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Biomedical subjects

M Riley

Publications and source records attributed to M Riley.

At least 109 records · Page 6Linked to original sources
PubMed1989

Complete sequence of the lamprey fibrinogen alpha chain.

The complete amino acid sequence of the lamprey fibrinogen alpha chain has been determined by a combination of peptide sequencing and cDNA and genomic cloning. The chain, which has an apparent molecular weight by dodecyl sulfate-polyacrylamide gel electrophoresis of ca. 100,000, is composed of 961 amino acid residues and has a calculated molecular weight of 96,722. It is distinguished by a large number of 18-residue repeats in a region where mammalian fibrinogens have 13-residue repeats. The data are in accord with our previous finding that the lamprey alpha chain has a distinctive amino acid composition, almost half the residues being glycine, serine, or threonine. The chain differs from mammalian alpha chains in that there are no cysteines in the carboxy-terminal half, and thus no intrachain loop, nor are there any RGD sequences in the lamprey alpha chain. Taken together with previous data on the sequences of the beta and gamma chains, the findings bear significantly on our understanding of fibrin formation. The alpha chain also provides an interesting case of structural convergence during evolution.

Amino Acid Sequence↗
PubMed1988

Analysis of the plasminogen activator activity of the human glomerulus.

An assay was developed to measure plasminogen activator activity from isolated human glomeruli. Activator was extracted from individual glomeruli with 0.2 M phosphate-buffered saline, pH 7.4 (PBS), containing 0.01% Triton X-100 and quantitated in 125I-fibrin films. Quenching studies using antibodies to tissue plasminogen activator and urokinase revealed that the extracted glomerular plasminogen activator activity contained both tissue plasminogen activator of urokinase. Monoclonal and polyclonal antibodies raised to tissue plasminogen activator demonstrated low-level inhibition of urokinase activity and monoclonal and polyclonal antibodies to urokinase demonstrated low-level inhibition of tissue plasminogen activator activity. The assay should be applicable to the study of glomerular plasminogen activator activity in experimental and human kidney diseases. The detection of antibody cross-reactivity to tissue plasminogen activator and urokinase may be related to the sensitivity of the 125I-fibrin assay and to the structural similarities of these activators.

Humans↗
PubMed1987

cDNA sequences of two apolipoproteins from lamprey.

The messages for two small but abundant apolipoproteins found in lamprey blood plasma were cloned with the aid of oligonucleotide probes based on amino-terminal sequences. In both cases, numerous clones were identified in a lamprey liver cDNA library, consistent with the great abundance of these proteins in lamprey blood. One of the cDNAs (LAL1) has a coding region of 105 amino acids that corresponds to a 21-residue signal peptide, a putative 8-residue propeptide, and the 76-residue mature protein found in blood. The other cDNA (LAL2) codes for a total of 191 residues, the first 23 of which constitute a signal peptide. The two proteins, which occur in the "high-density lipoprotein fraction" of ultracentrifuged plasma, have amino acid compositions similar to those of apolipoproteins found in mammalian blood; computer analysis indicates that the sequences are largely helix-permissive. When the sequences were searched against an amino acid sequence data base, rat apolipoprotein IV was the best matching candidate in both cases. Although a reasonable alignment can be made with that sequence and LAL1, definitive assignment of the two lamprey proteins to typical mammalian classes cannot be made at this point.

Amino Acid Sequence↗
PubMed1986

URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit.

The polypeptide encoded in URF6, the last unassigned reading frame of human mitochondrial DNA, has been identified with antibodies to peptides predicted from the DNA sequence. Antibodies prepared against highly purified respiratory chain NADH dehydrogenase from beef heart or against the cytoplasmically synthesized 49-kilodalton iron-sulfur subunit isolated from this enzyme complex, when added to a deoxycholate or a Triton X-100 mitochondrial lysate of HeLa cells, specifically precipitated the URF6 product together with the six other URF products previously identified as subunits of NADH dehydrogenase. These results strongly point to the URF6 product as being another subunit of this enzyme complex. Thus, almost 60% of the protein coding capacity of mammalian mitochondrial DNA is utilized for the assembly of the first enzyme complex of the respiratory chain. The absence of such information in yeast mitochondrial DNA dramatizes the variability in gene content of different mitochondrial genomes.

Amino Acid Sequence↗
PubMed1986

Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA.

In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH2-terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH2-terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells.

Antibodies↗
PubMed1985

Lamprey fibrinogen gamma chain: cloning, cDNA sequencing, and general characterization.

