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Biomedical subjects

C G Sahasrabuddhe

Publications and source records attributed to C G Sahasrabuddhe.

17 recordsLinked to original sources
PubMed1990

A novel method of human B cell activation by liposomes coated with Fab' fragments of anti-IgM.

A novel method for activating fresh human B cell, using liposomes decorated with Fab' fragments of anti-human IgM antibodies (immunoloposomes), is described. In our immunoliposome preparation, more than 80% of Fab' were efficiently bound to liposomes and over 95% of the vesicles were uniformly coated with Fab'. Immunoliposomes could activate fresh B cells from peripheral blood of normal donors at least 100-fold better than soluble F(ab')2 fragments, as detected by [3H]uridine uptake. Such activation was also reflected in the enlarged cell size and highly increased [3H]thymidine uptake by B cells when human B cell growth factor (BCGF) was added. Density of Fab' on the liposomes surface did not affect the efficacy of immunoliposomes. The activation of B cells by immunoliposomes was short lived (less than 3 days), due perhaps to rapid internalization of the vesicles by B cells.

Antibodies, Anti-Idiotypic↗
PubMed1989

Structural homology of interferon-gamma with B-cell growth factor and its proliferative effect on long term B-cell lines.

In this study, we have demonstrated that recombinant human interferon-gamma (IFN-gamma) acts as a B cell growth factor (BCGF) for long term B cell lines. Immuno dot blot analysis with polyclonal goat antibody against homogenously purified 60K intracellular BCGF (IC-BCGF) was shown to recognize IFN-gamma and IFN-alpha. Out of three E. coli derived recombinant IFN-gamma molecules tested, two containing the complete C-terminal were reactive with antibody and also exhibited BCGF activity, while the third lacking 15 amino acids from C-terminal did not exhibit BCGF activity nor any reaction to anti-IC-BCGF antibodies in immuno dot blot analysis. The proliferative effect of IFN-gamma on long term B cells could be blocked by anti-IC-BCGF antibodies in a dose dependent manner. These results indicated that this BCGF activity may be a result of structural homology between BCGF and C-terminal peptide of IFN-gamma.

Antibody Formation↗
PubMed1989

Isolation and characterization of growth factor(s) from a human B-cell lymphoma.

We demonstrate that human neoplastic B cells (Br cells) contain a cytoplasmic protein of molecular mass 60 Kd that exhibits B-cell growth factor (BCGF) activity on growth factor-dependent long-term human B cells as well as on autochthonous tumor cells. This 60-Kd protein is recognized by antibodies against a similar intracellular 60-Kd protein derived from normal human lymphocytes. These results demonstrate that the two proteins share epitope homology. Microculture bioassays indicate that neoplastic and normal 60-Kd proteins are capable of driving neoplastic B cells through S-phase. Western immunoblot analysis indicates that neoplastic B cells secrete 60- as well as 14-Kd protein. Immunoaffinity-purified proteins secreted by Br cells exhibit BCGF activity in anti-IgM or dextran sulfate-preactivated human B cells. In addition, a double-antibody immunofluorescence staining technique was used to demonstrate that Br cells express cell surface receptors for BCGF molecule(s). These studies provide support for the autocrine growth model for neoplastic human B cells and suggest that the autocrine growth factor derived from such tumor cells is similar if not identical to normal BCGF molecules.

Adult↗
PubMed1988

Immunological evidence for the relation between low MR secreted form of human B cell growth factor and an intracellular 60K protein.

Homogeneously purified intracellular high-Mr BCGF (IC-BCGF) has been used as an immunogen for the development of heterotypic anti-IC-BCGF antibodies. Anti-IC-BCGF immunoglobulin has been purified by affinity chromatography and used for constructing an immunoaffinity matrix. This matrix was shown to recognize specifically IC-BCGF and the extracellular low-Mr secreted form of human BCGF (EC-BCGF). In the cytosolic extracts of lectin activated human PBLs two other proteins of 50K and 30K were also recognized by these antibodies. Immuno dot blot and western blot analysis also demonstrated that anti-IC-BCGF antibodies recognized EC-BCGF. These antibodies weakly interacted with human interferons (alpha, gamma). Immunoaffinity-purified IC-BCGF was shown to have a molecular weight of 60K, and it exhibited BCGF activity in long-term BCGF-dependent B cell lines. The Mr of one-step affinity-purified EC-BCGF was found to be 14K by SDS-PAGE analysis. This material demonstrated BCGF activity in both long-term BCGF-dependent B cell lines and anti-IgM-activated GoB cells, yet it did not exhibit IL-1 and IL-2 activities. We conclude that EC-BCGF is immunologically related to IC-BCGF through common structural epitopes.

