Up-regulation of neuropeptide Y levels and modulation of glutamate release through neuropeptide Y receptors in the hippocampus of kainate-induced epileptic rats

Kainate-induced epilepsy has been shown to be associated with increased levels of neuropeptide Y (NPY) in the rat hippocampus. However, there is no information on how increased levels of this peptide might modulate excitation in kainate-induced epilepsy. In this work, we investigated the modulation of glutamate release by NPY receptors in hippocampal synaptosomes isolated from epileptic rats. In the acute phase of epilepsy, a transient decrease in the efficiency of NPY and selective NPY receptor agonists in inhibiting glutamate release was observed. Moreover, in the chronic epileptic hippocampus, a decrease in the efficiency of NPY and the Y(2) receptor agonist, NPY13-36, was also found. Simultaneously, we observed that the epileptic hippocampus expresses higher levels of NPY, which may account for an increased basal inhibition of glutamate release. Consistently, the blockade of Y(2) receptors increased KCl-evoked glutamate release, and there was an increase in Y(2) receptor mRNA levels 30 days after kainic acid injection, suggesting a basal effect of NPY through Y(2) receptors. Taken together, these results indicate that an increased function of the NPY modulatory system in the epileptic hippocampus may contribute to basal inhibition of glutamate release and control hyperexcitability.     

The tomato early fruit specific gene <Emphasis Type="Italic">Lefsm1</Emphasis> defines a novel class of plant-specific SANT/MYB domain proteins

We describe here a novel plant-specific gene, Lefsm1 (fruit SANT/MYB-like 1) harboring a single SANT/MYB domain. The expression of Lefsm1 is specific to the very early stages of tomato (Lycopersicon esculentum) fruit development. Ectopic expression of Lefsm1 results in severe developmental alterations manifested in retarded growth, and reduced apical dominance during tomato and Arabidopsis seedling development. A promoter sequence residing 1.0 kb upstream to the translation initiation codon confers the organ-specific expression of the gene. Lefsm1 belongs to a novel small gene family consisting of five to six members in tomato, Arabidopsis and rice. The SANT/MYB domain of LeFSM1 and its orthologs in Arabidopsis and rice differs from that of all other plant or animal MYB proteins and from the SANT domains found in part of the chromatin remodeling proteins. Together, our results indicate that Lefsm1 is a founding member of a small family of proteins containing a novel MYB/SANT domain which is likely to participate in the regulation of a plant-specific developmental program.     

Animal models for radiation injury, protection and therapy

Current events throughout the world underscore the growing threat of different forms of terrorism, including radiological or nuclear attack. Pharmaceutical products and other approaches are needed to protect the civilian population from radiation and to treat those with radiation-induced injuries. In the event of an attack, radiation exposures will be heterogeneous in terms of both dose and quality, depending on the type of device used and each victim's location relative to the radiation source. Therefore, methods are needed to protect against and treat a wide range of early and slowly developing radiation-induced injuries. Equally important is the development of rapid and accurate biodosimetry methods for estimating radiation doses to individuals and guiding clinical treatment decisions. Acute effects of high-dose radiation include hematopoietic cell loss, immune suppression, mucosal damage (gastrointestinal and oral), and potential injury to other sites such as the lung, kidney and central nervous system (CNS). Long-term effects, as a result of both high- and low-dose radiation, include dysfunction or fibrosis in a wide range of organs and tissues and cancer. The availability of appropriate types of animal models, as well as adequate numbers of animals, is likely to be a major bottleneck in the development of new or improved radioprotectors, mitigators and therapeutic agents to prevent or treat radiation injuries and of biodosimetry methods to measure radiation doses to individuals.     

Development of a recA gene-based identification approach for the entire Burkholderia genus

Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel Burkholderia recA sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of Burkholderia strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the Burkholderia recA gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this recA fragment differentiated both putative and known Burkholderia species and all members of the B. cepacia complex. In addition, it enabled the design of a Burkholderia genus-specific recA PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel recA genes enabled clarification of the taxonomic position of several important Burkholderia strains and revealed the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not be used as a means to differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of B. cepacia, B. stabilis, and B. ambifaria capable of residing in both niches.     

