PubMed Health⌕ Search

Biomedical subjects

V Volloch

Publications and source records attributed to V Volloch.

At least 19 recordsLinked to original sources
PubMed1998

Reduced thermotolerance in aged cells results from a loss of an hsp72-mediated control of JNK signaling pathway.

Aged organisms exhibit a greatly decreased ability to induce the major heat shock protein, Hsp72, in response to stresses, a phenomenon that can also be observed in cell cultures (Heydari AR, Takahashi R, Gutsmann A, You S and Richardson A (1994) Hsp70 and aging. Experientia 50: 1092-1098). Hsp72 was shown to protect cells from a variety of stresses. The protective function of Hsp72 has been commonly ascribed to its chaperoning ability. However, recently we showed that Hsp72 protects cells from heat shock by suppression of a stress-kinase JNK, an essential component of the heat-induced apoptotic pathway (Gabai VL, Meriin AB, Mosser DD, Caron AW, Rits S, Shifrin VI and Sherman MY (1997) Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J Biol Chem 272: 18033-18037). Here we demonstrate that because of the diminished inducibility of Hsp72 in aged cells, Hsp72-mediated control of JNK signaling pathway is compromised. This results in increased rate of apoptotic cell death following heat shock. We show that forced expression of Hsp72 in aged cells from an adenovirus-based vector completely suppresses activation of JNK by heat shock and consequently protects from heat-induced apoptosis. We also demonstrate for the first time that it is possible to restore endogenous expression of Hsp72 in aged cells. This can be achieved by treatment with the proteasome inhibitor MG132. Induction of Hsp72 in aged cells under these conditions leads to suppression of JNK activation by a heat shock and restoration of thermotolerance manifested in a lower rate of apoptosis.

Adenoviridae↗
PubMed1997

Possible mechanism for resistance to Alzheimer's disease (AD) in mice suggests a new approach to generate a mouse model for sporadic AD and may explain familial resistance to AD in man.

An overproduction of beta-amyloid (A beta) is associated with Alzheimer's disease (AD) and appears to be its primary cause. A model has been recently described which accounts for the overproduction of A beta in sporadic AD, this constituting the majority of all cases of AD. The proposed mechanism suggests the antisense RNA-Mediated generation of a 5'-truncated beta-amyloid precursor protein (beta APP) mRNA encoding a 12-kDa C-terminal fragment of beta APP, the immediate precursor of A beta. In the truncated mRNA, the first AUG codon, which contiguously precedes the A beta-coding segment, becomes the site of translation initiation of a polypeptide that can be further processed to generate A beta, this subsequently being secreted. Among the predictions of the proposed model is that mice and rats do not and indeed cannot develop sporadic AD because they lack the crucial component of the proposed mechanism, namely the ability of the beta APP antisense RNA to self-prime the synthesis of a new sense strand. According to the proposed model, however, mice could be rendered susceptible to AD by mutating the beta APP gene so as to confer self-priming ability on the antisense strand. In contrast to existing mouse models which by design are fundamentally unsuitable for study of the mechanism underlying sporadic AD, the AD pathology of the proposed model would be expected to faithfully reflect the human condition. The availability of such an acutely needed, experimental model would allow investigators to study not only the manifestation of the disease but, most significantly, also the factors triggering it. The proposed mouse model may explain familial resistance to AD in man, provide extremely valuable insights into the etiology of AD, and suggest means for its prevention.

Alzheimer Disease↗
PubMed1996

A mechanism for beta-amyloid overproduction in Alzheimer's disease: precursor-independent generation of beta-amyloid via antisense RNA-primed mRNA synthesis.

