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M Bina

Publications and source records attributed to M Bina.

At least 55 records · Page 3Linked to original sources
PubMed1987

The flexibility and topology of simian virus 40 DNA in minichromosomes.

The linking number of DNA in minichromosomes increases by 2 turns during SV40 assembly. Changes in temperature also influence the average linking number of the total intracellular forms of SV40 DNA. When the isolated minichromosomes assembled in vivo are incubated with topoisomerase I at 33 degrees C in vitro, the linking number of SV40 DNA decreases. This decrease is about: -1.1 turns for minichromosomes with an average nucleosome spacing of 198 base pairs (bp), wt776; and -0.6 turns for minichromosomes containing a shorter average nucleosome repeat (177 bp), tsC219. The difference between the average linking number of naked SV40 DNA relaxed with topoI at 33 degrees C and minichromosomes relaxed with the enzyme at the same temperature indicates that SV40 chromatin contains on the average 26 nucleosomes. However, the results of studies obtained both on DNA flexibility in chromatin and in naked DNA, and on the shape of the topoisomer distribution curves, indicate that all of the minichromosomes, regardless of their overall structure, do not contain the same number of nucleosomes; this heterogeneity may be as large as 8 nucleosomes. We find no apparent correlation between the amount of minichromosomes containing unconstrained torsional stress and the abundance of the molecules with a structure characteristic of transcriptionally active chromatin.

Chromatin↗
PubMed1986

Simian virus 40 protein VP1 is involved in spacing nucleosomes in minichromosomes.

We have investigated the average nucleosome spacing in the chromatin from several simian virus 40 virion assembly mutants temperature-sensitive in the major capsid protein VP1. Viral assembly intermediates that accumulate in cells infected with mutants that block virion assembly at the propagation step (tsB) have an average nucleosome repeat length similar to that of wild-type SV40 chromatin, approximately 198(+/- 4) base-pairs. This repeat length is longer than that of the host (BSC-40) cellular chromatin, which has a value of 187(+/- 4) base-pairs. In contrast, SV40 chromatin from cells infected with virus containing a mutation that blocks virion assembly at the initiation step (tsC) has a significantly shorter average repeat length of 177(+/- 4) base-pairs. At the permissive temperature (33 degrees C), tsC chromatin has a nucleosome spacing periodicity essentially the same as that of wild-type SV40 chromatin. In addition to possessing a chromatin structure with nucleosomes that are, on the average, closer together, tsC chromatin contains a nuclease-hypersensitive or open region in nearly all molecules, but apparently the same number of nucleosomes. These findings suggest that nucleosomes are deposited initially on newly replicated SV40 chromatin in such a way as to leave the DNA region containing the origin of replication and transcription enhancers uncovered. Subsequent interaction with capsid proteins appears to increase the average nucleosome spacing and consequently to cover the open region for encapsidation.

Animals↗
PubMed1986

A block in initiation of simian virus 40 assembly results in the accumulation of minichromosomes containing an exposed regulatory region.

The initiation of simian virus 40 assembly is blocked at the nonpermissive temperature in cells infected with the viral capsid protein VP1 mutant tsC219. Greater than 95% of the minichromosomes isolated from these cells are accessible to cleavage by Bgl I and Sph I, which recognize the sequences near the viral replication origin and in the transcription enhancer elements, respectively. The accessibility of the Ori region to Bgl I is considerably reduced when virion assembly is allowed to proceed in tsC219-infected cells at the permissive temperature. A reduced accessibility to Bgl I is also observed for chromatin isolated from cells infected with wt776, the wild-type parental strain of tsC219. For wt776 chromatin, variability to Bgl I sensitivity is observed and this can be correlated to the relative virion-to-chromatin yield. A similar correlation is not apparent for restriction endonucleases that recognize sequences within the coding region of simian virus 40 chromatin. These results, considered together, indicate that, when virion assembly initiation is blocked, nucleosomes are nonrandomly arranged with respect to the viral regulatory sequences. It appears that the open regulatory region in minichromosomes is established during replication and that a protected regulatory region is generated with the onset of virion assembly.

Animals↗
PubMed1985

Salt-stable association of simian virus 40 capsid with simian virus 40 DNA.

In 8 M CsCl, a fraction of the wild-type previrions and tsB228 nucleoprotein complexes lose their core histones but retain their capsid. These histone-depleted complexes appear in the electron microscope as a protein shell attached to supercoiled DNA. Consistent with this result, we find that in 1 M NaCl, the wild-type previrions dissociate into two populations of nucleoprotein complexes. One population sediments between 50 and 140 S and morphologically resembles the shell-DNA complexes isolated in CsCl gradients. The other population is comprised primarily of nucleoproteins which sediment at 40 S.