A cDNA library from lamprey liver was constructed in pBR322 and screened with a synthetic mixed oligonucleotide probe, the sequence of which was based on a partial amino acid sequence of the lamprey fibrinogen gamma chain determined by conventional procedures. Among the positive clones was one containing a 600-base insert that covered the carboxy-terminal third of the chain and another with a 1950-base insert that stretched more than full length. The two inserts were sequenced by the Maxam-Gilbert procedure. The DNA sequencing was corroborated by reference to the amino acid sequences of five cyanogen bromide peptides that compose the carboxy-terminal 130 amino acids, as well as to a number of tryptic peptides from elsewhere in the molecule. The clone with the smaller insert (6G) contained 594 nucleotides (not counting G and C tails), 435 of which are coding and correspond to residues 264-408 of the gamma chain. The remaining 159 nucleotides included the terminator codon followed by a noncoding segment. The larger clone (2E) coded for 408 amino acids that could be readily aligned with the 411-residue human gamma chain. A 24-residue signal peptide adjacent to the proposed amino terminal was also inferred. The amino acid sequence of the fibrinogen gamma chain has been differentially conserved during evolution, the lamprey and human sequences being more than 70% identical in certain key regions but dropping to less than 25% in other sections, including the segment thought to be a part of the "coiled coils". Overall, the resemblance amounts to 50% identity. Of the 10 cysteines found in mammalian chains, 9 are at identical positions, but the tenth, which in mammalian fibrinogens is a part of the interdimeric bridging, is absent in the lamprey.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence of Klebsiella pneumoniae lac genes.

The nucleotide sequences of the Klebsiella pneumoniae lacI and lacZ genes and part of the lacY gene were determined, and these genes were located and oriented relative to one another. The K. pneumoniae lac operon is divergent in that the lacI and lacZ genes are oriented head to head, and complementary strands are transcribed. Besides base substitutions, the lacZ genes of K. pneumoniae and Escherichia coli have suffered short distance shifts of reading frame caused by additions or deletions or both during evolutionary divergence from a common ancestral gene. Relative to corresponding E. coli sequences, the nucleotide sequences of the lacZ and lacY genes are 61 and 67% conserved, and the lacI genes are 49% conserved. A comparison of both nucleotide and amino acid sequences revealed that the K. pneumoniae and E. coli lacI genes and lac repressor proteins each are related to the galR gene and gal repressor of E. coli to about the same extent. In terms of evolutionary relationships, the divergence of the forerunner of the galR gene from an ancestral lac repressor gene preceded separation and differentiation of the K. pneumoniae and E. coli lac repressor genes.

Amino Acid Sequence↗
PubMed1985

Regulatory region of the divergent Klebsiella pneumoniae lac operon.

The chromosomal DNA that lies between the lacI and lacZ genes of Klebsiella pneumoniae constitutes a 196-base pair intercistronic region that contains regulatory sequences for both genes. The probable locations of specific regulatory elements for both lacI and lacZ genes were determined by analogy with the corresponding Escherichia coli sequences. A recombinational event in ancestral DNA evidently has inverted the transcriptional direction of lacI in K. pneumoniae relative to the transcriptional direction of lacI in E. coli. One end of the inversion was located within a 19-base pair sequence in the K. pneumoniae regulatory region. Sequences partially homologous to these 19 base pairs were found in two locations on either side of the E. coli lacI gene. The nucleotide sequence of the lac regulatory region in K. pneumoniae exhibits more than one possibility for folded tertiary structures. The spatial relationships of transcriptional binding sites differ in two possible structures. Associations of regulatory and transcriptional proteins with the DNA might affect conformation of the regulatory sequences and, as a consequence, transcription of the lac genes.

Base Sequence↗
PubMed1985

Pump and leak in regulation of fluid transport in rabbit cornea.

Rabbit corneas were isolated, denuded of epithelium, and perfused on the anterior and posterior surfaces with Krebs Ringer-bicarbonate with additions of 50 microM H2O2, 125 microM BCNU, or 100 microM ouabain. The permeability of the corneal endothelium to labelled mannitol and inulin was determined by adding these compounds to the endothelial perfusate and measuring the rate of appearance of radioactivity in the anterior perfusate. Both H2O2 and BCNU increased the flux of mannitol and inulin across the endothelium in a time dependent manner, but ouabain had no effect. Additions of glucose with H2O2 or of GSH with BCNU prevented the observed changes in permeability. ATPase activities in the endothelia of intact, isolated corneas were also determined following incubation in the same media. The only observable effects of H2O2 and BCNU were slight reductions in the activity of Na+ + K+ ATPase. It is concluded that permeability changes, the leak, are more critical than active transport processes, the pump, in determining the rate and extent of swelling that results from exposure of the cornea to these agents.

Adenosine Triphosphatases↗
PubMed1984

Physical map of Salmonella typhimurium LT2 DNA in the vicinity of the proA gene.

More than 55 kilobases of chromosomal DNA of Salmonella typhimurium LT2, including the gpt, proA, ataA, and newD genes, were cloned in plasmid vector pULB113. The locations of the genes and selected restriction endonuclease cleavage sites were established, and some of the restriction enzyme fragments were subcloned in plasmid vector pBR322.

Bacterial Proteins↗
PubMed1984

Location and analysis of nucleotide sequences at one end of a putative lac transposon in the Escherichia coli chromosome.

