Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle: EVIDENCE FOR PHYSICAL COMPLEX FORMATION*

Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450·P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR·CYP1A2, 2) CPR·CYP2B4, 3) a mixture of CPR·CYP1A2 vesicles with CPR·CYP2B4 vesicles, and 4) CPR·CYP1A2·CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.     

A blood based 12-miRNA signature of Alzheimer disease patients

Background

Alzheimer disease (AD) is the most common form of dementia but the identification of reliable, early and non-invasive biomarkers remains a major challenge. We present a novel miRNA-based signature for detecting AD from blood samples.

Results

We apply next-generation sequencing to miRNAs from blood samples of 48 AD patients and 22 unaffected controls, yielding a total of 140 unique mature miRNAs with significantly changed expression levels. Of these, 82 have higher and 58 have lower abundance in AD patient samples. We selected a panel of 12 miRNAs for an RT-qPCR analysis on a larger cohort of 202 samples, comprising not only AD patients and healthy controls but also patients with other CNS illnesses. These included mild cognitive impairment, which is assumed to represent a transitional period before the development of AD, as well as multiple sclerosis, Parkinson disease, major depression, bipolar disorder and schizophrenia. miRNA target enrichment analysis of the selected 12 miRNAs indicates an involvement of miRNAs in nervous system development, neuron projection, neuron projection development and neuron projection morphogenesis. Using this 12-miRNA signature, we differentiate between AD and controls with an accuracy of 93%, a specificity of 95% and a sensitivity of 92%. The differentiation of AD from other neurological diseases is possible with accuracies between 74% and 78%. The differentiation of the other CNS disorders from controls yields even higher accuracies.

Conclusions

The data indicate that deregulated miRNAs in blood might be used as biomarkers in the diagnosis of AD or other neurological diseases.     

Stuttered swallowing: Electric stimulation of the right insula interferes with water swallowing. A case report

Background  

Various functional resonance imaging, magnetoencephalographic and lesion studies suggest the involvement of the insular cortex in the control of swallowing. However, the exact location of insular activation during swallowing and its functional significance remain unclear.     

Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment

Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication, we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα), as being required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.     

MR angiography of collateral arteries in a hind limb ischemia model: comparison between blood pool agent Gadomer and small contrast agent Gd-DTPA

The objective of this study was to compare the blood pool agent Gadomer with a small contrast agent for the visualization of ultra-small, collateral arteries (diameter<1 mm) with high resolution steady-state MR angiography (SS-MRA) in a rabbit hind limb ischemia model. Ten rabbits underwent unilateral femoral artery ligation. On days 14 and 21, high resolution SS-MRA (voxel size 0.49×0.49×0.50 mm³) was performed on a 3 Tesla clinical system after administration of either Gadomer (dose: 0.10 mmol/kg) or a small contrast agent (gadopentetate dimeglumine (Gd-DTPA), dose: 0.20 mmol/kg). All animals received both contrast agents on separate days. Selective intra-arterial x-ray angiograms (XRAs) were obtained in the ligated limb as a reference. The number of collaterals was counted by two independent observers. Image quality was evaluated with the contrast-to-noise ratio (CNR) in the femoral artery and collateral arteries. CNR for Gadomer was higher in both the femoral artery (Gadomer: 73±5 (mean ± SE); Gd-DTPA: 40±3; p<0.01) and collateral arteries (Gadomer: 18±4; Gd-DTPA: 9±1; p = 0.04). Neither day of acquisition nor contrast agent used influenced the number of identified collateral arteries (p = 0.30 and p = 0.14, respectively). An average of 4.5±1.0 (day 14, mean ± SD) and 5.3±1.2 (day 21) collaterals was found, which was comparable to XRA (5.6±1.7, averaged over days 14 and 21; p>0.10). Inter-observer variation was 24% and 18% for Gadomer and Gd-DTPA, respectively. In conclusion, blood pool agent Gadomer improved vessel conspicuity compared to Gd-DTPA. Steady-state MRA can be considered as an excellent non-invasive alternative to intra-arterial XRA for the visualization of ultra-small collateral arteries.     

