The role of copper in topa quinone biogenesis and catalysis, as probed by azide inhibition of a copper amine oxidase from yeast

All known copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanine quinone (TPQ) as a redox cofactor. TPQ is derived posttranslationally from a specific tyrosine residue within the protein itself, and is utilized by the enzyme to oxidize amines to aldehydes. Several oxidative mechanisms for both turnover and the biogenesis of the cofactor have been proposed in recent years, which differ mainly in the nature of the interaction of oxygen with the enzyme. In this study, azide is used to probe the role of copper in catalysis and biogenesis, especially with respect to potential interactions between the metal and oxygen. During turnover, it is found that azide is a noncompetitive inhibitor with respect to O(2), most consistent mechanistically with oxygen binding off the metal prior to reaction. During biogenesis, it is found that azide likely prohibits ligation of the precursor tyrosine to the copper, thus preventing the formation of this key intermediate. This result is consistent with previous proposals, where the copper-tyrosine unit is the species that undergoes reaction with O(2). In addition, it is found that oxygen consumption is kinetically uncoupled from TPQ formation; this leads to an expanded kinetic model for biogenesis, with important implications for previous results.     

Fabry disease: recent advances in pathology, diagnosis, treatment and monitoring

Patent arterial duct (PAD) is a congenital heart abnormality defined as persistent patency in term infants older than three months. Isolated PAD is found in around 1 in 2000 full term infants. A higher prevalence is found in preterm infants, especially those with low birth weight. The female to male ratio is 2:1. Most patients are asymptomatic when the duct is small. With a moderate-to-large duct, a characteristic continuous heart murmur (loudest in the left upper chest or infraclavicular area) is typical. The precordium may be hyperactive and peripheral pulses are bounding with a wide pulse pressure. Tachycardia, exertional dyspnoea, laboured breathing, fatigue or poor growth are common. Large shunts may lead to failure to thrive, recurrent infection of the upper respiratory tract and congestive heart failure. In the majority of cases of PAD there is no identifiable cause. Persistence of the duct is associated with chromosomal aberrations, asphyxia at birth, birth at high altitude and congenital rubella. Occasional cases are associated with specific genetic defects (trisomy 21 and 18, and the Rubinstein-Taybi and CHARGE syndromes). Familial occurrence of PAD is uncommon and the usual mechanism of inheritance is considered to be polygenic with a recurrence risk of 3%. Rare families with isolated PAD have been described in which the mode of inheritance appears to be dominant or recessive. Familial incidence of PAD has also been linked to Char syndrome, familial thoracic aortic aneurysm/dissection associated with patent arterial duct, and familial patent arterial duct and bicuspid aortic valve associated with hand abnormalities. Diagnosis is based on clinical examination and confirmed with transthoracic echocardiography. Assessment of ductal blood flow can be made using colour flow mapping and pulsed wave Doppler. Antenatal diagnosis is not possible, as PAD is a normal structure during antenatal life. Conditions with signs and symptoms of pulmonary overcirculation secondary to a left-to-right shunt must be excluded. Coronary, systemic and pulmonary arteriovenous fistula, peripheral pulmonary stenosis and ventricular septal defect with aortic regurgitation and collateral vessels must be differentiated from PAD on echocardiogram. In preterm infants with symptomatic heart failure secondary to PAD, treatment may be achieved by surgical ligation or with medical therapy blocking prostaglandin synthesis (indomethacin or ibuprofen). Transcatheter closure of the duct is usually indicated in older children. PAD in preterm and low birth weight infants is associated with significant co-morbidity and mortality due to haemodynamic instability. Asymptomatic patients with a small duct have a normal vital prognosis but have a lifetime risk of endocarditis. Patients with moderate-to-large ducts with significant haemodynamic alterations may develop irreversible changes to pulmonary vascularity and pulmonary hypertension.     