The overproduction of beta-amyloid (A beta) appears to be a primary cause of Alzheimer's disease (AD). A beta can be generated by proteolytic cleavage of precursor protein (beta APP) at beta- and gamma-secretase sites in both disease and normal cells. There is, however, no evidence that proteolytic processing of beta APP in sporadic AD-affected tissues differs qualitatively or quantitatively from that occurring in normal cells, and additional pathways for the enhanced production of A beta in sporadic AD which constitutes the majority of all AD cases should be considered. The major factor limiting the production of A beta in normal cells is cleavage at the alpha-secretase site within the A beta sequence. But, whereas the intact beta APP is a substrate for cleavage at the alpha-secretase site, the immediate precursor of A beta, 12-kDa C-terminal beta APP fragment, is not susceptible to the alpha-secretase cleavage but it can be cleaved by gamma-secretase thus generating A beta. Moreover, the gamma-secretase cleavage is not the rate-limiting step in the production of A beta. Therefore, the increase in production of the 12-kDa C-terminal beta APP fragment may be an efficient way to overproduce A beta. A mechanism for the generation of the 12-kDa fragment independently of beta APP is proposed. It postulates an additional step of amplification of mRNA, namely the antisense RNA-mediated generation of a truncated mRNA encoding 12-kDa C-terminal fragment. Initiation of translation at the first AUG in the truncated mRNA results in a polypeptide that is cleaved by gamma-secretase generating A beta. The proposed model makes several verifiable predictions and suggests new directions of experimentation that may lead to a better understanding of the mechanisms involved in AD.

Alzheimer Disease↗
PubMed1996

Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense beta-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sense-strand globin mRNA.

Anemia↗
PubMed1994

Ligation-mediated amplification of RNA from murine erythroid cells reveals a novel class of beta globin mRNA with an extended 5'-untranslated region.

Ligation-mediated RNA amplification was developed as a tool for analysis and determination of the termini of RNA molecules [Volloch et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10671-10675]. In this approach, T4 RNA ligase is used to join cellular RNA with a defined ribo-oligonucleotide. Although several additional enzymatic steps are involved in this type of analysis, the reliability of the entire procedure is determined by the initial ligation step, which marks and preserves the termini of cellular RNA molecules. We applied this approach to the analysis of the 5' terminus of beta globin mRNA in various murine erythroid cells. As expected, we detected RNA molecules with 5' ends terminating at the regular cap site as well as globin RNA molecules truncated at the 5' end. Unexpectedly, we also detected a class of beta globin mRNA which is identical to regular beta globin mRNA in every respect but contains 17, 29, or 31 additional nucleotides 5' to the regular cap site. These extensions correspond precisely to the genomic segments just upstream of the regular cap site and are probably generated by initiation of transcription of the globin gene upstream from the regular cap site. It is likely that the extended globin RNA is transcribed not from the TATA promoter, which regulates the transcription of regular murine globin mRNA, but from the GATA regulatory element located 30 nucleotides upstream of the 31-nucleotide extension, in a position identical to that of the active GATA promoter of the TATA-less chicken beta globin gene. The evolutionary conservation of this relationship suggests the importance of the GATA promoter element of the mouse beta globin gene and its possible involvement in developmental regulation of expression of this gene.

Animals↗
PubMed1991

Identification of negative-strand complements to cytochrome oxidase subunit III RNA in Trypanosoma brucei.

A substantial amount of cytochrome oxidase subunit III (COIII) mRNA continues to be synthesized de novo in Trypanosoma brucei in the presence of actinomycin D, presumably by a DNA-independent transcription process. We describe the identification of negative-strand COIII RNA molecules, characterization of their termini, and the detection of RNA-dependent RNA polymerase activity. Three lines of evidence for the existence of negative-strand COIII RNA are presented: (i) hybridization with oligonucleotide probes with the same polarity as mRNA after preliminary enrichment for putative negative-strand RNA by affinity purification; (ii) cloning and sequencing of negative-strand complements for the unedited, edited, and partially edited COIII RNA; and (iii) exact correspondence of the terminal sequences of the putative negative-strand RNA molecules to the ends of COIII RNA. The presence of negative-strand complements of COIII RNA is consistent with the notion that a significant amount of mRNA in T. brucei is synthesized by an RNA-dependent RNA polymerase with negative-strand RNA as an intermediate template.

Animals↗
PubMed1991

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to exons.

