Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase from Clostridium aminobutyricum

4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of gamma-aminobutyrate in Clostridium aminobutyricum, represents approximately 15-25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg-1. The dehydratase catalyses the reversible conversion of 4-hydroxybutyryl-CoA (Km = 50 microM) to crotonyl-CoA and possesses a probably intrinsic vinylacetyl-CoA delta 3-delta 2-isomerase with a specific activity of 223 nkat mg-1. The equilibrium of the reversible dehydration was determined from both sides as K = [crotonyl-CoA]/[4-hydroxybutyryl-CoA] = 4.2 +/- 0.3. Cyclopropylcarboxyl-CoA was not converted to crotonyl-CoA. The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure. Under anaerobic conditions the active enzyme revealed a brown colour and contained 2 +/- 0.2 mol FAD (64 +/- 5% oxidized), 16 +/- 0.8 mol Fe and 14.4 +/- 1.2 mol inorganic sulfur, which probably form iron-sulfur clusters. Exposure to air resulted initially in a slight activation followed by irreversible inactivation. Concomitantly the vinylacetyl-CoA delta-isomerase activity was lost and the colour of the enzyme changed to yellow. Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanoferrate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration a non activated C-H bond at C-3 of 4-hydroxybutyryl-CoA has to be cleaved. A putative mechanism for 4-hydroxybutyryl-CoA dehydratase is proposed in which this cleavage is achieved by a FAD-dependent oxidation of 4-hydroxybutyryl-CoA to 4-hydroxycrotonyl-CoA. In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2' reaction leading to vinylacetyl-CoA. Finally isomerisation yields crotonyl-CoA. 4-Hydroxybutyryl-CoA dehydratase is quite distinct from 3-hydroxyacyl-CoA dehydratase (crotonase) and 2-hydroxyacyl-CoA dehydratases. Contrary to the latter enzyme [e.g. (R)-lactyl-CoA dehydratase and (R)-2-hydroxyglutaryl-CoA dehydratase] which are composed of three different subunits and similarly catalyse the cleavage of a non activated C-H bond at C-3, 4-hydroxybutyryl-CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity. Furthermore 4-hydroxybutyryl-CoA dehydratase is not inactivated by oxidants such as 5 mM 4-nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.     

白血病和淋巴瘤患者ABO血型分布及地区性差异分析

目的 探讨ABO血型与白血病、淋巴瘤的相关性和地区性差异.方法 采用病例对照研究方法,调查不同类型白血病、淋巴瘤患者和健康对照组ABO血型分布特征,分析不同地区白血病、淋巴瘤患者的ABO血型分布情况.结果 急性非淋巴细胞白血病、急性淋巴细胞白血病、非霍奇金淋巴瘤患者的ABO血型分布与健康人群分布差异有统计学意义(χ2=21.23、χ2=8.36、χ2=9.39,均P<0.05).国内不同地区的白血病、淋巴瘤ABO血型分布有差异,其中白血病ABO血型分布差异具有统计学意义(χ2=50.65,P<0.05).结论 ABO血型可能是白血病、淋巴瘤的遗传易感因素,但地理因素可能是主要影响因素之一.     

L-threonine dehydrogenase. Purification and properties of the homogeneous enzyme from Escherichia coli K-12

L-Threonine dehydrogenase, which catalyzes the conversion of L-threonine to aminoacetone + CO2 presumably via the intermediate formation of alpha-amino-beta-ketobutyrate, has been purified to apparent homogeneity from extracts of a mutant of Escherichia coli K-12 which has constitutively derepressed levels of the enzyme. Three fractionation steps were used including controlled heat denaturation, DEAE-Sephadex chromatography, and blue dextran-Sepharose affinity chromatography. The purified enzyme migrated as a single band, coincident with dehydrogenase activity, when electrophoresed on polyacrylamide gels at pH 8.0 and 9.5. Electrophoresis in 1% sodium dodecyl sulfate also showed one band and a single schlieren peak was seen during sedimentation velocity centrifugation. The enzyme has an apparent molecular weight of 140,000 +/- 4,000 as determined by sucrose density and sedimentation equilibrium centrifugation. Based on electrophoresis in 1% sodium dodecyl sulfate, sedimentation equilibrium centrifugation in 6 M guanidine.HCl, and cross-linking with dimethyl suberimidate, the molecule is a tetramer consisting of identical (or nearly identical) subunits with Mr approximately equal to 35,000. L-Threonine dehydrogenase is specific for NAD+ or NAD+ analogs and utilizes L-threonine, D-allothreonine, or L-threonine amide as the best substrates. In 50 mM Tris.HCl buffer (pH 8.4) and 37 degrees C, the Km values for L-threonine and NAD+ are 1.43 and 0.19 mM, respectively. The enzyme has a pH optimum of 10.3, is activated by Mn2+, and shows a substantial loss of activity when treated with certain sulfhydryl-reacting reagents.     

