Biological oxidation of metallic copper by Acidithiobacillus ferrooxidans

This is a report on the kinetic aspects and the analytical study of the bioproducts of the oxidation of zero-valence copper by immobilized Acidithiobacillus ferrooxidans. Two different mechanisms of oxidation were considered: direct and indirect. A custom-built bioreactor was used to grow A. ferrooxidans in an iron-free media, which was required for the study of the direct mechanism. X-ray microdiffraction analysis of the copper after biooxidation in the sulfate-free medium revealed the presence of the copper sulfate, piypite, K(2)Cu(2)O(SO(4))(2), which indicates biooxidation of Cu metal has occurred. It was shown that the direct oxidation exists, but it is relatively slow, as compared to the indirect mechanism.     

The drivers of avian‐haemosporidian prevalence in tropical lowland forests of New Guinea in three dimensions

Haemosporidians are among the most common parasites of birds and often negatively impact host fitness. A multitude of biotic and abiotic factors influence these associations, but the magnitude of these factors can differ by spatial scales (i.e., local, regional and global). Consequently, to better understand global and regional drivers of avian‐haemosporidian associations, it is key to investigate these associations at smaller (local) spatial scales. Thus, here, we explore the effect of abiotic variables (e.g., temperature, forest structure, and anthropogenic disturbances) on haemosporidian prevalence and host–parasite networks on a horizontal spatial scale, comparing four fragmented forests and five localities within a continuous forest in Papua New Guinea. Additionally, we investigate if prevalence and host–parasite networks differ between the canopy and the understory (vertical stratification) in one forest patch. We found that the majority of Haemosporidian infections were caused by the genus Haemoproteus and that avian‐haemosporidian networks were more specialized in continuous forests. At the community level, only forest greenness was negatively associated with Haemoproteus infections, while the effects of abiotic variables on parasite prevalence differed between bird species. Haemoproteus prevalence levels were significantly higher in the canopy, and an opposite trend was observed for Plasmodium. This implies that birds experience distinct parasite pressures depending on the stratum they inhabit, likely driven by vector community differences. These three‐dimensional spatial analyses of avian‐haemosporidians at horizontal and vertical scales suggest that the effect of abiotic variables on haemosporidian infections are species specific, so that factors influencing community‐level infections are primarily driven by host community composition.     

Lysine 219 participates in NADPH specificity in a flavin-containing monooxygenase from Saccharomyces cerevisiae

The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) uses NADPH and O(2) to oxidize thiol containing substrates such as GSH and thereby generates the oxidizing potential for the ER. The enzyme uses NADPH 12 times more efficiently than NADH. Amino acid sequence analysis suggests that Lys 219 and/or Lys 227 may act as counterions to the 2' phosphate of NADPH and to help determine the preference for pyridine nucleotides. Site directed mutations show that Lys 219 makes the greater contribution to cosubstrate recognition. Conversion of Lys 219 to Ala reduces NADPH dependent activity 90-fold, but has no effect on NADH-dependent activity. Conversion of Lys 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent activity threefold. Dissociation constants for NADP(+) to oxidized yFMO were measured spectroscopically. K(d) is 12 microM for the wild-type enzyme and 243 microM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding.     

Oligonucleotides in human urine do not contain 8-oxo-7,8-dihydrodeoxyguanosine

The promutagenic DNA modification 8-oxo-7,8-dihydrodeoxyguanosine is the most frequently used marker for oxidative stress to DNA. The unmodified base and nucleoside and the 8-hydroxylated guanine base and nucleoside are found in urine, the latter used as a global measure of oxidative stress to DNA. Nucleotide excision repair (NER) excises a 27- to 29-mer oligonucleotide with oxidative lesions, and if found in urine, it could be used as a measure of DNA repair in vivo. Enzymatic hydrolysis of human urines followed by HPLC-tandem mass spectrometry was not able to reveal oligonucleotides and/or mononucleotides with the 8-oxo-7,8-dihydrodeoxyguanosine modification. The recovery of a synthetic oligonucleotide with the modification was complete (95% confidence limits: 98-124%). These experiments show that oligonucleotides are excreted into urine, but that 8-oxo-7,8-dihydrodeoxyguanosine is found only as the mononucleoside and is not present in any significant amounts in oligonucleotides. We conclude that oligonucleotides are excreted into urine, and they do not contain oxidized lesions. Either NER products are degraded after excision or NER functions differently in vivo in humans compared with cellular systems.     

Influence of oxygen tension on myocardial performance. Evaluation by tissue Doppler imaging

Background

Endothelial function in hypercholesterolemic rabbits is usually evaluated ex vivo on isolated aortic rings. In vivo evaluation requires invasive imaging procedures that cannot be repeated serially.

Aim

We evaluated a non-invasive ultrasound technique to assess early endothelial function in rabbits and compare data with ex vivo measurements.

Methods

Twenty-four rabbits (fed with a cholesterol diet (0.5%) for 2 to 8 weeks) were given progressive infusions of acetylcholine (0.05–0.5 μg/kg/min) and their endothelial function was assessed in vivo by transcutaneous vascular ultrasound of the abdominal aorta. Ex vivo endothelial function was evaluated on isolated aortic rings and compared to in vivo data.

Results

Significant endothelial dysfunction was demonstrated in hypercholesterolemic animals as early as 2 weeks after beginning the cholesterol diet (aortic cross-sectional area variation: -2.9% vs. +4% for controls, p < 0.05). Unexpectedly, response to acetylcholine at 8 weeks was more variable. Endothelial function improved in 5 rabbits while 2 rabbits regained a normal endothelial function. These data corroborated well with ex vivo results.