Fluctuation and linkage relations in macromolecular solution

It is shown in the appendix that the derivatives of the excess free energy of a macromolecule in solution, with respect to the activities of other solution components, lead to fluctuation and linkage relations among these other components. Solution fluctuation theory is used, but it is specialized to the fluctuations and correlations associated with the presence of a macromolecule, and is developed with a modified ensemble. The relations of the appendix are used to analyze the interaction of two solution components, A and B, with the macromolecule and with one another. Three cases are considered: (1) A and B are ligands that bind stoichiometrically to the macromolecule. This case reduces to Wyman's binding polynomial analysis. (2) A and B are two substances at high concentration that interact selectively with the macromolecule. (3) A is a species that binds stoichiometrically to the macromolecule, while B is a component at high concentration that interacts weakly with the macromolecule.     

Application of a non-hazardous vital dye for cell counting with automated cell counters

Recent advances in automated cell counters enable us to count cells more easily with consistency. However, the wide use of the traditional vital dye trypan blue (TB) raises environmental and health concerns due to its potential teratogenic effects. To avoid this chemical hazard, it is of importance to introduce an alternative non-hazardous vital dye that is compatible with automated cell counters. Erythrosin B (EB) is a vital dye that is impermeable to biological membranes and is used as a food additive. Similarly to TB, EB stains only nonviable cells with disintegrated membranes. However, EB is less popular than TB and is seldom used with automated cell counters. We found that cell counting accuracy with EB was comparable to that with TB. EB was found to be an effective dye for accurate counting of cells with different viabilities across three different automated cell counters. In contrast to TB, EB was less toxic to cultured HL-60 cells during the cell counting process. These results indicate that replacing TB with EB for use with automated cell counters will significantly reduce the hazardous risk while producing comparable results.     

Changes in mammary uptake and metabolic fate of glucose with once-daily milking and feed restriction in dairy cows

The aim of this review is to better understand the regulation of milk yield in response to once-daily milking and feed restriction. Glucose is the principal precursor for the synthesis of lactose (a major osmotic agent in milk), and participates in determining the milk volume produced. When applying these two breeding factors, reductions in milk yield are associated with a reduction in milk lactose yield and in the arterial flow of glucose, due to a decrease in the mammary blood flow. The ability of the udder to extract glucose is altered with once-daily milking but not necessarily with feed restriction. Lactose synthesis is down-regulated in response to once-daily milking and feed restriction but the percentage of the extracted glucose which is converted into lactose is differently affected in response to treatments. No marked change is observed with once daily milking whereas this would be increased with feed restriction and in contrast, depressed with fasting.     

The gene 1.2 protein of bacteriophage T7 interacts with the Escherichia coli dGTP triphosphohydrolase to form a GTP-binding protein

Escherichia coli encodes a dGTP triphosphohydrolase (dGTPase) that cleaves dGTP to deoxyguanosine and tripolyphosphate. dGTP is hydrolyzed with a Michaelis constant (Km) of 5 microM and a maximal velocity (Vmax) of 1.8 mumols/min/mg. The ribonucleotide GTP is a poor substrate with a much lower affinity. It is hydrolyzed with a Km of 150 microM and Vmax of 0.07 mumols/min/mg. Bacteriophage T7 encodes a specific inhibitor of dGTPase, the gene 1.2 protein, that forms a tight complex with the enzyme. The enzyme-inhibitor complex binds dGTP with a dissociation constant (KD) of 1.5 microM, but the bound dGTP is not hydrolyzed. It remains stably bound to the complex with a half-life of approximately 5 min. In contrast, dGTP is unable to bind to gene 1.2 protein alone, and dGTP bound to dGTPase alone is quickly hydrolyzed and released. Surprisingly, the dGTPase-gene 1.2 protein complex has a higher affinity for GTP than for dGTP. GTP is stably bound to the dGTPase-gene 1.2 protein complex with a half-life greater than 30 min and KD of 0.8 microM; GTP is not stably bound to either dGTPase or gene 1.2 protein alone. Both GTP and dGTP bind to and stabilize the dGTPase-gene 1.2 protein complex, inhibiting its dissociation. Although the presence of dGTP induces conformation changes in dGTPase so that it is unable to associate with the gene 1.2 protein, saturating concentrations of GTP have no such effect. The enzyme efficiently associates with its inhibitor in the presence of GTP. These results indicate that E. coli dGTPase and gene 1.2 protein interact to form a high affinity GTP-binding site. dGTP is most effective in preventing the association of the enzyme with the inhibitor whereas GTP is most effective in preventing the dissociation of the enzyme-inhibitor complex.     