Drug-induced pseudolymphoma and hypersensitivity syndrome. Two different clinical entities

OBJECTIVE: To test the hypothesis that drug-induced pseudolymphoma and hypersensitivity syndrome are 2 distinct clinical entities. DESIGN: Retrospective study from 1980 to 1993. SETTING: Departments of dermatology and medicine of 5 referral universitary hospitals. PATIENTS: Twenty-four patients who met arbitrary criteria selected as being suggestive of lymphoma, with probable drug cause. Patients with other definite cutaneous drug-induced eruptions were excluded. INTERVENTION: None. MAIN OUTCOME MEASURES: Suspect drugs; clinical, biological, and pathological findings; and evolution of each case and of 110 published case reports. RESULTS: Two groups were separated according to their mode of onset and clinical aspect. Three patients (and 15 cases in the literature) had subacute papulonodular or infiltrated plaques, without visceral involvement. Skin biopsy specimens showed a dense lymphocytic infiltrate mimicking lymphoma. Healing was constant when the drug was stopped. The 21 remaining patients (and 95 published cases) had an acute widespread eruption, with fever, enlarged lymph nodes, and multivisceral involvement. Lymphocytosis, atypical lymphocytes, eosinophilia, hepatitis, and high levels of lactate dehydrogenase were frequent. Skin biopsy findings were usually not specific (lymphocytic infiltrate and keratinocyte necrosis) but sometimes mimicked lymphoma. Severe forms and relapses occurred, even after the drug was stopped. The inducing drugs were the same in the 2 groups. CONCLUSIONS: These 2 groups correspond to drug-induced pseudolymphoma and hypersensitivity syndrome. We think that they are 2 distinct entities with different clinical and biological features and outcome, even if the pathological findings are sometimes similar. Prospective studies are needed to confirm these facts, to evaluate the therapy, and to follow up patients.     

Semantic differential placement of attributions and dimensions in four different groups.

Validated the classifications of 50 undergraduate education majors, 50 graduate education majors, 50 undergraduate psychology students, and 50 naive Ss (over 35 yrs of age) of 11 attributions (mood, skill, knowledge, chance, effort, competence, help, ability, task, bias, luck) according to dimensions of locus, stability, controllability, predictability, and globality. Attributions were placed on separate pages of a questionnaire, followed by a random ordering of the 5 dimensions presented on 7-point scales. Results show that Ss' dimensional assignment of 5 of the original 8 attributions of B. Weiner (see record 1979-28688-001) differed significantly from Weiner's assignment of those attributions. In 62% of all possible instances, the results of the present study were in agreement with the theoretical assignments of attributions from the literature. (19 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wild-type and mutant enzymes

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha is a protein with two different functions. We have overexpressed and purified the human wild-type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768-774]. The dehydratase behaves as a tetramer on gel filtration, while cross-linking experiments showed mono-, di-, tri-, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl-modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild-type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay approximately 10-fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihydropterin-4a-carbinolamine approximately 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved three-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032-2042].     

Epidemiology of trauma: Frequency and impact of different potentially traumatic events on different demographic groups.

Examined the frequency and impact of 10 potentially traumatic events in a sample of 1,000 adults. Drawn from 4 southeastern cities, the sample was half Black, half White, half male, half female, and evenly divided among younger, middle-aged, and older adults. Over their lifetimes, 69% of the sample experienced at least 1 of the events, as did 21% in the past year alone. The 10 events varied in importance, with tragic death occurring most often, sexual assault yielding the highest rate of posttraumatic stress disorder (PTSD), and motor vehicle crash presenting the most adverse combination of frequency and impact. Numerous differences were observed in the epidemiology of these events across demographic groups. Lifetime exposure was higher among Whites and men than among Blacks and women; past-year exposure was highest among younger adults. When impact was analyzed as a continuous variable (perceived stress), Black men appeared to be most vulnerable to the effects of events, but young people showed the highest rates of PTSD. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Remembering and knowing: Two different expressions of declarative memory.

