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.     

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.     

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.     

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.     

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.     

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.     

Anti-CTLA-4 monoclonal antibody: a major step in the treatment of metastatic melanoma

The anti-CTLA-4 Mab ipilimumab is an efficient treatment of metastatic melanoma as a single agent and combined with dacarbazine chemotherapy. The benefit is observed in 10 to 20?% of the patients but it is the only drug to demonstrate an increase in overall survival of patients with metastatic melanoma. The pattern of response is new with delayed and prolonged responses over time. New evaluation criteria have been proposed to evaluate the efficacy of this new therapy. The safety profile is new also with frequent and potentially severe side effects related to the activation of the immune system. The challenges are now to identify biomarkers able to predict ipilimumab benefit and to know how to use ipilimumab in combination with new targeted therapies of melanoma in order to optimize the treatment efficacy.     

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.     

Interaction of Cd2+ with the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of human erythrocyte ghosts

Treatment of erythrocyte ghosts with micromolar concentrations of Cd2+ results in a noncompetitive inhibition of the calmodulin-dependent (Ca2+ + Mg2+)-ATPase activity. Higher concentrations of Cd2+ are required for inhibition of the (Ca2+ + Mg2+)-ATPase activity of calmodulin-depleted ghosts. The interaction of Cd2+ is time-dependent with an apparent rate constant around 0.12/min. The inhibition is relieved by addition of EGTA with a rate constant around 0.15/min. If Cd2+ is allowed to interact with calmodulin prior to the association of the protein with the ghosts, the inhibition is mainly competitive. The results suggest that the inhibitory effect caused by Cd2+ is due to an interaction with calmodulin. The slow interaction of Cd2+ suggests that calmodulin bound to the (Ca2+ + Mg2+)-ATPase is inaccessible to Cd2+.     

Fluorimetric microassay of DNA using a modified thiobarbituric acid assay

A method for the rapid, specific measurement of minute amounts of DNA is presented. The method is sensitive to about 20 ng of DNA, which is equivalent to approximately 3000 mammalian cells. The method employs enzymatic liberation of desoxyribose with DNase and phosphodiesterase. The liberated desoxyribose is reacted with thiobarbituric acid to yield a fluorescing compound with an excitation maximum at 532 nm and an emission maximum at 549 nm, measured with a fluorimeter. The entire procedure is accomplished within a few hours. The result is linear up to about 1 μg DNA or 3 × 10⁵ lymphocytes.     

ATP-induced cell contraction in dermal fibroblasts: effects of cAMP and myosin light-chain kinase

When 1 mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol-permeabilized DF with alkaline phosphatase before adding 1 mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light-chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin-dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction.     

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.     

Measures of association between disease and genotype.

This paper is concerned with the statistical aspects of the phenomenon of disease occurring more frequently in individuals with some genotypes than in individuals with others. A correlation coefficient is defined to quantify association between disease and genotype. A distinction is made between the concepts of independence of allele and disease and independence of genotype and disease. This distinction is used to define two components of association which describe separate aspects of association of disease with genotype. One component is a measure of the association of disease with allele; the other a measure of the effect of allele interaction on association of disease and genotype. One aspect of the usefulness of the partition into components which is discussed is in expressing the recurrence risk of disease for a relative of an affected individual. A chi-squared analysis is provided to test hypotheses about the components of association and other hypotheses of genetic interest. This analysis is illustrated using a study done to determine the effect of the sex-linked dwarfing gene in male chickens on resistance to E. coli infection. This analysis shows a significant allele interaction effect on resistance to disease but no association of disease with alleles. In conclusion, some extensions and limitations of the proposed concepts and procedures are discussed.     

The organization of the sarcoplasmic reticulum in teleost ventricular myocardial cells

Summary The morphology of the sarcoplasmic reticulum in myocardial cells of the ventricle of the trout heart is described as the result of an investigation with the electron microscope. The sarcoplasmic reticulum is sparse in distribution compared to that of birds or mammals but shows a fundamentally similar organization. A very loose network of fine tubules is in intimate contact with the myofibrils but with no local modification with respect to the arrangement of myofilaments within the sarcomeres. There is no special association of the sarcoplasmic reticulum with the Z-bands. Some tubules pass to the cell periphery where they expand to form subsarcolemmal cisternae in which electron-dense matter is often seen. The occurrence of the subsarcolemmal cisternae (peripheral couplings) is random and they are not observed in the vicinity of intercalated discs. The sarcoplasmic reticulum is discussed in relation to excitation-contraction coupling in teleost myocardial cells, and in comparison with that of other vertebrates.I am grateful to Professor J. D. Lever for making certain facilities available, and to Mr. P. F. Hire for photographic assistance.     