Antibody Specificity↗
PubMed1986

Production of B cell growth factor(s) by neoplastic B cells from hairy cell leukemia patients.

Recent studies have shown that normal human T cells contain a high-molecular-weight (mol wt) protein exhibiting B cell growth factor (BCGF) activity. Other studies have shown that virally transformed human B cells also secrete a high-mol-wt BCGF-like molecule in vitro. We have studied neoplastic B cells from patients with untreated hairy cell leukemia (HCL) to ascertain whether such cytoplasmic BCGF activity is present in the tumor cells. Studies on HCL cells from four patients indicated that BCGF-like activity was in fact present in the cytosolic extracts when tested on autochthonous HCL cells as well as on normal BCGF-dependent human B cell lines. Chromatographic analysis indicated that the BCGF activity from HCL cells was similar in mol wt as well as function to the normal T cell-derived cytosolic BCGF activity. These studies suggest that HCL cells contain and, in some cases, secrete a high-mol-wt growth factor that can be autostimulatory and appears to resemble a similar growth factor molecule found in normal human T cells.

B-Lymphocytes↗
PubMed1986

Purification and partial characterization of human intracellular B cell growth factor.

In the present report a protocol for the purification of an intracellular protein, exhibiting BCGF activity has been described. This protein is obtained from lectin stimulated human peripheral blood mononuclear cells and has been previously shown to be present only in the cytosolic extract of normal human T-lymphocytes. The isoelectric point (pI) of the purified protein is 6.3, as determined by both sucrose gradient isoelectric focusing and chromatofocusing. The molecular weight of the chromatofocused protein, determined by SDS-PAGE, is 60 kD. Thirty-three percent of the BCGF activity contained in the initial crude extract is recovered as a homogenously purified protein, with an estimated specific activity of 10(5) U/mg.

B-Lymphocytes↗
PubMed1985

Establishment of stable human T-T hybridomas.

Human T-T hybridomas potentially provide an invaluable resource for a variety of immunoregulatory molecules that modulate the immune response. To date, success in this technology, using human cell populations, has been hampered by several problems associated with proliferative and functional instability of the hybrid cells. These forms of instability are the result of a multifactorial process, with 1 parameter of importance being the chromosome number of the malignant parent cell line used for fusion. The present studies describe the production of a stable human T-T hybridoma generated by fusing a near diploid (modal chromosome number of 48) aminopterin-sensitive T cell line, CEM TG E11, and lectin-stimulated human peripheral blood lymphocytes. The rapidly growing hybrid cells have been clonally selected for the production of a B cell growth factor. Hybridization was documented by the presence of HLA phenotypes reflecting the combined antigens of the fusion partners. Fusions with 4 other partners besides CEM TG E11, where the majority of the cells had modal chromosome numbers ranging from 78 to 94, were proliferatively unstable. To date, hybrid cells derived from the CEM TG E11 fusion have been doubling approximately every 48 h for greater than 12 months, and selected clones constitutively produce B cell growth factor.

B-Lymphocytes↗
PubMed1985

Partial purification and characterization of mitotic factors from HeLa cells.

Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.

Alkaline Phosphatase↗
PubMed1985

Growth factor-mediated proliferation in B cell non-Hodgkin's lymphomas.

The non-Hodgkin's lymphomas (NHLs) are a heterogeneous group of human lymphoid tumors, primarily of B cell lineage, which appear to represent arrested stages in B lymphocyte differentiation. Control of cell proliferation is a fundamentally important but poorly understood area of study in these tumors. We have studied a representative group of B cell NHLs to assess their potential for growth factor-mediated proliferation in vitro. Our results show that purified monoclonal NHL B cells of the small cell (well-differentiated lymphocytic lymphoma, nodular poorly differentiated lymphocytic lymphoma, etc) type, that were positive for the human malignancy-associated nucleolar antigen could be stimulated by human B cell growth factor (BCGF) to proliferate in vitro. Other B cell activators such as insoluble anti-Ig and the mitogen protein A also could stimulate thymidine incorporation in the lymphoma cell populations. In vitro lymphoma cell growth could be maintained in the presence of the growth factor for up to five weeks. The large B cell type NHL, however, appeared to be refractory to in vitro stimulation by BCGF as well as other stimulators of normal B cells. These studies suggest that human B cell lymphoid tumors are not only phenotypically similar to their normal B lymphocyte counterparts, but are also sensitive in some cases, to the same types of immunoregulatory molecules that control normal lymphoid cell growth.