Conclusion

Endothelial function can be evaluated non-invasively in vivo by transcutaneous vascular ultrasound of the abdominal aorta in the rabbit and results correlate well with ex vivo data.     

Sensitivity of molecular dynamics simulations to the choice of the X-ray structure used to model an enzymatic reaction

A subject of great practical importance that has not received much attention is the question of the sensitivity of molecular dynamics simulations to the initial X-ray structure used to set up the calculation. We have found two cases in which seemingly similar structures lead to quite different results, and in this article we present a detailed analysis of these cases. The first case is acyl-CoA dehydrogenase, and the chief difference of the two structures is attributed to a slight shift in a backbone carbonyl that causes a key residue (the proton-abstracting base) to be in a bad conformation for reaction. The second case is xylose isomerase, and the chief difference of the two structures appears to be the ligand sphere of a Mg2+ metal cofactor that plays an active role in catalysis.     

LNA guanine and 2,6-diaminopurine. Synthesis, characterization and hybridization properties of LNA 2,6-diaminopurine containing oligonucleotides

LNA guanine and 2,6-diaminopurine (D) phosphoramidites have been synthesized as building blocks for antisense oligonucleotides (ON). The effects of incorporating LNA D into ON were investigated. As expected, LNA D containing ON showed increased affinity towards complementary DNA (Delta Tm +1.6 to +3.0 degrees C) and RNA (Delta Tm +2.6 to +4.6 degrees C) ON. To evaluate if LNA D containing ON have an enhanced mismatch sensitivity compared to their complementary LNA A containing ON thermal denaturation experiments towards singly mismatched DNA and RNA ON were undertaken. Replacing one LNA A residue with LNA D, in fully LNA modified ON, resulted in higher mismatch sensitivity towards DNA ON (Delta Delta Tm -4 to >-17 degrees C). The same trend was observed towards singly mismatched RNA ON (Delta Delta Tm D-a = -8.7 degrees C and D-g = -4.5 degrees C) however, the effect was less clearcut and LNA A showed a better mismatch sensitivity than LNA D towards cytosine (Delta Tm +5.5 degrees C).     

Effects of truncated angiotensins in humans after double blockade of the renin system

Angiotensins different from ANG II exhibit biological activities, possibly mediated via receptors other than ANG II receptors. We studied the effects of 3-h infusions of ANG III, ANG-(1-7), and ANG IV in doses equimolar to physiological amounts of ANG II (3 pmol. kg-1. min-1), in six men on low-sodium diet (30 mmol/day). The subjects were acutely pretreated with canrenoate and captopril to inhibit aldosterone actions and ANG II synthesis, respectively. ANG II infusion increased plasma angiotensin immunoreactivity to 53 +/- 6 pg/ml (+490%), plasma aldosterone to 342 +/- 38 pg/ml (+109%), and blood pressure by 27%. Glomerular filtration rate decreased by 16%. Concomitantly, clearance of endogenous lithium fell by 66%, and fractional proximal reabsorption of sodium increased from 77 to 92%; absolute proximal reabsorption rate of sodium remained constant. ANG II decreased sodium excretion by 70%, potassium excretion by 50%, and urine flow by 80%, whereas urine osmolality increased. ANG III also increased plasma aldosterone markedly (+45%), however, without measurable changes in angiotensin immunoreactivity, glomerular filtration rate, or renal excretion rates. During vehicle infusion, plasma renin activity decreased markedly ( approximately 700 to approximately 200 mIU/l); only ANG II enhanced this decrease. ANG-(1-7) and ANG IV did not change any of the measured variables persistently. It is concluded that 1) ANG III and ANG IV are cleared much faster from plasma than ANG II, 2) ANG II causes hypofiltration, urinary concentration, and sodium and potassium retention at constant plasma concentrations of vasopressin and atrial natriuretic peptide, and 3) a very small increase in the concentration of ANG III, undetectable by usual techniques, may increase aldosterone secretion substantially.     

Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic beta-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.     

The structure and specificity of Escherichia coli maltose acetyltransferase give new insight into the LacA family of acyltransferases

The crystallographic three-dimensional structure of the Escherichia coli maa gene product, previously identified as a maltose O-acetyltransferase (MAT) [Brand, B., and Boos, W. (1991) J. Biol. Chem. 266, 14113-14118] has been determined to 2.15 A resolution by the single anomalous dispersion method using data from a crystal cocrystallized with trimethyllead acetate. It is shown here that MAT acetylates glucose exclusively at the C6 position and maltose at the C6 position of the nonreducing end glucosyl moiety. Furthermore, MAT shows higher affinity toward artificial substrates containing an alkyl or hydrophobic chain as well as a glucosyl unit. The presence of a long hydrophobic patch near the acceptor site provides the structural explanation for this preference. The three-dimensional structure reveals the expected trimeric left-handed parallel beta-helix structure found in all other known hexapeptide repeat enzymes. In particular, the structure shows similarities both overall and at the putative active site to the recently determined structure of galactoside acetyltransferase (GAT), the lacA gene product [Wang, X.-G., Olsen, L. R., and Roderick, S. L. (2002) Structure 10, 581-588]. The structure, together with the new biochemical data, suggests that GAT and MAT are more closely related than previously thought and might have similar cellular functions. However, while GAT is specific for acetylation of galactosyl units, MAT is specific for glucosyl units and is able to acetylate maltooligosaccharides, an important property for biotechnological applications. Structural differences at the acceptor site reflect the differences in substrate specificity.