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Biomedical subjects
D L Robertson
Publications and source records attributed to D L Robertson.
Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing.
Rous sarcoma virus was shown to induce in chicken embryo fibroblasts (CEF) a 4.1-kilobase mRNA (designated CEF-147) encoding a 603-amino acid protein. Analysis of the protein sequence showed that it shared 59% amino acid identity with sheep prostaglandin G/H synthase, the enzyme that catalyzes the rate-limiting steps in the production of prostaglandins. Significant differences, at both the protein and mRNA levels, existed between the src oncogene product-inducible prostaglandin synthase and the protein isolated and cloned from sheep seminal vesicle, suggesting that the src-inducible prostaglandin synthase may be a new form of the enzyme. A distinguishing feature of src-inducible prostaglandin synthase mRNA is its low abundance in nonproliferating chicken embryo fibroblasts and its relatively high abundance in src-transformed cells. Additionally, the majority of the src-inducible prostaglandin synthase RNA present in nonproliferating cells was found to be nonfunctional because of the presence of an unspliced intron that separated the signal peptide from the remainder of the protein. Upon mitogenic stimulation, this intron was removed, resulting in the induction of fully-spliced CEF-147 mRNA.
Amino Acid Sequence↗
Complete cloning of the chloroplast genome of safflower in lambda EMBL3 and mapping of 23S and 16S rRNA genes.
A recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of lambda EMBL3. Seventeen lambda recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.
Bacteriophage lambda↗
Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA.
The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties. We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro. Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution experiments to be carried out in response to artificially imposed selection constraints. We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. We selected for variant forms of the enzyme that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.
Animals↗
Nucleotide sequence and analysis of the lethal factor gene (lef) from Bacillus anthracis.
The nucleotide sequence of the Bacillus anthracis lethal factor (LF) gene (lef) has been determined. LF is part of the tripartite protein exotoxin of B. anthracis along with protective antigen (PA) and edema factor (EF). The apparent ATG start codon, which is located immediately upstream from codons which specify the first 16 amino acids (aa) of the mature secreted LF, is preceded by an AAAGGAG sequence, which is its probable ribosome-binding site. This ATG codon begins a continuous 2427-bp open reading frame which encodes the 809-aa LF-precursor protein with an Mr of 93,798. The mature secreted protein (776 aa; Mr 90,237) was preceded by a 33-aa signal peptide which has characteristics in common with leader peptides for other secreted proteins of the Bacillus species. The codon usage of the LF gene reflects its high (70%) A + T content. The N-terminus of LF (first 300 aa) shared extensive homology with the N-terminus of the anthrax EF protein. Since LF and EF each bind PA at the same site, these homologous regions probably represent their common PA-binding domains.
Amino Acid Sequence↗
Relationships between the calmodulin-dependent adenylate cyclases produced by Bacillus anthracis and Bordetella pertussis.
The nucleotide sequences for the calmodulin-dependent adenylate cyclases produced by Bordetella pertussis and Bacillus anthracis have recently been determined. The GC% for the B. pertussis and B. anthracis cyclase genes are about 65% and 29%, respectively. Despite this difference in nucleotide composition, these cyclases possess three highly conserved amino acid domains and share some nucleotide sequence homology. One of these conserved domains appears to be involved in ATP binding and is related to the consensus amino acid sequences present in many eukaryotic and prokaryotic ATP and GTP binding proteins. The possible relationship between these cyclases and eukaryotic calmodulin-dependent adenylate cyclases is discussed.
Adenosine Triphosphate↗
Nucleotide sequence of the Bacillus anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase.
The nucleotide sequence of the Bacillus anthracis edema factor (EF) gene (cya), which encodes a calmodulin-dependent adenylate cyclase, has been determined. EF is part of the tripartite protein exotoxin of B. anthracis. An ATG start codon, immediately upstream from codons which specify the first 15 amino acids (aa) of EF, was preceded by an AAAGGAGGT sequence which is its probable ribosome-binding site. Starting at this ATG codon, there was a continuous 2400-bp open reading frame which encodes the 800-aa EF-precursor protein with a Mr of 92,464. The mature, secreted protein (767 aa; Mr 88,808) was preceded by a 33-aa signal peptide which has characteristics in common with leader peptides for other secreted proteins of the Bacillus species. A consensus amino acid sequence (Gly-X-X-X-X-Gly-Lys-Ser,X = any aa), which was part of the presumed ATP binding site for EF, was also present. The codon usage of the EF gene reflected the high A + T (71%) base composition for its DNA. B. anthracis EF was not related to the Escherichia coli or yeast adenylate cyclases, but was related to the Bordetella pertussis calmodulin-dependent adenylate cyclase.
Amino Acid Sequence↗
Molecular cloning and expression of the Bacillus anthracis edema factor toxin gene: a calmodulin-dependent adenylate cyclase.
