Biomedical subjects

B H Brownstein

Publications and source records attributed to B H Brownstein.

30 records · Page 2Linked to original sources

PubMed1989

Isolation of single-copy human genes from a library of yeast artificial chromosome clones.

A recently developed cloning system based on the propagation of large DNA molecules as linear, artificial chromosomes in the yeast Saccharomyces cerevisiae provides a potential method of cloning the entire human genome in segments of several hundred kilobase pairs. Most application of this system will require the ability to recover specific sequences from libraries of yeast artificial chromosome clones and to propagate these sequences in yeast without alterations. Two single-copy genes have now been cloned from a library of yeast artificial chromosome clones that was prepared from total human DNA. Multiple, independent isolates were obtained of the genes encoding factor IX and plasminogen activator inhibitor type 2. The clones, which ranged in size from 60 to 650 kilobases, were stable on prolonged propagation in yeast and appear to contain faithful replicas of human DNA.

Chromosomes, Fungal↗

PubMed1989

Rapid dot blot quantitation of viral DNA and amplified genes in less than 1,000 human cells.

The copy number of intracellular DNA sequences can be quantitated rapidly with great sensitivity in 100 to 1,000 cells as starting material. The method applies DNA from lysed cells to a charged nylon membrane that permits successive hybridizations with probes for different genes or DNA sequences. This method was tested with eight types of human cells, including leukemic cells, and has detected Epstein-Barr virus (DNA virus) in immortalized cells, integrated HTLV-I (RNA retrovirus) in infected cells, and determined copy numbers of the amplified multiple drug-resistant gene in human cells resistant to various cytotoxic agents. It could also be used for estimating copy number of transfected DNA in human or other mammalian cells. The described method is not as sensitive as polymerase chain reaction may potentially prove, but is easily quantitated for accurate clinical diagnosis where sensitive and quantitative assays must be carried out on a limited number of cells. Examples of the method's clinical application are the staging of human neuroblastomas and the evaluation of oncogene amplification, which has prognostic value for both overall survival and relapse time in breast cancer patients.

Cell Line↗

PubMed1989

Heat shock proteins in normal and leukemic blood cells.

Heat shock proteins (HSPs) are though to represent a ubiquitous cellular response to heat or stress. We tested whether HSPs can be induced in hairy cells, other human leukemic cells, and normal lymphocytes, and whether there are additive or synergistic effects between heat shock and interferon-alpha 2b (IFN-alpha 2b) on these cells. We analyzed lysates of cells from 22 patients (6 with hairy cell leukemia, 12 with other acute and chronic leukemias and lymphocytes of 4 normals) after exposure to a heat shock and/or IFN-alpha 2b by one-dimensional polyacrylamide gel electrophoresis. In all cells a pattern of HSPs was readily induced with prominent bands identified at approximately 115, 90, and 65 kD. None of these major proteins appeared to represent the previously described IFN-alpha 2b induced band at 80 kD. IFN-alpha 2b by itself was not found to induce HSPs. We conclude that a pattern of HSPs can readily be induced in a variety of normal and leukemic human blood cells. IFN-alpha 2b is not a HSP inducer in these cells. The previously described IFN-induced p80 is apparently not a HSP.

Dactinomycin↗

PubMed1987

Action of interferons in hairy cell leukemia.

We have investigated the direct effects of interferon (IFN) on hairy cells (HCs) isolated from patients with hairy cell leukemia using one- and two-dimensional gel electrophoresis. We have previously characterized the induction of synthesis of 10-16 specific proteins by IFN-alpha 2b in HCs, as analyzed by one-dimensional electrophoresis. By two-dimensional electrophoresis, we have now confirmed this induction and shown that the synthesis of the same number of specific proteins is down-regulated in HCs exposed to IFN-alpha 2b. When compared to HCs, fewer proteins are induced by IFN-alpha 2b in other normal, or neoplastic, lymphoid cells. We also report that protein induction occurs in HCs exposed in vivo to IFN-alpha 2b. We have demonstrated the presence of tubuloreticular structures in the cytoplasm of HCs exposed to IFN-alpha 2b in vitro, using transmission electron microscopy. We now report that these too are seen in HCs exposed to IFN in vivo during therapy. We investigated the effects of IFN-gamma on HCs and found that it also induces specific proteins. The pattern of induced proteins is distinctly different after IFN-gamma exposure in vitro. The fact that such induction occurs suggests that HCs possess also a receptor for IFN-gamma. These results demonstrate that there are direct effects of IFN-alpha on HCs and that such direct effects might be important in the antitumor activity of IFN-alpha in hairy cell leukemia.

