Activation of thiamin diphosphate and FAD in the phosphatedependent pyruvate oxidase from Lactobacillus plantarum

The phosphate- and oxygen-dependent pyruvate oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10(-2) s-1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s-1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in pyruvate oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-pyruvate oxidase ternary complex requires a second step, which was detected at low temperature.     

Activation of the alternative complement pathway and production of factor H by skeletal myotubes

Recent negative findings regarding differences in blood pressure (BP) and heart rate (HR) reactivity at work between Type A and Type B workers may stem from the failure to note the stressors to which they were exposed. In this naturalistic study, an objective work stressor--varying levels of ambient noise--was individually monitored among 123 normotensive males. We examined the relationship of Type A behavior (assessed by the Thurstone Temperament Schedule) to ambulatory BP and HR reactivity under high and low noise conditions. We also tested the hypothesized mediating function of tension arousal. Results of multiple regression analyses indicated that when workers were exposed to high noise levels (> or = 80 dB(A)), Type A behavior was positively related to diastolic BP (p = 0.029) and HR reactivity (p = 0.0001), even after adjusting for clinic values, time of measurement, worker activities, and body position during measurement. When the same workers were exposed to low noise levels, the results were not significant. This suggests that noise constitutes a stressor for Type A individuals, and highlights the importance of noting the actual stressor-exposure condition. In addition, Type A individuals reported high tension, noted concurrently with the ambulatory measurements. Tension arousal was also related to BP and HR reactivity. Adding the tension variable to the above regressions weakened the relationship between Type A behavior and BP and HR reactivity. Thus, the tension experienced by Type A workers exposed to noise stress may have served as a mediator of cardiovascular reactivity.     

Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria

Import of the synthetic precursor of the alternative oxidase from soybean was shown to be dependent on a membrane potential and ATP. The membrane potential in soybean mitochondria may be formed either by respiration through the cytochrome pathway, or through the alternative oxidase pathway with NAD(+)-linked substrates. Import of the alternative oxidase precursor in the presence of succinate as respiratory substrate was inhibited by KCN. Import in the presence of malate was insensitive to KCN and SHAM added separately, but was inhibited by KCN and SHAM added together (inhibitors of the cytochrome and alternative oxidases respectively). Import of the alternative oxidase was accompanied by processing of the precursor to a single 32 kDa product in both cotyledon and root mitochondria. This product had a different mobility than the two alternative oxidase bands detected by immunological means (34 and 36 kDa), suggesting that the enzyme had been modified in situ. When the cDNA clone of the alternative oxidase was modified by a single mutation (-2 Arg changed to -2 Gly), the processing of the precursor was inhibited.     

Immunological identification of the alternative oxidase of Acanthamoeba castellanii mitochondria

The exchange energy in the long-range near-dissociation region is analyzed for the ground electronic states of homo- and heteronuclear alkali diatomic molecules. The analysis is based on the use of the functional form Ae-ar to represent this exchange term. A period effect is observed in the values of both parameters and the exponent parameter a appears to approach an asymptotic limit for the heavier molecules. The results obtained in this study also suggest that some care must be taken in using even this function to represent the exchange energy. Copyright 1997 Academic Press. Copyright 1997Academic Press     

Activation of the alternative pathway of human complement by apoptotic human umbilical vein endothelial cells

Complement activation generally does not occur on homologous cells. We observed C3 deposition on cultured human umbilical vein endothelial cells (HUVEC) when those which had died of apoptosis were treated with human serum. The C3 deposition on apoptotic HUVEC required Mg2+ but not Ca2+. In addition, the incubation of apoptotic HUVEC with purified C3, B, and D in the presence of Mg2+ resulted in C3 deposition. These results indicated that the C3 deposition on apoptotic HUVEC is mediated by the activation of the alternative complement pathway. C3 contains an intrachain thioester bond in the alpha chain (110 kDa) and upon activation to C3b, binds with membrane molecules by forming an ester or amide bond. Western blotting of reduced C3b-membrane molecule complexes, isolated from serum-treated apoptotic HUVEC by immunoprecipitation with anti-C3, revealed the covalent binding of C3b to several membrane molecules. Most of the C3b-membrane molecule complexes were cleaved by hydroxylamine, suggesting covalent binding via an ester bond. The molecular mass of the major alpha chain fragment released by hydroxylamine treatment was not 105 kDa but 68 kDa, which corresponds to the alpha 1 fragment of iC3b. These results indicate that most of the C3b on HUVEC was cleaved at its alpha' chain to yield iC3b, which consists of three disulfide-linked polypeptide chains and is a ligand of the complement receptor type 3 (CR3) of phagocytes. These results suggest that apoptotic HUVEC can activate the alternative pathway of the homologous complement and that the complement is related to the clearance of apoptotic cells by phagocytes.     

Cloning and molecular characterization of plant aldehyde oxidase

Primary structural information of a plant aldehyde oxidase (AO), which was purified from maize coleoptiles using indole-3-acetaldehyde as a substrate, was obtained by sequencing a series of cleavage peptides, permitting the cloning of the corresponding cDNA (zmAO-1). The complete nucleotide sequence was determined; the deduced amino acid sequence encodes a protein of 1358 amino acid residues of Mr 146,681, which is consistent with the size of the AO monomeric subunit. There is a significant similarity with AO from mammals and xanthine dehydrogenases from various sources. The maize AO polypeptide contains consensus sequences for iron-sulfur centers and a putative molybdopterin cofactor-binding domain. In addition, another cDNA (zmAO-2), highly homologous to zmAO-1 at both the nucleotide and amino acid sequence levels, was cloned. zmAO-2 would encode a protein of 1349 amino acid residues of Mr 145,173 and has molecular characteristics similar to those of zmAO-1. zmAO-1 was expressed at a high level in roots, which was closely correlated with immunoblotting results using antiserum raised against the purified maize AO protein, whereas zmAO-2 was expressed at a higher level in coleoptiles than in roots. We propose each zmAO may have a unique function during plant development.     