Capsid↗
PubMed1985

Simian virus 40 maturation in cells harboring mutants deleted in the agnogene.

The predominant leader region of the late 16 S mRNAs of simian virus 40 encodes a histone-like, 61-amino acid, DNA-binding protein called the agnoprotein or LP1. To test the hypothesis that this protein facilitates assembly of viral minichromosomes into virions, we have studied the synthesis of virions in cells infected with mutants deleted in this region of the SV40 genome. We found that 220 S mature virions, indistinguishable from those of wild type, were produced in cells infected with these mutants. As in wild-type-infected cells, no assembly intermediates other than 75 S chromatin were observed. However, data obtained from both steady-state and pulse-chase labeling experiments indicated that cells infected with agnogene deletion mutants produced virions more slowly than cells infected with wild-type virus. Taken together with data showing that similar levels of virion proteins were present in the wild-type- and mutant-infected cells, these findings strongly suggest that LP1 plays a role in expediting virion assembly.

Animals↗
PubMed1985

DNA sequence alterations responsible for the synthesis of thermosensitive VP1 in temperature-sensitive BC mutants of simian virus 40.

The segment of simian virus 40 (SV40) genome which is recognized as the BC domain encodes for the COOH-terminal end of the SV40 major capsid protein VP1. Mutations in this domain lead to the synthesis of a thermosensitive VP1 which fails to assemble mature SV40 at the nonpermissive temperature. We determined the DNA sequences of eight BC mutants and compared them with the DNA sequences of wild-type SV40, polyomavirus, and BK virus. We found that BC11 and BC223 mutations result from changes in nucleotide residues 2367 (A to C) and 2084 (C to T), respectively. The others (i.e., BC208, BC214, BC216, BC217, BC248, and BC274) share the same point mutation at nucleotide 2354 (C to T). These mutations resulted in the following changes: Lys to Thr, His to Tyr, and Pro to Ser at VP1 amino acid residues 290, 196, and 286, respectively.

Base Sequence↗
PubMed1984

A model chromatin assembly system. Factors affecting nucleosome spacing.

Poly[d(A-T)].poly[d(A-T)], when reconstituted with chicken erythrocyte core histones and subsequently incubated with sufficient histone H5 in a solution containing polyglutamic acid, forms structures resembling chromatin. H5 induces nucleosome alignment in about two hours at physiological ionic strength and 37 degrees C. The nucleosome spacing and apparent linker heterogeneity in the assembled nucleoprotein are very similar to those in chicken erythrocyte chromatin. Also, condensed chromatin-like fibers on the polynucleotide can be visualized. The binding of one mole of H5 per mole of core octamer is necessary to generate the physiological nucleosome spacing, which remains constant with the addition of more H5. The nucleosome repeat length is not a function of the core histone to poly[d(A-T)] ratio for values lower than the physiological ratio. With increasing ratios, in excess of the physiological value, nucleosome spacing first becomes non-uniform, and then takes on the close packing limit of approximately 165 base-pairs. In addition to eliminating possible base sequence effects on nucleosome positioning, poly[d(A-T)] allows nucleosomes to slide more readily than does DNA, thereby facilitating alignment. Evidence is presented that polyglutamic acid facilitates the nucleosome spacing activity of histone H5, primarily by keeping the nucleoprotein soluble. This model system should be useful for understanding how different repeat lengths arise in chromatin.

Animals↗
PubMed1984

The structure of triple helical poly(U).poly(A).poly(U) studied by Raman spectroscopy.

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U).poly(A).poly(U) triple helix. We compared the Raman spectra of poly(U).poly(A).poly(U) in H2O and D2O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A).poly(U). The presence of a Raman band at 863 cm-1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3' endo; that of the second poly(U) chain may be C2' endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A).poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.

Base Composition↗
PubMed1984

Temperature-sensitive BC mutants of simian virus 40: block in virion assembly and accumulation of capsid-chromatin complexes.

We examined the morphology, protein composition, and stability of the nucleoprotein complexes assembled in cells infected with simian virus 40 mutants belonging to the BC complementation group (tsBC11, tsBC208, tsBC214, tsB216, tsBC217, tsBC248, tsBC223, and tsBC274). We found that the 220S virions were not assembled in tsBC-infected cells under restrictive conditions. This block in assembly resulted in the accumulation of 75S chromatin in tsBC11-infected cells, as previously observed by Garber et al. (E.A. Garber, M.M. Seidman, and A.J. Levine, Virology 107:389-401, 1980). In cells infected with any other mutant listed above, the block in assembly resulted in the accumulation of 75S chromatin as well as nucleoprotein complexes sedimenting from 90 to 140S. Biochemical analysis revealed that these latter complexes contained the capsid proteins in addition to simian virus 40 DNA and the cellular core histones. Electron microscopic analysis clearly showed the association of the capsid proteins with the viral chromatin. Our results suggest that these proteins interact with simian virus 40 chromatin in the course of virion maturation and may thus play an active role in controlling simian virus 40 functions.

