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51.
GJ Mancini-Samuelson V Kieweg KM Sabaj S Ghisla MT Stankovich 《Canadian Metallurgical Quarterly》1998,37(41):14605-14612
The modulation of the electron-transfer properties of human medium-chain acyl-CoA dehydrogenase (hwtMCADH) has been studied using wild-type and site-directed mutants by determining their midpoint potentials at various pH values and estimating the involved pKs. The mutants used were E376D, in which the negative charge is retained; E376Q, in which one negative charge (pKa approximately 6. 0) is removed from the active center; E99G, in which a different negative charge (pKa approximately 7.3) also is affected; and E376H (pKa approximately 9.3) in which a positive charge is present. Em for hwtMCADH at pH 7.6 is -0.114 V. Results for the site-directed mutants indicate that loss of a negative charge in the active site causes a +0.033 V potential shift. This is consistent with the assumption that electrostatic interactions (as in the case of flavodoxins) and specific charges are important in the modulation of the electron-transfer properties of this class of dehydrogenases. Specifically, these charge interactions appear to correlate with the positive Em shift observed upon binding of substrate/product couple to MCADH [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715], which coincides with a pK increase of Glu376-COOH from approximately 6 to 8-9 [Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445]. From the pH dependence of the midpoint potentials of hwtMCADH two mechanistically important ionizations are estimated. The pKa value of approximately 6.0 is assigned to the catalytic base, Glu376-COOH, in the oxidized enzyme based on comparison with the pH behavior of the E376H mutant, it thus coincides with the pK value recently estimated [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The pKa of approximately 7.1 is assigned to Glu376-COOH in reduced hwtMCADH. Comparable values for these pKas for Glu376-COOH in pig kidney MCADH are pKox = 6.5 and pKred = 7.9. The Em measured for K304E-MCADH (a major mutant resulting in a deficiency syndrome) is essentially identical to that of hwtMCADH, indicating that the disordered enzyme has an intact active site. 相似文献
52.
X Meng X Lu Z Li ED Green H Massa BJ Trask CA Morris MT Keating 《Canadian Metallurgical Quarterly》1998,103(5):590-599
Williams syndrome (WS) is a contiguous gene deletion disorder caused by haploinsufficiency of genes at 7q11.23. We have shown that hemizygosity of elastin is responsible for one feature of WS, supravalvular aortic stenosis (SVAS). We have also implicated LIM-kinase 1 hemizygosity as a contributing factor to impaired visual-spatial constructive cognition in WS. However, the common WS deletion region has not been completely characterized, and genes for additional features of WS, including mental retardation, infantile hypercalcemia, and unique personality profile, are yet to be discovered. Here, we present a physical map encompassing 1.5 Mb DNA that is commonly deleted in individuals with WS. Fluorescence in situ hybridization analysis of 200 WS individuals shows that WS individuals have the consistent deletion interval. In addition, we identify three novel genes from the common deletion region: WS-betaTRP, WS-bHLH, and BCL7B. WS-betaTRP has four putative beta-transducin (WD40) repeats, and WS-bHLH is a novel basic helix-loop-helix leucine zipper (bHLHZip) gene. BCL7B belongs to a novel family of highly conserved genes. We describe the expression profile and genomic structure for each of these genes. Hemizygous deletion of one or more of these genes may contribute to developmental defects in WS. 相似文献
53.
NS Bora PS Bora MT Tandhasetti TP Cirrito HJ Kaplan 《Canadian Metallurgical Quarterly》1996,37(9):1877-1883
PURPOSE: Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein. METHODS: A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells. RESULTS: The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein. CONCLUSIONS: The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36. 相似文献
54.