The Bacillus anthracis exotoxin is composed of a lethal factor, a protective antigen, and an edema factor (EF). EF is a calmodulin-dependent adenylate cyclase which elevates cyclic AMP levels within cells. The entire EF gene (cya) has been cloned in Escherichia coli, but EF gene expression by its own B. anthracis promoter could not be detected in E. coli. However, when the EF gene was placed downstream from the lac or the T7 promoter, enzymatically active EF was produced. The EF gene, like the protective antigen (pag) and lethal factor (lef) genes, was present on the large B. anthracis toxin plasmid pXO1.
Anthrax↗
Purification and physical analysis of Bacillus anthracis plasmids pXO1 and pXO2.
Virulent strains of Bacillus anthracis contain two large plasmids. pXO1 encodes the three component protein exotoxin and pXO2 is necessary for synthesis of the poly-D-glutamic acid capsule. A procedure for the isolation of these plasmids which yields high quantities of pure DNA is described. Restriction endonuclease analysis of these plasmids shows that they are not related. pXO1 is 174 kilobase pairs and pXO2 is 95 kilobase pairs. From their bouyant densities and melting temperatures we also determined their GC contents. pXO1 contains 31.1% GC base pairs and pXO2 is 31.4% GC. Both of these values are close to the GC content of B. anthracis genomic DNA which is 32.2%.
Bacillus anthracis↗
Molecular cloning and expression in Escherichia coli of the lethal factor gene of Bacillus anthracis.
We have cloned and expressed in Escherichia coli the lethal factor (LF) gene of Bacillus anthracis. At least two of the six LF recombinant plasmids produce full-length LF protein. Transcription of the LF gene in E. coli appears to be under the control of its own B. anthracis promoter. Recombinant LF protein produced in E. coli remains intracellular and is not secreted. However, this LF protein is biochemically active and displays the same lethal effects as LF secreted by B. anthracis in the mouse macrophage assay. The LF gene, like that of the protective antigen gene, is present on the large B. anthracis toxin plasmid pXO1.
Antigens, Bacterial↗
The effect of non-receptor-mediated uptake of cholesterol on intracellular cholesterol metabolism in human skin fibroblasts.
Unilamellar lipid vesicles of various cholesterol:phosphatidylcholine molar ratios were used to alter, via passive exchange at the plasma membrane, the cellular free cholesterol content of cultured human skin fibroblasts which had been preincubated in lipoprotein-deficient serum. The effects of these net surface transfers of cholesterol on cellular cholesterol biosynthesis, cholesterol esterification and low density lipoprotein (LDL) binding were determined and were compared with the effects of cholesterol delivered to the cell interior via the receptor-mediated endocytosis of LDL. Both LDL and cholesterol-rich lipid vesicles increased cell cholesterol within 6 h. Cells exposed to LDL also showed, within 6 h, decreased cholesterol synthesis, decreased LDL binding and increased cholesterol esterification. Cells incubated with the cholesterol-rich vesicles showed similar changes but these were delayed and did not occur until 24 h. Fibroblasts incubated with cholesterol-free phosphatidylcholine vesicles had decreased cell cholesterol, increased cholesterol synthesis, increased LDL binding, and decreased esterification, but only after 24 h of incubation. These results suggest that passive net transfers of cholesterol occurring at the cell surface can with time modulate intracellular cholesterol metabolism. These findings are consistent with the idea that the movement of cholesterol from the cell surface to the cell interior is a limited and relatively slow process.
Cells, Cultured↗
Anatomy and physiology of the pharynx.
Radiographic evaluation of the passage of a bolus from the mouth through the pharynx into the esophagus is based upon identification of specific anatomical landmarks and the integrated motion accomplished by the oral and pharyngeal muscles during swallowing. Twenty-six muscles and 6 cranial nerves must be coordinated to enable the safe performance of the complex physiological task of transporting liquids and firm food from the mouth into the esophagus. The following discussion and illustrations of pharyngeal anatomy and physiology are pertinent to an understanding of normal swallowing function.
Deglutition↗
Dexamethasone-stimulated expression of a proviral copy of mouse mammary tumor virus env mRNA.
The kinetics of dexamethasone induction of mouse mammary tumor virus RNAs in a cell line (designated line 8) which contains a deleted proviral DNA has been examined. This DNA is an apparent reverse transcription product of the env mRNA. A detectable increase in the 3.8-kilobase RNA (mouse mammary tumor virus env mRNA) was present within 15 min after hormone addition. The 2.5-kilobase RNA detected previously during hormone induction studies was also observed in these cells. Both RNAs increased in concentration up to about 4 h after hormone addition and thereafter attained the steady-state level. Pr73env was detected by immunoprecipitation and appeared to be identical to bona fide Pr73env isolated from wild-type mouse mammary tumor virus-infected cells. An additional 5.7 kilobases of RNA was detected in augmented quantities after hormone addition, but the structure of this RNA is not known.
