Search PubMed⌕ Search

Biomedical subjects

B Schweitzer

Publications and source records attributed to B Schweitzer.

At least 37 records · Page 2Linked to original sources
PubMed1996

Molecular approaches towards the isolation of tyrosine phosphatases expressed in the peripheral nervous system.

Protein tyrosine phosphatases (PTP) are increasingly appreciated to play a crucial function in the development, homeostasis and regeneration of the nervous system. In an attempt to determine the set of PTPs that are expressed in the PNS, we have employed polymerase chain reaction (PCR)-based strategies. Degenerate oligonucleotide primers which were designed based on PTP-consensus motifs were used on substrate cDNAs derived from sciatic nerves of either 3, 10 or 60 days-old rats as well as from cultured rat Schwann cells. The resulting partial PTP clones were used for low-stringency hybridization screening of a cDNA library constructed from the sciatic nerves of 7 to 8 days-old rats. The combined approaches yielded three known rat PTPs and at least three potential rat species homologues of previously identified mouse PTPs. Furthermore, several partial clones encoding potentially novel PTPs have been isolated. The suitability of our experimental approaches for the identification and characterization of PNS-expressed PTPs will be discussed.

Aging↗
PubMed1995

Transcription of the 5'-terminal cap nucleotide by RNA-dependent DNA polymerase: possible involvement in retroviral reverse transcription.

The possible transcription of the 5'-terminal cap nucleotide of mRNA by RNA-dependent DNA polymerase was examined by a single-step assay, based on the generation of a hairpin structure during reverse transcription of globin mRNA. Using this approach, we demonstrated that the 5'-terminal cap nucleotide of mRNA is indeed transcribed by RNA-dependent DNA polymerase into a 3'-terminal residue of cDNA and were able to measure the extent of such transcription. The observed transcription of the cap nucleotide raises a number of questions that may be addressed using the relatively simple single-step assay employed in the present study. Cap nucleotide transcription by RNA-dependent DNA polymerase may have important implications for our understanding of the mechanism of action of reverse transcriptases. It may represent a selection mechanism for only partial transcription of the 5' repeat element of viral RNA genome, thus generating a RNA fragment that may play a role in priming of second (plus) DNA strand during retroviral reverse transcription. Moreover, the demonstrated ability of complementary nucleotides to form hydrogen bonds, even when in a parallel orientation, may have interesting and important consequences.

Animals↗
PubMed1994

Evolutionarily conserved elements in the 5' untranslated region of beta globin mRNA mediate site-specific priming of a unique hairpin structure during cDNA synthesis.

Generation of double-stranded cDNA during reverse transcription of a variety of mRNA molecules is well known to involve the formation of covalently linked antisense and sense strands in a hairpin configuration. In the present study we have examined the sequence of molecular events which occurs during cDNA synthesis from mouse beta globin mRNA, in particular the self-priming event that initiates synthesis of sense-strand DNA. Upon completion of reverse transcription of globin mRNA and the removal of RNA template by RNase H activity associated with reverse transcriptase, the 3' end of cDNA snaps back to form a stable double-stranded structure, which is extended by reverse transcriptase to generate the sense DNA strand. Surprisingly, the fourteen 3' terminal nucleotides of the beta globin antisense DNA strand (cDNA) have strong complementarity with an internal segment of the same molecule corresponding to a portion of the 5'-untranslated region of the mRNA located just upstream of the translation start site. Efficient second strand cDNA synthesis appears to require the occurrence within the cDNA molecule of these two complementary elements, one of which must be 3'-terminal. A second surprising feature is that the strong complementarity between the terminal and the internal portions of the molecule exists in the antisense DNA and not in the sense mRNA strand. This is because A:C mismatches on the sense strand correspond to relatively stable T:G base pairs on the antisense strand. Such an extended region of complementarity within the segment of cDNA corresponding to the short 5' untranslated region of beta globin mRNA is unlikely to occur purely by chance, suggesting some underlying function. In this regard it is of interest that cDNAs of adult beta globin mRNAs from other mammalian species show a very similar arrangement of complementary elements, and that complementarity is heavily conserved, even when there are substitutions in nucleotide sequence.

Animals↗
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↗
PubMed1994

Stress and burnout in junior doctors.