Human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) is a clinicopathologic entity that includes proteinuria, azotemia, focal segmental glomerulosclerosis or mesangial hyperplasia, and tubulointerstitial disease. The incidence of HIVAN is increased in black patients and variable depending on the age and geographic area. The objective of this study was to describe relevant clinical and pathological findings in 30 children with HIVAN followed at the Children's National Medical Center in Washington, D.C. Our experience of the last 12 years showed a spectrum of HIVAN that seems to be coincident with the degree of acquired immunodeficiency syndrome (AIDS) symptomatology. By renal sonograms and frequent urinalysis, we identified children undergoing the early stages of HIVAN with enlarged echogenic kidneys, proteinuria, and "urine microcysts". HIVAN did not necessarily progress rapidly to end-stage renal disease. Nephrotic syndrome or chronic renal insufficiency were late manifestations of HIVAN. Children with HIVAN were likely to develop transient electrolyte disorders, heavy proteinuria, and acute renal failure due to systemic infectious episodes or nephrotoxic drugs. HIVAN was associated with other HIV-induced illnesses and high mortality rates. Early detection and careful clinical follow-up of children with HIVAN may reduce the incidence of renal-cardiovascular complications and improve their quality of life. 相似文献
55.
L Dupclay MT Rupp RW Bennett TM Jarnagin 《Canadian Metallurgical Quarterly》1999,39(1):74-81; quiz 105-6
OBJECTIVE: To measure grocery chain pharmacists' work-related behaviors to assess the impact of a Pharmaceutical Care Certificate Program (PCCP) and other future interventions intended to alter pharmacists' practice behaviors. DESIGN: This study used multidimensional work sampling (MWS), a work measurement methodology that breaks "work" into three components: activity (what was done), contact (with whom the activity was performed), and function (the purpose or objective of the activity). Pharmacists were signaled at random intervals during the workday by a random signal generator. A selection was made from a list of items in each of the three dimensions of work to form an activity-contact-function combination code that described the work-related behavior at that point in time. SETTING AND PARTICIPANTS: Pharmacists in 15 grocery chain stores in the Indianapolis area; 20 pharmacists were enrolled in Purdue University's PCCP and 10 served as controls. Data were collected for a period of six weeks during April through June 1997 before the beginning of the PCCP program. MAIN OUTCOME MEASURES: Pharmacists' work-related behaviors. RESULTS: Writing/keyboarding was the most frequently recorded activity (22%), followed by one-to-one meetings (21.6%), and drug preparation (18%). Pharmacists spent most of their time working alone (62.9%), while a smaller but still substantial proportion of time was spent interacting with patients (17.9%). The most frequently recorded purpose (i.e., function) of pharmacists' activities was drug distribution (23.9%), followed by personal time (12.4%), receiving or transferring a medication order (10.2%), and patient counseling (6.6%). Out of a possible, 1,760 activity-contact-function combinations, 10 accounted for 46.3% of all reported observations, with "Prepare drug-Self-Drug distribution" representing the most frequently recorded activity-contact-function combination (15.7%). CONCLUSION: MWS is useful in helping grocery chain management better understand how pharmacy personnel are currently being utilized. This study provides a baseline for evaluating the impact of training programs or other alterations in the practice environment on pharmacists' work-related behaviors. 相似文献
56.
Retrospective analyses of data from the authors' program of research on hypnosis and memory are presented, with special emphasis on effects observed among low hypnotizable individuals. In Experiment 1, participants completed seven forced-recall trials in an attempt to remember a series of pictures that had been shown 1 week earlier. For half the participants, the middle five trials were carried out using hypnotic procedures; the remaining participants performed all recall attempts in a motivated waking condition. Hypnosis failed to enhance correct recall for either high or low hypnotizable participants beyond the hypermnesia and reminiscence effects associated with repeated retrieval attempts over time. However, whereas high hypnotizable participants produced substantial numbers of confident recall errors (i.e., intrusions) independent of the use of hypnosis, low hypnotizable participants exposed to hypnotic procedures reported significantly more intrusions than their counterparts in the waking condition. In Experiment 2, participants were asked to identify whether specific recollections, reported during two forced-interrogatory recall tests conducted 1 week earlier, had originated in the first or second of those tests. A general bias to misattribute previously reported recollections to the first of two recall occasions was observed; however, the effect was greatest among low hypnotizables who had undergone the second recall attempt in hypnosis. The findings imply that highly hypnotizable individuals are not unique in their vulnerability to distortions of memory induced by hypnotic techniques. Individuals of lesser hypnotic capacity also manifest memory alterations when exposed to such procedures. 相似文献
57.
58.
