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Biomedical subjects

W R Jacobs

Publications and source records attributed to W R Jacobs.

At least 127 records · Page 7Linked to original sources
PubMed1989

Genetic relationships among Mycobacterium leprae, Mycobacterium tuberculosis, and candidate leprosy vaccine strains determined by DNA hybridization: identification of an M. leprae-specific repetitive sequence.

Comparative DNA hybridization studies of genomic DNA indicated that, while different isolates of armadillo-derived Mycobacterium leprae have a high degree of homology, binding of M. leprae genomic DNA to DNA of other species of mycobacteria or to corynebacteria was low, establishing that M. leprae is only remotely genetically related to any of the species examined. Several candidate leprosy vaccine mycobacterial strains were similarly found to have little genetic similarity to M. leprae. In contrast, the DNAs of the slow-growing mycobacteria M. tuberculosis, M. africanum, M. bovis, and M. microti were found to be very closely related. In the course of these studies, an M. leprae-specific repetitive sequence, greater than 15-fold per genome equivalent, was identified that might be useful for diagnostic and epidemiological studies.

Antigens, Bacterial↗
PubMed1989

Development of genetic systems for the mycobacteria.

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system which allows for the transfer, mutation, and expression of specific genes. Genetic analysis of mycobacteria has been exceedingly difficult since the mycobacteria grow slowly and no natural efficient method of gene transfer within the pathogenic has thus far been found. Using a molecular genetic approach, we have developed both the vectors and the methodology for efficient gene transfer in the mycobacteria. Initially, a novel of type of mycobacteriophage vector was developed, termed a shuttle phasmid. This hybrid shuttle vector replicates in Escherichia coli as a plasmid and in mycobacteria as a phage, capable of introducing foreign DNA into a wide variety of mycobacterial species. A set of shuttle phasmids, constructed from a temperate mycobacteriophage, retained their ability to lysogenize their mycobacterial hosts and could thus introduce foreign DNA stably into mycobacterial cells. An E. coli gene conferring kanamycin-resistance was cloned into these vectors and shown to express in the mycobacteria, thus providing the first selectable marker gene for subsequent genetic studies. Using kanamycin-resistance gene as a selection, the M. fortuitum plasmid pAL5000 replicon, and electroporation; a plasmid transformation system has been developed for both M. smegmatis and BCG. We now plan to use these phage and plasmid systems to analyze, genetically, the virulence attributes of the pathogenic mycobacteria. In addition, by introducing and expressing foreign antigens in BCG, we hope to develop a novel recombinant multi-vaccine vehicle capable of conferring immunity to a variety of bacterial, viral, and parasitic pathogens.

BCG Vaccine↗
PubMed1988

Lysogeny and transformation in mycobacteria: stable expression of foreign genes.

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system that allows for the transfer, mutation, and expression of specific genes. Because of the continuing importance of tuberculosis and leprosy worldwide, we initiated studies to develop a genetic system in mycobacteria and here report the use of two complementary strategies to introduce and express selectable genetic markers. First, an Escherichia coli cosmid was inserted into the temperate mycobacteriophage L1, generating shuttle phasmids replicating as plasmids in E. coli and phage capable of lysogenizing the mycobacterial host. These temperate shuttle phasmids form turbid plaques on Mycobacterium smegmatis and, upon lysogenization, confer resistance to superinfection and integrate within the mycobacterial chromosome. When an L1 shuttle phasmid containing a cloned gene conferring kanamycin resistance in E. coli was introduced into M. smegmatis, stable kanamycin-resistant colonies--i.e., lysogens--were obtained. Second, to develop a plasmid transformation system in mycobacteria, M. fortuitum/E. coli hybrid plasmids containing mycobacterial and E. coli replicons and a kanamycin-resistance gene were constructed. When introduced into M. smegmatis or BCG (Mycobacterium tuberculosis typus bovinus var. Bacille-Calmette-Guérin) by electroporation, these shuttle plasmids conferred stable kanamycin resistance upon transformants. These systems should facilitate genetic analyses of mycobacterial pathogenesis and the development of recombinant mycobacterial vaccines.

Cloning, Molecular↗
PubMed1987

Effect of norepinephrine on renal tubular Na-K-ATPase and oxygen consumption.

Previous studies from this laboratory have demonstrated that norepinephrine (NE) increases sodium dependent phenylalanine uptake by in vitro rat cortical tubules. In the present study, we have examined whether the observed increase in proximal tubular sodium transport, during NE treatment, is related to changes in membrane Na-K-ATPase and cellular oxygen consumption. Treatment of intact tubules with NE increased microsomal Na-K-ATPase activity but had no effect on cellular oxygen consumption or ouabain inhibitable oxygen consumption. The increased Na-K-ATPase activity is consistent with the observed increase in sodium transport while the lack of a detectable effect on oxygen consumption suggests that the increased transport does not require additional oxygen utilization.

