Activation of particulate guanylyl cyclase by Vibrio vulnificus hemolysin

Recently we reported that Vibrio vulnificus hemolysin, an exotoxin produced by V. vulnificus, dilates rat thoracic aorta via elevated cGMP levels without affecting nitric oxide synthase. We investigated the mechanism further by observing the guanylyl cyclase activities in cytosolic, membrane, unfractionated, or reconstituted preparations. Hemolysin did not activate guanylyl cyclase in the membrane or cytosolic fraction, while it activated guanylyl cyclase in unfractionated or reconstituted preparation. The increased activity was not inhibited by the HS-142-1, a microbial polysaccharide which antagonizes atrial natriuretic peptide receptor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor. However, it was attenuated by 6-(phenylamino)-5,8-quinolinedione (LY 83.583), which inhibits the catalytic domain of both guanylyl cyclases, and by cholesterol, which blocks hemolysin-incorporation into the membrane. Removing ATP, a cofactor of particulate guanylyl cyclase, attenuated the activation and ATPgammaS, a non-phosphorylating analog, restored it. These results suggest that V. vulnificus hemolysin activates particulate guanylyl cyclase via hemolysin incorporation into the vascular smooth muscle cell membrane in cooperation with certain unidentified cytosolic component(s).     

Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos

The impact of oxidative stress on the in vitro development of bovine embryos in synthetic oviduct fluid medium (mSOF) was assessed by using H2O2 as a stress inducer. In a preliminary experiment, a chemiluminescent method was used to measure the antioxidative capacity of the mSOF culture medium. Pyruvate was the mSOF component displaying the highest H2O2 degrading ability. Essential and nonessential amino acids also significantly reduced the H2O2 concentration, whereas lactate and glutamine were ineffective. The effect on further development of a short exposure of zygotes, 9-16-cell stage embryos and blastocysts to 0 M; 10(-7) M ; 10(-6) M, and 10(-5) M H2O2 in pyruvate-free mSOF was evaluated. Developmental rates of the H2O2-treated zygotes to the 5-8-cell or blastocyst stages and survival of H2O2-treated blastocysts were reduced in a dose-dependent manner whereas the 9-16-cell embryos were unaffected by those treatments. Blastocysts treated with H2O2 also tended to have lower numbers of bisbenzimide-stained nuclei and showed increased nuclear fragmentation. Including pyruvate in the mSOF culture medium during a 10(-5) M H2O2 pulse highly reduced the H2O2 concentration as measured by chemiluminescence and improved zygote and blastocyst development, but failed to prevent blastocyst nuclei degradation. These experiments suggest that bovine embryos show developmental change in sensitivity to exogenous H2O2, the 9-16-cell embryos being more resistant than zygotes and blastocysts and that H2O2 and its toxic effects can be attenuated by including pyruvate in the medium.     

An in vitro tissue culture bilayer model to examine early events in Mycobacterium tuberculosis infection

A tissue culture bilayer system that mimics some aspects of early alveolar infection by Mycobacterium tuberculosis was developed. This model incorporates human lung epithelial type II pneumocyte (A549) (upper chamber) and endothelial cell (lower chamber) layers separated by a microporous membrane. This construction makes it possible to observe and quantify the passage of bacteria through the two layers, to observe the interaction of the bacteria with the various cell types, and to examine the basic mechanisms of immune cell recruitment to the site of infection. After 10(7) organisms were added to the upper chamber we microscopically observed large numbers of bacteria attached to and within the pneumocytes and we determined by viable-cell counting that a small percentage of the inoculum (0.02 to 0.43%) passed through the bilayer into the lower chamber. When peripheral blood mononuclear cells were added to the lower chamber, microscopic examination indicated a migration of the mononuclear cells through the bilayer to the apical surface, where they were seen associated with the mycobacteria on the pneumocytes. The added complexity of the bilayer system offers an opportunity to define more precisely the roles of the various lung cell types in the pathogenesis of early tuberculosis.     

Nonlinear gas turbine modeling using NARMAX structures

The estimation of a nonlinear autoregressive moving average with exogenous inputs (NARMAX) model of an aircraft gas turbine is presented. A method is proposed whereby periodic signals with certain harmonic content are used to qualify the nature of the nonlinearity of the engine in the frequency domain. The static behavior of the engine is investigated in the time domain to approximate the order of nonlinearity and this information is used a priori to restrict the search space of the potential NARMAX models. A forward-regression orthogonal estimation algorithm is then employed to select the model terms using the error reduction ratio. The performance of the estimated NARMAX model is illustrated against a range of small- and large-signal engine tests     

Unrelated donor bone marrow transplantation for chronic myelogenous leukemia: a decision analysis

BACKGROUND: Chronic myelogenous leukemia (CML) is an indolent but ultimately fatal disease. Because the natural history of CML varies and quality of life with CML may be excellent until shortly before death, deciding whether and when to pursue unrelated donor bone marrow transplantation is often difficult. OBJECTIVE: To compare early transplantation, delayed transplantation, and no transplantation for patients with chronic-phase CML on the basis of discounted, quality-adjusted life expectancy. DESIGN: A markov model comparing different strategies was constructed. This model considers patient age, quality of life, risk aversion, and the competing risks for CML progression and transplant toxicity. SETTING: Therapeutic decision at the time of diagnosis of CML. PATIENTS: The base case is a 35-year-old patient with intermediate-prognosis CML. Younger and older patients with better and worse prognoses are also evaluated. INTERVENTION: Early transplantation, delayed transplantation, and no transplantation. MEASUREMENTS: Quality-adjusted, discounted life expectancy. RESULTS: For patients with newly diagnosed CML, transplantation within the first year provides the greatest quality-adjusted expected survival, although this benefit decreases with increasing patient age. For a 35-year-old patient with intermediate-prognosis CML, transplantation within the first year results in 53 more discounted, quality-adjusted years of life expectancy than does no transplantation. This finding is robust even with varying baseline assumptions. CONCLUSIONS: These results support the use of early unrelated donor bone marrow transplantation for most patients with CML.