A taxonomic study of the Subantarctic Piptoporus (Polyporaceae, Basidiomycetes) II

Morphological and cultural studies of Polyporus maculatissimus and P. portentosus are presented. Neolentiporus gen. nov. is proposed to accommodate P. maculatissimus , a species with a striking macromorphological similarity with the north temperate P. squamosus . The new genus is characterized by medium to large stipitate fruitbodies with a poroid hymenophore, circular to flabellate pilei with a scaly surface and an excentric or lateral stipe that sometimes is reduced to a short, robust umbo. The hyphal system is dimitic with clamped, irregularly thick-walled generative hyphae that do not react with cresyl-blue, and terminal, unbranched, thick-walled skeletal hyphae that are strongly metachromatic in cresyl-blue. Spores are cylindric, hyaline, thin-walled, inamyloid and binucleate. The sexuality is bipolar, the nuclear behavior is astatocoeno-cytic and the associated wood-rot is brown. The new genus is regarded as the poroid counterpart of the agaricoid Neolentinus. Polyporus portenrosus is included in Laetiporus on the basis of its yellowish fruitbodies, its soft, punky context, its hyphal system composed of simple septate generative hyphae and binding hyphae, its holocoenocytic nuclear behavior and its associated brown wood-rot. A new code symbol, i.e. '9s' is proposed to codify the presence, in cultures, of simple-septate generative hyphae with irregularly thickened walls.     

Uncommon pneumonia associated with acute poststreptococcal glomerulonephritis

184 cases of acute poststreptococcal glomerulonephritis were investigated and six of these were associated with a peculiar, uncommon pneumonia, and another one had a lethal course. The clinicoradiological and especially pathological data summarized in this study attempt to demonstrate the individuality of this type of pneumonia. Pneumonia associated with acute poststreptococcal glomerulonephritis is similar or identical to rheumatic pneumonia. In both entities, pneumonia and pulmonary edema may and do coexist, and the differentiation of pneumonia from congestive heart failure is difficult and often impossible without pathological evidence. The most attractive pathogenic interpretation is the hypothesis of an immune mechanism in the induction of pneumonia. Authors attributed the lung changes, consecutive to a vascular damage, to a hypersensitivity phenomenon, with accumulation of fibrinogen in alveoli where it is converted to fibrin. Then, the hyaline membrane lining the alveoli, an important feature of pneumonia associated with glomerulonephritis is built up. It is, however, plausible that the effect of the immune reaction is associated with those of hydrosaline retention, arterial hypertension and congestive heart failure. Analogous to rheumatic pneumonia, the "peculiar pneumonia" associated with acute poststreptococcal glomerulonephritis should be named nephritic pneumonia.     

Reversible reaction of cyanate with a reactive sulfhydryl group at the glutamine binding site of carbamyl phosphate synthetase.

Carbamyl phosphate synthetase from Escherichia coli reacts stoichiometrically (one to one) with [14C]cyanate to give a 14C-labeled complex which can be isolated by gel filtration. The formation of the complex is prevented if L-glutamine is present or if the enzyme is first reacted with 2-amino-4-oxo-5-chloropentanoic acid, a chloro ketone analog of glutamine which has been shown to react with a specific SH group in the glutamine binding site. The rate of complex formation is increased by ADP and decreased by ATP and HCO3-. The isolated complex is inactive with respect to glutamine-dependent synthetase activity. However, the reaction of cyanate with the enzyme is reversible. The rate of dissociation of the isolated complex is not affected by pH (over the pH range 6-10), is greatly increased by ATP and HCO3-, and is decreased by ADP. The allosteric effectors ornithine and UMP have no effect on either the rate of formation or the rate of dissociation of the complex; however, the apparent affinity of the enzyme for ATP is decreased by UMP and increased by ornithine. The site of reaction of cyanate with carbamyl phosphate synthetase, which is composed of a light and a heavy subunit, is with an SH group in the light subunit to give an S-carbamylcysteine residue. The binding of L-[14C]glutamine to the enzyme and the inhibition of glutamine-dependent synthetase activity by the chloroketone analog are both prevented by the presence of cyanate. The reaction with cyanate is considered to be with the same essential SH group which is located in the glutamine binding site and is alkylated by 2-amino-4-oxo-5-chloropentanoic acid. The bicarbonate-dependent effects of ATP suggest that formation of the activated carbon dioxide intermediate is accompanied by changes in the heavy subunit which functionally alter the properties of the glutamine binding site on the light subunit. The allosteric effects of ornithine and UMP are probably not related to this intersubunit interaction.     

