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Biomedical subjects

K E Reed

Publications and source records attributed to K E Reed.

31 records · Page 2Linked to original sources
PubMed1993

Proximal femur of Australopithecus africanus from Member 4, Makapansgat, South Africa.

A left proximal femur (MLD 46) from Member 4, Makapansgat, South Africa is described and analyzed. It consists of the head, neck, and a small segment of the shaft that extends to just below the lesser trochanter. The femur exhibits degenerative joint disease in the form of marginal osteophyte formation and thus its taxonomic identity has been somewhat obscured. Consideration of all like-sized mammalian femora from Makapansgat suggests that the femur is that of either a felid or hominid. Comparison of MLD 46 to femora of extent and extinct felids reveals that MLD 46 does not possess two morphological features that are characteristic of felids, namely a deep, prolonged trochanteric fossa and a high neck-shaft angle. Simple shape variables (ratios) and multivariate analyses consistently place MLD 46 with modern and fossil hominids, and most closely align it with the australopithecines. We conclude that the femur is most reasonably attributable to Australopithecus africanus, which is the only hominid yet identified from Makapansgat. Despite its pathological condition, MLD 46 is the most complete proximal femur known for A. africanus, thereby permitting further morphological comparisons with homologues of A. afarensis and Paranthropus. Marginal osteophytes of mammalian femoral heads characteristically occur in individuals of advanced age, suggesting that MLD 46 may have lived some time with the disease. Finally, MLD 46 is considerably larger than the previously described specimen, Sts 14, from Sterkfontein Member 4. There may be as great a contrast in body size in A. africanus as there is between the large and small specimens of A. afarensis.

Animals↗
PubMed1993

Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes.

Two genes, lipA and lipB, involved in lipoic acid biosynthesis or metabolism were characterized by DNA sequence analysis. The translational initiation site of the lipA gene was established, and the lipB gene product was identified as a 25-kDa protein. Overproduction of LipA resulted in the formation of inclusion bodies, from which the protein was readily purified. Cells grown under strictly anaerobic conditions required the lipA and lipB gene products for the synthesis of a functional glycine cleavage system. Mutants carrying a null mutation in the lipB gene retained a partial ability to synthesize lipoic acid and produced low levels of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase activities. The lipA gene product failed to convert protein-bound octanoic acid moieties to lipoic acid moieties in vivo; however, the growth of both lipA and lipB mutants was supported by either 6-thiooctanoic acid or 8-thiooctanoic acid in place of lipoic acid. These data suggest that LipA is required for the insertion of the first sulfur into the octanoic acid backbone. LipB functions downstream of LipA, but its role in lipoic acid metabolism remains unclear.

Aerobiosis↗
PubMed1993

Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.

Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins.

Alcohols↗
PubMed1992

Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle.

Gap junctions contain intercellular channels which are formed by members of a group of related proteins called connexins. Connexins contain conserved transmembrane and extracellular domains, but unique cytoplasmic regions which may provide connexin-specific physiologic properties. We used polymerase chain reaction (PCR) amplification and cDNA library screening to clone DNA encoding a novel member of this gene family, rat connexin40 (Cx40). The derived rat Cx40 polypeptide contains 356 amino acids, with a predicted molecular mass of 40,233 Da. Sequence comparisons suggest that Cx40 is the mammalian homologue of chick connexin42, but it has predicted cytoplasmic regions that differ from previously described mammalian connexins. Southern blots of rat genomic DNA suggest that Cx40 is encoded by a single copy gene containing no introns within its coding region. Northern blots demonstrate that Cx40 is expressed in multiple tissues (including lung, heart, uterus, ovary, and blood vessels) and in primary cultures and established lines of vascular smooth muscle cells. Cx40 is coexpressed with connexin43 in several cell types, including A7r5 cells, which contain two physiologically distinct gap junctional channels. To demonstrate that Cx40 could form functional channels, we stably transfected communication-deficient Neuro2A cells with Cx40 DNA. These Cx40-transfected cells showed intercellular passage of microinjected Lucifer yellow CH. The expression of multiple connexins (such as Cx40 and Cx43) by a single cell may provide a mechanism by which cells regulate intercellular coupling through the formation of multiple channels.

