Kinetic characterization of wild-type and proton transfer-impaired variants of beta-carbonic anhydrase from Arabidopsis thaliana

We have cloned and overexpressed a truncated, recombinant form of beta-carbonic anhydrase from Arabidopsis thaliana. The wild-type enzyme and two site-directed variants, H216N and Y212F, have been kinetically characterized both at steady state by stopped-flow spectrophotometry and at chemical equilibrium by (18)O isotope exchange methods. The wild-type enzyme has a maximal k(cat) for CO2 hydration of 320 ms(-1) and is rate limited by proton transfer involving two residues with apparent pK(a) values of 6.0 and 8.7. The mutant enzyme H216N has a maximal k(cat) at high pH that is 43% that of wild type, but is only 5% that of wild type at pH 7.0. (18)O exchange studies reveal that the effect of the mutations H216N or Y212F is primarily on proton transfer steps in the catalytic mechanism and not in the rate of CO2-HCO3- exchange. These results suggest that residues His-216 and Tyr-212 are both important for efficient proton transfer in A. thaliana carbonic anhydrase.     

The Chemical Basis of Membrane Bioenergetics

All organisms rely on chemiosmotic membrane systems for energy transduction; the great variety of participating proteins and pathways can be reduced to a few universal principles of operation. This chemical basis of bioenergetics is reviewed with respect to the origin and early evolution of life. For several of the cofactors which play important roles in bioenergetic reactions, plausible prebiotic sources have been proposed, and it seems likely that these cofactors were present before elaborate protein structures. In particular, the hydrophobic quinones require only a membrane-enclosed compartment to yield a minimum chemiosmotic system, since they can couple electron transport and proton translocation in a simple way. It is argued that the central features of modern bioenergetics, such as the coupling of redox reactions and ion translocation at the cytoplasmic membrane, probably are ancient features which arose early during the process of biogenesis. The notion of a thermophile root of the universal phylogenetic tree has been discussed controversially, nevertheless, thermophiles are interesting model organisms for reconstructing the origin of chemiosmotic systems, since they are often acidophiles and anaerobic respirers exploiting iron–sulfur chemistry. This perspective can help to explain the prominent role of iron–sulfur proteins in extant biochemistry as well as the origin of both respiration and proton extrusion within the context of a possible origin of life in the vicinity of hot vents. Received: 6 June 2001 / Accepted: 16 October 2001     

Evidence for a wide occurrence of proton-translocating pyrophosphatase genes in parasitic and free-living protozoa

The BAZF gene has recently been identified as a novel homologue of the BCL6 oncogene. Here we cloned the human BAZF gene using murine BAZF as a probe. The predicted amino acid sequence was 91% identical to that of murine BAZF. The BTB/POZ and zinc finger domains were almost completely conserved between human and murine BAZF. Fluorescence in situ hybridization analysis revealed that the human BAZF gene is located on chromosome 17p13.1. Although expression of human BAZF mRNA was ubiquitously detected in human tissues, abundant expression was detected in heart and placenta. BAZF mRNA was expressed in some immature B cell lines and erythroleukemia cell lines. The expression in a human erythroleukemia cell line, HEL cells, was upregulated during megakaryocytic differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate. These expression patterns of BAZF mRNA suggest that BAZF may regulate differentiation in stages or lineages that are different from those regulated by BCL6.     

Multigene DNA prime-boost vaccines for SHIV89.6P

We assessed four prime-boost vaccine regimens with a Gene Gun component for SHIV89.6P in Macaca nemestrina. A dosing experiment using beta-galactosidase plasmid showed that 30 or 45 shots per dose elicited higher titer antibody than smaller doses. For SHIV89.6P, we administered a six-plasmid vaccine capable of producing non-infectious virions in vivo in combination with either vaccinia recombinants or inactivated virus. DNA prime/vaccinia boost, or the reverse, elicited strong immune responses. The SHIV89.6P challenge virus was grown in M. nemestrina peripheral blood mononuclear cells and titered in vivo intrarectally. As has been observed for SHIV89.6P in M. mulatta, the infected M. nemestrina experienced rapid and severe loss of circulating CD4+ T cells. Vaccinated macaques were challenged three weeks after the last boost. DNA prime/vaccina boost or vaccina prime/DNA boost protected 11/12 animals from acute CD4+ T cell depletion and disease, while other regimens were not effective.     