MT Manley WN Capello JA D''Antonio AA Edidin RG Geesink 《Canadian Metallurgical Quarterly》1998,80(8):1175-1185
OBJECTIVE: To investigate the roles of brain angiotensin II and C-type natriuretic peptide (CNP) in the hypertensive mechanism of deoxycorticosterone acetate (DOCA)-salt hypertension. METHODS: We injected 50 microg/kg CV-11 974, an angiotensin II type-1 receptors antagonist, 30 nmol/kg CNP-22, or the vehicle (artificial cerebrospinal fluid) into the cerebral ventricle or intravenously 5 min before the intracerebroventricular infusion of 1.5 mol/I NaCl solution for 30 min into either male normotensive Wistar rats or DOCA-salt hypertensive rats anesthetized with urethane, and their arterial pressures and heart rates were continuously recorded. Blood (2 ml) was collected at the end of the infusion for the measurement of plasma concentration of arginine vasopressin. We infused 10 or 50 microg/kg per day CV-11 974, 10 or 50 nmol/kg per day CNP-22, or the vehicle (1 microl/h) into the cerebral ventricles of DOCA-salt hypertensive rats for 7 days by using osmotic minipumps, and measured their systolic arterial pressures, pulse rates, and urinary excretions of vasopressin. RESULTS: Intracerebroventricular pre-administrations of CV-11 974 and of CNP-22 inhibited increases in mean arterial pressure, heart rate, and plasma vasopressin concentration induced by intracerebroventricular infusion of 1.5 mol/l NaCl into normotensive rats; increases in hemodynamics and plasma level of vasopressin induced by intracerebroventricular infusion of 1.5 mol/l NaCl were suppressed by intracerebroventricular pre-injections of CV-11 974, but not of CNP-22, into DOCA-salt hypertensive rats. Continuous intracerebroventricular infusions of 50 microg/kg per day CV-11 974 attenuated hypertension in DOCA-salt treated rats, accompanied by a reduction in urinary excretion of vasopressin. Continuous intracerebroventricular infusions of 50 nmol/kg per day CNP-22, however, affected neither hypertension nor urinary excretion of vasopressin in DOCA-salt hypertensive rats. CONCLUSION: Brain angiotensin II could play a role in the pressor mechanism of DOCA-salt hypertension by increasing release of vasopressin via type 1 receptors. That brain CNP has an inhibitory effect on release of vasopressin in acute experiments indicates that the impairment of this inhibitory effect of brain CNP on secretion of vasopressin could be involved in the pathogenesis of DOCA-salt hypertension in rats. 相似文献
59.
PURPOSE: Ischemic neurodegeneration contributes to many retinal diseases. An isolated retina model has been used to examine the neuronal cell death induced by deprivation of oxygen and glucose (simulated ischemia) as a model for ischemic disease. METHODS: Neurodegeneration in the isolated chick embryo retina was induced by simulated ischemia and assessed using biochemical (lactate dehydrogenase release) and morphologic (light microscopy) techniques. RESULTS: Simulated ischemia led to lactate dehydrogenase release gradually in a period of 6 to 24 hours. Light microscopic observations demonstrated morphologic cell degeneration well before lactate dehydrogenase release occurred. N-Methyl-D-aspartate (NMDA) and non-NMDA receptor blockers individually provided partial protection, and the combination was fully protective. No protection was provided if the antagonists were added after simulated ischemia. When NMDA receptors were blocked by MK-801, cyclothiazide, an inhibitor of desensitization at non-NMDA receptors, enhanced lactate dehydrogenase released after 1 or 2 hours of simulated ischemia. Low concentrations of glucose effectively prevented lactate dehydrogenase release, despite anoxic conditions. CONCLUSIONS: The isolated retina provided a convenient system to characterize quantitatively ischemic cell death. Retinal ischemic neurodegeneration is an excitotoxic process that involves overactivation of NMDA and non-NMDA glutamate receptors. Blockade of both of these receptor subtypes was necessary for complete neuroprotection. Receptor desensitization played a protective role. If even low concentrations of glucose were delivered to an ischemic retina in vitro, substantial neuroprotection could be achieved. This may have implications for the management of acute retinal ischemic episodes. 相似文献
60.