The objective of the experiments described in this paper was to determine the feasibility of inhibition of pre-mRNA splicing by antisense RNA in vitro. Three different types of antisense RNA were utilized: antisense RNA complementary to the spliced RNA molecule; antisense RNA complementary to the unprocessed mRNA precursor molecule; and antisense RNA complementary to the 5' and 3' splice junctions. Whereas antisense RNA complementary to mRNA had little effect on splicing, antisense RNAs complementary to mRNA precursor or to splice junctions strongly inhibited splicing of pre-mRNA molecule. The results obtained indicate that the inhibitory effect is most likely due to hybrid formation between pre-mRNA and antisense RNA molecules and that antisense RNA complementary to the exon portion but not to the intron portion of splice junction exhibit an inhibitory effect. This inhibition can be overcome by bringing together 5' and 3' splice junctions via hybrid formation with antisense RNA complementary to the spliced RNA molecule.

Animals↗
PubMed1991

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to introns.

The objective of the experiments described in this paper was to test the potential of antisense RNAs complementary to the internal portion of an intron to inhibit the splicing process and to determine the mechanism of such inhibition. The results obtained indicate that RNA fragments complementary to the internal portion of an intron can effectively inhibit the splicing of pre-mRNA. Inhibition was observed only with antisense RNA complementary to pre-mRNA suggesting that the inhibitory effect was due to the formation of a hybrid with the corresponding portion of the pre-mRNA's intron. The observed inhibition was not due to interference with possible intron elements essential for the splicing process, for the deletion of the sequences complementary to inhibitory antisense RNA from the corresponding pre-mRNA molecule did not affect the efficiency of a splicing reaction, and the addition of antisense RNA to pre-mRNA mutants carrying such deletions did not result in any inhibition. Our results indicate that the observed inhibition is a function of the length of the antisense RNA expressed as a fraction of an intron with which it interacts when antisense RNA is modified by incorporation of a "hinge" element, it loses its inhibitory potential suggesting that the inhibitory effect is probably due to limitation of conformational flexibility of an intron.

Animals↗
PubMed1990

Uncoupling of the synthesis of edited and unedited COIII RNA in Trypanosoma brucei.

RNA editing, a novel and unexpected type of information processing, was first demonstrated in the kinetoplasts of certain protozoans. It is a remarkable phenomenon: certain species of messenger RNA have nucleotide sequences that differ greatly from the sequences of the genes from which they are presumably transcribed. The differences are usually due to addition of uridylate residues, but occasionally also to their deletion. The most spectacular case of editing known occurs in the mRNA of the mitochondrial gene for subunit III of cytochrome oxidase (COIII) in Trypanosoma brucei. This mRNA is twice the length of its gene owing to the addition of several hundred uridylate residues and a few deletions, spread over the entire length of the RNA molecule. Whereas unedited RNA molecules, the nucleotide sequences of which correspond to the genomic sequence, have been isolated, no DNA template corresponding to the edited RNA sequence has been detected in either the mitochondrial or nuclear genome. It was suggested, therefore, that unedited mRNAs are transcribed from mitochondrial DNA and then edited post-transcriptionally by endonucleolytic cleavage of the primary transcript at specific sites, followed by insertion or deletion of uridylate residues and religation. We have now examined the general nature of the RNA editing process to determine whether it involves the insertion of nucleotide residues into pre-existing molecules or the continuous de novo synthesis of edited mRNA. The results of our experiments rule out an insertion mechanism and strongly indicate that edited mRNA is synthesized as a unit.

Animals↗
PubMed1987

Messenger RNA changes during differentiation of murine erythroleukemia cells.