Electrophoresis, Polyacrylamide Gel↗

PubMed1987

Different proteins are induced by alpha- and gamma-interferon in hairy cell leukemia.

Despite the striking antiproliferative effect of alpha-interferon (alpha-IFN) in hairy cell leukemia, gamma-interferon (gamma-IFN) does not appear to have such an effect. We have previously demonstrated the induction of synthesis of specific proteins by alpha-IFN, both in vitro and in vivo. We have now shown that gamma-IFN induces synthesis of specific proteins, but the pattern differs from that seen after alpha-IFN exposure. The prominent 80,000-dalton protein induced by alpha-IFN was not induced by gamma-IFN, and the prominent 62,000-dalton protein induced by gamma-IFN was only rarely induced by alpha-IFN. Two other proteins were induced by both alpha-IFN and gamma-IFN. The other proteins induced by alpha-IFN were not induced by gamma-IFN. These differences may be related to the different biological response of hairy cells to the two types of IFN. We also showed for both alpha-IFN and gamma-IFN that some IFN-induced proteins are probably transported to the nucleus of the hairy cell, although the majority of proteins induced by both alpha-IFN and gamma-IFN were in the cytosol/membrane fraction of the cell. We have therefore demonstrated that gamma-IFN does have a biochemical effect on hairy cells in terms of induction of specific protein synthesis, leading to the inference that hairy cells must possess another receptor at least functionally analogous to the type II IFN receptors on fibroblasts, with which gamma-IFN interacts.

Cell Membrane↗

PubMed1987

In vivo induction of proteins during therapy of hairy cell leukemia with alpha-interferon.

The mechanism of the antineoplastic effect of interferon (IFN) is not known and may result from direct effects on the neoplastic cells themselves, activation of intermediary effector cells, or a combination of these effects. The synthesis of specific proteins is induced in hairy cells when they are exposed to alpha-IFN in vitro. In particular, the called p80, is markedly induced. We investigated this effect in the hairy cells of seven patients in the leukemic phase of hairy cell leukemia who were being treated with subcutaneous (SC) IFN alpha 2b (r-Hu-IFN-alpha 2). Polyacrylamide gel electrophoresis (PAGE) was carried out on [35S]methionine-labeled whole cell lysates visualized by autoradiography and silver staining. Within 2 days of starting IFN therapy, induction of specific protein synthesis, including p80 was seen by [35S]methionine labeling in freshly isolated circulating hairy cells from 6 of 6 patients tested. Therefore, alpha-IFN has a direct biochemical effect on hairy cells in vivo that is similar to in vitro effects, at least with regard to p80 synthesis, although the kinetics of this effect may vary in the two situations.

Humans↗

PubMed1986

In vitro induction of proteins by alpha-interferon in hairy cell leukemia.

The striking clinical response of hairy cell leukemia to alpha-interferon led us to investigate the effects of interferon on hairy cells in vitro. We examined the nature of protein induction by interferon in the target hairy cells. To do this, we analyzed whole cell lysates of hairy cells from 11 patients by one-dimensional polyacrylamide gel electrophoresis. With this method, we showed the induction of 16 proteins, which ranged from Mr 140,000 to 12,000. Exposure to alpha-interferon caused very rapid induction of specific proteins in hairy cells, and induction continued for at least 9 days. Proteins were induced at extremely low interferon concentrations, and a dose-response effect was seen with increasing concentrations. A larger number of proteins was induced in hairy cells than in other lymphoid cells under the same conditions. Induction of most proteins was inhibited by actinomycin D, showing that new messenger RNA synthesis was required for their induction to occur. The number or pattern of induced proteins, or their prolonged induction, may be relevant to a change in the target hairy cells which is part of the process resulting in the antiproliferative effect of interferon in hairy cell leukemia.

Dactinomycin↗

PubMed1985

Terminal sequences of the bacteriophage phi 6 segmented dsRNA genome and its messenger RNAs.

The ends of the three dsRNA genome segments (L, M, and S) of bacteriophage phi 6 (strand separated and/or intact) and the 5' ends of the middle and small single-strand messenger RNAs have been sequenced by base-specific partial enzymatic digestion. Terminal sequences for the large and middle dsRNA strands extend about 60 bases. The three dsRNA segments have 18 homologous bases at the left end except for position 2, which differs in the L segment. A 17-base homology defines the right ends of L and M dsRNAs and probably S dsRNA as well. The 5' ends of middle and small messenger RNAs are identical to the corresponding viral (+) strands.