广西地区2型糖尿病患者直接卫生费用研究

目的分析广西地区近8年2型糖尿病患者的住院费用情况。方法收集广西医科大学第一附属医院2004年4月~2011年12月出院的2型糖尿病患者的病案资料,按有无并发症分为无并发症组2050例,有并发症组4795例,用两样本比较的秩和检验比较两组住院费用的差异。按并发症个数将有并发症组分为3个亚组,即B组（1个并发症组2478例）、C组（2个并发症1444例）、D组（3个及3个以上并发症组873例）,用多个样本比较Kruskal-Wallis H检验,组间比较用扩增t检验。按有无大血管病变将患者分为2型糖尿病无大血管病变组5711例,2型糖尿病并大血管病变组1134例,采用秩和检验比较两组住院费用的差异。结果 2型糖尿病患者中位住院费用为7009.55元;无并发症组的中位住院费用为5702.2元,有并发症组的中位住院费用为7726.65元;1个并发症组中位住院费用为6999.89元,2个并发症组的中位住院费用为8060.78元,3个及3个以上并发症组中位住院费用为10396.24元;2型糖尿病无大血管病变中位住院费用为6525.84元,2型糖尿病并大血管病变中位住院费用为11215.13元。结论有并发症较无并发症住院费用更高,有大血管病变较无大血管病变的2型糖尿病费用更高,并发症个数越多,费用越高。     

腰椎间盘突出症阶梯式手术治疗方案的临床疗效和患者心理状况调查

目的:探讨阶梯式手术治疗方案对腰椎间盘突出的临床疗效和患者对临床疗效的心理接受情况。方法:回顾性分析2014年至2016年本院骨科确诊为腰椎间盘突出症的50例患者,经椎间孔镜下行髓核摘除微创手术(Percutaneous transforaminal endoscopic discectomy,PTED),术后1天和3个月采用视觉疼痛模拟评分(VAS)和改良Mac Nab疗效评定、术后3个月采用Oswestry功能障碍指数(ODI)评价手术效果,以及相应调查时间点患者对手术效果心理接受程度调查;对于7例微创手术疗效不佳患者进一步采用传统的开窗髓核摘除术(Fenestration discectomy,FD),采用同样方法评价手术效果和调查患者心理接受情况。结果:50例微创术后1天及术后3个月的VAS评分和ODI指数均显著低于术前,差异具有显著性(P均0.05);改良Mac Nab疗效评定:术后1天优良率为84%,术后3个月为86%;患者对术后疗效心理满意度调查:术后1天,满意并接受为84%,不满意但接受为16%,不满意难接受为0;术后3个月,满意并接受为86%,不满意但接受为14%,不满意难接受为0。对于微创术后效果欠佳7例患者行FD手术,术后1天及3个月的VAS评分和ODI指数均显著低于术前,差异具有显著性(P均0.05);改良Mac Nab疗效评定:术后1天优良率为71%,术后3个月为100%;患者对开窗术后疗效心理满意度调查:术后1天,满意并接受为71%,不满意但接受为0,不满意难接受为29%;术后3个月,满意并接受为100%,不满意但接受为0,不满意难接受为0。结论:腰椎间盘突出症阶梯式手术治疗方案临床效果和患者心理接受情况均值得临床推荐。     

BIOASSAY SYSTEMS FOR THE PYRETHRINS

The special properties of pyrethrum assays with crawling insects by the film technique lend themselves to the twin cross-over design, hitherto restricted to assays with graded responses. The conditions under which this design is appropriate, and the treatment of control responses, are discussed. The design is particularly efficient with laboratory cultures of insects with fairly stable tolerance levels, and is also useful with heterogeneous populations collected in the field. Examples of the increased precision afforded are considered. The reversibility of the primary responses of grain weevils to the pyrethrins is also demonstrated.     

