PubMed Health⌕ Search

Biomedical subjects

P Rosteck

Publications and source records attributed to P Rosteck.

9 recordsLinked to original sources
PubMed1997

Cloning and expression of a rat brain interleukin-1beta-converting enzyme (ICE)-related protease (IRP) and its possible role in apoptosis of cultured cerebellar granule neurons.

Several members of the IL-1beta-converting enzyme (ICE) family of proteases recently have been implicated in the intracellular cascade mediating the apoptotic death of various cell types. It is unclear, however, whether ICE-related proteases are involved in apoptosis of mammalian neurons and, if so, how they are activated. Here we report the cloning of an ICE-related protease (IRP) from rat brain, which displays strong sequence identity to human CPP32. In situ hybridization histochemistry reveals that this IRP mRNA is expressed in neuron-enriched regions of the developing and adult rat brain but is profoundly downregulated in the adult (compared with developing) brain. To investigate whether this IRP is involved in the death of neurons in the developing brain, we studied IRP expression in cultured cerebellar granule neurons. In cultured cerebellar granule neurons, reduction of extracellular K+ reliably induces apoptosis and stimulates overexpression of IRP mRNA. The latter is especially prominent 4 hr after switching from high K+ to low K+ medium. The expression of IRP mRNA was maintained at this level for at least 8 hr and was followed by apoptotic death of these neurons. Induction of IRP mRNA and cell death are blocked completely by adding depolarizing concentrations of K+ </=90 min after switching to low K+ medium (i.e., before the commitment point for apoptosis) and partially blocked by brain-derived neurotrophic factor (BDNF), which also partially rescues granule neurons from low K+-induced apoptosis. In addition, overexpression of IRP cDNA in HeLa cells results in cell death accompanied by strong internucleosomal cleavage of DNA, a typical feature of apoptosis. Finally, we detected cleavage of the putative death substrate poly (ADP-ribose) polymerase (PARP), beginning 8 hr after changing from high K+ to low K+ medium, coinciding with the time course of induced expression of the IRP gene. Our data suggest that transcriptional activation of IRP could be one of the mechanisms involved in the apoptotic death of cerebellar granule neurons.

Amino Acid Sequence↗
PubMed1994

The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase: cloning, sequence and high-level expression in Escherichia coli.

p-Nitrobenzyl esters serve as protecting groups on intermediates in the manufacture of clinically important oral beta-lactam antibiotics; de-esterification of the intermediates is required for synthesis of the final product. A Bacillus subtilis PNB carboxy-esterase (PNBCE) catalyzes hydrolysis of several beta-lactam antibiotic PNB esters to the corresponding free acid and PNB alcohol. This communication (i) describes cloning the pnbA gene, which encodes PNBCE, (ii) provides the nucleotide sequence of the pnbA open reading frame (ORF) and (iii) describes a method for efficiently expressing the ORF in Escherichia coli. The amino acid (aa) sequence, deduced from the nucleotide sequence of the pnbA ORF, matched an experimentally determined N-terminal aa sequence of B. subtilis PNBCE and also matched an active site sequence previously identified by biochemical analyses. Specific activity of PNBCE in crude extracts was more than 90-fold greater in recombinant E. coli, as compared to B. subtilis. This increase in expression led to more than a 500-fold improvement in the efficiency of purification of PNBCE.

Amino Acid Sequence↗
PubMed1994

Improved expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum.

A hybrid cefE gene, encoding penicillin N expandase, was constructed by fusing the promoter sequences, Pcp, and terminator sequences, Pct from the Penicillium chrysogenum pcbC gene to the open reading frame (orf), cefEorf, from the Streptomyces clavuligerus cefE gene. The resulting hybrid gene, Pcp/cefE'orf/Pct, differed from a previously reported hybrid cefE gene contained on plasmid pPS65. The latter gene, Pcp/cefE'orf/Sct, contained the Pcp sequences fused to the S. clavuligerus cefE orf still attached to the S. clavuligerus terminator sequences, Sct. The new hybrid gene was transformed into P. chrysogenum on plasmid vector pRH6. Transformants were selected by phleomycin resistance conferred by a hybrid ble gene present on plasmid pRH6. The hybrid ble gene was formed by attaching Pcp sequences to the ble orf. Among transformants obtained with pRH6, one exhibited a 70-fold higher level of activity of penicillin N expandase than the best transformant previously obtained from a 10-fold larger population of pPS65 transformants. The penicillin N expandase activity in pRH6 transformant, 9EN-5-1, was fourfold higher than the activity in the S. clavuligerus strain used as the source of the cefE orf and 75% of the activity observed in an industrial strain of Cephalosporium acremonium. Sequencing of the junctions of the heterologous DNA in Pcp/cefEorf/Pct uncovered a modification of the cefE open reading frame introduced during construction of the hybrid gene; the modified open reading frame is designated cefE'orf.

Base Sequence↗
PubMed1992

Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins.

The three macrolide-resistance-encoding genes, tlrC from Streptomyces fradiae, srmB from Streptomyces ambofaciens, and carA from Streptomyces thermotolerans, encode proteins that possess significant sequence similarity to ATP-dependent transport proteins. The N-terminal and C-terminal halves of these proteins are very similar to each other and contain highly conserved regions that resemble ATP-binding domains typically present within the superfamily of ATP-dependent transport proteins. These observations suggest that the mechanism by which these genes confer resistance to macrolides is due to export of the antibiotics, a process that is driven by energy derived from ATP hydrolysis.

Adenosine Triphosphate↗
PubMed1980

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library.

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.

Animals↗
PubMed1980

Unusual sequence homology at the 5-ends of the developmentally regulated beta A-, beta C-, and gamma-globin genes of the goat.

We have constructed a recombinant library of goat DNA and have isolated clones containing the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene. These genes are switched on and off during development and, thus, provide a model system for the study of gene regulation. To identify regions that may be involved in this switch, we determined the sequence of the areas surrounding the 5' ends of the beta A-, beta C- and gamma-globin genes. Surprisingly, the sequences of the beta A-, and beta C-globin genes are identical, beginning with the translation initiation codon and extending 131 base pairs (bp) in the 5' direction. At this point, two nucleotide differences are seen and only six additional differences are encountered when the sequences are extended 144 bp further in the 5' direction. Furthermore, the nucleotide sequence of the 5' end of the gamma-globin gene is very similar to those of the beta A-0, and beta C-globin genes. Only three nucleotide differences are found in the gamma-globin gene within the 131-bp region in which the beta A- and beta C-globin genes are identical. We conclude that this identical region cannot contain regulatory signals that mediate the switch from beta C to beta A expression.

Animals↗
PubMed1978

The location of the globin mRNA sequence within its 16S precursor.

The coding sequence of globin mRNA has been located at or very close to the 3' end of its poly(A)-containing 16S precursor. The 16S RNA was annealed to globin cDNA and the hybrid digested with ribonuclease H. The undigested fragment did not bind to oligo(dT)-cellulose and its size was that expected for the intact 5' portion of the precursor.

Animals↗