Establishment of a Gene Expression System in Ochrobactrum anthropi

Genetic studies of Ochrobactrum anthropi are hindered by the lack of a suitable gene expression system. We constructed a set of vectors containing several promoters and a His tag fusion in the N terminus to facilitate protein detection and purification. The new vectors should significantly enhance the genetic manipulation and characterization of O. anthropi.     

In-depth, longitudinal analysis of viral quasispecies from an individual triply infected with late-stage human immunodeficiency virus type 1, using a multiple PCR primer approach

Co-infections with more than one human immunodeficiency virus type 1 (HIV-1) subtype appear to be the source of new recombinant strains and may be commonplace in high-risk cohorts exposed to multiple subtypes. Many potential dual infections have been identified during the HIV Superinfection Study in Mbeya, Tanzania, where 600 female bar workers who are highly exposed to subtypes A, C, and D have been evaluated every 3 months for over 3 years by use of the MHAacd HIV-1 genotyping assay. Here we describe an in-depth, longitudinal analysis of the viral quasispecies in a woman who was triply infected with HIV-1 and who developed AIDS and passed away 15 months after enrollment. The MHA results obtained at 0, 3, 6, 9, and 12 months revealed dual-probe reactivities and shifts in subtype over time, indicating a potential dual infection and prompting further investigation. The multiple infection was confirmed by PCR amplification of three genome regions by a multiple primer approach, followed by molecular cloning and sequencing. A highly complex viral quasispecies was found, including several recombinant forms, with vpu/gp120 being the most diverse region. A significant fluctuation in molecular forms over time was observed, showing that the serial sample format is highly desirable, if not essential, for the identification of multiple infections. In a separate experiment, we confirmed that the detection of co-infections is more efficient with the use of multiple amplification primers to overcome the primer bias that results from the enormous diversity in the HIV-1 genome.     

L-lactate measures in brain tissue with ceramic-based multisite microelectrodes

A newly developed multisite array microelectrode for in vivo measurements of L-lactate is presented. The resulting microelectrode is composed of three functional layers. First, Nafion is used to repel interfering electroactive anions, such as ascorbate. Second, L-lactate oxidase immobilized onto the recording sites is used to convert L-lactate to hydrogen peroxide. The H2O2 produced is proportional to L-lactate concentrations and is quantified at the platinum recording sites. Third, a layer of polyurethane is coated over the L-lactate oxidase to adjust the linear range of the microelectrode to one that is compatible with in vivo measurements. This layer reduces the amount of L-lactate that diffuses to the enzyme while not significantly limiting oxygen diffusion. The resulting L-lactate microelectrodes were linear to 20 mM (R2 = 0.997 +/- 0.001) and beyond in some cases with detection limits of 0.078 +/- 0.013 mM (n = 12). The selectivity and response time of these electrodes make them suitable for in vivo measurements in brain tissue. Self-referencing recordings may be utilized to further improve the selectivity of the recordings. However this is not necessary for most applications in the brain, because the resting and stimulated levels of dopamine (DA), norepinephrine (NE), and other potentially interfering cations are two to three orders of magnitude lower than that of in vivo L-lactate, which is in the millimolar range. Preliminary in vivo measures of L-lactate in the brain of anesthetized rats support that the microelectrodes are capable of measuring rapid endogenous changes in vivo.     

6-hydroxydopamine lesions in anuran amphibians: a new model system for Parkinson's disease?

We investigated the effects of dopamine depletion on acoustically guided behavior of anurans by conducting phonotaxis experiments with female gray treefrogs (Hyla versicolor) before and 90 min after bilateral injections of 3, 6, or 12 microg 6-hydroxydopamine (6-OHDA) into the telencephalic ventricles. In experiments with one loudspeaker playing back a standard artificial mating call, we analyzed the effects of 6-OHDA on phonotactic response time. In choice tests we measured the degree of distraction from the standard call (20 pulses/s) by three different variants with altered pulse-rate (30/s, 40/s, 60/s). Five days after experiments, brains were immunostained for tyrosine hydroxylase. Labeled neurons were counted in the suprachiasmatic nucleus, posterior tuberculum, interpeduncular nucleus, and locus coeruleus, and correlation between neuronal numbers and behavioral scores was tested. Response times increased together with 6-OHDA concentrations, which was mainly due to longer immobile periods before the animals started movement. In choice tests the most irrelevant stimulus (60/s) distracted 6-OHDA injected females from the standard stimulus, while sham injected controls were undistracted. The number of catecholaminergic neurons decreased with increasing 6-OHDA concentration in the suprachiasmatic nucleus, posterior tuberculum, and interpeduncular nucleus. The normalized number of immunoreactive neurons in the posterior tuberculum was positively correlated with phonotaxis scores in the one-speaker test, demonstrating that motor deficits are a function of tubercular cell loss. We conclude that bilateral 6-OHDA lesions in anuran amphibians cause motor (difficulty to start movements) as well as cognitive symptoms (higher distraction by irrelevant stimuli) that have also been described for human Parkinson patients.     

Zinc fluxes into developing barley grains: use of stable Zn isotopes to separate root uptake from remobilization in plants with contrasting Zn status

Background and Aims

Zn imported into developing cereal grains originates from either de novo Zn uptake by the roots or remobilization of Zn from vegetative tissues. The present study was focused on revealing the quantitative importance of the two pathways for grain Zn loading and how their relative contribution varies with the overall plant Zn status.