Activation of homologous complement via the alternative complement pathway by quail cells transformed by Rous sarcoma virus

After incubation of the cells with fresh quail serum, deposition of the third component of complement (C3) was demonstrated on the cell surface of various quail cell lines transformed by Rous sarcoma virus (RSV) as well as on that of primary quail embryo (QE) cells transformed by RSV. The C3 deposition occurred irrespective of virus production. On the other hand, the C-3 deposition was not observed on two quail cell lines transformed by a chemical carcinogen, QE cells infected with avian leukosis virus or normal QE cells. Moreover, QE cells infected with a temperature-sensitive mutant of RSV activated the complement at 37 degrees C but not at 41 degrees C. Since the progeny virus was generated even at 41 degrees C, viral molecules on the cell surface may not play an essential role for the activation. The activation of complement was blocked by EDTA but not by EGTA-Mg++. Therefore, the complement activation on the transformed cells appears to be mediated via the alternative complement pathway (ACP). Similar results were obtained with the complement consumption test; the residual cytolytic activity of fresh quail serum via ACP was markedly reduced by pre-incubation of the serum with transformed cells.     

Regulation of the cyanide-resistant alternative oxidase of plant mitochondria. Identification of the cysteine residue involved in alpha-keto acid stimulation and intersubunit disulfide bond formation

The cyanide-resistant alternative oxidase of plant mitochondria is a homodimeric protein whose activity can be regulated by a redox-sensitive intersubunit sulfhydryl/disulfide system and by alpha-keto acids. After determining that the Arabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesis of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala. Using diamide oxidation and chemical cross-linking of the protein expressed in Escherichia coli, Cys-78 was shown to be: 1) the Cys residue involved in the sulfhydryl/disulfide system; and 2) not required for subunit dimerization. The C128A mutant was stimulated by pyruvate, while the C78A mutant protein had little activity and displayed no stimulation by pyruvate. Mutating Cys-78 to Glu produced an active enzyme which was insensitive to pyruvate, consistent with alpha-keto acid activation occurring through a thiohemiacetal. These results indicate that Cys-78 serves as both the regulatory sulfhydryl/disulfide and the site of activation by alpha-keto acids. In light of these results, the previously observed effects of sulfhydryl reagents on the alternative oxidase of isolated soybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of alpha-keto acid activation and of the regulatory sulfhydryl/disulfide system.     

Motivation levels and the marihuana high.

Assigned randomly 112 male 18–30 yr olds to 8 groups, with 14 Ss per group. For each of 2 drug conditions (no drug and marihuana), there were 4 motivation conditions. In the 1st motivation group, the Ss were merely given instructions concerning how to perform on each dependent measure. The 2nd motivation group was given the additional instructions to "try as hard as possible" on each measure. The 3rd motivation group could earn a small amount of money, contingent on the performance of the dependent measures. The 4th group could earn a substantial amount of money contingent upon task performance. Time perception, choice reaction time, and a paired-associate memory task were used as dependent measures. Results indicate a significant, detrimental drug effect on all measures and a significant motivation effect on the reaction time measure. Examination of the data suggests that the drug effect occurred because of the ineffectiveness of the motivation manipulation with the marihuana Ss. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Slot technique--an alternative method of scatter reduction in radiography

The most common method of scatter reduction is the use of an antiscatter grid. Its disadvantage is the absorption of a certain percentage of primary radiation in the lead strips of the grid and the fact that due to the limited thickness of the lead strips their scatter absorption is also limited. A possibility for avoiding this disadvantage is offered by the so-called slot technique, ie, the successive exposure of the subject with a narrow fan beam provided by slots in rather thick lead plates. In this paper the results of a comparison between grid and slot technique regarding dose to the patient, scatter reduction, image quality and the effect of automatic exposure control are reported.     

高压氢还原法制备Ni包SiC粉末的研究

采用高压氢还原法制备了Ni包SiC复合粉末,研究了反应温度、氢分压、表面活性剂及包覆次数对包覆效果的影响,并且对该包覆粉末的热稳定性进行了评价。结果表明:在反应温度为150~160℃、氢分压为1.5~2.5MPa、活性剂的质量浓度为0.05g/L、搅拌速度为800r/min的条件下,包覆二次可以获得连续均匀的包覆层。包覆粉末在1 150℃左右会发生反应,生成Ni2Si和C。     

高还原势气氛下烧结矿低温还原粉化试验研究

在高还原势气氛下进行烧结矿的低温还原粉化试验,考察RDI＋6.3、RDI＋3.15和RDI-0.5的变化情况,分析不同还原气氛对烧结矿低温还原粉化的影响规律,探究烧结矿在氧气高炉上部的粉化行为.结果表明：烧结矿在氧气高炉气氛下的低温还原粉化率高于在传统高炉气氛下的低温还原粉化率,但其RDI＋3.15仍高达82.7%;随着还原气氛中CO和H2含量的提高,烧结矿的低温还原粉化率明显上升.