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.     

The second to fourth digit ratio and variation in the androgen receptor gene

The second to fourth digit ratio (2D:4D) is sexually dimorphic, with lower mean values in males compared to females. It has been suggested that the sex difference in 2D:4D is determined prenatally, 2D:4D is negatively related to prenatal testosterone and positively to prenatal oestrogen, and that 2D:4D is a marker for levels of sex steroids during brain organisation. There is growing evidence that many sex-dependent behaviours are correlated with 2D:4D. However, there is no direct evidence for an effect of prenatal sex steroids on the digit ratio. The response to prenatal testosterone is dependent on the amount produced and the foetal sensitivity to the hormone. Variation in the X-linked androgen receptor gene (AR) determines sensitivity to testosterone. Alleles of AR with low numbers of CAG triplets respond to testosterone with high transactivational activity, while high numbers of CAG's are associated with increased insensitivity to testosterone. We show in a sample of 50 men (49 Caucasian subjects, 1 Caucasian/Chinese subject) that 2D:4D is a phenotypic correlate of AR structure. Right-hand 2D:4D was positively correlated with CAG number and individuals with low 2D:4D in their right hand compared to left hand had AR alleles with low CAG numbers. We discuss the implications of our findings for our understanding of the aetiology of 2D:4D, its relationships with sex-dependent behaviours, and the evolutionary implications of variation in 2D:4D and AR.     

Clinical evaluation of antibiotic therapy of NAG-infection]

It is not concluded yet whether it is expedient to use antibiotic therapy with respect to patients and vibrio-carries with NAG-infection. Observation of a group of patients with acute gastro-intestinal infections caused by NAG-vibrio and carriers of NAG-vibrioes showed that the rate of vibrio isolation after a course of antibiotic therapy (tetracycline, levomycetin) significantly decreased as compared to that in the group of the patients subjected only to symptomatic therapy. The data of the study provided recommendation of antibacterial therapy with respect to patients with NAG-infection especially in cases with accompanying infections or invasions. As for "asymptomic" carriers antibiotic therapy is required only with respect to persons with repeated vibrio isolation.     

Interaction of gene 5 protein of phage f1 with single and double-stranded DNA and polynucleotides

The fluorescence method was used to reveal some differences in the interaction of gene 5 protein of phage f1 with single- and double-stranded polynucleotides (DNA). The binding with the duplexes is non-cooperative and the Kapp is twice lower than that for the cooperative formation of the complex with single-stranded structures. In the complex with a double-stranded polynucleotide (DNA) the protein cover 3 nucleotide pairs. The complex dissociates with a lower concentration of salt and the contribution of the energy of nonelectrostatic interactions to the total energy of complex formation for it is lower than for the complex with single-stranded DNA. In the complex of protein with single-stranded structure the fluorescence of the tyrosine (Tyr) residues is quenched to a greater degree and their accessibility to the external quencher is lower than that of the complex with double-stranded polynucleotides (DNA). The suggestion is made that in destabilization of nucleic double helices by gene 5 protein of phage f1, a great role belongs to Tyr residues because of their high affinity to single-stranded structures and because of their different localization in the complexes with single- and double-stranded polynucleotides.     

Quantitative determination of factor VIII antigen with an enzyme immunoassay

We describe an enzyme-immunoassay for the determination of factor VIII antigen. After representation of the isolation of proteins the enzyme-immunoassay is presented. The principle of the method is the following: Test plasma is mixed with rabbit antibody in excess and incubated at 37 degrees C. The incubation mixture is added to polystyrene tubes, which are coated with human factor VIII. The rabbit antibody is available to adhere to factor VIII coating the tube and can be detected with an enzyme-labeled antibody to rabbit IgG. This method is sensitive to 7.8 . 10(-3) U/ml factor VIII antigen; the variation coefficient is 10.9%.     

Observations on the Kinetics of Uranyl Acetate and Phosphotungstic Acid Staining of Chromatin in thin Sections for Electron Microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO₄) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with gutaraldehyde only, but stain uptake is reduced following a long wash with distilled water to a level similar to that seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO₄ increases UAc uptake by a factor of about 3.

The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO₄; similar levels for final PTA uptake are also observed.

An increase in the resin content of embedded chromatin postfixed with OsO₄ is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.