NADP-Isocitrate dehydrogenase from Pseudomonas nautica: kinetic constant determination and carbon limitation effects on the pool of intracellular substrates

Variations of intracellular concentrations of isocitrate and NADP+ were measured throughout all growth phases of the marine bacterium Pseudomonas nautica. The intracellular isocitrate concentration tracked the intracellular protein concentration throughout all phases of growth. It rapidly increased in early exponential phase to a maximum and fell to nearly zero in parallel with pyruvate exhaustion in the culture medium. The intracellular NADP+ and protein concentrations increased in parallel during the exponential phase but were poorly correlated. Even after carbon exhaustion, the intracellular NADP+ concentration stayed high, as did protein levels. The results demonstrated that the intracellular isocitrate concentration, but not the intracellular NADP+ concentration, was affected by the carbon availability in the culture. They also suggest that, because of its variability, isocitrate, but not NADP+, plays the larger role in the control of the respiratory CO2 production rate (RCO2). From initial rate studies, bisubstrate Michaelis constants and the dissociation constant were determined for NADP+-specific isocitrate dehydrogenase (IDH) from P. nautica. These studies support the hypothesis that the mechanism of IDH's activity involves the ordered addition of the substrates, D-isocitrate and NADP+. Furthermore, the results support the use of a bisubstrate enzyme kinetic equation to model RCO2 in P. nautica.     

Vertebral artery dissection

Vertebral artery dissections (VADs) following a variety of minor traumatic mechanisms have been previously reported. This article reports 2 cases of VAD with delayed recognition following motor vehicle collisions (MVCs). The first VAD patient developed major neurologic abnormalities 28 hours after an MVC. The second VAD patient presented with 3 weeks of neck and head pain beginning 8 weeks after an MVC and subsequent chiropractic manipulation. The anatomy and pathophysiology of VAD are reviewed. Early ED recognition prior to the onset of major neurologic deficits (e.g., paresis, dysarthria, ataxia, or altered mental status) is emphasized. An algorithm for the ED management of the entity is suggested.     

Blocking interleukin-6 activity with chimeric anti-IL6 monoclonal antibodies in multiple myeloma: effects on soluble IL6 receptor and soluble gp130

Interleukin-6 (IL6) plays a major role in the pathogenesis of multiple myeloma. In patients with monoclonal gammopathy serum levels of sIL6R have been found to be increased. The role of IL6 in the regulation of soluble receptors is still unclear. In a phase I/II study we treated 12 myeloma patients with high-affinity chimeric anti-IL6 monoclonal antibodies. This treatment resulted in a total in vivo blockage of IL6 activity and as a result we had an unique opportunity to gain insight into the possible regulation effects of IL6 on these soluble IL6 receptors. Pre-treatment sIL6R levels were elevated in 9 of the 12 patients; pre-treatment sgp130 levels were significantly increased in all patients. Total blockage of IL6 activity by the high-affinity cMab did not influence sIL6R in 10 of these 12 patients and sgp130 levels remained stable in all patients. Of the 2 patients whose sIL6R levels increased during therapy, one had progressive disease and the other developed an acute infection. We conclude that in most end-stage myeloma patients sIL6R and sgp130 serum levels are elevated, but that there is no relation between IL6 activity and sIL6R or sgp130 levels.     

Prepubic dermoid sinus: possible variant of dorsal urethral duplication (Stephens type 3)

The authors present the case of 4-month-old girl with a midline prepubic sinus extending from the skin overlying the pubis to the superior surface of the bladder, and continuing to the umbilicus via the median umbilical ligament. The distal portion of the exicised sinus was surrounded by concentric bundles of collagen and smooth muscle with minimal inflammatory infiltrates, which suggests a developmental origin. According to Stephens' classification, the sinus appears to be a variant of dorsal urethral duplication of Stephens type 3.     

Long-range and short-range oxidative damage to DNA: photoinduced damage to guanines in ethidium-DNA assemblies

Short-range and long-range photoreactions between ethidium and DNA have been characterized. While no DNA reaction is observed upon excitation into the visible absorption band of ethidium, higher-energy irradiation (313-340 nm) leads both to direct strand cleavage at the 5'-G of 5'-GG-3' doublets and to piperidine-sensitive lesions at guanine. This reactivity is not consistent with oxidation of guanine by either electron transfer or singlet oxygen as shown by comparison with reactions of a rhodium intercalator and methylene blue, respectively. By covalently tethering ethidium to one end of a DNA duplex, we demonstrate the presence of two distinct reactions, one short-range and the other long-range. The short-range reaction involves a covalent modification of guanine by ethidium, based upon HPLC analysis of the nucleoside products and studies with ethidium derivatives. The long-range reaction is entirely consistent with oxidation of guanine by DNA-mediated electron transfer. The yield of this electron-transfer reaction is not attenuated with distance; equal yields of guanine damage are observed at a proximal (17 A Et-GG separation) and distal (44 A Et-GG separation) site. These results are quite similar to those previously observed with a covalently tethered rhodium photooxidant and underscore the unique ability of the DNA base stack to facilitate long-range electron transfer so as to effect oxidative damage from a distance.     

Cross-reactivity of anti-10kD heat shock protein antibodies in leprosy and tuberculosis patients

The response to recombinant 10-kD heat shock protein (HSP) of Mycobacterium leprae (rML10) was evaluated by indirect ELISA in sera from leprosy patients, household contacts, tuberculosis patients and healthy controls in a leprosy-endemic area in the North East of Argentina. Some technical parameters were analyzed: within-assay and between-assay variability, dose-response curves and detectability indexes (specificity and sensitivity) of ELISA applied to measure anti-10 kDa antibodies. High levels of these antibodies have already been reported in positive bacilloscopy patients; herein we have also demonstrated that tuberculosis patients sera cross-react with this M. leprae antigen. This test seems to have a low sensitivity and specificity for leprosy detection; it confirms that antibodies against highly conserved HSP antigens are important in the polyclonal response against mycobacterial epitopes in leprosy as well as in tuberculosis.     

Protozoa and the decline of Rhizobium populations added to soil

A fall in Rhizobium abundance occurred in nonsterile soil inoculated with large numbers of the root-nodule bacteria, but many of the rhizobia still survived. No such decline was evident in sterile soil. Protozoa feeding on these bacteria were isolated from soil and other environments. As the abundance of Rhizobium meliloti and a cowpea Rhizobium strain in soil decreased, the protozoan density increased. The inability of the predators to eliminate their prey from soil was not the result of the presence of organisms feeding on the protozoa because many rhizobia survived in sterile soil inoculated with the prey and cultures of individual protozoa, nor was it the result of the rapid multiplication of the bacteria to replace those consumed because survivors were still numerous in essentially organic matter free soil in which the bacteria did not grow appreciably. The lack of elimination also was not associated with a protective effect of soil particles because survivors were still abundant in solutions inoculated with protozoa and bacteria. It is suggested that the size of the prey population diminishes until a density is attained at which the energy used by the predator in hunting for the survivors equals that obtained from the feeding.