively.

Cell Separation↗
PubMed1984

Human sperm swimming in flow.

To study the movement of human sperm, we have developed a microflow cell by miniaturizing our design for a preparative fractionation flow column. The microflow cell enabled us to view the movement of sperm over periods as long as 2 min. Sequential steps of filming, editing, and analysis revealed that the curved swimming patterns of sperm swimming in stagnant fluid become nearly straight tracks when the flow velocity is increased. However, the net swimming speed remained unchanged. Motile sperm accumulated near solid wall surfaces surrounding the fluid and oriented against the direction of the current; the velocity gradient was steepest in these regions. A laminar-flow preparative column separated motile sperm from dead sperm by carrying the nonmotile sperm and debris with the stream while leaving the motile sperm near the surrounding walls.

Adult↗
PubMed1984

Error-prone mutagenesis detected in mammalian cells by a shuttle vector containing the supF gene of Escherichia coli.

When a shuttle vector containing a tyrosine suppressor tRNA (supF) gene as a target for mutagenesis replicated in a monkey kidney cell line, the frequency of SupF+ mutations was 2.3 +/- 0.5 x 10(-3). When the host cells were treated with ethyl methanesulfonate 40 h before transfection, a 10-fold increase in SupF+ mutation frequency was observed. These results supported the hypothesis that a damage-inducible mutagenic pathway exists in mammalian cells and also demonstrated the utility of this shuttle vector for the study of mutagenesis in mammalian cells.

Animals↗
PubMed1983

The cytoplasmic 4 S translation inhibitory RNA species of chick embryonic muscle. Effect on mRNA binding to 43 S initiation complex.

A cytoplasmic 10 S ribonucleoprotein (iRNP) isolated from chick embryonic muscle is a potent inhibitor of mRNA translation in vitro and contains a 4 S translation inhibitory RNA species (iRNA) (Sarkar, S., Mukherjee, A. K., and Guha, C. (1981) J. Biol. Chem. 256, 5077-5086). Using an in vitro assay system, we show that the iRNA has no effect on the elongation phase of peptide synthesis. iRNA inhibits translation at the initiation step by inhibiting mRNA binding to 43 S initiation complexes. The iRNA does not inhibit the binding of Met-tRNAf to the 40 S ribosomal subunit, but rather causes an increase in the level of 43 S initiation complexes in the reticulocyte lysate. The formation of the 80 S initiation complex from the 43 S complex is specifically blocked in the presence of iRNA. The significance of these results in relation to biological function of iRNA is discussed.

Animals↗
PubMed1982

Correlation between the protein and mRNA levels for myosin light chains and tropomyosin subunits during chick fast muscle development in vivo.

Using myosin light chains and tropomyosin subunits as representative myofibrillar proteins, we have characterized their isoprotein forms and also correlated them with the accumulation of the corresponding mRNAs during development of a fast muscle in chicken, viz, pectoralis. Both slow and fast myosin light chain isoforms, except fast myosin light chain LC3, and the two subunits of tropomyosin are present in early embryonic muscle. During development, the slow myosin light chains and beta-tropomyosin appear in reduced amounts in pectoralis muscle and finally they disappear in adult muscle. Translation studies with total cellular RNA from developing muscle indicates that while the protein levels of the above isoforms, in general, correlate with the accumulation of corresponding mRNAs, for LC3, additional post-transcriptional control appears to modulate the expression of this isoprotein skeletal muscle development in vivo.

Aging↗
PubMed1982

Association of cap binding protein-related polypeptides with cytoplasmic RNP particles of chick embryonic muscle.

Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (Mr 78 000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low-Mr immunoreactive peptides (Mr 43 000 and Mr 29 000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive Mr 43 000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed.

Animals↗
PubMed1982

Quantitation of muscle-specific mRNAs by using cDNA probes during chicken embryonic muscle development in ovo.

The emergence of abundant-class mRNAs specific for contractile muscle proteins and their distribution between polysomal and free mRNP fractions were studied in skeletal muscle excised from chicken embryos during the transition from myoblasts (day 9) to myotubes (day 18). Muscle-specific cDNA was selectively prepared by hybridizing cDNA to template RNA (polysomal poly(A)+ mRNA) from day-14 embryos followed by isolation of the abundant class, which represents approximately 20% of total mRNA. The specificity of the cDNA probe for this class was confirmed by the differential degree of hybridization to cytoplasmic RNA from cultured myotube and myoblast cells and by its inability to hybridize with mRNA from nonmuscle cells such as liver. Except for muscle from day-9 embryos, the concentrations of the abundant-class muscle-specific mRNAs were higher in polysomes than in free mRNP fractions. Furthermore, the levels of these mRNAs in polysomes increased 12-fold from day 9 (myoblast) to day 14 (intermediate) with a further 3.6-fold increase from day 14 to day 18 (myotube). In contrast to this 45-fold net increase in the polysomal level of these mRNAs from day 9 to day 18, the levels in the free mRNP fraction showed only a 3-fold decrease during this period. Because the amount of mRNA lost from the mRNP fraction is much less than the net increase in the polysome fraction, mRNP does not serve as a reservoir of untranslated muscle-specific mRNA for transfer to polysomes. Consequently, the emergence of muscle-specific polysomal mRNA for contractile proteins during myogenesis in ovo appears to be regulated primarily by transcriptional control.

Age Factors↗
PubMed1981

Nonselective inhibition of messenger RNA translation by highly purified low molecular weight RNA species from ribosomal salt wash of chick embryonic muscle.