Amino Acid Sequence↗
PubMed1991

Escherichia coli exports previously folded and biotinated protein domains.

Biotination of proteins is a post-translational modification that requires a folded acceptor domain. We previously showed that an acceptor domain fused to the carboxyl terminus of several cytosolic proteins results in biotinated fusion proteins in vivo. We now show that proteins encoded by translational gene fusions of two periplasmic proteins, alkaline phosphatase and TEM beta-lactamase, to carboxyl-terminal biotin-accepting sequences are biotinated and exported by Escherichia coli. Expression of the alkaline phosphatase fusion protein in wild type strains resulted in inefficient biotination of the fusion product. This result was due to the rapid export of the acceptor protein before biotination could occur since a very large increase in biotinated fusion protein levels was observed in strains lacking the SecB chaperone protein. The beta-lactamase fusion protein was biotinated but was only stable in strains lacking the DegP periplasmic protease. Both biotinated fusion proteins accumulated in the culture medium in strains possessing defective outer membranes. These results indicate that the export machinery can accommodate both a post-translational modification and a protein domain previously folded into its mature conformation in vivo.

Alkaline Phosphatase↗
PubMed1991

Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system.

We report the isolation and genetic characterization of novel Tn10dTc and Tn1000dKn insertion mutations in and near the lip locus of the Escherichia coli chromosome. The Tn10dTc and Tn1000dKn mutations define two genes, lipA and lipB, involved in lipoic acid biosynthesis. Two representative alleles (lip-2 and lip-9) from the previously reported genetic class of lipoic acid auxotrophic mutants (A. A. Herbert and J. R. Guest, J. Gen. Microbiol. 53:363-381, 1968) were assigned to the lipA complementation group. We have cloned the E. coli lip locus and developed a recombinant plasmid-based genetic system for fine-structure physical-genetic mapping of mutations in this region of the E. coli chromosome. We also report that a recombinant plasmid containing a 5.2-kbp PvuII restriction fragment from the E. coli lip locus produced three proteins of approximately 8, 12, and 36 kDa by using either a maxicell or in vitro transcription translation expression system. The 36-kDa protein was identified as the gene product encoded by the lipA locus. Finally, we have identified a previously unreported lipoylated protein that functions in the glycine cleavage system of E. coli.

Alleles↗
PubMed1989

Mechanism of penicillin killing in the absence of bacterial lysis.

Exposure of group A streptococci (a nonlytic-death phenotype) to benzylpenicillin (penicillin G) produced a dose-dependent, rapid, and extensive hydrolysis of total cellular RNA, with the subsequent loss of hydrolysis products from the cell. This loss of RNA correlated well with loss of viability and was not accompanied by solubilization of the cell wall or comparable losses of either protein or DNA. Simultaneous treatment with penicillin G and either chloramphenicol or rifampin resulted in reduced levels of killing and the complete inhibition of RNA loss. These findings define a new mechanism of penicillin G-induced killing in the absence of cell wall disruption and suggest a basis for drug-induced antagonism of penicillin G-mediated nonlytic death.

Bacterial Proteins↗
PubMed1989

Effects of medium composition on penicillin-induced hydrolysis and loss of RNA and culture turbidity in group A streptococci.

Exposure to penicillin G of exponentially growing cultures of group A streptococci growing in chemically defined medium (CDM) can lead to extensive loss of culture turbidity. Significant reductions in culture turbidity did not accompany comparable treatments of group A streptococci growing in Todd-Hewitt broth (THB). Studies with THB and a high-molecular-weight (greater than 12,000) fraction of THB demonstrated that components in this complex medium inhibited the efflux of RNA hydrolysis products from otherwise intact cells. Hydrolysis products accumulated intracellularly and inhibited the extensive hydrolysis of RNA and consequently the loss of culture turbidity. Results of survival studies with cultures of group A streptococci exposed to penicillin G in THB demonstrated that this treatment protocol produces conditions of phenotypic tolerance relative to exposure in CDM. In combination, these findings provide further support for the hypothesis of RNA hydrolysis as the bactericidal mechanism of penicillin G action in this nonlytic death phenotype.