Actinomycin D binding to DNA and chromatin: A colorimetric procedure suitable for the analysis of turbid preparations and for simultaneous processing of several samples

A simple, sensitive, and inexpensive method is presented for measuring the binding of actinomycin D (AMD) to either chromatin or DNA. The procedure gives results which are comparable with those obtained by the optical titration procedure, but is superior to that method in that it does not require continuous attention, can be used with turbid chromatin preparations, and allows simultaneous processing of several samples. Chromatin or DNA that has been allowed to react with AMD is subjected to sucrose density gradient centrifugation to remove unbound antibiotic. The AMD-DNA or AMD-chromatin complex is then dissociated by incubation with 5m urea, and the released AMD is extracted with ether and estimated spectrophotometrically.     

Continuum reaction field calculation of dielectric constant and vapor pressures for water and carbon disulfide.

Continuum reaction field theory is applied to calculations of dielectric constant, contribution of intermolecular interactions to the free energy of a liquid, and heat of vaporization. Introduction of repulsive interactions and the use of one adjustable parameter, the free volume, enables prediction of vapor pressures. The calculations are illustrated for a simple nonpolar liquid, carbon disulfide, and for liquid water. It is shown that when Onsager's equation is rearranged to a quadratic equation, and a recently found value of the polarizability is employed, its solutions for liquid water yield good agreement with experimental values throughout the whole temperature range. The decrease of the dielectric constant with temperature is essentially linear with the inverse of absolute temperature, but there is additional significant decrease due to the decrease of density with temperature. The relatively high value of the heat of vaporization of liquid water is expressed in terms of large dipolar interaction of a water molecule with the environment, which is due to polarization effects.     

Ruhemann's purple from ninhydrin,ascorbate, and nitrite

Ruhemann's purple is formed from nitrite, ascorbate, and ninhydrin with the formation of variable amounts of ammonia. The reaction begins with the nitrosation of ninhydrin to form nitrosoninhydrin, which will release nitric oxide. The nitrosoninhydrin is reduced by ascorbate to diketohydrindamine, which then couples with a second molecule of ninhydrin to form the pigment. The concentration of pigment is linear with nitrite over the range 1 to 10 mm, but ammonia production is not stoichiometric with initial nitrite.     

Economics, Biology, and Naturalism: Three Problems Concerning the Question of Individuality

The paper examines the ramifications of naturalism with regard to the question of individuality in economics and biology. Economic theory has to deal with whether households, firms, and states are individuals or are mere entities such as clubs, networks, and coalitions. Biological theory has to deal with the same question with regard to cells, organisms, family packs, and colonies. To wit, the question of individuality in both disciplines involves three separate problems: the metaphysical, phenomenist, and ontological. The metaphysical problem is concerned with purposeful action: Is the firm or organism exclusively the product of efficient causality (optimization) or is it motivated by final causality (purposefulness)? The phenomenist problem is interested in the substantiality of essences: Is the firm's or organism's scheme of institutions/traits deep or is it extraneous to identity? The ontological problem is related to the issue of reductionism: Is the behavior of lower-level organization governed by a pre-constituted entities or is it context-sensitive? The paper finds that theoretical differences run along the naturalist/anti-naturalist divide rather than along disciplinary specialization. Also, the paper finds that it is not inconsistent for the same theorist to be naturalist with regard to one problem and anti-naturalist with respect to the other two problems.     

Identification of Fluorescent-Antibody Labeled Group A Streptococci by Fluorometry

A quantitative system, amenable to automation, for determining the presence of group A streptococci in broth culture is described. After separation from the broth, the cells' protein coats are removed by digestion with 0.1% trypsin, and they are then stained with anti-A fluorescent antibody (FA). Excess FA is removed, and bound FA is put into solution by dissociation with demineralized distilled water. The amount of FA bound to the cells is quantitated by fluorometry of the solution. The level of nonspecific staining is measured by staining the cells with fluorescein-conjugated normal rabbit globulin absorbed with group A cells, dissociating, and quantifying, as above. The two quantities are subtracted to measure specific binding of FA to group A cells. A clinical trial showed 92% agreement with microscopists.     

pH effects in plasmin-catalysed hydrolysis of alpha-N-benzoyl-L-arginine compounds.

