Fluorometric TLC assay for evaluation of protein kinase inhibitors

A fluorometric assay for measuring protein kinase activity has been developed. The assay is based on the separation of fluorescently marked substrate 5-carboxytetramethylrhodamine-kemptide (5-TAMRA-kemptide) from its phosphorylated counterpart by TLC and quantification of the product ratiometrically by fluorescence imaging. The utility of the assay was demonstrated by measuring the activity of cAMP-dependent protein kinase. 5-TAMRA-kemptide was characterized as a substrate of this kinase by the kinetic parameters K(m)(app) and V(max). The attachment of 5-TAMRA dye to the N terminal of kemptide decreased the K(m)(app) value but did not have a significant effect on the rate and stoichiometry of the phosphorylation reaction. The inhibitory potency of three known inhibitors was evaluated with the new assay. The closeness of the obtained inhibitory activities of the compounds to the activities determined with the phosphocellulose paper-binding assay, as well as the Z' factor value of 0.5, demonstrates the reliability of the new assay for evaluation of inhibitors of protein kinases.     

Involvement of the C-terminal tail of Arthrobacter ureafaciens sialidase isoenzyme M in cleavage of the internal sialic acid of ganglioside GM1

Arthrobacter ureafaciens sialidase comprises four isoenzymes, L, M1, M2 and S, of which L, M1, and M2, but not S, have the unique ability to cleave GM1 ganglioside, but the hydrolysis of GM3 and colominic acid by S occurs at a higher rate than that by L, M1 and M2. Since the N-terminal amino acid sequences of L, M1, M2 and S were shown to be identical on protein sequencing, they were suggested to have arisen from the same protein through truncation at different C-terminal sites. A DNA segment containing an open reading frame was cloned from a genomic library, and the structural gene was found to comprise 2,970 bp encoding a protein of 990 amino acids including a signal peptide at the N-terminus, a conserved FYRIP-region and four Asp boxes. The molecular weights of the isoenzymes determined by MALDI-TOFMS revealed that L, M1, M2 and S should comprise amino acids 39-773, 39-653, 39-655 and 39-528, respectively. In fact, recombinant enzymes M2 and S prepared in Escherichia coli exhibited identical substrate specificities toward gangliosides as those of the purified enzymes. Consequently, the C-terminal tail of isoenzyme M2 might be involved in the hydrolysis of the internal sialic acid of GM1.     

Alphav-integrin utilization in human beta-cell adhesion, spreading, and motility

The role of individual integrins in human beta-cell development and function is largely unknown. This study describes the contribution of alpha(v)-integrins to human beta-cell adhesion, spreading, and motility. Developmental differences in alpha(v)-integrin utilization are addressed by comparing the responses of adult and fetal beta-cells, and vitronectin is used as a substrate based on its unique pattern of expression in the developing pancreas. Fetal and adult beta-cells attached equally to vitronectin and integrin alpha(v)beta(5) was found to support the adhesion of both mature and immature beta-cell populations. Fetal beta-cells were also observed to spread and migrate on vitronectin, and integrin alpha(v)beta(1) was found to be essential for these responses. In contrast to their fetal counterparts, adult beta-cells failed to either spread or migrate and this deficit was associated with a marked down-regulation of alpha(v)beta(1) expression in adult islet preparations. The integrin alpha(v)beta(3) was not found to support significant beta-cell attachment or migration. Based on our findings, we conclude that integrins alpha(v)beta(5) and alpha(v)beta(1) are important mediators of human beta-cell adhesion and motility, respectively. By supporting fetal beta-cell migration, alpha(v)beta(1) could play an important role in early motile processes required for islet neogenesis.     