Bacteriophages↗

PubMed1980

Displacement of parental RNA strands during in vitro transcription by bacteriophage phi 6 nucleocapsids.

We have studied the mode of transcription of the three double-stranded RNA segments found in bacteriophage phi 6. Stable transcription intermediates, isolated following in vitro incorporation of nucleoside triphosphates by phi 6 nucleocapsids, were examined by electron microscopy. Specimens were either spread and shadowed or deposited on polylysine film and stained. In either case, branched molecules with one or more single-stranded arms were seen. The single-stranded arm, in all molecules observed, has about half the contour length of one double-stranded arm. The branched molecules are stable in high salt or hot phenol, resistant to proteinase K, but sensitive to RNAase A in high salt, yielding fragments of double-stranded RNA. These results are consistent with a transcription mechanism in which each new transcript displaces one of the parental RNA strands. From the rate of movement of the branch point, we found transcription rates in vitro of similar to or approximately 25 nucleotides per sec at 30 degrees C and 19 nucleotides per sec at 25 degrees C. Based on the spacing between branches in multiply branched molecules, initiation occurs approximately once every 40 sec at 30 degrees C on M or S RNA templates and about 6 times less frequently on L RNA.

Bacteriophages↗

PubMed1978

Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.

Bacillus megaterium↗

PubMed1976

Structure, synthesis, and post-transcriptional modification of ribosomal ribonucleic acid in Bdellovibrio bacteriovorus.

The structure, synthesis, and post-transcriptional modifications of 23-S and 16-S ribosomal RNAs (rRNAs) have been studied in the facultatively parasitic bacterium, Bdellovibrio bacteriovorus. The mature 23-S and 16-S type of rRNAs in Bdellovibrio are larger than the analogous molecules in Escherichia coli by at least 1.0 - 10(5) and 0.5 - 10(5) daltons, respectively, and have a conformation different from E. coli rRNAs as judged by relative electrophoretic mobilities in polyacrylamide gels with and without denaturing conditions. Studies on the kinetics of synthesis and maturation of ribosomal RNA in Bdellovibrio show that precursor forms analogous to p23-S and p16-S in E. coli are synthesized. In addition, some earlier precursor rRNAs in Bdellovibrio are seen that appear analogous to the 25S and 17.5-S pre-rRNAs that have only been observed in the RNAase III deficient mutant of E. coli strain AB301-105 (Nikolaev, Birenbaum, M. and Schlessinger, D. (1975) Biocheim, Biophys. Acta 395, 478-489). These early precursor stages have not been observed in other procaryotic species, including E. coli that have normal levels of RNAase III. The results from the Bdellovibrio system provide that the 25-s and 17.5-S pre-rRNAs are normal stages of rRNA modification and are part of a multiple step maturation process, and therefore are not aberrations associated with the RNase III deficient mutation.

Bdellovibrio↗

PubMed1976

Ribosomal precursor particles of Bacillus megaterium.

Pulse-labeled cells of Bacillus megaterium were converted to protoplasts, and lysates of the protoplasts were analyzed by sucrose gradient sedimentation. Precursor ribonucleoprotein (RNP) particles then appeared predominantly as 50S and 30S precursor ribosomal subunits. Polyacrylamide gel electrophoresis of the ribosomal ribonucleic acid from the 50S and 30S RNP particles confirmed their precursor nature since they were shown to contain precursor 23S and 16S ribosomal ribonucleic acid, respectively. Treatment of protoplast lysates with 0.5% deoxycholate prior to sedimentation analysis resulted in a markedly different radioactivity profile. The 50S RNP particles were no longer present, but 43S particles were observed in addition to increased amounts of pulse-labeled material sedimenting at 30S and slower. Extracts from cells broken in a French press showed a profile from sucrose gradient sedimentation similar to that of the deoxycholate-treated protoplast lysate. These data suggest that the nature of the precursor ribosomal particles appears to be a function of the method of cell disruption or detergent treatment of the cell extract preparation. The observed 50S and 30S RNP particles may be the major precursor ribosomal subunits in vivo; the slower-sedimenting species could result from some form of breakdown or change in the configuration of the 50S and 30S precursors.

Bacillus megaterium↗