Animals↗
Determination of hydrazine in pharmaceuticals III: hydralazine and isoniazid using GLC.
A GLC procedure has been developed for the determination of hydrazine in hydralazine and isoniazid drug raw materials, single and multicomponent tablets, injectables, and syrups. The method is based on the derivatization of hydrazine with benzaldehyde to form benzalazine. The minimum detectable amount of hydrazine in hydralazine and isoniazid raw materials and formulations is approximately 0.0003%. No hydrazine was found in the hydralazine raw material specimens examined. Traces of hydrazine (approximately 0.0003%) were found in some tablet lots and approximately 0.02% was found in an injectable product. A trace of hydrazine was found in one lot of isoniazid raw material and low levels (0.0012 and 0.0029%) were found in isoniazid tablet products. An isoniazid syrup contained approximately 0.2% hydrazine.
Chemistry, Pharmaceutical↗
Hydrazine levels in formulations of hydralazine, isoniazid, and phenelzine over a 2-year period.
Hydrazine levels in formulations of hydralazine, isoniazid, and phenelzine have been measured over a 2-year period under ambient conditions and under temperature and humidity stress. Hydralazine tablets are stable under ambient conditions, but the hydrazine level in an injectable formulation increased from 4.5 to 10 micrograms/ml over a 23-month period. Isoniazid tablets are also stable, but hydrazine levels in an elixir and a pyridoxine combination product doubled to 44 micrograms/ml and 19 micrograms/tablet, respectively. Levels in phenelzine tablets appeared to remain constant at approximately 60 micrograms/tablet, with considerable tablet-to-tablet variation.
Drug Contamination↗
Residual coding in ICHPPC: the size of the problem and its impact on research.
In initial field trials of the International Classification of Health Problems in Primary Care (ICHPPC), the number of problems coded in residual (;other') categories was <5 per cent. Since then, there have been no published analyses of the residual category. Problems have been coded and computerized for over eight years at the Medical University of South Carolina Department of Family Medicine model practice. An analysis of that experience demonstrates that 14.3 per cent of the 85,000 problems were coded as residuals. Many classes or subclasses had >40 per cent coded as residuals. A residual as large as this is a major deterrent for research or practice analysis in a residency setting in anything more than a general category or descriptive manner. If many residuals also occur in other settings, this aspect of the code as well as the coding itself will need further study and improvement.
Adolescent↗
Dexamethasone induction of the intracellular RNAs of mouse mammary tumor virus.
We have studied the kinetics of dexamethasone induction of mouse mammary tumor virus (MMTV) RNAs and proteins in virus-infected rat XC cells and GR mouse mammary tumor cells. A detectable increase in viral RNA in infected XC cells was present within 10 min after hormone addition, and half-maximal induction was achieved in less than 2 h. The increase in viral RNA concentration was apparent first in nuclear RNA and later in the cytoplasm. Within the first 15 min of induction, only genome-sized RNA (35S, 7.8 kilobases) was present in augmented amounts, whereas the major subgenomic RNA (24S, 3.8 kilobases) did not appear until at least 30 to 60 min postinduction. The sequential appearance of these RNAs, the probable mRNA's for the gag and env proteins, paralleled the order of appearance of the gag and env proteins, respectively, after hormone treatment. An additional species of viral RNA (20S, 2.5 kilobases) was detected during these induction experiments, but the role of this RNA is not known. Both subgenomic RNAs contain sequences derived from both the 5' and 3' termini of genomic RNA and are presumably spliced. After dexamethasone induction of infected XC cells, we detected two smaller env-related proteins which were not found in full hormone induction. The functional role of these smaller proteins is not known. A previously reported smaller species of RNA (13S, 1.0 kilobase) did not appear to be induced and was shown to be cellular rather than viral in origin. In the fully induced infected XC and GR mammary tumor cells, the only viral RNAs present were the 35S and 24S RNAs. In addition, mammary tumors contained only these two viral RNAs. Thus, tumor cells appear to contain only the viral RNAs which direct the synthesis of the gag, pol, and env proteins of the virion.
Animals↗
Symptoms encountered during a three-year family practice residency.
Family practice research so far has placed a heavy emphasis upon diagnoses (or problems). There are no published descriptive studies of symptoms collected in a family practice in the United States. This study is a collection of the symptoms encountered by a family medicine resident during his three years in a model unit. Three hundred four patients were seen, offering 1,377 complaints in 956 visits (1.44 symptom per visit). Morbidity related complaints consisted of 59 percent of the total, 9 percent of the total being low back or extremity complaints, while 41 percent were nonsymptomatic reasons for a visit. The majority of morbidity complaints dealt with pain. Three percent of the total consisted of delayed complaints, presented only after the resident previously had dealt with other minor complaints in that visit. The results corresponded closely to those found in the National Ambulatory Medical Care Survey.
Adolescent↗