Bauhinia bauhinioides plasma kallikrein inhibitor: interaction with synthetic peptides and fluorogenic peptide substrates related to the reactive site sequence

A serine proteinase inhibitor was purified from Bauhinia bauhinioides seeds after extraction with 0.15M NaCl by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q chromatography or alternatively by affinity chromatography on trypsin- Sepharose. The inhibitor is a single polypeptide chain with molecular mass 20 kDa by gel filtration on Superose 12, but was resolved into two peaks by ion - exchange chromatography on Mono Q (FPLC system). The main eluted peak inhibits trypsin (Ki = 0.6 nM), plasma kallikrein (Ki = 0.35 nM), plasmin (Ki = 33.1 nM), and weakly chymotrypsin (Ki = 2,700 nM), being the most effective plasma kallikrein inhibitor isolated from Bauhinia seeds. Therefore, it was denominated Bauhinia bauhinioides kallikrein inhibitor (BbKI). Activity is thermolabile and on trypsin inhibition optimum pH is 8.0. BbKI displays high homology to other plant Kunitz inhibitors, except for the absence of disulfide bridges, and the only cysteine residue is at the C-terminal position (residue 154) characterizes a distinct member of the Kunitz family. The affinity of the inhibitor to trypsin was confirmed by adsorption to trypsin-Sepharose resin and by isolation of the trypsin-inhibitor complex by gel filtration. Peptides with variations around the reactive site of BbKI (GLPVRFESPLRINIIKESY) were synthesized containing a quenched fluorogenic group. Trypsin but not plasma kallikrein substrates, these peptides strongly inhibited plasma kallikrein.     

Laminar specific alterations of thalamocortical projections in organotypic cultures following layer 4 disruption in ferret somatosensory cortex

The developing neocortex influences the growth of thalamocortical projections. Layer 4 in particular receives the majority of input from the thalamus and is important in instructing thalamic afferents to terminate. Previous in vivo experiments demonstrated that disruption of layer 4 during corticogenesis in ferret somatosensory cortex by application of methylazoxy methanol acetate (MAM) prevents proper termination of thalamic afferents in appropriate cortical regions. To further explore the role of layer 4 in thalamocortical development, we prepared organotypic cocultures consisting of normal gestational day 0 (P0) ferret thalamus paired with normal, embryonic day 33 (E33), or E38 MAM-treated cortex obtained from ferrets at either P0 or P7. Injection of MAM on E33 disrupts layer 4 formation, whereas similar injections on E38 interfere with layer 2 formation. The cocultures grew together for a number of days, then discrete injections of either fluorescent dextrans or 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) were made into the thalamic piece. The labeled thalamic afferents that grew into the cortical slice were analysed and the sites of their terminations quantified after 3, 5, or 7-10 days in culture (DIC). Our results varied somewhat with the amount of time in culture, but the preponderance of thalamic fibers in normal cortex terminated in layer 4, whereas their counterparts in E33 MAM-treated cortex grew beyond the cortical plate and many fibers terminated inappropriately within lower cortical layers or white matter. Terminal distribution of thalamic fibers in E38 MAM-treated cortex looked similar to normal. These results demonstrate that the cells of layer 4 provide thalamic afferents with important positional and termination cues.     

Anthropometry and body composition in ethnic Japanese and Caucasian adolescent boys

Background: The effect of environmental conditions on development, including growth, maturation and the fulfillment of genetic potential, can be identified through the study of the variations found among different ethnic groups in the same population. The objectives of the present study were: (i) to compare the various anthropometric and body composition parameters based on ethnicity and maturation stage in 31 Japanese and 99 Caucasian prepubescent boys and 50 Japanese and 98 Caucasian post‐pubescent boys; and (ii) to assess body mass index (BMI) and its relationship with other methods of body fat evaluation. Methods: The percentage of body fat was measured using bioelectrical impedance, near‐infrared interactance and Slaughter cutaneous skinfold equations. Results: Weight and height were statistically lower for the Japanese than the Caucasian subjects. There were no differences in body fat between the ethnic groups, but the Japanese subjects had statistically lower levels of fat‐free mass. The gain in fat‐free mass and the loss in body fat when attaining maturation were greater in the Caucasian subjects. The agreement of BMI with other methods was good in all of the groups but lower for the Japanese than for the Caucasian subjects. Conclusion: Height and weight differences between the ethnic groups indicated distinct genetic potential ranges. The body fat mass did not differ between the ethnic groups, but the degree of changes when attaining maturation in the Caucasian subjects was greater. If this difference were to be maintained between the groups then years later there would be a greater accumulation of fat in the Japanese subjects.     

Action of anticytoskeletal compounds on in vitro cytopathic effect, phagocytosis, and adhesiveness of Trichomonas vaginalis.

The cytopathic effects of Trichomonas vaginalis treated with inhibitory concentrations of anticytoskeletal compounds (mebendazole, griseofulvin, colchicine, taxol, and cytochalasin B) were studied in mouse CLID fibroblast cultures. The evaluation, at different times, of cell numbers and morphological alterations showed that cytopathic effect was considerably reduced when protozoa were pretreated with mebendazole and griseofulvin, whereas colchicine, taxol, and cytochalasin B had less effect. Furthermore, treatment with mebendazole, griseofulvin, and colchicine decreased adhesiveness of the protozoan, whereas treatment with cytochalasin B and colchicine completely inhibited its phagocytic activity. From these results it may be concluded that alterations induced in the trichomonal cytoskeleton may affect its adhesiveness and its in vitro cytopathic effect, but there is no direct correlation between protozoan phagocytosis and its in vitro pathogenic effect.     