Amnesic patients and a control group were given a recognition test 10 min after studying words. For each recognized word, participants indicated whether they remembered it (R) or whether simply they knew that the word was presented but had no recollections about it (K). The patients were impaired for both R and K responses, performing like a control group tested after 1 week. Another control group was tested both 10 min and 1 week after study. The proportion of words initially eliciting an R response and later eliciting a K response exceeded the proportion of K responses that shifted to R responses. These data are accounted for if items initially eliciting R responses can also elicit K responses. We conclude that the R–K distinction does not reflect the operation of explicit and implicit memory but reflects instead a distinction within declarative memory. Thus, K responses depend on brain structures damaged in amnesia; R responses depend on these same structures and also on the frontal lobes for contextual information. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

188Re- and 99mTc-MAG3 as prosthetic groups for labeling amines and peptides: approaches with pre- and postconjugate labeling

Either radiolabeled Tc-99m- or Re-188-labeled MAG3-4-nitrophenylester or unlabeled Bz-MAG3-4-nitrophenylester was reacted with amines and peptides to follow a pre- or a postconjugate radiolabeling route, respectively. The model compounds were N'-t-butyloxycarbonyl-1,6-diaminohexane (DH-Boc) and a Lys-protected derivative of the somatostatin analog RC-160 (cyclic D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2). In the case of labeling DH-Boc, both the preconjugate labeling and the postconjugate labeling were found by using analytical HPLC to provide identical radiolabeled compounds regardless whether Re-188 or Tc-99m was used. The results are supported by infrared and mass-spectral data obtained from compounds synthesized using stable rhenium. The 188Re- or 99mTc-MAG3-RC-160 somatostatin analog were synthesized following the preconjugate labeling route and subsequent removal of the protecting group. Biodistributions of 188Re-and 99mTc-MAG3-RC-160 were evaluated in normal and tumor-bearing mice, and were similar to those of radioiodinated 131-RC-160. All radiolabeled analogs of RC-160 were rapidly cleared from the blood and were excreted through the hepatobiliary system with very little normal organ uptake. The tumor uptake (PC-3, human prostate adenocarcinoma) of systemically administered Re-188-MAG3-RC160 was very low, and it reached only 0.28% injected dose/g (%IDg) at 24 h postinjection, similar to what was obtained with I-131-RC-160. Intratumor injections resulted in significant tumor retentions (9.3% ID/g at 24 h).     

Enterococci with acquired vancomycin resistance in pigs and chickens of different age groups

Results of isolation of glycopeptide-resistant enterococci from fecal samples of pigs and chickens were found to differ strongly depending upon the type and age of animals and isolation technique (direct selective plate or broth enrichment). Isolations were frequent in broiler chickens and in sows but rare in layer chickens.     

Two siblings of type 3 GM1 gangliosidosis with different clinical features and different ages of onset

We experienced two siblings of type 3 GM1 gangliosidosis. A 33-year-old woman developed dysarthria, dysbasia and bradykinesia at around the age of 30. Her 28-year-old brother showed locomotor retardation and skeletal deformity in infancy. He lost the ability to stand walk at childhood, and developed progressive dystonia. The major neurologic manifestations were parkinsonian symptoms in the elder sister, and progressive dystonia in her brother. Both had markedly reduced beta-galactosidase activity in peripheral blood lymphocyte and were diagnosed as having type 3 GM1 gangliosidosis. Gene analysis revealed that these patients were homozygotes of the adult type mutant gene. The two siblings are unique in that the clinical manifestations and the age of onset of symptoms differed markedly between them despite the same mutant gene in both cases.     

Performance of different diagnostic groups on the tachistoscopic and copy phases of the Bender-Gestalt.

Neurotics, alcoholics, organics, and controls (25 Ss in each group) were administered the Bender designs by tachistoscopic exposure (tach phase) and by the standard copying technique (copy phase). Records were scored by blind analysis with the Pascal and Suttell criteria. The copy phase discriminated the diagnostic groups from the controls at the .01 level, but did not discriminate the diagnostic groups from each other. On the tach phase, control Ss were discriminated from alcoholics and organics but not from neurotics; however, the scores of the neurotic group differed from the organic group at the .02 level. (18 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Analytic methods for modeling longitudinal data from rolling therapy groups with membership turnover.

