NADPH oxidase is an O sub(2) times super(-)-generating enzyme found in phagocytes such as neutrophils. It is composed of a membrane-bound cytochrome b, the cytosolic proteins p67 super(phox), p47 ...super(phox), p40 super(phox), and the G-protein p21rac. The system is dormant in resting cells but acquires catalytic activity on exposure to appropriate stimuli. Cytochrome b, p67 super(phox), p47 super(phox), and rac2 associate with the cytoskeleton and membrane skeleton of activated neutrophils. It is not known whether p40 super(phox) associates with the cytoskeleton. The purpose of this study was to analyze the subcellular distribution of p40 super(phox). When resting neutrophils were lysed in Triton X-100 or octyl glucoside buffer and separated into detergent-soluble and detergent-insoluble fractions, p40 super(phox) and p67 super(phox) were mainly associated with the detergent-insoluble fraction (defined as the cytoskeleton), whereas p47 super(phox) was mainly found in the soluble fraction. Neutrophil activation by phorbol myristate acetate (PMA) induced p47 super(phox) translocation to the cytoskeleton but did not affect the distribution of p40 super(phox) or p67 super(phox). Using immunofluorescence confocal microscopy, we found that p40 super(phox) colocalized with filamentous actin. In neutrophils from a p67 super(phox)-deficient patient with detectable p40 super(phox), p40 super(phox) associated with the cytoskeleton only after activation by PMA. A complex containing the three proteins was isolated from the cytoskeleton of activated neutrophils. When activated membranes were treated with Triton X-100 buffer, p40 super(phox), p47 super(phox), and p67 super(phox) were found in the membrane skeleton enriched in NADPH-oxidase activity; some p40 super(phox) and p47 super(phox) was found in the soluble membrane fraction, but no p67 super(phox) was detected. These findings show that p40 super(phox), like p67 super(phox) and p47 super(phox), binds to the cytoskeleton and membrane skeleton. In addition, p40 super(phox) can dissociate from p67 super(phox) in activated membranes.
Previous experiments showed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot ...structure that exists in equilibrium with two stem-loops and to trap the ribosome on its mRNA loading site in a transitory state. In this study, we investigated the effect of mutations in the translational operator on: the binding of protein S15, the formation of the 30S/mRNA/tRNA(fMet) ternary initiation complex, the ability of S15 to inhibit the formation of this ternary complex. The results were compared to in vivo expression and repression rates. The results show that (1) the pseudoknot is required for S15 recognition and translational control; (2) mRNA and 16S rRNA efficiently compete for S15 binding and 16S rRNA suppresses the ability of S15 to inhibit the formation of the active ternary complex; (3) the ribosome binds more efficiently to the pseudoknot than to the stem-loop; (4) sequences located between nucleotides 12 to 47 of the S15 coding phase enhances the efficiency of ribosome binding in vitro; this is correlated with enhanced in vivo expression and regulation rates.
Previous experiments showed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot ...structure that exists in equilibrium with two stem-loops and to trap the ribosome on its mRNA loading site in a transitory state. We investigated the effect of mutations in the translational operator on: the binding of protein S15, the formation of the 30S/mRNA/tRNA sub(f) super(Met) ternary initiation complex, the ability of S15 to inhibit the formation of this ternary complex. The results were compared to in vivo expression and repression rates. The results show that (1) the pseudoknot is required for S15 recognition and translational control; (2) mRNA and 16S rRNA efficiently compete for S15 binding and 16S rRNA suppresses the ability of S15 to inhibit the formation of the active ternary complex; (3) the ribosome binds more efficiently to the pseudoknot than to the stem-loop; (4) sequences located between nucleotides 12 to 47 of the S15 coding phase enhances the efficiency of ribosome binding in vitro; this is correlated with enhanced in vivo expression and regulation rates.