Animals↗
PubMed1987

Renal metabolism and acute renal failure.

We briefly review what appear to be the most important elements responsible for renal cell injury during and after oxygen deprivation. Recent studies in numerous laboratories have vastly improved our understanding of the changes in cell function that occur during ischemia and yet, the underlying mechanisms by which tubule damage and cell death occur remain elusive. We attempt to separate the effects that occur during ischemia or anoxia from those occurring during reperfusion (reoxygenation). These are not always separable, especially because it appears that ischemia initiates a series of complex events that may only become manifested during reperfusion. Ischemia-induced renal dysfunctions are clearly multifactorial events that will require major efforts to unravel.

Acute Kidney Injury↗
PubMed1987

Fluorescent measurements of intracellular free calcium in isolated toad urinary bladder epithelial cells.

Sodium-calcium exchange has been suggested to play a pivotal role in the regulation of cytosolic free calcium (Caf) by epithelial cells. Using isolated epithelial cells from the toad urinary bladder, Caf has been measured using the intracellular Ca-sensitive fluorescent dyes Fura 2 and Quin 2. Dye loading did not alter cell viability as assessed by measurements of ATP and ADP content or cell oxygen consumption. When basal Caf was examined over a wide range of cell dye content (from 0.04 to 180 nmol dye/mg protein) an inverse relationship was observed. At low dye content, Caf was 300-380 nM and, as dye content was increased, Caf progressively fell to 60 nM. Using low dye content cells, in which minimal alteration in Ca steady state would be expected, the role for plasma membrane Na-Ca exchange was examined using either medium sodium substitution or ouabain. While medium sodium substitution increased Caf, prolonged treatment with ouabain had no effect on Caf despite a clear increase in cell sodium content. The lack of effect of ouabain suggests that Na-Ca exchange-mediated Ca efflux plays a minimal role in the regulation of basal Caf. However, exchange-mediated Ca efflux may play a role in Caf regulation when cytosolic calcium is elevated.

Animals↗
PubMed1986

Evidence for the presence of functional beta-adrenoceptor along the proximal tubule of the rat kidney.

Evidence for the presence of beta adrenoceptors on proximal tubules from the rat kidney has been obtained using enriched tubule suspensions prepared by Percoll centrifugation. Intact tubules demonstrated simultaneous enrichment of parathyroid hormone and isoproterenol sensitive cAMP production with no enrichment of antidiuretic hormone sensitive cAMP production. Both norepinephrine and epinephrine were less potent than isoproterenol and the stimulatory effect of catecholamines could be blocked with propranolol but not phentolamine. The stimulatory effect of norepinephrine on cellular phenylalanine uptake is blunted by co-addition of isoproterenol suggesting that the beta receptor may modulatory catecholamine stimulated transport.

Animals↗
PubMed1986

Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K-12.

Genomic libraries of Mycobacterium leprae DNA partially digested with Pst I were constructed in the expression vector pYA626, which contains the promoter region from the Streptococcus mutans gene encoding aspartate beta-semialdehyde dehydrogenase, which is very efficiently expressed in Escherichia coli. We have detected several clones that complement a mutation in the citrate synthase gene of E. coli. Southern blot analysis demonstrated that the complementing DNA was M. leprae DNA. Sodium dodecyl sulfate/polyacrylamide gel analysis of polypeptides produced by minicells containing the citrate synthase-complementing recombinant molecules demonstrated the production of a 46-kDa polypeptide. When the citrate synthase-complementing fragment was cloned in pYA626 in the reverse orientation, the recombinant molecule was no longer able to complement the mutation in the citrate synthase gene and no longer produced the 46-kDa polypeptide. When the DNA fragment was cloned in the Pst I site of pHC79, so as to allow expression from the beta-lactamase promoter, the resulting recombinant failed to complement the mutation in the E. coli citrate synthase gene yet still produced the 46-kDa polypeptide, but in one-fourth the amount than when expressed from the S. mutans asd promoters. This demonstrates that M. leprae translational sequences can be recognized by E. coli translational machinery. Promoter expression vectors can be used to obtain expression of protein antigens to be used for early diagnosis of leprosy or components of a vaccine and proteins that are targets of potential antileprosy drugs.

Aspartate-Semialdehyde Dehydrogenase↗
PubMed1986

In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries.