Questionnaires were sent to doctors who had graduated 2 1/2 years previously from two English-medium universities in South Africa. Findings showed that 77.8% of doctors had experienced symptoms consistent with burnout since graduating. Incidence of burnout was found to be related to an inability to communicate freely with patients in their own language. Although those doctors who saw more than forty patients a day reported more burnout, the difference was not statistically significant. The highest incidence of burnout was among doctors working in day hospitals and clinics, followed by those in hospital posts. Doctors working in their own practices experienced least burnout. Sixty-three per cent of doctors felt that a support group would be helpful.

Adaptation, Psychological↗
PubMed1992

NPK1, a nonessential protein kinase gene in Saccharomyces cerevisiae with similarity to Aspergillus nidulans nimA.

A new protein kinase gene [called NPK1 (for nonessential protein kinase)] has been found on chromosome I of Saccharomyces cerevisiae between CDC15 and ADE1. The 435 amino acid/48 kDa gene product is very similar to known protein kinases. It is most closely related to the nimA protein of Aspergillus nidulans, displaying 45.9% identity and 63.5% similarity in the protein kinase domain. A 1.4 kb transcript of the NPK1 gene was detected. Disruption of the NPK1 gene impedes neither growth on glucose or a variety of other carbon sources, nor mating or sporulation.

Amino Acid Sequence↗
PubMed1992

Enzymes of the thymidylate cycle as targets for chemotherapeutic agents: mechanisms of resistance.

This brief review should serve to indicate that it is possible to assess tumor sensitivity to antifolates using fresh human tissue in short-term culture. The assays described differ from general assays of tumor sensitivity, such as the clonogenic assay or assays that measure 3H-thymidine incorporation as an indicator of cell viability (32-34), in that the effect of a drug on a human specific target (dihydrofolate reductase) is measured. These specific assays also may prove to be extremely useful in the detection of acquired drug resistance and for new analog drug development. In a general sense, this type of assay may eventually also be useful for guiding and selecting treatment for individual patients with other drugs with known mechanisms of action. Knowledge of the basis of tumor resistance is essential to develop new approaches to treatment, such as the use of other folate analogs that may still be effective, and to devise ways in which to selectively inhibit tumor cell growth using new analogs (trimetrexate) and leucovorin.

Antimetabolites, Antineoplastic↗
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↗
PubMed1991

Prediction of antifolate efficacy in a rat sarcoma model.

A methylcholanthrene-induced rat sarcoma propagated both in vitro and in vivo was used to examine the usefulness of a rapid biochemical in situ assay that measures thymidylate synthase (TS) activity in whole cells to predict sensitivity and resistance to the folate antagonists methotrexate (MTX), 10-ethyl-10-deazaaminopterin (10-EDAM) and trimetrexate (TMTX). There was an excellent correlation between the effects of these drugs on inhibiting TS and cytotoxicity as measured by a clonogenic assay, when the exposure time was 4 hr and those when the rat sarcoma cell line was employed (p less than 0.001). When tumor-cell suspensions were prepared from the rat sarcoma propagated in vivo, they were less sensitive to these antifolates, but the relative effectiveness of the 3 antifolates was similar: TMTX much greater than 10-EDAM greater than MTX. As expected, continuous exposure (10 to 12 days) produced cytotoxicity at a much lower dose of these drugs. When the 3 drugs were tested for anti-tumor effectiveness in rats bearing this tumor, the tumor regression measured was best with TMTX; 10-EDAM was intermediate in effectiveness as compared with MTX. These results indicate that the in situ TS assay and clonogenic assay may be used to predict anti-tumor efficacy of antifolates in vivo in this rat sarcoma.

Aminopterin↗
PubMed1991

CDC15, an essential cell cycle gene in Saccharomyces cerevisiae, encodes a protein kinase domain.

The cell division cycle gene CDC15 is essential for the late nuclear division in the yeast Saccharomyces cerevisiae. The amino acid sequence of the 974 amino acids/110 kDa CDC15 gene product, as deduced from the nucleotide sequence, includes an aminoterminal protein kinase domain which contains a primary sequence mosaic showing patterns specific for protein serine/threonine kinases besides those for protein tyrosine kinases. Many protein kinases non-essential for growth are known. CDC15 represents an essential protein kinase like CDC7 and CDC28. A carboxyterminal deletion of 32 amino acids renders the protein inactive.

Amino Acid Sequence↗
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↗