Leishmania: role of P glycoprotein in drug resistance and reversion strategies

Protozoan parasites are important causative agents of morbidity and mortality throughout the world--a problem further complicated by the emergence of drug resistance in these parasites. Mechanisms of drug resistance include the following: decreased uptake of the drug into the cell, loss of drug activation, alterations in the drug target, and over-expression of a well-known multiple drug transporter proteins. In this review, two critical components of resistance are stressed: (1) the role of ATP binding cassette proteins, such as P-glycoproteins, in mediating drug resistance in Leishmania and other protozoans, followed by development of cross-resistance to many structurally and functionally unrelated drugs, and (2) some concepts concerning the reversal mechanism of multidrug resistance by drugs and natural products. Several modulators or chemosensitizers alter the capacity of P-glycoproteins to maintain subtoxic intracellular drug concentrations. Calcium channel blockers such as verapamil act in this mode; however, high concentrations are required for an efficient and effective inhibition and, in addition, produce undesirable side effects. The discovery of new, natural product modulators of P-glycoproteins is stressed. This category of modulators offer potentially improved efficacy and lowered toxicity for the mammalian host.     

Common receptor for Bacillus thuringiensis toxins Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera, Helicoverpa zea, and Spodoptera exigua

Binding studies using (125)I-Cry1Ac and biotinylated Cry1Fa toxins indicate the occurrence of a common receptor for Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera, Helicoverpa zea, and Spodoptera exigua. Our results, along with previous binding data and the observed cases of cross-resistance, suggest that this pattern seems to be widespread among lepidopteran species.     

Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies

Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating the NMR analysis of larger proteins. Here we demonstrate that segmental isotopic labeling of proteins can be conveniently achieved in Escherichia coli using intein-based protein ligation. Our method is based on a dual expression system that allows sequential expression of two precursor fragments in media enriched with different isotopes. Using this in vivo approach, unlabeled protein tags can be incorporated into isotopically labeled target proteins to improve protein stability and solubility for study by solution NMR spectroscopy.     

Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli

Protoplast fusion has been used to combine genes from different organisms to create strains with desired properties. A recently developed variant on this approach, genome shuffling, involves generation of a genetically heterogeneous population of a single organism, followed by recursive protoplast fusion to allow recombination of mutations within the fused protoplasts. These are powerful techniques for engineering of microbial strains for desirable industrial properties. However, there is a prevailing opinion that it will be difficult to use these methods for engineering of Gram-negative bacteria because the outer membrane makes protoplast fusion more difficult. Here we describe the successful use of protoplast fusion in Escherichia coli. Using two auxotrophic strains of E. coli, we obtained prototrophic strains by recombination in fused protoplasts at frequencies of 0.05-0.7% based on the number of protoplasts subjected to fusion. This frequency is three-four orders of magnitude better than those previously reported for recombination in fused protoplasts of Gram-negative bacteria such as E. coli and Providencia alcalifaciens.     

Expression of Sara2 human gene in erythroid progenitors

A human homologue of Sar1, named Sara2, was shown to be preferentially expressed during erythropoiesis in a culture stimulated by EPO. Previous studies, in yeast, have shown that secretion-associated and Ras-related protein (Sar1p) plays an essential role in protein transport from the endoplasmic reticulum to the Golgi apparatus. Here, we report the molecular analysis of Sara2 in erythroid cell culture. A 1250 bp long cDNA, encoding a 198 amino-acid protein very similar to Sar1 proteins from other organisms, was obtained. Furthermore, we also report a functional study of Sara2 with Real-time quantitative PCR analysis, demonstrating that expression of Sara2 mRNA increases during the initial stages of erythroid differentiation with EPO and that a two-fold increase in expression occurs following the addition of hydroxyurea (HU). In K562 cells, Sara2 mRNA was observed to have a constant expression and the addition of HU also up-regulated the expression in these cells. Our results suggest that Sara2 is an important gene in processes involving proliferation and differentiation and could be valuable for understanding the vesicular transport system during erythropoiesis.