Animals↗
PubMed1984

Presence of complement-fixing anti-endothelial cell antibodies in systemic lupus erythematosus.

Vasculitis in systemic lupus erythematosus (SLE) is associated with the deposition of IgG and complement in blood vessel walls. However, it is not known whether immune injury to endothelial cells is a part of this process. Therefore, we used a solid phase radioimmunoassay to study the ability of IgG from normal human sera and sera from patients with SLE to bind to endothelial cells. In this assay, cultured human umbilical venous endothelial cells were sequentially incubated with normal or SLE sera, goat anti-human IgG, and 125I-labeled staphylococcal protein A (*SPA). After exposure to normal sera, 2.5 +/- 0.5% (mean +/- SD) of the added *SPA bound to the cells, whereas after exposure to SLE sera 13.8 +/- 7.6% of the added *SPA bound to these cells. This difference in binding was highly significant (P less than 0.001). Binding was partially reduced when SLE sera were preincubated with B-lymphocytes or monocytes, but not after exposure to erythrocytes, platelets, or T lymphocytes. Incubation of endothelial cells with the 7S fraction of SLE sera or with the F(ab')2 fragment of SLE-IgG resulted in the deposition of greater than 80% as much IgG as was deposited on endothelial cells by whole serum. However, since higher molecular weight fractions (greater than 7S) of SLE sera were also active, we tested the capacity of endothelial cells to bind IgG complexes. Endothelial cells bound heat-aggregated IgG (HA-IgG) in a saturable manner at one log concentration below the binding of normal monomeric IgG. Binding of HA-IgG to endothelial cells was markedly enhanced by preincubation with a serum source of complement. Both HA-IgG and SLE-IgG also bound to freshly obtained endothelial cells in suspension, as detected by automated fluorescence flow cytometry. Binding of SLE-IgG and HA-IgG to endothelium initiated complement activation, deposition of the third component of complement, and disruption of the monolayer. In addition, SLE-IgG and HA-IgG caused endothelial cells to secrete prostacyclin and caused the adherence of platelets, confirmed by scanning electron microscopy. These studies demonstrate that IgG anti-endothelial antibodies are present in the sera of patients with active SLE. These sera may also contain IgG complexes that are capable of binding to endothelial cells. The association of IgG and complement with endothelial cells may initiate vascular injury in SLE and other human disorders.

Antibody Specificity↗
PubMed1983

Simian virus 40 morphogenetic pathway. An analysis of assembly-defective tsB201 DNA protein complexes.

Under restrictive conditions, the 220 S SV40 virions are not assembled in tsB201-infected cells. Instead, a new class of SV40 DNA-containing particles is isolated in addition to the 75 S chromatin. This new class of nucleoprotein complex sediments heterogeneously between 100 to 160 S with a peak at 130 S. Under an electron microscope, these complexes appear predominantly as SV40 chromatin associated with a shell-like protein cluster. These structures resemble the wild type assembly intermediates previously observed by Coca-Prados and Hsu (Coca-Prados, N., and Hsu, M.-T. (1979) J. Virol. 31, 199-208). Like the wild type assembly intermediates, the tsB201 DNA-protein complexes are unstable in high salt. In CsCl, they yield a protein species with a density characteristic of empty shells. In 1 M NaCl, they release heterogeneous 55-110 S protein polymers which consist of the capsid proteins VP1, VP2, and VP3. Our results indicate that the tsB201 nucleoproteins consist of capsid proteins, with varying extents of polymerization, held to chromatin by electrostatic bonds. The accumulation of these nucleoproteins is consistent with a simian virus 40 morphogenetic pathway wherein the capsid proteins are added gradually to the 75 S chromatin.

Cell Transformation, Neoplastic↗
PubMed1983

Simian virus 40 chromatin interaction with the capsid proteins.

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40 degrees C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100-160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100-160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40 degrees C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.

Capsid↗
PubMed1982

The pH-dependent structure of calf thymus DNA studied by Raman spectroscopy.

