Oxygen metabolism by the anaerobic bacteriumVeillonella alcalescens

Veillonella alcalescens contained a membrane-bound lactate oxidase system. Studies on the effect of inhibitors on lactate oxidase showed the participation of non-heme iron, quinone and cytochromesb andd. Superoxide anion radicals ( ) and H₂O₂ were shown to be formed at lactate oxidation and presumably arose from cyanide- and azide-resistant side chains of the respiratory system. The H⁺/O ratio withL-lactate as a hydrogen donor was 2.3. When an anaerobic culture growing on lactate was shifted to a high dissolved oxygen tension (d.o.t.=15 kPa) rapid inhibition of growth and lactate conversion occurred. This could be correlated with a rapid inactivation of lactate dehydrogenase. The effects of high d.o.t.'s on lactate dehydrogenase, lactate conversion and growth were reversible. After a shift to low d.o.t.'s (<2.5 kPa) growth ofV. alcalescens continued for one or two doublings whereafter lysis did occur. Acetate and pyruvate were the main fermentation products. P/O ratio's were calculated from molar growth yields and fermentation balances. A P/O value of 0.66 was found after a shift to a very low oxygen supply at which the d.o.t. presumably was zero. Shifts to higher d. o. t.'s gave much lower growth yields. Presumably, under these conditions uncoupling between growth and energy production occurred. Accumulation of toxic oxygen compounds was given as an explanation for the behaviour ofV. alcalescens at low d.o.t.'s.Abbreviations HQNO 2-n-heptyl-4-hydroxy-quinoline-N-oxide - CCCP carbonyl cyanide m-chlorophenyl-hydrazone - ABTS 2,2-azino-di-3-ethyl-benzthiazoline sulfonate - DCPIP 2,6 dichlorophenolindophenol - PMS phenazine methosulfate - NBT p-nitro blue tetrazolium chloride - d.o.t. dissolved oxygen tension - SOD superoxide dismutase     

Huntingtin fragments that aggregate go their separate ways

N-terminal region of mutant huntingtin forms intranuclear and cytoplasmic aggregates in neurons that may contribute to neuronal death in Huntington's disease. show that different endoprotease-cleaved huntingtin fragments form nuclear and cytoplasmic inclusions.     

Gene and QTL detection in a three-way barley cross under selection by a mixed model with kinship information using SNPs

Quantitative trait locus (QTL) detection is commonly performed by analysis of designed segregating populations derived from two inbred parental lines, where absence of selection, mutation and genetic drift is assumed. Even for designed populations, selection cannot always be avoided, with as consequence varying correlation between genotypes instead of uniform correlation. Akin to linkage disequilibrium mapping, ignoring this type of genetic relatedness will increase the rate of false-positives. In this paper, we advocate using mixed models including genetic relatedness, or ‘kinship’ information for QTL detection in populations where selection forces operated. We demonstrate our case with a three-way barley cross, designed to segregate for dwarfing, vernalization and spike morphology genes, in which selection occurred. The population of 161 inbred lines was screened with 1,536 single nucleotide polymorphisms (SNPs), and used for gene and QTL detection. The coefficient of coancestry matrix was estimated based on the SNPs and imposed to structure the distribution of random genotypic effects. The model incorporating kinship, coancestry, information was consistently superior to the one without kinship (according to the Akaike information criterion). We show, for three traits, that ignoring the coancestry information results in an unrealistically high number of marker–trait associations, without providing clear conclusions about QTL locations. We used a number of widely recognized dwarfing and vernalization genes known to segregate in the studied population as landmarks or references to assess the agreement of the mapping results with a priori candidate gene expectations. Additional QTLs to the major genes were detected for all traits as well.     

Podoplanin Immunopositive Lymphatic Vessels at the Implant Interface in a Rat Model of Osteoporotic Fractures

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.     

Genome-wide Screen for Modulation of Hepatic Apolipoprotein A-I (ApoA-I) Secretion

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25–40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.     

Kinetic modulation of bacterial hydrolases by microbial community structure in coastal waters

In this study, we hypothesized that shifts in the kinetic parameters of extracellular hydrolytic enzymes may occur as a consequence of seasonal environmental disturbances and would reflect the level of adaptation of the bacterial community to the organic matter of the ecosystem. We measured the activities of enzymes that play a key role in the bacterial growth (leucine aminopeptidase, β- and α-glucosidases) in surface coastal waters of the Eastern Cantabrian Sea and determined their kinetic parameters by computing kinetic models of distinct complexity. Our results revealed the existence of two clearly distinct enzymatic systems operating at different substrate concentrations: a high-affinity system prevailing at low substrate concentrations and a low-affinity system characteristic of high substrate concentrations. These findings could be the result of distinct functional bacterial assemblages growing concurrently under sharp gradients of high-molecular-weight compounds. We constructed an ecological network based on contemporaneous and time-delayed correlations to explore the associations between the kinetic parameters and the environmental variables. The analysis revealed that the recurring phytoplankton blooms registered throughout the seasonal cycle trigger the wax and wane of those members of the bacterial community able to synthesize and secrete specific enzymes.