Culture Media↗
PubMed1989

Talk-back regulation: a regulatory response to the inhibitions of cell surface growth.

The net accumulation of RNA and protein was studied in exponentially growing cultures of Streptococcus rattus FA-1 (formerly S. mutans FA-1) during periods of antibiotic induced inhibition of cell surface growth. Disruption of either the cytoplasmic, membrane, or final assembly steps of peptidoglycan (PG) synthesis following exposure of cultures to D-cycloserine plus beta-chloro-D-alanine (CS-CA), vancomycin (Vanco) or benzylpenicillin (Pen G) respectively, was accompanied by a rapid inhibition of RNA accumulation followed by an inhibition of protein accumulation. At physiologically comparable concentrations, CS-CA and Vanco inhibited RNA more efficiently than Pen G. CS-CA inhibited RNA and PG to the same degree while RNA accumulation was clearly more sensitive to Vanco than was PG. Simultaneous treatment with chloramphenicol (CAP), relaxed the cell wall antibiotic induced inhibition of RNA to a degree and with kinetics similar to those observed during CAP relaxation of the stringent response in amino acid deprived cultures. The degree of survival following exposure to the PG inhibitors varied directly with the efficiency of the antibiotics to inhibit RNA accumulation. CAP and tetracycline acted synergistically with CS-CA and to a lesser degree with Vanco while marginally antagonizing the killing effects of Pen G. On the basis of these and other observations it is proposed that the assembly status of the cell wall can be communicated, i.e. 'talk back', to the cytoplasmic processes of macromolecular synthesis via a specific regulatory type circuit. The efficiency of induction of the talk-back response appears to be directly related to survival.

Amino Acids↗
PubMedDate not supplied

Early hominid evolution and ecological change through the African Plio-Pleistocene.

The habitats in which extinct hominids existed has been a key issue in addressing the origin and extinction of early hominids, as well as in understanding various morphological and behavioral adaptations. Many researchers postulated that early hominids lived in an open savanna (Dart, 1925; Robinson, 1963; Howell, 1978). However, Vrba (1985, 1988) has noted that a major global climatic and environmental shift from mesic, closed to xeric, open habitats occurred in the late African Pliocene (approximately 2.5 m.y.a.), thus implying that the earliest hominids existed in these mesic, wooded environs. This climatic shift is also suggested to have contributed to a pulse in speciation events with turnovers of many bovid and possibly hominid species. Previous environmental reconstructions of hominid localities have concentrated on taxonomic identities and taxonomic uniformitarianism to provide habitat reconstructions (e.g., Vrba, 1975; Shipman & Harris, 1988). In addition, relative abundances of species are often used to reconstruct a particular environment, when in fact taphonomic factors could be affecting the proportions of taxa. This study uses the morphological adaptations of mammalian assemblages found with early hominids to reconstruct the habitat based on each species' ecological adaptations, thus minimizing problems introduced by taxonomy and taphonomy. Research presented here compares east and south African Plio-Pleistocene mammalian fossil assemblages with 31 extant mammalian communities from eight different habitat types. All communities are analyzed through ecological diversity methods, that is, each species trophic and locomotor adaptations are used to reconstruct an ecological community and derive its vegetative habitat. Reconstructed habitats show that Australopithecus species existed in fairly wooded, well-watered regions. Paranthropus species lived in similar environs and also in more open regions, but always in habitats that include wetlands. Homo is the first hominid to exist in areas of fairly open, arid grassland. This change from closed to open habitats occurs gradually from about 4 m.y.a. until about 2 m.y.a. when there is a major increase in arid and grazing adapted mammals. Therefore, the appearance of open savannas do not appear to have influenced the origination or adaptations of the earliest hominids, but could have contributed to their demise. As Stanley (1992) hypothesized, Homo species appear the first to be adapted to open, arid environments.

Africa↗
PubMedDate not supplied

Promoting clinical scholarship through scholarly writing.

To promote clinical scholarship, critical thinking, problem-solving ability, and effective writing and research skills in their students, faculty replaced their major care plan with a capstone scholarly paper. The authors discuss how faculty, who serve as mentors, guide their students through the development of the scholarly paper.

Education, Nursing, Associate↗