An analysis of the proteomic profile for Thermoanaerobacter tengcongensis under optimal culture conditions

The genome of Thermoanaerobacter tengcongensis is estimated to encode 2588 theoretical proteins. In this study, we have vitalized approximately 46% of the theoretical proteome experimentally using a proteomic strategy that combines three different methods, shotgun digestion plus high-performance liquid chromatography (HPLC) with ion-trap tandem mass spectrometry (shotgun-liquid chromatography (LC)/MS), one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus HPLC with ion-trap tandem mass spectrometry (one-dimensional electrophoresis (1DE)-LC/MS), and two-dimensional gel electrophoresis plus matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (2DE-MALDI-TOF-MS). Of the 1200 proteins identified, as few as 76 proteins were globally found by all three approaches, and notably, most of these proteins were in the soluble fraction. However, there were a number of unique proteins detected by one method only, suggesting that our strategy provides a means toward obtaining a comprehensive view of protein expression profile. Proteins from the major metabolic pathways are strongly represented on the map, and a number of these enzymes were identified by more than one proteomic method. Based upon the proteins identified in the present study, we are able to broaden the understanding of how T. tengcongensis survives under high temperature environment, whereas several of its properties can not be fully explained by genome data.     

Ca2+ -dependence and inhibition of transformation by trifluoperazine and chlorpromazine in Thermoactinomyces vulgaris

Ca(2+) enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no. 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no. 1227). The number of transformants showed a marked increase with increasing concentration of CaCl(2) upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly. Antipsychotic drugs that are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01-0.04 mM along with optimal CaCl(2) concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca(2+)-stimulated transformation. The results of present investigation suggest the involvement of a Ca(2+)-dependent protein activator in the development of Ca(2+)-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic spore forming thermophilic actinomycete.     

Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons

Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, we generated a mouse model bearing a germline disruption in parkin. Parkin-/- mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin-/- mice. Intracellular recordings of medium-sized striatal spiny neurons showed that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin-/- mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin-/- mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and alpha-synuclein, which were identified previously as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin-/- brains. Together these findings provide the first evidence for a novel role of parkin in dopamine regulation and nigrostriatal function, and a non-essential role of parkin in the survival of nigral neurons in mice.     

A medium-throughput functional assay of KCNQ2 potassium channels using rubidium efflux and atomic absorption spectrometry

Heterologous expression of KCNQ2 (Kv7.2) results in the formation of a slowly activating, noninactivating, voltage-gated potassium channel. Using a cell line that stably expresses KCNQ2, we developed a rubidium flux assay to measure the functional activity and pharmacological modulation of this ion channel. Rubidium flux was performed in a 96-well microtiter plate format; rubidium was quantified using an automated atomic absorption spectrometer to enable screening of 1000 data points/day. Cells accumulated rubidium at 37 degrees C in a monoexponential manner with t(1/2)=40min. Treating cells with elevated extracellular potassium caused membrane depolarization and stimulation of rubidium efflux through KCNQ2. The rate of rubidium efflux increased with increasing extracellular potassium: the t(1/2) at 50mM potassium was 5.1 min. Potassium-stimulated efflux was potentiated by the anticonvulsant drug retigabine (EC(50)=0.5 microM). Both potassium-induced and retigabine-facilitated efflux were blocked by TEA (IC(50)s=0.4 and 0.3mM, respectively) and the neurotransmitter release enhancers and putative cognition enhancers linopirdine (IC(50)s=2.3 and 7.1 microM, respectively) and XE991 (IC(50)s=0.3 and 0.9 microM, respectively). Screening a collection of ion channel modulators revealed additional inhibitors including clofilium (IC(50) = 27 microM). These studies extend the pharmacological profile of KCNQ2 and demonstrate the feasibility of using this assay system to rapidly screen for compounds that modulate the function of KCNQ2.     

Protein kinase C-dependent phosphorylation and mitochondrial translocation of aldose reductase

Although aldose reductase (AR) is a critical participant in osmoregulation, and the metabolism of glucose and aldehydes derived from lipid peroxidation, post-translational mechanisms regulating its activity have not been identified. In this paper, we report that stimulation of protein kinase C (PKC) in several cell types induces phosphorylation of AR and translocation of the phosphorylated protein to the mitochondria. In vitro, recombinant AR was directly phosphorylated by activated PKC, suggesting that AR may be an in vivo PKC substrate. Together, these observations reveal a novel link between PKC activation and the regulation of glucose and aldehyde metabolism.     

Dothistroma pini,a forest pathogen,contains homologs of aflatoxin biosynthetic pathway genes

Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatoxin and sterigmatocystin gene clusters with predicted activities of a ketoreductase (dotA), oxidase (dotB), major facilitator superfamily transporter (dotC), and thioesterase (dotD). A D. pini dotA mutant was made by targeted gene replacement and shown to be severely impaired in dothistromin production, confirming that dotA is involved in dothistromin biosynthesis. Accumulation of versicolorin A (a precursor of aflatoxin) by the dotA mutant confirms that the dotA gene product is involved in an aflatoxin-like biosynthetic pathway. Since toxin genes have been found to be clustered in fungi in every case analyzed so far, it is speculated that the four dot genes may comprise part of a dothistromin biosynthetic gene cluster. A fifth gene, ddhA, is not a homolog of aflatoxin genes and could be at one end of the dothistromin cluster. These genes will allow comparative biochemical and genetic studies of the aflatoxin and dothistromin biosynthetic pathways and may also lead to new ways to control Dothistroma needle blight.     

Neurotoxicity of fluoride: neurodegeneration in hippocampus of female mice

Light microscopic study of hippocampal sub-regions demonstrated significant number of degenerated nerve cell bodies in the CA3, CA4 and dentate gyrus(Dg) areas of sodium fluoride administered adult female mice. Ultrastructural studies revealed neurodegenrative characteristics like involution of cell membranes, swelling of mitochondria, clumping of chromatin material etc, can be observed in cell bodies of CA3, CA4 and dentate gyrus (Dg). Fluoride intoxicated animals also performed poorly in motor co-ordination tests and maze tests. Inability to perform well increased with higher fluoride concentration in drinking water.     

Reversal of fluoride induced cell injury through elimination of fluoride and consumption of diet rich in essential nutrients and antioxidants

The objective of the present communication is to address the issues concerning reversal of fluoride induced cell injury and disease (i.e. fluorosis) through the elimination of fluoride and consumption of a diet containing essential nutrients and antioxidants. Humans afflicted with fluorosis, as a result of consuming fluoride contaminated water or food, have been investigated. Hospital based diagnostic procedure for early detection of fluorosis, through retrieval of history, clinical complaints, testing of blood, urine and drinking water for fluoride using ion selective electrode technology, along with X-ray of the forearm have been carried out. Confirmed cases of fluorosis were introduced to an intervention protocol consisting of (1) provision of safe drinking water with fluoride levels less than 1 mg/L and (2) counselling on nutritional supplementation with focus on adequate intake of calcium, vitamins C, E and antioxidants. The patients were monitored at frequent intervals up to one year and the results are reported. With a standardized early diagnosis, elimination of fluoride intake and supplementation of a diet rich in essential nutrients and antioxidants, we have shown that the fluorosis can be reversed.