Antigens, Surface↗
PubMed1985

Intracellular human IL-1: a precursor for the secreted monokine.

An intracellular monocyte derived protein possessing interleukin 1 (IL-1) activity has been compared with the secreted from of IL-1. Our results indicated that this intracellular IL-1 had an apparent size greater than that of extracellular IL-1. Data are presented to show that a rabbit heterotypic antiserum prepared against epitopes on the secreted form of human IL-1 blocked the biologic activity of intracellular IL-1 in a dose dependent manner. Intracellular IL-1 had a mass of 30-40 kDa as determined by gel filtration using sephacryl-200 gel.

Cytoplasm↗
PubMed1978

Responses of mammalian metaphase chromosomes to endonuclease digestion.

Digestion of fixed metaphase chromosomes by endonucleases (micrococcal nuclease and DNase II) under optimal digestion conditions followed by Giemsa staining produces sharp banding patterns identical to G-bands. In 3H-thymidine labeled, synchronized metaphase cells of the chinese hamster (CHO line), the band induction is accompanied by the removal of DNA. The single strand specific nuclease S1 and DNase I do not produce such banding patterns.

Animals↗
PubMed1978

Transcription of nucleosomes from human chromatin.

Nucleosomes (chromatin subunits) prepared by micrococcal nuclease digestion of human nuclei are similar in histone content but substantially reduced in non-histone proteins as compared to undigested chromatin. Chromatin transcription experiments indicate that the DNA in the nucleosomes is accessible to DNA-dependent RNA polymerase in vitro. The template capacities of chromatin and nucleosomes are 1.5 and 10%, respectively, relative to high molecular weight DNA, with intermediate values for oligonucleosomes. Three distinct sizes of transcripts, 150, 120 and 95 nucleotides in length, are obtained when nucleosomes are used as templates. However, when nucleosomal DNA is used as a template, the predominant size of transcripts is 150 nucleotides. When oligonucleosomes are used as templates longer transcripts are obtained. This indicates that RNA polymerase can transcribe the DNA contained in the nucleosomes.

Binding Sites↗
PubMed1977

Salt-induced structural changes in nucleosomes.

Nucleosomes and oligonucleosomes were prepared by digestion of human placental nuclei with staphlococcal nuclease and fractionated by gel filtration chromatography. The effect of increasing salt on the structure of nucleosomes was examined in the presence and absence of 10 mM MgCl2. Nucleosomes and oligonucleosomes are insoluble over a broad range of salt concentration. Nucleosomes are insoluble in larger than or equal to 120 mM (NH4)2SO4 containing 10 mM MgCl2 allowing analyses of changes in nucleosomal DNA by C.D. spectroscopy. Nucleosomes are insoluble in less than or equal to 120 mM (NH4)2SO4 containing 10 mM MgCl2 as demonstrated by turbidity measurements. We conclude that the insolubility of nucleosomes accompanies salt-induced structural changes possibly due to individual particle condensation. As the salt concentration is increased the nucleosomes condense and then relax at higher salt concentrations.

Ammonium Sulfate↗
PubMed1976

Presence of messenger specifying sequences in the DNA of chromatin subunits.

Messenger-specifying sequences in subunits of human lymphocyte chromatin were detected by hybridization of DNA complementary to cytoplasmic polyadenylylated RNA with DNA isolated from the subunits. Comparison of the kinetics and extents of hybridization of complementary DNA with chromatin subunit DNA and with nuclear DNA showed that most of the repetitive sequences and single copy sequences in mRNA are present in chromatin subunits. This result indicates that inclusion of a DNA sequence into the subunit does not prevent its transcription in vivo.

Chromatin↗
PubMedDate not supplied

Cytogenetic characterization of eight human lymphoblastoid cell lines.

Cytogenetic analyses of eight human lymphoblastoid cell lines that were established by a new procedure using B-cell growth factor (BCGF) and interleukin-4 (IL-4) revealed that three cell lines (37.5%) showed structural anomalies in more than 97% of their metaphases, whereas others were predominantly normal diploid. Our results indicate that a) the structural anomalies seen in three cell lines were not induced by culture technique but were constitutional defects present in donors' peripheral blood samples; b) one donor, whose blood gave rise to cell line BCDI, is a constitutional mosaic because both normal diploid and altered metaphases were present; and c) genetic instability should be monitored in regular blood donors because some of them may harbor blood-mediated clastogen or chromosome breakage factor(s) that could induce higher rates of recombinations in somatic cells of some recipients and thus may potentiate such individuals to develop certain types of malignancies.

B-Lymphocytes↗