Verapamil and cyclosporin A modulate doxorubicin toxicity by distinct mechanisms

Cyclosporin A has been reported to enhance the sensitivity of cells displaying multiple drug resistance to anthracyclines. However, the mechanism of action of cyclosporin A in modulating drug resistance is still controversial. This study compares the effects of cyclosporin A and verapamil on doxorubicin resistance in chinese hamster ovary cells (CHRC5) using several criteria including in vitro cytotoxicity, drug accumulation, intracellular distribution by video microscopy, and nuclear DNA damage. Our results demonstrate that verapamil modulation of doxorubicin resistance was paralleled by cellular accumulation of doxorubicin, altered intracellular distribution of doxorubicin from cytoplasm to nucleus, and an increase in the formation of doxorubicin related DNA strand breaks. In contrast, the modulating effect of cyclosporin was qualitatively different. High concentrations of cyclosporin (5 micrograms/ml) increased doxorubicin accumulation and caused partial redistribution to the nucleus. However, with low concentrations of cyclosporin (1 microgram/ml) increased doxorubicin sensitivity was observed without changes in net accumulation of doxorubicin or intracellular distribution, and without enhanced doxorubicin induced DNA breakage. These results suggest that cyclosporin A can modulate doxorubicin cytotoxicity by means other than interference with the P-glycoprotein drug efflux system.     

Drug-Resistant Malaria: The Era of ACT

As drug-resistant falciparum malaria has continued to evolve and spread worldwide, artemisinin-based combination therapies (ACT) have become the centerpiece of global malaria control over the past decade. This review discusses how advances in antimalarial drug resistance monitoring and rational use of the array of ACTs now available can maximize the impact of this highly efficacious therapy, even as resistance to artemisinins is emerging in Southeast Asia.     

Effect of chitosan malate on viability and cytoskeletal structures morphology of Caco-2 cells

The aim of this work was to evaluate the effects of the treatment with chitosan malate (CM) on viability of Caco-2 cells and on the morphology and the integrity of their cytoskeletal structures (microtubules, microfilaments). Cytotoxicity of CM, both as a solution and as microparticles obtained by spray drying, was evaluated by using the reduction of MTT reagent; microtubule and microfilaments organization of Caco-2 cells treated with CM solution was examined with immunofluorescence techniques in monolayers fixed with the glutaraldehyde-borohydride method.CM as a solution displayed a concentration-dependent cytotoxicity towards Caco-2 cells, with viability percentages of 5 ± 2%, 7 ± 3% and 31 ± 15% at 15, 10 and 5 mg/mL, respectively, while at 2.5 mg/mL or less cell viability was 90% or higher. CM microparticles also produced a remarkable cytotoxic effect (cell viability 84 ± 17%, 16 ± 8% and 5 ± 6% after treatment with 1, 5 and 10 mg CM per well, respectively), resulting more toxic than CM solution.Microtubules pattern of Caco-2 cells, which is a network regularly arranged around the nucleus, appeared deeply modified by CM treatment in a concentration-dependent way, with progressive microtubule changes in length and spatial disposition and deposition of fluorescent aggregates at the periphery of the cells. Furthermore, after treatment with 5-15 mg/mL CM, remarkable alterations of actin organization were observed, with a progressive disruption of the network of stress fibers and the appearance of actin aggregates inside the cell cytoplasm.In conclusion, viability and the cytoskeletal pattern of Caco-2 cells are modified by treatment with CM at high concentrations, which might be locally reached in vivo after application of drug-loaded chitosan microparticles onto mucosal cells.     

Kinetic characterization of l-[H]glutamate uptake inhibition and increase oxidative damage induced by glutaric acid in striatal synaptosomes of rats

Glutaric acidemia type I (GA-I) is an inherited metabolic disease characterized by accumulation of glutaric acid (GA) and striatal degeneration. Although growing evidence suggests that excitotoxicity and oxidative stress play central role in the neuropathogenesis of this disease, mechanism underlying striatal damage in this disorder is not well established. Thus, we decided to investigate the in vitro effects of GA 10 nM (a low concentration that can be present initial development this disorder) on l-[³H]glutamate uptake and reactive oxygen species (ROS) generation in synaptosomes from striatum of rats. GA reduced l-[³H]glutamate uptake in synaptosomes from 1 up to 30 min after its addition. Furthermore, we also provided some evidence that GA competes with the glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC), suggesting a possible interaction of GA with glutamate transporters on synaptosomes. Moreover, GA produced a significant decrease in the V_MAX of l-[³H]glutamate uptake, but did not affect the K_D value. Although the GA did not show oxidant activity per se, it increased the ROS generation in striatal synaptosomes. To evaluate the involvement of reactive species generation in the GA-induced l-[³H]glutamate uptake inhibition, trolox (0.3, 0.6 and 6 μM) was added on the incubation medium. Statistical analysis showed that trolox did not decrease inhibition of GA-induced l-[³H]glutamate uptake, but decreased GA-induced reactive species formation in striatal synaptosomes (1, 3, 5, 10, 15 and 30 min), suggesting that ROS generation appears to occur secondarily to glutamatergic overstimulation in this model of organic acidemia. Since GA induced DCFH oxidation increase, we evaluate the involvement of glutamate receptor antagonists in oxidative stress, showing that CNQX, but not MK-801, decreased the DCFH oxidation increase in striatal synaptosomes. Furthermore, the results presented in this report suggest that excitotoxicity elicited by low concentration of GA, could be in part by maintaining this excitatory neurotransmitter in the synaptic cleft by non-competitive inhibition of glutamate uptake. Thus the present data may explain, at least partly, initial striatal damage at birth, as evidenced by acute bilateral destruction of caudate and putamen observed in children with GA-I.