During differentiation of murine erythroleukemia cells, the levels of certain mRNA were observed to change. To characterize the various patterns of changes that occur during differentiation, cDNA libraries made from RNA isolated from uninduced and differentiating cells were screened with labeled cDNA or RNA labeled in vivo for different periods of time. cDNA clones that corresponded to individual mRNAs whose level remained constant, increased, or decreased during differentiation were identified. These clones were used to analyze Northern blots containing RNA from uninduced and differentiated cells. A number of characteristic changes in individual mRNAs in differentiating murine erythroleukemia cells could be identified, such as no change, increase in concentration, increase in concentration and slight change in size, decrease in concentration, decrease in concentration and change in size, appearance of new band(s) of entirely different size, and change in relative concentrations of two related mRNAs. Measurements of rates of mRNA synthesis and degradation suggest that both parameters change during differentiation and that these changes are instrumental in establishing cellular concentration of specific mRNAs. It seems that the changes in mRNA stability observed in differentiating murine erythroleukemia cells may be associated with changes in the primary structure of the transcribed portion of mRNA. The observation that specific mRNA synthesized before and after induction may have very different stabilities at the same point in differentiation supports this hypothesis.

Animals↗
PubMed1987

Synthesis of globin RNA in enucleated differentiating murine erythroleukemia cells.

In an earlier report (Volloch, V., 1986, Proc. Natl. Acad. Sci. USA., 83:1208-1212) we had presented evidence for the occurrence of the cytoplasmic synthesis of globin mRNA and of RNA complementary to globin mRNA which differed from DNA-dependent transcription by its insensitivity to actinomycin D. In this paper, we describe the use of enucleated differentiating mouse erythroleukemia cells to demonstrate directly the occurrence of cytoplasmic synthesis of both positive- and negative-strand globin RNA. For this purpose, we developed an enucleation procedure which yielded pure cytoplasts from differentiated mouse erythroleukemia cells in the absence of cytochalasin B and selectively permeabilized the cytoplasts to small molecules by treatment with dextran sulfate and saponin. The permeabilized cytoplasts incorporated [3H]dUTP into positive- and negative-strand globin RNA and experiments with mercurated nucleotide substrate suggested that this process involved de novo RNA synthesis rather than limited terminal nucleotide addition. Globin RNA synthesis required Mg++, was inhibited by Mn++, and was unaffected by the addition of Zn++. Studies of its response to inhibitors of DNA-dependent RNA synthesis showed that it differed from that process in its insensitivity to actinomycin D and alpha-amanitin, but that like many other macromolecular biosynthetic reactions it was inhibited by rifamycin AF/ABDP and aurintricarboxylic acid. These observations provide additional evidence for the occurrence of cytoplasmic RNA-dependent RNA synthesis in differentiated cells and show permeabilized enucleated cells to be a useful experimental system for further studies of the characteristics of that process.

Animals↗
PubMed1986

Cytoplasmic synthesis of globin RNA in differentiated murine erythroleukemia cells: possible involvement of RNA-dependent RNA polymerase.

Three lines of evidence indicate that RNA-dependent RNA synthesis occurs in mouse erythroleukemia cells. The first involves labeling studies with [3H]uridine and shows a greater initial labeling rate of globin RNA in the cytoplasm than in the nucleus. Labeled globin RNA found in the cytoplasm after a very short pulse with tritiated uridine is of the "mature" 9S size while labeled globin RNA in the nuclei is exclusively in the form of 15S precursor molecules, suggesting that cytoplasmic globin RNA is not of nuclear origin. A high concentration of actinomycin D has no effect on the initial rate of labeling of cytoplasmic globin RNA, supporting this conclusion. Other experiments showed that the labeling of cytoplasmic globin RNA does not involve end addition to preexisting globin RNA. The second line of evidence is the identification of globin RNA minus strand in the cytoplasm of differentiated murine erythroleukemia cells by hybridization with single-stranded DNA probes containing the strand of the same sense as globin mRNA. This material has the same electrophoretic mobility as globin RNA and hybridizes with probes containing only the 5' part or only the 3' part of the gene suggesting that it is a full size copy of globin RNA. Finally, in murine erythroleukemia cells an RNA-dependent RNA polymerase activity is detected by using poly(A) . oligo(U) as a template-primer combination. This activity increases significantly after induction, suggesting that it is differentiation specific.