A low molecular weight RNA species, in the 70-90 nucleotide size range (iRNA), has been purified from the ribosomal salt wash of chick embryonic muscle by a combination of DEAE-cellulose and hydroxyapatite chromatography. This method yields iRNA free from contaminating tRNA and gives better and more reproducible yields than those obtained with our previous method involving lengthy dialysis of the salt wash. The iRNA at the concentration of 20-80 ng range strongly inhibits the translation of homologous and heterologous mRNAs i.e. chick muscle poly(A)+mRNA and rabbit globin mRNA; uncapped mRNA; and poly(A)-mRNA in micrococcal nuclease-treated reticulocyte lysate indicating that inhibition by iRNA is nonselective in nature. The translation of endogenous globin mRNA and polysomes in the lysate is strikingly less sensitive to iRNA suggesting that the initiation step is primarily affected by iRNA. The iRNA does not appear to be double-stranded RNA. It is concluded that iRNA is distinct from other low molecular weight RNA species described in the literature which modulate protein synthesis in cell-free systems.

Animals↗
PubMed1981

The translational inhibitor 10 S cytoplasmic ribonucleoprotein of chick embryonic muscle. Dissociation and reassociation.

Cytoplasmic 10S ribonucleoprotein (iRNP) is a potent inhibitor of mRNA translation in vitro and contains a 4 S translation inhibitory RNA species (iRNA) (Sarkar, S., Mukherjee, A. K., and Guha, C. (1981) J. Biol. Chem. 256, 5077-5086). This ribonucleoprotein has now been resolved into protein and RNA components by DEAE-cellulose chromatography in the absence of both K+ and Mg2+ ions. These cations are required for maintaining the nucleoprotein structure of iRNP. Incubation of the dissociated protein and RNA components in the presence of K+ and Mg2+ at 35 degrees C reconstitutes a 10 S particle which is indistinguishable from native iRNP with respect to the elution profile by gel filtration, UV spectra, buoyant density, resistance to pancreatic RNase, and ability to inhibit exogenous mRNA translation in vitro. Chick muscle tRNA and globin mRNA could not form an RNP complex with the protein moieties of iRNP. The separated proteins, unlike iRNA and iRNP, do not inhibit mRNA translation. Their function may be to protect iRNA from ribonuclease digestion, since iRNP is ribonuclease-resistant. The ability to dissociate the iRNP particle and to specifically reconstitute it from the separated components indicates that it is a unique cellular entity which is distinct from other ribonucleoproteins.

Animals↗
PubMed1981

A ribonuclease-resistant cytoplasmic 10 S ribonucleoprotein of chick embryonic muscle. A potent inhibitor of cell-free protein synthesis.

A ribonucleoprotein (RNP) particle sedimenting at 10 S in sucrose gradients had been isolated from the post-polysomal fraction of homogenates of 14-day-old chick embryonic leg and breast muscle by sucrose gradient fractionation and gel filtration. The 10 S RNP contains a 4 S RNA species (base composition: AMP, .3%; GMP, 22.2%; CMP, 24.2%; and UMP, 23.2%), and shows three major bands in the 70-90-nucleotide size range by polyacrylamide gel electrophoresis in 99% formamide. The 4 S RNA does not contain oligo(U)- and oligo(A)-rich tracts. The RNP has a characteristic buoyant density of 1.410 g/ml, which corresponds to an RNA/protein ratio of about 1:4. The UV absorption spectra of the RNP is very distinct from that of its RNA component. Both 4 S RNA and the 10 S RNP are potent inhibitors of translation of a variety of mRNAs such as chick muscle poly(A)+ mRNA, rabbit globin mRNA, EMC virus RNA, and poly(A)- and mRNA of rat liver in micrococcal nuclease-treated rabbit reticulocyte lysate. The inhibitory action of the RNA and the RNP on mRNA translation appears to involve the initiation process. The RNA and RNP do not have a nuclease activity associated with them. The hyperchromicity profile of the inhibitory RNA with increasing temperature indicates that it does not contain a significant amount of double-stranded structure. This is also supported by the complete loss of biological activity of the RNA by treatment with pancreatic RNase. In contrast, the inhibitory activity of the RNP was resistant to RNase. Electrophoresis of the protein moieties of the inhibitory RNP using both one- and two-dimensional gel techniques in the presence of sodium dodecyl sulfate shows a complex pattern of polypeptides of Mr = 12,000-150,000. The protein pattern of the 10 S particle is quite different from those of free and polysomal mRNP and poly(A)-protein complexes of chick embryonic muscles, indicating that most, if not all of the mRNA-associated proteins, are absent in the 19 S RNP. The properties of the inhibitory RNA indicate that it is different from the various low molecular weight RNA species which are involved in the modulation of protein synthesis in cell-free systems. It is concluded that the 10 S particle represents a novel class of RNP, which may be involved in posttranscriptional regulation of protein synthesis in embryonic muscles.

Animals↗
PubMed1981

DMBA is ineffective in inducing breast cancer in feminized male Holtzman rats.

Mammary tissue of male rats "feminized" by in utero treatment with cyproterone acetate, accrues the capacity to convert circulating androgens to estrogens at the mammary tissue level in adult life. DMBA-induced mammary tumorigenesis was studied in these feminized males to find out whether or not this altered steroid metabolism leading to heightened estrogenicity would increase mammary tumor yield when compared to that seen in normal males, where such conversion is significantly low. Results on the incidence of mammary tumors, tumor pathology and latency period of tumor appearance in these 2 groups, however, do not differ significantly. From observations on mammary morphology of these feminized males it appears that in vivo conversion of testosterone to estrogen probably is not total and that the available testosterone inhibits tumor induction.

9,10-Dimethyl-1,2-benzanthracene↗