Strains of Escherichia coli K-12 were constructed that permitted the amplification of in vitro-packaged recombinant cosmid-transducing particles by in vivo repackaging of recombinant cosmid molecules. Thermal induction of these thermoinducible, excision-defective lysogens containing recombinant cosmid molecules yielded high titers of packaged recombinant cosmids and low levels of PFU. These strains were used to amplify packaged recombinant cosmid libraries of Mycobacterium leprae, Mycobacterium vaccae, Salmonella typhimurium, and Streptococcus mutans DNA. Contiguous and noncontiguous libraries were compared for the successful identification of cloned genes. Construction of noncontiguous libraries allowed the dissociation of desired genes from genes that were deleterious to the survival of a cosmid recombinant and permitted selection for unlinked traits that resulted in a selected phenotype. In vivo repackaging of recombinant cosmids permitted amplification of the original in vitro-packaged collection of transducing particles, storage of cosmid libraries as phage lysates, facilitation of complementation screening, expression analysis of repackaged recombinant cosmids after UV-irradiated cells were infected, in situ enzyme or immunological screening, and facilitation of recovery of recombinant cosmid molecules containing transposon inserts.

ATP-Dependent Proteases↗
PubMed1985

Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae.

Molecular analysis of DNA from Mycobacterium leprae, "Mycobacterium lufu," and Mycobacterium vaccae has demonstrated that the G + C (guanine plus cytosine) contents of the DNAs are 56, 61, and 65%, respectively, and that the genome sizes are 2.2 X 10(9), 3.1 X 10(9), and 3.1 X 10(9) daltons, respectively. Because of the significant differences in both G + C content and genome size among M. leprae, "M. lufu," and M. vaccae DNAs, these species are not related, although hybridization experiments under nonstringent conditions, with two separate cloned M. leprae DNA inserts as probes, indicate that there are some conserved sequences among the DNAs. The G + C content of Dasypus novemcinctus (armadillo, the animal of choice for cultivating M. leprae) DNA was determined to be 36%. Genomic libraries potentially representing more than 99.99% of each genome were prepared by cloning into the cosmid vector, pHC79, in Escherichia coli K-12. A genomic library representing approximately 95% of the genome of M. vaccae was prepared in pBR322. M. leprae DNA was subcloned from the pHC79::M. leprae library into an expression vector, pYA626. This vector is a 3.8-kilobase derivative of pBR322 in which the promoter region of the asd (aspartate semialdehyde dehydrogenase) gene from Streptococcus mutans has been inserted in place of the EcoRI-to-PstI fragment of pBR322. Several (44% of those tested) pYA626::M. leprae recombinants and one pBR322::M. vaccae recombinant synthesized new polypeptides in minicells of E. coli, indicating that mycobacterial DNA can be expressed in E. coli K-12, although expression is probably dependent upon use of nonmycobacterial promoters recognized by the E. coli transcription-translation apparatus.

Bacterial Proteins↗
PubMed1979

Echocardiographic aortic ejection area as a reflection of left ventricular stroke volume.

Systolic cusp separation of normal aortic valves has been shown by echocardiography and by angiography to be related to left ventricular stroke volume. In this study, stroke volume was compared with echocardiographic area between the aortic cusps in systole (aortic ejection area). An excellent correlation was observed (r = 0.93) over a wide range of stroke volumes, even in the presence of mitral regurgitation. Stroke volume appears to correlate more closely with aortic ejection area than with angiographic or echocardiographic measurements of peak systolic cusp separation.

Aortic Valve↗
PubMed1979

Lack of ouabain effect on pacing-induced myocardial ischemia in patients with coronary artery disease.

Sixteen patients with significant two and three vessel coronary artery disease but without clinical congestive heart failure were studied during rapid atrial pacing before and after infusion of 0.015 mg/kg of ouabain. Seven patients with a decreased (less than 50 percent) ejection fraction and nine patients with a normal ejection fraction had a significant (P less than 0.05) increase in resting arterial systolic pressure after the administration of ouabain. However, resting values for coronary sinus flow, coronary vascular resistance, myocardial oxygen consumption and myocardial lactate extraction did not change significantly in either group. During pacing, patients with a decreased ejection fraction demonstrated more ischemia than patients with a normal ejection fraction; however, the administration of ouabain did not significantly alter pacing-related changes in coronary sinus flow, myocardial oxygen consumption, myocardial lactate extraction, ischemic electrocardiographic changes or onset of chest pain in either group. The administration of ouabain has a negligible effect on coronary hemodynamics, myocardial metabolism or clinical signs of ischemia in patients with coronary artery disease with normal or abnormal left ventricular function.

Adult↗