A segment of Escherichia coli DNA that contained a discontinuity of homology with Salmonella typhimurium DNA was isolated. The segment, 1,430 base pairs long, was derived from one end of the lac "loop," a region of about 12 kilobase pairs of E. coli DNA, including the lac operon which has no detectable homology with S. typhimurium DNA (K. Lampel and M. Riley, Mol. Gen. Genet. 186:82-86, 1982). The nucleotide sequence of the 1,430-base-pair segment of DNA was determined. The location of the junction of discontinuity of homology within the segment was established by hybridization experiments. Nucleotide sequences at or near the junction were determined to be similar to sequences that are involved in site-specific inversion in S. typhimurium, E. coli, phage P1, and phage Mu. Similar sequences are also present within the terminal inverted repeat sequences of transposon Tn5 and at the V-D-J joining sequences of eucaryotic immunoglobulin genes. Therefore, the lac operon, together with flanking DNA, may have been inserted into the E. coli chromosome at one time via a site-specific recombination event. Rearrangement events of this kind undoubtedly have played a significant role in the evolutionary divergence of chromosomal DNAs.

Base Composition↗
PubMed1983

Diazepam increases GABA mediated inhibition in the olfactory cortex slice.

The effect of diazepam on inhibition has been examined using an in vitro preparation of the guinea-pig olfactory cortex. Diazepam (0.03-30 mumol/l) doubled the intensity and duration of the recurrent inhibitory conductance. Diazepam had no effect on single evoked excitatory post-synaptic potential (e.p.s.p.) nor any effects on the action potential or membrane electrical constants. Diazepam (0.003-100 mumol/l) also reduced the multisynaptic e.p.s.p. generated through a recurrent pathway directed at the soma when elicited during the time-course of the inhibitory conductance. Diazepam had a comparitively small effect on the monosynaptic e.p.s.p. generated on the distal dendrite. Pentobarbitone had a similar though more intense effect over a narrow concentration range (10-200 mumol/l). The inhibitory coductance is thought to be GABA-mediated. Diazepam doubled the potency of the GABA analogue, muscimol, when applied via the bathing solution, whereas a modest 50 mumol/l pentobarbitone increased muscimol potency by about four-fold. The mild but selective effect of diazepam contrasts with the more intense and general effects of pentobarbitone and supports the idea that these drugs act through different mechanisms at the GABA receptor/channel complex.

Animals↗
PubMed1983

Cloning the Escherichia coli K-12 argD gene specifying acetylornithine delta-transaminase.

The argD gene of Escherichia coli was shown to be present in plasmids pLC2-28 and pLC3-11 of the collection of Clarke and Carbon [Cell 9 (1976) 91-99]. The gene was cloned into pBR322 as a 6.3-kb BamHI fragment. Enzyme determination showed that the cloned DNA contains the structural gene for acetylornithine delta-transaminase. The argD DNA was used as a probe in hybridization experiments which indicated that the argM gene resides in a duplicated portion of E. coli DNA that is highly similar to the argD region.

Arginine↗
PubMed1983

Cloning chromosomal lac genes of Klebsiella pneumoniae.

The chromosomal gene for beta-galactosidase from Klebsiella pneumoniae strain T17R1 and associated regulatory genes have been cloned as a 5-kb HindIII fragment in the pBR322 plasmid vector. The beta-galactoside permease gene is not present in a functional form in the 5-kb fragment. The K. pneumoniae genes are expressed in an Escherichia coli host. The synthesis of beta-galactosidase is inducible by isopropyl-beta-D-galactosidase (IPTG) and is sensitive to catabolite repression. There appears to be greater homology between the K. pneumoniae and E. coli structural genes for beta-galactosidase than there is between the respective repressor genes.

Cloning, Molecular↗
PubMed1983

Identification of the polypeptides encoded in the ATPase 6 gene and in the unassigned reading frames 1 and 3 of human mtDNA.

Antibodies prepared against chemically synthesized peptides predicted from the DNA sequence have been used to identify the polypeptides encoded in the ATPase 6 gene and in unidentified reading frames (URFs) 1 and 3 of human mtDNA. In particular, antibodies directed against the COOH-terminal nonapeptide of the putative polypeptide encoded in the ATPase 6 reading frame immunoprecipitated specifically component 17 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal undecapeptide of the putative URF1 product or against the COOH-terminal heptapeptide of the presumptive URF3 product were effective in immunoprecipitating specifically component 12 or, respectively, component 24 of the mitochondrial translation products. The sizes of proteins 17, 12, and 24, as estimated from their electrophoretic mobilities, are compatible with their being the products of the ATPase 6 gene, URF1, and URF3, respectively.

Adenosine Triphosphatases↗
PubMed1983

Employee performance reviews that work.

Employee performance reviews can often be a frustrating, time-consuming and unproductive process for both the employee and the manager. This article outlines three steps that will make an employee performance review system more effective. According to the author, EPRs can be used to increase productivity, improve morale, and create better communication.

Employee Performance Appraisal↗