Methods

The stable isotope ⁶⁷Zn was used to trace Zn uptake and remobilization fluxes in barley (Hordeum vulgare L.) plants growing in hydroponics at 0.1?μM (low Zn), 1.5?μM (medium Zn) or 5?μM Zn (high Zn). When grain development reached 15?days after pollination the Zn source was changed to an enriched ⁶⁷Zn isotope and plants were harvested after 6 to 48?h. Zn concentrations and isotope ratios were determined using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).

Results

Plants with low Zn status absorbed 3-fold more Zn than plants with medium or high Zn status when roots were exposed to an external concentration of 1.5?μM ⁶⁷Zn. Stems and ears were the primary recipients of the de novo incorporated Zn with preferential allocation to the developing grains over time. The leaves received in all cases a very small proportion (<5?%) of the newly absorbed Zn and the proportion did not increase over time. Zn fluxes derived from uptake and remobilization were almost equal in plants with low Zn status, while at high Zn status remobilization delivered 4 times more Zn to the developing grains than did root Zn uptake.

Conclusions

Stable isotopes in combination with ICP-MS provided a strong tool for quantification of Zn fluxes in intact plants. The importance of Zn remobilization compared to de novo root absorption of Zn increased with increasing plant Zn status. Very little de novo absorbed Zn was translocated to the leaves during generative growth stages.     

Reverse crosstalk of TGFβ and PPARβ/δ signaling identified by transcriptional profiling

Previous work has provided strong evidence for a role of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and transforming growth factor-β (TGFβ) in inflammation and tumor stroma function, raising the possibility that both signaling pathways are interconnected. We have addressed this hypothesis by microarray analyses of human diploid fibroblasts induced to myofibroblastic differentiation, which revealed a substantial, mostly reverse crosstalk of both pathways and identified distinct classes of genes. A major class encompasses classical PPAR target genes, including ANGPTL4, CPT1A, ADRP and PDK4. These genes are repressed by TGFβ, which is counteracted by PPARβ/δ activation. This is mediated, at least in part, by the TGFβ-induced recruitment of the corepressor SMRT to PPAR response elements, and its release by PPARβ/δ ligands, indicating that TGFβ and PPARβ/δ signals are integrated by chromatin-associated complexes. A second class represents TGFβ-induced genes that are downregulated by PPARβ/δ agonists, exemplified by CD274 and IL6, which is consistent with the anti-inflammatory properties of PPARβ/δ ligands. Finally, cooperative regulation by both ligands was observed for a minor group of genes, including several regulators of cell proliferation. These observations indicate that PPARβ/δ is able to influence the expression of distinct sets of both TGFβ-repressed and TGFβ-activated genes in both directions.     

Crystal structure of an archaeal pentameric riboflavin synthase in complex with a substrate analog inhibitor: stereochemical implications

Whereas eubacterial and eukaryotic riboflavin synthases form homotrimers, archaeal riboflavin synthases from Methanocaldococcus jannaschii and Methanothermobacter thermoautrophicus are homopentamers with sequence similarity to the 6,7-dimethyl-8-ribityllumazine synthase catalyzing the penultimate step in riboflavin biosynthesis. Recently it could be shown that the complex dismutation reaction catalyzed by the pentameric M. jannaschii riboflavin synthase generates riboflavin with the same regiochemistry as observed for trimeric riboflavin synthases. Here we present crystal structures of the pentameric riboflavin synthase from M. jannaschii and its complex with the substrate analog inhibitor, 6,7-dioxo-8-ribityllumazine. The complex structure shows five active sites located between adjacent monomers of the pentamer. Each active site can accommodate two substrate analog molecules in anti-parallel orientation. The topology of the two bound ligands at the active site is well in line with the known stereochemistry of a pentacyclic adduct of 6,7-dimethyl-8-ribityllumazine that has been shown to serve as a kinetically competent intermediate. The pentacyclic intermediates of trimeric and pentameric riboflavin synthases are diastereomers.     

Dialysis continuous process for ammonium lactate fermentation: Simulated and experimental dialysate-feed,immobilized-cell systems

A generalized mathematical model, previously developed and experimentally validated, was modified and used to computer-simulate two dialysate-feed systems for operating a dialysis continuous process for the ammonium lactate fermentation. The simulations predicted that the feeding of substrate into the dialysate circuit and thence into the fermentor circuit via dialysis should greatly improve the production of cell mass and metabolite product. Experiments were conducted to test the system in which the fermentor is operated without an effluent, thus immobilizing the cells. Dried cheese whey ultrafiltrate was rehydrated to contain a normal concentration of lactose (62 mg/ml), supplemented with yeast with an adapted culture of Lactobacillus bulgaricus. The system was operated without interruption for 26 days. Results during steady-state conditions showed that the system is a new and useful way to immobilize living cells for the purpose of producing a metabolite at a high rate for a prolonged time. The substrate consumed by the cells is converted to product via maintenance metabolism only and is sterilized by dialysis.     

Density, porosity, and structure of dried cell walls isolated from Bacillus megaterium and Saccharomyces cerevisiae.

Helium displacement and nitrogen adsorption techniques were used to determine the density and porosity, respectively, of freeze-dried cell walls isolated from Bacillus megaterium KM and Saccharomyces cerevisiae. The densities were 1.302 and 1.180 g/cm3, respectively, suggesting noncrystalline solids. The porosities were extremely small, indicating that the cell walls had collapsed and become essentially impervious upon lyophilization.