Effects of artificial codon changes in the movement protein gene on adaptation of a hybrid bromovirus to cowpea

A hybrid Cowpea chlorotic mottle virus(CCMV) with the movement protein (MP) gene replaced with that of the closely related Brome mosaic virus cannot infect cowpea systemically. Twenty-nine spontaneous mutants from the hybrid CCMV capable of systemic infection in cowpea appeared through biased codon changes that resulted in Lys or Arg at five specific positions in the MP gene. In this study, we report that systemic infection of cowpea with the hybrid CCMV can be achieved by artificial codon changes that do not result in Lys or Arg. We discuss mechanisms that restrict the occurrence of cowpea-adapted mutants in nature.     

Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana

The 3a movement protein (MP) plays a central role in the movement of the RNA plant virus, Brome mosaic virus (BMV). To identify host factor genes involved in viral movement, a cDNA library of Nicotiana benthamiana, a systemic host for BMV, was screened with far-Western blotting using a recombinant BMV MP as probe. One positive clone encoded a protein with sequence similarity to the alpha chain of nascent-polypeptide-associated complex from various organisms, which is proposed to contribute to the fidelity of translocation of newly synthesized proteins. The orthologous gene from N. benthamiana was designated NbNACa1. The binding of NbNACa1 to BMV MP was confirmed in vivo with an agroinfiltration-immunoprecipitation assay. To investigate the involvement of NbNACa1 in BMV multiplication, NbNACa1-silenced (GSNAC) transgenic N. benthamiana plants were produced. Downregulation of NbNACa1 expression reduced virus accumulation in inoculated leaves but not in protoplasts. A microprojectile bombardment assay to monitor BMV-MP-assisted viral movement demonstrated reduced virus spread in GSNAC plants. The localization to the cell wall of BMV MP fused to green fluorescent protein was delayed in GSNAC plants. From these results, we propose that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.     

Peptide-Based Delivery of Oligonucleotides Across Blood–Brain Barrier Model

Delivery of pharmaceutical agents across a blood–brain barrier (BBB) is a challenge for brain cancer therapy. In this study, an in vitro BBB model was utilized to study the delivery of oligonucleotides across brain endothelial cells targeting to glioma cells in a Transwell? setup. A series of novel peptides were synthesized by covalent conjugation of cell-penetrating peptides with targeting peptides for delivery of gene-based therapeutics. These peptides were screened for passage across the Transwell? and we found the most efficient peptide PepFect32 from originating PepFect 14 coupled with the targeting peptide angiopep-2. PepFect32/pDNA nanocomplexes exhibited high transcytosis across the BBB in vitro model and the highest transfection efficiency to glioma cells. In conclusion, PepFect32 revealed the most efficient peptide-based vector for pDNA delivery across in vitro BBB model.     

Effects of exercise on immune functions of undernourished mice.

Regular moderate exercise may modulate the response to a stressor and thus improve immune functions in conditions commonly associated with immunodepression and elevated levels of stress hormones. For example, anorexia nervosa patients, many of whom engage in regular aerobic exercise, generally have normal immune function and viral disease resistance in spite of their severe undernutrition. To test the hypothesis that exercise can prevent undernutrition-induced immunodepression, mice were fed a nutritionally complete, semi-purified diet, either ad libitum or in restricted quantities to induce 25% loss of initial weight over 3 weeks. Half the animals from each dietary group were run on a treadmill for 30 min/day, 5 days/week. Exercise had no effect on several measures of nutritional status. Spleen weight and blastogenic response to lipopolysaccharide were significantly increased by exercise in undernourished mice. In vivo antibody response to sheep red blood cells, and in vitro splenic responses to concanavalin A and phytohemagglutin were not significantly affected by exercise. Serum corticosterone level was increased by food restriction and significantly decreased by exercise in the undernourished mice. Within a treatment group there were no significant correlations between serum corticosterone level and any immune system measure. Hypothalamic concentration of uric acid was increased in food restriction groups and concentration of norepinephrine was increased in exercise groups. The results suggest that regular exercise may help prevent undernutrition-induced immunodepression, possibly through modulation of the stress response.