Zebrafish atoh1 genes: classic proneural activity in the inner ear and regulation by Fgf and Notch

Hair cells of the inner ear develop from an equivalence group marked by expression of the proneural gene Atoh1. In mouse, Atoh1 is necessary for hair cell differentiation, but its role in specifying the equivalence group (proneural function) has been questioned and little is known about its upstream activators. We have addressed these issues in zebrafish. Two zebrafish homologs, atoh1a and atoh1b, are together necessary for hair cell development. These genes crossregulate each other but are differentially required during distinct developmental periods, first in the preotic placode and later in the otic vesicle. Interactions with the Notch pathway confirm that atoh1 genes have early proneural function. Fgf3 and Fgf8 are upstream activators of atoh1 genes during both phases, and foxi1, pax8 and dlx genes regulate atoh1b in the preplacode. A model is presented in which zebrafish atoh1 genes operate in a complex network leading to hair cell development.     

Beef cattle selected for increased muscularity have a reduced muscle response and increased adipose tissue response to adrenaline

The aim of this experiment was to evaluate the impact of selection for greater muscling on the adrenaline responsiveness of muscle, adipose and liver tissue, as reflected by changes in plasma levels of the intermediary metabolites lactate, non-esterified fatty acids (NEFA) and glucose. This study used 18-month-old steers from an Angus herd visually assessed and selected for divergence in muscling for over 15 years. Ten low muscled (Low), 11 high muscled (High) and 3 high muscled heterozygotes for myostatin mutation (High(Het)) steers were challenged with adrenaline doses ranging between 0.2 to 3.0 μg/kg live weight. For each challenge, 16 blood samples were taken between -30 and 130 min relative to adrenaline administration. Plasma was analysed for NEFA, lactate and glucose concentration and area under curve (AUC) over time was calculated to reflect the tissue responses to adrenaline. Sixteen basal plasma samples from each animal were also assayed for growth hormone. Muscle glycogen and lactate concentration were analysed from four muscle biopsies taken from the semimembranosus, semitendinosus and longissimus thoracis et lumborum of each animal at 14, 90 and 150 days on an ad libitum grain-based diet and at slaughter on day 157. In response to the adrenaline challenges, the High steers had 30% lower lactate AUC than the Low steers at challenges greater than 2 μg/kg live weight, indicating lower muscle responsiveness at the highest adrenaline doses. Aligning with this decrease in muscle response in the High animals were the muscle glycogen concentrations which were 6.1% higher in the High steers. These results suggest that selection for muscling could reduce the incidence of dark, firm, dry meat that is caused by low levels of glycogen at slaughter. At all levels of adrenaline challenge, the High steers had at least 30% greater NEFA AUC, indicating that their adipose tissue was more responsive to adrenaline, resulting in greater lipolysis. In agreement with this response, the High steers had a higher plasma growth hormone concentration, which is likely to have contributed to the increased lipolysis evident in these animals in response to adrenaline. This difference in lipolysis may in part explain the reduced fatness of muscular cattle. There was no effect of selection for muscling on liver responsiveness to adrenaline.     

Regulation of the JNK3 Signaling Pathway during Islet Isolation: JNK3 and c-fos as New Markers of Islet Quality for Transplantation