Acetamides↗
PubMed1982

Dissociation of hemoglobin accumulation and commitment during murine erythroleukemia cell differentiation by treatment with imidazole.

The effect of imidazole on DMSO-induced murine erythroleukemia (MEL) cell differentiation has been examined. While imidazole does inhibit heme, globin mRNA, and hemoglobin accumulation in DMSO-induced MEL cells, it does not affect the commitment of MEL cells to the specific limitation of proliferative capacity associated with the in vitro differentiation program. Furthermore, imidazole treatment does not affect DMSO-induced changes in cell volume, in the relative proportion of nuclear protein IP25, and in the specific activity of the enzyme cytidine deaminase. A clonal analysis in the presence of imidazole indicated that the drug prevents heme accumulation even in MEL cells already committed to terminal differentiation. These observations suggest that imidazole effectively dissociates two aspects of the erythroid differentiation program of MEL cells: globin gene expression and commitment to loss of proliferative capacity.

Animals↗
PubMed1982

Terminal differentiation of murine erythroleukemia cells: physical stabilization of end-stage cells.

An important limitation in the use of the murine erythroleukenia (MEL) cell system as an in vitro system for the study of terminal erythroid differentiation has been the inability to produce significant numbers of cells which represent the end-point of the pathway in vitro. We show here that a major reason for the failure to observe end-stage cells in vitro is that such cells are physically unstable under the standard culture conditions used for MEL cell differentiation. Modification of these culture conditions by the addition of either bovine serum albumin or Ficoll leads to physical stabilization of end-stage cells. Under such culture conditions, uniform cultures of terminally differentiated MEL cells with morphological characteristics similar to those of normal mouse orthochromatophilic erythroblasts and reticulocytes are observed. Examination of physical and biochemical parameters of these cell populations give values which are similar to values characteristic of mouse reticulocytes. A physically stabilized MEL cell shows a narrow cell volume distribution with an average value of approximately 100 mum(3), similar to the cell volume distribution observed for mouse reticulocytes, while a typical MEL cell culture treated with DMSO but without a stabilizing agent exhibits a broader, more heterogeneous cell volume distribution with an average value of approximately 500 mum(3). Globin mRNA levels and levels of globin synthesis reach values almost equal to those in mouse reticulocytes in cultures of physically stabilized MEL cells while differentiating cultures not treated with a stabilizing agent reach substantially lower values for these parameters. We suggest that the ability to produce populations of MEL cells which undergo complete terminal erythroid differentiation in vitro will allow the analysis of the molecular mechanisms which control the terminal stages of the erythroid differentiation process.

Animals↗
PubMed1981

Stability of globin mRNA in terminally differentiating murine erythroleukemia cells.

The stability of globin mRNA is terminally differentiating MEL cells has been reevaluated. Previously, it had been reported that globin mRNA has a half-life of approximately 17 hr in terminally differentiating MEL cells. We show that the previous measurements of this parameter were confounded by physical instability of differentiating MEL cells. By using culture conditions that physically stabilize end-stage cells we show that the stability of globin mRNA in terminally differentiating MEL cells is equal to the value observed for ribosomal RNA, a half-life greater than 60 hr.

Animals↗
PubMed1980

Hemin does not cause commitment of murine erythroleukemia (MEL) cells to terminal differentiation.

The effect of hemin on the differentiation program of murine erythroleukemia (MEL) cells has been investigated. While hemin treatment does induce increased levels of globin mRNA and hemoglobin, it fails to lead to other biochemical changes associated with MEL cell differentiation induced by DMSO and thioguanine. These include increased levels of the nuclear protein IP25 and of the enzyme cytidine deaminase. Clonal analysis of hemin-treated cells revealed that unlike other inducers, hemin does not cause a reprogramming of MEL cells to a specific limitation of proliferative capacity. These observations suggest that hemin differs from DMSO and thioguanine in that it exerts specific effects on globin expression in MEL cells without triggering commitment to the terminal differentiation program.

Animals↗