Genome-wide miRNA signatures of human longevity

Little is known about the functions of miRNAs in human longevity. Here, we present the first genome-wide miRNA study in long-lived individuals (LLI) who are considered a model for healthy aging. Using a microarray with 863 miRNAs, we compared the expression profiles obtained from blood samples of 15 centenarians and nonagenarians (mean age 96.4 years) with those of 55 younger individuals (mean age 45.9 years). Eighty miRNAs showed aging-associated expression changes, with 16 miRNAs being up-regulated and 64 down-regulated in the LLI relative to the younger probands. Seven of the eight selected aging-related biomarkers were technically validated using quantitative RT-PCR, confirming the microarray data. Three of the eight miRNAs were further investigated in independent samples of 15 LLI and 17 younger participants (mean age 101.5 and 36.9 years, respectively). Our screening confirmed previously published miRNAs of human aging, thus reflecting the utility of the applied approach. The hierarchical clustering analysis of the miRNA microarray expression data revealed a distinct separation between the LLI and the younger controls (P-value < 10(-5) ). The down-regulated miRNAs appeared as a cluster and were more often reported in the context of diseases than the up-regulated miRNAs. Moreover, many of the differentially regulated miRNAs are known to exhibit contrasting expression patterns in major age-related diseases. Further in silico analyses showed enrichment of potential targets of the down-regulated miRNAs in p53 and other cancer pathways. Altogether, synchronized miRNA-p53 activities could be involved in the prevention of tumorigenesis and the maintenance of genomic integrity during aging.     

New solvent-producing Clostridium sp. strains, hydrolyzing a wide range of polysaccharides, are closely related to Clostridium butyricum

Thirteen new Clostridium strains, previously isolated from soil and found to produce high amounts of solvents from glucose, hydrolyzed a great variety of α- and β-glycans, including raw starch, xylan, pectin, inulin and cellulose. The sequences of the PCR-amplified DNA fragments containing the variable 3′ part of one of the 16S rRNA genes were 99.5% identical. The macrorestriction pattern of two endonucleolytic digests of chromosomal DNA in the pulsed-field gel electrophoresis (PFGE) confirmed their high homogeneity on the DNA level. The complete 16S rRNA gene sequence of three selected strains was 99.8% identical to the 16S rRNA gene sequence from Clostridium butyricum and separates them from C. acetobutylicum. To the closely related four species of solventogenic clostridia a new group of strains has to be added, which has a great potential for the direct fermentation of biomass. Journal of Industrial Microbiology & Biotechnology (2001) 27, 329–335. Received 12 September 2000/ Accepted in revised form 25 July 2001     

Biochemical analysis of the 20 S proteasome of Trypanosoma brucei

We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven alpha- and seven beta-subunits. Of the seven beta-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three beta-subunits were labeled by the affinity reagents, only two beta-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to beta2 and beta5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal beta1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.     

Production and characterization of Penicillium brasilianum pectinases with regard to industrial application

The objective of this study was to evaluate the production of pectinase by an isolated strain of Penicillium brasilianum in a bioreactor and to consider its potential for industrial applications (i.e. fruit juice). The optimization of production was achieved through experimental design. The maximum exo-polygalacturonase (Exo-PG) production in the bioreactor was 53.8?U mL^?1 under the conditions of 180?rpm, an aeration rate of 1.5 vvm, 30?°C, pH_initial of 5.5, 5?×?10⁶ spores mL^?1, 32?g L^?1 pectin, 10?g L^?1 of yeast extract and 0.5?g L^?1 magnesium sulfate and bioproduction for 36?h. The production of Exo-PG in the bioreactor was 1.3 times higher than that obtained in shake flasks, with aeration (1.5 vvm) and agitation (180?rpm) control. The crude enzyme complex, beyond the pectinolytic activity of Exo-PG (53.8?U mL^?1), also contained activity pectin methylesterase (6.0?U mL^?1) and pectin lyase (6.61?U mL^?1). At a crude enzyme complex with a concentration of 0.5% (v/v), viscosity of peach juice was reduced by 11.66%, turbidity was reduced by 13.71% and clarification was increased by 26.92%. Based on the present results, we can conclude that the new strain of isolated P. brasilianum produced high amounts of pectinases in a bioreactor with mechanical agitation, and has the potential to be applied to in the clarification of juices.