Influence of ethanol on the activities of 3-hydroxy-3-methylglutaryl-coenzyme A-reductase and squalene-hopene-cyclase in Zymomonas mobilis

Summary The influence of different primary aliphatic alcohols on the activities of two key enzymes in hopanoid biosynthesis of Zymomonas mobilis was investigated. By use of ¹⁴C- and ³H-labelled substrates the enzymes 3-hydroxy-3-methylglutaryl-CoA-reductase and squalene-hopenecyclase were detected with activities of 1.6 pmol x (min x mg protein)^-1 and 2.3 pmol x- (min x mg protein)^-1, respectively. Cells grown in the presence of 6% (v/v) ethanol did not show higher activities of these enzymes than cells grown in the presence of 1% (v/v) ethanol. Furthermore, 3-hydroxy-3-methylglutaryl-CoA-reductase was not activated by ethanol. However, ethanol activated the squalene-hopene-cyclase when added to the enzyme test system. Besides ethanol, propanol also had a positive effect on the squalene-hopene-cyclase: the enzyme's activity increased 1.7-fold in the presence of either alcohol at a concentration of 6% (v/v). This corresponded with a similar increase of hopanoid content of whole cells when grown in the presence of 6% (v/v) added ethanol or propanol. These results indicated that the squalene-hopene-cyclase has a regulatory function in the alcohol dependent hopanoid biosynthesis of Z. mobilis.Abbreviation HMG-CoA-reductase 3-hydroxy-3-methylglutaryl-coenzyme A-reductase     

Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8(-). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.     

Animal models of ischemic stroke: balancing experimental aims and animal care

Animal models of ischemic stroke are examples of an induced model that can present challenges from the perspectives of protocol review and animal management. The review presented here will include a brief summary of the current state of knowledge about clinical stroke; a general synopsis of important unanswered research questions that justify use of animal stroke models; an overview of various animal models of ischemic stroke, including strengths and limitations; and a discussion of animal care issues relative to ischemic stroke models. Good communication and interactive education among primary investigators, laboratory animal veterinarians and caretakers, and institutional animal care and use committee members are critical in achieving a balance between research objectives and animal care issues when using animal stroke models.     

Genetic determinants of cell type-specific poliovirus propagation in HEK 293 cells

The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.     

Indirect competitive immunoassay for detection of aflatoxin B1 in corn and nut products using the array biosensor

Because of the potential health risks of aflatoxin B₁ (AFB₁), it is essential to monitor the level of this mycotoxin in a variety of foods. An indirect competitive immunoassay has been developed using the NRL array biosensor, offering rapid, sensitive detection and quantification of AFB₁ in buffer, corn and nut products. AFB₁-spiked foods were extracted with methanol and Cy5-anti-AFB₁ added to the resulting sample. The extracted sample/antibody mix was passed over a waveguide surface patterned with immobilized AFB₁. The resulting fluorescence signal decreased as the concentration of AFB₁ in the sample increased. The limit of detection for AFB₁ in buffer, 0.3 ng/ml, was found to increase to between 1.5 and 5.1 ng/g and 0.6 and 1.4 ng/g when measured in various corn and nut products, respectively.     

Translation initiator EIF4G1 mutations in familial Parkinson disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.     

How accessible are coral reefs to people? A global assessment based on travel time

The depletion of natural resources has become a major issue in many parts of the world, with the most accessible resources being most at risk. In the terrestrial realm, resource depletion has classically been related to accessibility through road networks. In contrast, in the marine realm, the impact on living resources is often framed into the Malthusian theory of human density around ecosystems. Here, we develop a new framework to estimate the accessibility of global coral reefs using potential travel time from the nearest human settlement or market. We show that 58% of coral reefs are located < 30 min from the nearest human settlement. We use a case study from New Caledonia to demonstrate that travel time from the market is a strong predictor of fish biomass on coral reefs. We also highlight a relative deficit of protection on coral reef areas near people, with disproportional protection on reefs far from people. This suggests that conservation efforts are targeting low‐conflict reefs or places that may already be receiving de facto protection due to their isolation. Our global assessment of accessibility in the marine realm is a critical step to better understand the interplay between humans and resources.     

Limited influence of local and landscape factors on finescale gene flow in two pond‐breeding amphibians

Dispersal and gene flow within animal populations are influenced by the composition and configuration of the landscape. In this study, we evaluated hypotheses about the impact of natural and anthropogenic factors on genetic differentiation in two amphibian species, the spotted salamander (Ambystoma maculatum) and the wood frog (Lithobates sylvaticus) in a commercial forest in central Maine. We conducted this analysis at two scales: a local level, focused on factors measured at each breeding pond, and a landscape level, focused on factors measured between ponds. We investigated the effects of a number of environmental factors in six categories including Productivity, Physical, Land Composition, Land Configuration, Isolation and Location. Embryos were sampled from 56 spotted salamander breeding ponds and 39 wood frog breeding ponds. We used a hierarchical Bayesian approach in the program GESTE at each breeding pond and a random forest algorithm in conjunction with a network analysis between the ponds. We found overall high genetic connectivity across distances up to 17 km for both species and a limited effect of natural and anthropogenic factors on gene flow. We found the null models best explained patterns of genetic differentiation at a local level and found several factors at the landscape level that weakly influenced gene flow. This research indicates multiscale investigations that incorporate local and landscape factors are valuable for understanding patterns of gene flow. Our findings suggest that dispersal rates in this system are high enough to minimize genetic structuring and that current forestry practices do not significantly impede dispersal.