Effect of proton pump inhibitor alone or in combination with clarithromycin on mycobacterial growth in human alveolar macrophages

The effect of omeprazole, a clinically used proton pump inhibitor, alone or in combination with clarithromycin was evaluated against Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium tuberculosis, using a human alveolar macrophage model of infection. Omeprazole exhibited no significant effect on the growth of the two M. avium complex strains or on the mycobactericidal activity of clarithromycin against them. In contrast, omeprazole significantly promoted the growth of Mycobacterium tuberculosis and the anti-mycobacterial activity of clarithromycin against it in human alveolar macrophages. It was speculated that intracellular acidic milieu around M. tuberculosis might be one reason for the lower activity of clarithromycin in the treatment of human tuberculosis.     

Accounting for Missing Data in Noncancer Risk Assessment

Noncancer risk assessments are generally forced to rely on animal bioassay data to estimate a Tolerable Daily Intake or Reference Dose, as a proxy for the threshold of human response. In cases where animal bioassays are missing from a complete data base, the critical NOAEL (no-observed-adverse-effect level) needs to be adjusted to account for the impact of the missing bioassay(s). This paper presents two approaches for making such adjustments. One is based on regression analysis and seeks to provide a point estimate of the adjustment needed. The other relies on non-parametric analysis and is intended to provide a distributional estimate of the needed adjustment. The adjustment needed is dependent on the definition of a complete data base, the number of bioassays missing, the specific bioassays which are missing, and the method used for interspecies scaling. The results from either approach can be used in conjunction with current practices for computing the TDI or RfD, or as an element of distributional approaches for estimating the human population threshold.     

Dynamic and structural properties of glucose oxidase enzyme

The catalytic oxidation of β-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I′ band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH₂ does not change the amount of α-helix present in the protein outer shell, but reorganises a fraction of random coil to β-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism. Received: 27 December 1996 / Accepted: 18 July 1997     

Resampling in Multiple‐Dose Factorial Designs

Multiple‐dose factorial designs may provide confirmatory evidence that (fixed) combination drugs are superior to either component drug alone. Moreover, a useful and safe range of dose combinations may be identified. In our study, we focus on (A) adjustments of the overall significance level made necessary by multiple testing, (B) improvement of conventional statistical methods with respect to power, distributional assumptions and dimensionality, and (C) construction of corresponding simultaneous confidence intervals. We propose novel resampling algorithms, which in a simple way take the correlation of multiple test statistics into account, thus improving power. Moreover, these algorithms can easily be extended to combinations of more than two component drugs and binary outcome data. Published data summaries from a blood pressure reduction trial are analysed and presented as a worked example. An implementation of the proposed methods is available online as an R package.     

Knockdown of miR-210 decreases hypoxic glioma stem cells stemness and radioresistance

Glioma contains abundant hypoxic regions which provide niches to promote the maintenance and expansion of glioma stem cells (GSCs), which are resistant to conventional therapies and responsible for recurrence. Given the fact that miR-210 plays a vital role in cellular adaption to hypoxia and in stem cell survival and stemness maintenance, strategies correcting the aberrantly expressed miR-210 might open up a new therapeutic avenue to hypoxia GSCs. In the present study, to explore the possibility of miR-210 as an effective therapeutic target to hypoxic GSCs, we employed a lentiviral-mediated anti-sense miR-210 gene transfer technique to knockdown miR-210 expression and analyze phenotypic changes in hypoxic U87s and SHG44s cells. We found that hypoxia led to an increased HIF-2α mRNA expression and miR-210 expression in GSCs. Knockdown of miR-210 decreased neurosphere formation capacity, stem cell marker expression and cell viability, and induced differentiation and G0/G1 arrest in hypoxic GSCs by partially rescued Myc antagonist (MNT) protein expression. Knockdown of MNT could reverse the gene expression changes and the growth inhibition resulting from knockdown of miR-210 in hypoxic GSCs. Moreover, knockdown of miR-210 led to increased apoptotic rate and Caspase-3/7 activity and decreased invasive capacity, reactive oxygen species (ROS) and lactate production and radioresistance in hypoxic GSCs. These findings suggest that miR-210 might be a potential therapeutic target to eliminate GSCs located in hypoxic niches.