Interventions for a variety of emotional and behavioral problems are commonly delivered in the context of treatment groups, with many using rolling admission to sustain membership (i.e., admission, dropout, and discharge from group are perpetual and ongoing). The authors present an overview of the analytic challenges inherent in rolling group data and outline commonly used (but flawed) analytic and design approaches to addressing (or sidestepping) these issues. Moreover, the authors propose use of latent class pattern mixture models (LCPMMs) as a statistically and conceptually defensible approach for modeling treatment data from rolling groups. The LCPMM approach is illustrated with rolling group data from a group-based alcoholism pilot treatment trial (N = 128). Different inferences were made with regard to treatment efficacy under LCPMM vs. the commonly used standard group-clustered latent growth model (LGM); coupled with other preliminary findings in this area, inferences from LGMs may be overly liberal when applied to data from rolling groups. Continued work on data analytic difficulties in groups with membership turnover is critical for furthering the ecological validity of research on behavioral treatments. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Cytosolic enzymes with a mitochondrial ancestry from the anaerobic chytrid Piromyces sp. E2

The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen-producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N-terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria-bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N-terminal leader sequences.     

Comparison of work groups with stable and unstable membership.

Compared 151 employees in work groups with unstable membership to 582 employees in similar groups with stable membership. Contrary predictions concerning the effects of unstable membership were derived from the R. Likert and the W. G. Bennis conceptions of organization structure. Employees in unstable work groups reported less within-group peer leadership, but more favorable organizational context. Contrary to the prediction derived from Likert's theory, there were no differences on measures of group-referrent intervening variables and end-result variables, suggesting the viability of organic-adaptive structure described by Bennis. Increased lateral linkage among work groups of unstable membership is suggested as the mechanism that appears to compensate for the decreased peer leadership evident in unstable work groups. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evaluation of enzyme-linked immunosorbent assay for the diagnosis of Helicobacter pylori infection in children from different age groups with and without duodenal ulcer

BACKGROUND: Myocardial ischemia induced by 5-fluorouracil (5-FU) is a relatively rare, but potentially serious, occurrence. Some case reports have indicated that recurrent ischemia may be prevented if 5-FU is resumed after pretreatment with antianginal therapy. METHODS: A 54-year old woman was diagnosed with stage IIA squamous cell carcinoma of the anus. Treatment with concurrent radiation and chemotherapy (mitomycin-C and 5-FU) was initiated with curative intent. RESULTS: The patient had no evidence of underlying coronary artery disease based on history, physical examination or ECG. Approximately 48 h after initiation of 5-FU infusion the patient developed anginal pain associated with ECG changes compatible with ischemia. After resolution of ischemia and ruling out of myocardial infarction, coronary arteriography demonstrated normal coronary arteries. In an attempt to prevent myocardial ischemia, calcium channel blocker and nitrate therapy was started, but anginal pain with ECG change recurred when 5-FU was resumed. This necessitated selection of an alternative chemotherapy regimen. CONCLUSIONS: Even in the presence of normal coronary arteries, antianginal therapy may not preclude the occurrence of potentially serious 5-FU induced myocardial ischemia. For patients who experience 5-FU-induced myocardial ischemia, development of alternative chemotherapy regimens should be considered.     

Properties of free and bound glycolytic enzymes from scorpionfish brain. II. Lactate dehydrogenase

It is known that a part of lactate dehydrogenase in animal tissues is bound to the structural elements. However, if in the tissues of higher animals this part is insignificant, in fish tissues it can make tens of percent. The work is the continuation of a series of experiments on the comparative characteristic of the free and bound glycolytic enzymes in fish tissues, in this case-brain of the sea teleost scorpion fish. It was found that the sensitivity of LDH activity to different pH values and to the inhibition by high pyruvate concentrations were different for the both forms. The enzyme affinity for pyruvate and NADH was 4-6 times lower in free enzyme. Possible physiological significance of bound LDH form existence in the energy supply of the brain is discussed.     

Purification and properties of two fibrinolytic enzymes from Fusarium oxysporum N.R.C.1

OBJECTIVE: To find differences in effect on sperm motility of agents that increase intracellular cAMP: manganese ion, methyl-isobutyl-xanthine (MIX), 2-deoxyadenosine, glucose, and Mn-MIX and Mn-glucose. DESIGN: Nine men with asthenozoospermia vs. fertile donors. METHODS: Sperm was washed in Hepes-buffered saline, motility tested by laser-Doppler technique. RESULTS: Best activation was obtained with Mn and 2-deoxyadenosine; generally poor response to MIX or glucose. CONCLUSIONS: Usually, poor endogenous stimulation of adenylyl cyclase, and probably not limited energy supply, is the cause of impaired motility.