Diazepam administration after prolonged status epilepticus reduces neurodegeneration in the amygdala but not in the hippocampus during epileptogenesis

An episode of status epilepticus (SE), if left untreated, can lead to death, or brain damage with long-term neurological consequences, including the development of epilepsy. The most common first-line treatment of SE is administration of benzodiazepines (BZs). However, the efficacy of BZs in terminating seizures is reduced with time after the onset of SE; this is accompanied by a reduced efficacy in protecting the hippocampus against neuronal damage, and is associated with impaired function and internalization of hippocampal GABA_A receptors. In the present study, using Fluoro-Jade C staining, we found that administration of diazepam to rats at 3 h after the onset of kainic acid-induced SE, at a dose sufficient to terminate SE, had no protective effect on the hippocampus, but produced a significant reduction in neuronal degeneration in the amygdala, piriform cortex, and endopiriform nucleus, examined on days 7–9 after SE. Thus, in contrast to the hippocampus, the amygdala and other limbic structures are responsive to neuroprotection by BZs after prolonged SE, suggesting that GABA_A receptors are not significantly altered in these structures during SE.     

Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi

Aims: The purpose of this study was to investigate the role of H₂O₂ and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani. Methods and Results: Using the H₂O₂‐specific scopoletin fluorometric assay and the CAT‐dependent H₂O₂ consumption assays, it was found that the production rate of intra/extracellular H₂O₂ and CAT levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their nondifferentiating counterpart strains. They peaked in the transition between the undifferentiated and the differentiated state of the sclerotiogenic strains, suggesting both a cell proliferative and differentiative role. In addition, the indirect indicator of oxidative stress, lipid peroxidation, was substantially decreased in the nondifferentiating strains. Conclusions: These findings suggest that the differentiative role of H₂O₂ is expressed via induction of higher oxidative stress in the sclerotiogenic filamentous phytopathogenic fungi. Significance and Impact of the Study: This study shows that the direct marker of oxidative stress H₂O₂ is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii, S. minor, S. sclerotiorum and R. solani, which could have potential biotechnological implications in terms of developing antifungal strategies by regulating intracellular H₂O₂ levels.     

Expression and membrane topology of Anopheles gambiae odorant receptors in lepidopteran insect cells

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel 'topology screen' assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties.     

High-throughput screening of enzyme libraries

Directed evolution is becoming a widely used technique for modifying or enhancing protein performance. Ultimately, the success of directed protein evolution experiments hinges on the efficiency of the methods used to screen libraries for mutants with properties of interest. Although there is still a paucity of general methods for enzyme library screening, in recent years a number of promising strategies have emerged and are increasingly being used to explore challenging issues in protein engineering.     

Fuzzy polynucleotide spaces and metrics

The study of genetic sequences is of great importance in biology and medicine. Mathematics is playing an important role in the study of genetic sequences and, generally, in bioinformatics. In this paper, we extend the work concerning the Fuzzy Polynuclotide Space (FPS) introduced in Torres, A., Nieto, J.J., 2003. The fuzzy polynuclotide Space: Basic properties. Bioinformatics 19(5); 587–592 and Nieto, J.J., Torres, A., Vazquez-Trasande, M.M. 2003. A metric space to study differences between polynucleotides. Appl. Math. Lett. 27:1289–1294: by studying distances between nucleotides and some complete genomes using several metrics. We also present new results concerning the notions of similarity, difference and equality between polynucleotides. The results are encouraging since they demonstrate how the notions of distance and similarity between polynucleotides in the FPS can be employed in the analysis of genetic material.     

Effect of alkaline medium on the production and excretion of B-lactamase byEscherichia coli

Summary The effects of pH on the production of a secreted protein were investigated. Growth ofEscherichia coli in alkaline media caused a large decrease in the specific activity of -lactamase but had little effect on the level of other cytoplasmic and periplasmic enzymes. Proteolytic degradation was at least partly responsible for the low -lactamase activity. The pH of the fermentation medium also affected the permeability of the outer membrane causing an increase in the excretion of periplasmic enzymes and lipopolysaccharide into the culture medium.     

Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase.

High-level synthesis of the periplasmic protein beta-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. beta-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature beta-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor.     

Functional and physical characteristics of rat Leydig cell populations isolated by metrizamide and Percoll gradient centrifugation

The physical and functional properties of Leydig cell populations obtained by centrifugation of testicular cells in two different density gradient media, Percoll and Metrizamide, were compared. Percoll-gradient centrifugation yielded two Leydig cell bands (Peak I and Peak II) that were comparable, as to their density and testosterone-producing capacity, to the respective Leydig cell bands, Population I and Population II, isolated in a Metrizamide gradient. The denser Leydig cell band (II) had a greater capacity for testosterone production than the less dense band (I), regardless of the type of gradient used for its isolation. Metrizamide gradient centrifugation separated the majority of germ cells from the "light" (Population I) Leydig cells, whereas in the Percoll gradient, germ cells comigrated with Peak I Leydig cells. Leydig cell separation by Percoll gradients was highly dependent on the presence of Ca2+ and Mg2+ in the medium, while these cations had no effect on the separation of Leydig cells by Metrizamide. In conclusion, Metrizamide gradient centrifugation yielded two Leydig cell populations of similar functional and physical properties to the respective populations isolated in Percoll gradients.     

Identification of the cydC locus required for expression of the functional form of the cytochrome d terminal oxidase complex in Escherichia coli.

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases which are differentially regulated. The cytochrome o complex predominates under growth conditions of high aeration, whereas the cytochrome d complex predominates when the oxygen tension is low. Either terminal oxidase will support aerobic growth. The goal of the work presented in this paper was to identify genes required for the expression of the functional form of the cytochrome d complex, other than the genes encoding the polypeptide components of the oxidase complex (cyd locus). A strain lacking the cytochrome o complex (cyo mutant strain) was mutagenized by using a lambda-Mu hybrid hopper bacteriophage, lambda placMu53, which inserts randomly into the chromosome and carries a kanamycin resistance marker. Strains were isolated and examined which were unable to grow aerobically, i.e., which lacked functional cytochrome d complex, and which could not be complemented by introduction of the cyd gene on F-prime episomes. One strain was selected for characterization. The phage insert was mapped to min 18.9 on the genetic linkage map, defining a new genetic locus, cydC. Evidence described in the text suggests that the gene product is probably required for the synthesis of the unique heme d component of the cytochrome d complex.     

Analysis of receptor clustering on cell surfaces by imaging fluorescent particles.

Fluorescently labeled low density lipoproteins (LDL) and influenza virus particles were bound to the surface of human fibroblasts and imaged with a cooled slow-scan CCD camera attached to a fluorescence microscope. Particles were also imaged after attachment to polylysine-coated microscope slides. The digital images were analyzed by fitting data points in the region of fluorescent spots by a two-dimensional Gaussian function, thus obtaining a measure of spot intensity with correction for local background. The intensity distributions for particles bound to polylysine slides were mainly accounted for by particle size distributions as determined by electron microscopy. In the case of LDL, the intensity distributions for particles bound to fibroblasts were considerably broadened, indicative of clustering. The on-cell intensity distributions were deconvolved into 1-particle, 2-particle, 3-particle, etc. components using the data obtained with LDL bound to polylysine-coated slides as an empirical measure of the single particle intensity distribution. This procedure yielded a reasonably accurate measure of the proportion of single particles, but large errors were encountered in the proportions of larger cluster sizes. The possibility of studying the dynamics of clustering was investigated by binding LDL to cells at 4 degrees C and observing changes in the intensity distribution with time after warming to 20 degrees C.