The steady-state kinetics of plasmin (EC 3.4.21.7) catalysed reactions with some alpha-N-benzoyl-L-arginine compounds is investigated in the pH range 5.8--9.0. The results are interpreted in terms of a three-step mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. Alpha-N-Benzoyl-L-arginine methyl ester and ethyl ester show the same pH behaviour. The kinetic parameter kc/Km is influenced by two groups with pK values of 6.5 and 8.4, respectively. kc is affected only by the group with pK equal to 6.5 and Km only by the group with pK equal to 8.4. It is suggested that the group with pK equal to 6.5 is the 1-chloro-3-tosyl-amido-7-amino-2-heptanone-sensitive histidine residue in the active site and that the group with pK equal to 8.4 is perhaps the alpha-amino group of the N-terminus in analogy to trypsin and chymotrypsin. alpha-N-Benzoyl-L-arginine amide is not hydrolysed by plasmin, but proves to be a competitive inhibitor, Ki = 12.8 +/- 1.8 mM, pH = 7.8. Also the product alpha-N-benzoyl-L-arginine is a competitive inhibitor, Ki = 26 +/- 3.1 mM, pH = 7.8. Estimates of individual rate constants are compared with similar trypsin data.     

Ponderal indices at birth

At birth, the index weight/height is the highest correlated with weight, height and cephalic perimeter, for the index of Quételet (BMI) the correlations are also significant although lower. In both cases, the variation with age is significant. For the Rohrer index, the situation is different, the changes with age being almost absent. In front of these results, can we decide which index would be adequate to establish the nutritional status at birth? For Cole (1986), for such a ponderal index the correlation with height would have to be absent. Rolland-Cachera (1982) proposed also that the correlations with height would be absent but would be high with weight. In the case of the newborns of our study the only index being not correlated to height is the Rohrer index with low correlation also with the cephalic perimeter and high correlation with weight (although here the correlation is even higher with BMI and W/H). During the utero-placentary disfunction the foetal weight is affected but not height and cephalic perimeter: in this case, the advantage is also to an index correlated with weight but not with height.     

Thioester enolate stabilization in the acyl-CoA dehydrogenases: the effect of 5-deaza-flavin substitution

The redox-inactive thioester analog 3-thia-octanoyl-CoA blocks transfer of a hydride equivalent to the flavin prosthetic group of the medium-chain acyl-CoA dehydrogenase with the accumulation of a stable enolate intermediate not encountered with normal substrates. Substitution of the normal flavin with 5-deaza-FAD would thus be expected to lead to enolate formation with both normal and 3-thia-substrate analogs, because reduction of the 5-deaza-enzyme is thermodynamically highly unfavorable. However, spectrophotometric titrations show that neither ligand forms significant enolate species with the 5-deaza-FAD enzyme. Similarly, the substituted dehydrogenase catalyzes undetectable alpha-proton exchange with octanoyl-CoA and ca. 1% of the corresponding rate with 3-thia-octanoyl-CoA when compared to the native enzyme. This inability to stabilize enolate species is not simply due to impaired binding of CoA-thioester analogs, because binding of a range of ligands is weakened by only 2- to 10-fold with the 5-deaza-enzyme. 4-Thia-trans-2-enoyl-CoA product is polarized normally on binding to the substituted protein, showing that this critical aspect of catalysis is apparently normal. These data, together with studies with CoA-persulfide and acetoacetyl- and p-nitrophenylacetyl-CoA, suggest that 5-deaza-FAD substitution exerts subtle, unanticipated, effects on the reductive half-reaction of the medium-chain acyl-CoA dehydrogenase. The involvement of charge-transfer interactions in the acidification of weakly acidic acyl-CoA thioesters is discussed.