The pH-dependent structure of calf thymus DNA is analyzed using Raman spectroscopy. The Raman spectra in the acidic region demonstrate that denaturation occurs in several steps. The binding of H+ to adenine and cytosine residues is accompanied by a decrease in the percentage of DNA in the B-conformation and a concurrent increase in a conformation most probably related to the C-form. The denaturation of DNA is observed at pH 3.3 and parallels the protonation of guanine bases. The Raman spectra of calf thymus DNA in the basic region (above pH 10) show that guanine residues are deprotonated at lower pH value than are thymine residues. In addition, Raman spectra in the basic region detect conformational changes of the phosphate backbone different from those found in the acidic region.

Animals↗
PubMed1982

Fc and C3 receptors induced by herpes simplex virus on cultured human endothelial cells.

The mechanism by which immune complexes deposit in vascular tissue is uncertain. Several human viruses, including herpes simplex virus, have recently been demonstrated to replicate in human endothelial cells. Such viruses may injure vascular tissue and could play a role in the pathogenesis of immune complex deposition. Therefore, we studied the expression of receptors for immune complexes containing IgG and C3 on endothelial cells after infection with herpes simplex virus type I.Human umbilical vein endothelial cells were incubated with (51)Cr-labeled sheep erythrocytes sensitized with IgG, IgM, or IgM plus complement. Preferential binding of IgG or complement-coated erythrocytes to uninfected endothelial monolayers was not observed. In contrast, significant binding of erythrocytes coated with IgG or IgM plus complement was observed after viral infection. Phase-contrast and scanning electron microscopy demonstrated erythrocyte adherence around the infected endothelial cells in a rosette pattern. Binding of IgG-coated erythrocytes was fully inhibited by Fc (0.31 mg/ml) but not Fab' fragments of nonimmune IgG. Binding of complement-coated cells was unaffected by the presence of IgG (1 mg/ml). With purified individual components, binding of complement-coated erythrocytes depended on the presence and was proportional to the concentration of C3. Binding of IgG-or C3-coated cells was detected beginning 4 h after infection. These studies indicate that herpes simplex virus type I infection can induce IgG and C3 receptors on human endothelial cells. These receptors may promote the deposition of immune complexes in vascular tissue after certain viral infections.

Culture Techniques↗
PubMed1982

Heavy metal-nucleotide interactions. 15. Reactions of calf thymus DNA with the electrophiles methylmercury(II) nitrate, cis-dichlorodiammineplatinum(II), and trans-dichlorodiammineplatinum(II) studied using Raman difference spectroscopy. Evidence for the formation of c-DNA upon metalation.

This study details the reactions of the electrophiles CH3Hg(NO3), cis-[PtCl2(NH3)2] (cis-DDP) and trans-[PtCl2(NH3)2] (trans-DDP) with calf thymus DNA using Raman and Raman difference spectroscopy. The order of CH3Hg(II) binding to calf thymus DNA is G > T > C > A. The electrophilic attack of cis- and trans-DDP on calf thymus DNA produces different orders of binding: cis-DDP-G>C approximately AT, trans-DDP-G approximately C approximately AT. The reaction of CH3Hg(II) with DNA results in a decrease in the percentage of B-form DNA. whereas the reactions of cis- and trans-DDP with DNA decrease the percentage of B-DNA and cause the formation of C-DNA structure.

Journal Article↗
PubMed1982

Granulocyte-associated IgG in neutropenic disorders.

We applied a radiolabeled antiglobulin test to a study of patients with a variety of neutropenic disorders. After defining the nature of the interaction of radiolabeled anti-IgG with the neutrophil, we studied 16 patients with neutropenia of uncertain etiology and adequate bone marrow granulocyte precursors. Twelve of these 16 patients had increased neutrophil-associated IgG (PMN-IgG). Patients with the highest levels of PMN-IgG had the lowest neutrophil counts. The majority of patients with neutropenia and increased PMN-IgG had an underlying immunologic disorder that included immune thrombocytopenic purpura in 5 patients and autoimmune hemolytic anemia in 1 patient. In some patients, elevated PMN-IgG preceded other evidence for immunologic disease. The direct antiglobulin test helped to distinguish neutropenic patients with increased PMN-IgG both from patients with neutropenia due to a known nonimmune disorder and from nonneutropenic patients with rheumatoid arthritis or systemic lupus erythematosis. Each of four patients with increased neutrophil-associated IgG treated with systemic corticosteroids responded clinically with an associated fall in neutrophil IgG and a rise in the circulating neutrophil count. The radiolabeled antiglobulin test appears useful in defining a subpopulation of patients with neutropenia due to an underlying immunologic disorder.

Adrenal Cortex Hormones↗