Stress conditions generated throughout pancreatic islet processing initiate the activation of pro-inflammatory pathways and beta-cell destruction. Our goal is to identify relevant and preferably beta-specific markers to assess the activation of beta-cell stress and apoptotic mechanisms, and therefore the general quality of the islet preparation prior to transplantation. Protein expression and activation were analyzed by Western blotting and kinase assays. ATP measurements were performed by a luminescence-based assay. Oxygen consumption rate (OCR) was measured based on standard protocols using fiber optic sensors. Total RNA was used for gene expression analyzes. Our results indicate that pancreas digestion initiates a potent stress response in the islets by activating two stress kinases, c-Jun N-terminal Kinase (JNK) and p38. JNK1 protein levels remained unchanged between different islet preparations and following culture. In contrast, levels of JNK3 increased after islet culture, but varied markedly, with a subset of preparations bearing low JNK3 expression. The observed changes in JNK3 protein content strongly correlated with OCR measurements as determined by the Spearman''s rank correlation coefficient rho in the matching islet samples, while inversely correlating with c-fos mRNA expression . In conclusion, pancreas digestion recruits JNK and p38 kinases that are known to participate to beta-cell apoptosis. Concomitantly, the islet isolation alters JNK3 and c-fos expression, both strongly correlating with OCR. Thus, a comparative analysis of JNK3 and c-fos expression before and after culture may provide for novel markers to assess islet quality prior to transplantation. JNK3 has the advantage over all other proposed markers to be islet-specific, and thus to provide for a marker independent of non-beta cell contamination.     

Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.     

Analysis of streptomycin-resistance of Escherichia coli mutants.

We previously reported about Escherichia coli transformation experiments yielding streptomycin-resistant cells carrying a C912 to T transition in a plasmid-born 16S rRNA gene. These experiments were based on results obtained with streptomycin-resistant Euglena chloroplasts bearing an equivalent mutation in the single chloroplast 16S rRNA gene. We extended this study and transformed E. coli with plasmid constructs having a mutated 16S rRNA gene at position 914 (A to C) or a double mutation at positions 912 and 888 (C to T:G to A) or a mutation in the S12 gene (Lys-42 to Thr). We tested the transformed cells before and after a screening procedure in the presence of streptomycin. We find that the plasmid-born mutations protect colonies against a short streptomycin exposure, but ribosomes carrying mutated 16S rRNA do not significantly reduce codon misreading in vitro. However, ribosomes isolated from transformed cells after the screening procedure resist misreading. These ribosomes have acquired a second mutation in the S12 protein as shown in one case by sequencing and by transformation experiments. Furthermore, we show that the A914 to C mutation prevents (strongly reduces) base methylation in the central domain of 16S rRNA.     

Mesozooplankton of the Kosi Bay lakes,South Africa

The Kosi coastal lake system, a chain of four interconnected basins, is located in the subtropical north-eastern corner of South Africa. Little information is available on zooplankton of the system and the main aim of this study is to report on zooplankton samples collected during 2002 and 2003. The set of samples consists of seasonal, subsurface mesozooplankton samples that were collected during nighttime in each of the lakes. A well-developed salinity gradient was evident along the interconnected lakes in the subsurface water during all seasons, ranging from freshwater in the upper lake Amanzamnyama to a maximum of 22 recorded in Lake Makhawulani. The zooplankton community structures of the lakes reflected the salinity gradient of the system, with some coastal marine taxa recorded in the lakes closer to the mouth and only freshwater taxa recorded in Lake Amanzamnyama. Mesozooplankton diversity and abundance were relatively low compared to other estuarine systems along the eastern coast of South Africa. The dominant taxa were calanoid copepods Acartiella natalensis and Pseudodiaptomus stuhlmanni and the mysid Mesopodopsis africana in the lower lakes, whereas cyclopoids Mesocyclops sp. and Thermocyclops sp. dominated the freshwater lake Amanzamnyama.     

Acute graft-versus-host disease after allogeneic bone marrow transplantation.

In 25 patients receiving allogeneic bone marrow transplants methotrexate was used to prevent acute graft-versus-host disease (GVHD). Acute GVHD, grades 2 to 4, developed in only 5 (20%) of the patients. The incidence of acute GVHD in other series of recipients of bone marrow transplants has ranged from 5% to 76%. A review of the literature suggests that this variation cannot be completely accounted for by age, type of disease treated by transplantation or type of GVHD prophylaxis. However, transfusion of allogeneic lymphocytes that have not been completely inactivated by irradiation (e.g., in platelet and granulocyte preparations) and inadequate isolation-decontamination procedures may increase the probability of GVHD following bone marrow transplantation.