Ultrafiltration coefficient and glomerular capillary resistance in a model of immune complex glomerulonephritis. Decreased ultrafiltration coefficient, LpA or Kf, was documented previously in ...micropuncture studies of glomerulonephritis in rats. Observations were made immediately following an injection of antiglomerular basement membrane (anti-GBM) antibody, later in the course of glomerulonephritis, and during the chronic phase of Heymann nephritis. To gain further insight into the basis of reduced glomerular filtration rate in immune-complex glomerulonephritis, we studied the anatomic, physiologic, and rheologic properties of isolated glomeruli from female Buffalo rats with nephritis which developed during infection withTrypanosoma rhodesiense. Immune-complex mediated glomerulonephritis was present 2 weeks after inoculation and progressed throughout the 4 weeks of study. Renal insufficiency occurred, with serum creatinine concentrations rising to 5 to 10 times control values by week 4. Mesangial hypercellularity, mesangial electron dense deposits, and endothelial cell swelling were observed. Increased numbers of mononuclear cells were present within the glomerulus. Total glomerular water volume was greater in nephritic than in normal animals. Increased cell volume accounted for most of the volume increment. When filtration into the capillaries was induced in vitro by imposing an oncotic gradient of 6.5mm Hg or greater across the capillary wall, rapid and uniform erythrocyte movement occurred within the capillaries of control glomeruli and erythrocytes were ejected into the medium. In contrast, a transcapillary gradient of 30 to 40mm Hg was required to produce erythrocyte movement in glomeruli from nephritic animals studied 4 weeks after inoculation. The ultrafiltration coefficient of nephritic glomeruli was estimated in vitro and was not different from that of control glomeruli (5.81 ± 0.35 vs. 6.21 ± 0.49 nl/minmm Hg). An impairment of capillary perfusion may be responsible for the decreased rate of glomerular filtration observed in this model of glomerulonephritis.
Evaluation in vitro du coefficient d'ultrafiltration et de la résistance capillaire glomérulaire dans un modèle de glomérulonéphrite des comples immuns. La diminution du coefficient d'ultrafiltration, LpA ou Kf, a été établie précédement au cours de travaux utilisant les microponctions chez des rats atteints de glomérulonéphrite, immédiatement après l'injection d'anti-corps anti-membrane basale glomérulaire (anti-GBM) et, ultérieurement, au cours de l'évolution de glomérulonéphrite et durant la phase chronique de la néphrite de Heymann. Afin d'obtenir plus d'informations sur les fondements de la diminution du débit de filtration glomérulaire au cours de la néphrite des complexes immuns, nous avons étudié les propriétés anatomiques, physiologiques, et biologiques des glomérules isolés de rats femelles de la souche Buffalo atteints de néphrite développée au cours de l'infection parTrypanosoma rhodesiense. Une glomérulonéphrite des complexes immuns existait deux semaines après l'inoculation et évoluait pendant les 4 semaines de l'étude. Il existait une insuffisance rénale et la créatinine sérique atteignait des valeurs 5 à 10 fois plus grandes que les contrôles à la 4 semaine. L'hypercellularité mésangiale, sous la forme de dépôts denses mésangiaux en microscopie électronique, et le gonflement des cellules endothéliales ont été observés. Le nombre des cellules mononucléés du glomérule était augmenté. Le volume total d'eau du glomérule était plus grand chez les animaux atteints de néphrite que chez les contrôles. L'augmentation du volume cellulaire rendait compte de la plus grande partie de l'augmentation de volume. Quand la filtration dans les capillaires a été declenchée par l'imposition d'un gradient oncotique de 6,5mm Hg ou plus à travers la paroi capillaire, un mouvement rapide et uniforme des érythrocytes est apparu et les érythrocytes ont été éjectés dans le milieu. Par contre, pour les glomérules provenant d'animaux néphritiques, étudiés quatre semaines après l'inoculation, un gradient de 30 à 40mm Hg était nécessaire pour produire un mouvement des érythrocytes. Le coefficient d'ultrafiltration des glomérules d'animaux néphritiques a été évalué in vitro et n'est pas différent de celui des animaux contrôles (5,81 ± 0,35 vs. 6,21 ± 0,49 nl/minmm Hg). L'altération de la perfusion capillaire est responsable de la diminution du débit de filtration glomérulaire observée dans ce modèle de glomérulonéphrite.
In this retrospective study of 36 patients and 58 feet, the "L" shaped osteotomy of the calcaneus body was investigated. The procedure, designed to add intrinsic stability to the shape of the ...osteotomy, was performed to correct triplane deformities of the heel in a variety of pathologic foot types. A retrospective analysis of clinical and radiographic data suggests that this procedure is a valuable alternative for surgical treatment of deformities of the calcaneus. Advantages appear to be predictability, stability, and versatility with respect to triplane reduction of deformities. A reliable method of evaluating calcaneal position both perioperatively and intraoperatively is also presented.
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