Evidence that proteosome inhibitors and chemical chaperones can rescue the activity of retinol dehydrogenase 12 mutant T49M

Retinol dehydrogenase 12 (RDH12) is a microsomal enzyme that catalyzes the reduction of all-trans-retinaldehyde to all-trans-retinol when expressed in cells. Mutations in RDH12 cause severe retinal degeneration; however, some of the disease-associated RDH12 mutants retain significant catalytic activity. Our previous study (Lee et al., 2010 [9]) demonstrated that the catalytically active T49M and I51N variants of RDH12 undergo accelerated degradation through the ubiquitin-proteosome system, which results in reduced levels of these proteins in the cells. Here, we investigated whether the stabilization of T49M or I51N RDH12 protein levels through the inhibition of proteosome activity or improved folding could rescue their retinaldehyde reductase activity. For the T49M variant, the inhibition of proteosome activity resulted in an increased level of T49M protein in the microsomal fraction. The higher level of the T49M variant in microsomes correlated with the higher microsomal retinaldehyde reductase activity. T49M-expressing living cells treated with the inhibitors of proteosome activity or with dimethyl sulfoxide exhibited an increase in the conversion of retinaldehyde to retinol, consistent with the recovery of functional RDH12 protein. On the other hand, accumulation of the I51N variant in the microsomes did not result in higher retinaldehyde reductase activity of the microsomes or cells. These results provide a proof of concept that, at least in the case of the T49M variant, the prevention of accelerated degradation could lead to restoration of its function in the cells. This finding justifies further search for more efficient and clinically relevant compounds for stabilizing the T49M variant activity.     

WCI,a novel wheat chymotrypsin inhibitor: purification,primary structure,inhibitory properties and heterologous expression

A novel chymotrypsin inhibitor, detected in the endosperm of Triticum aestivum, was purified and characterized with respect to the main physical–chemical properties. On the basis of its specificity, this inhibitor was named WCI (wheat chymotrypsin inhibitor). WCI is a monomeric neutral protein made up of 119 residues and molecular mass value of 12,933.40 Da. Automated sequence and mass spectrometry analyses, carried out on several samples of purified inhibitor, evidenced an intrinsic molecular heterogeneity due to the presence of the isoform [des-(Thr)WCI], accounting for about 40% of the total sample. In vitro, WCI acted as a strong inhibitor of bovine pancreatic chymotrypsin as well as of chymotryptic-like activities isolated from the midgut of two phytophagous insects, Helicoverpa armigera (Hüb.) and Tenebrio molitor L., respectively. No inhibitory activities were detected against bacterial subtilisins, bovine pancreatic trypsin, porcine pancreatic elastase or human leukocyte elastase. The primary structure of WCI was significantly similar (45.7–89.1%) to those of several proteins belonging to the cereal trypsin/α-amylase inhibitor super-family and showed the typical sequence motif of this crowed protein group. The cDNA of the inhibitor (wci-cDNA) was isolated from wheat immature caryopses and employed to obtain a recombinant product in E. coli. Experimental evidences indicated that the recombinant inhibitor was localized in the inclusion bodies from which it was recovered as soluble and partially active protein by applying an appropriate refolding procedure. WCI reactive site localization, as well as its inhibitory specificity, was investigated by molecular modeling approach.     

Male hosts drive infracommunity structure of ectoparasites

We studied the co-occurrence of flea species in infracommunities of 16 rodents from four regions (South Africa, Tanzania, central Europe and western Siberia) using null models, and predicted that flea co-occurrences will be expressed more strongly in male than in female hosts. We examined patterns of co-occurrence (measured as the C score) in infracommunities of fleas that are parasitic on male and female hosts by comparing co-occurrence frequencies with those expected by chance. When a significant degree of nonrandomness in flea co-occurrences was detected, it indicated aggregative infracommunity structure. In Tanzanian rodents, no significant flea co-occurrences were detected in either male or female hosts. In a South African rodent, significant flea co-occurrences were not detected in males, but were found in females in some localities. In Palaearctic rodents, significant nonrandomness was detected either equally for males and females or more frequently in males than in females. Meta-analyses demonstrated that the frequency of the detection of nonrandomness in flea co-occurrences was significantly higher in male than in female hosts. The values of the standardized effect size (SES) for the C score differed significantly among host species, but not between host genders. When the Palaearctic hosts were analyzed separately, the effects of both host gender and species appeared to be significant, with the SES values for the C score in males being smaller than those in females. The strength of the gender difference in the manifestation of flea community structure increased with increasing gender difference in flea species richness, and with decreasing gender difference in flea prevalence for the Palaearctic hosts. We conclude that male hosts are the main drivers of flea infracommunity structure. However, the manifestation of gender bias in flea community structure varies among host species, and is likely determined by the pattern of species-specific spatial behavior.     

Contrasting community compensatory trends in alternative successional pathways in central Amazonia

Based on eight years of annual censuses in secondary forests in central Amazonia, we compared successional dynamics in areas presenting alternative states due to different land use histories. Sites that had been clearcut without subsequent use are dominated by the pioneer genus Cecropia, but their understory is characterized by a diverse species assemblage. In contrast, areas clearcut and then used for pasture are dominated by the genus Vismia, forming nearly monogeneric stands. We evaluated whether such patterns were the outcome of differences in community compensatory trends, leading to a dynamic system of sequential replacement of species in Cecropia stands, and to a persistent stage of succession in Vismia stands. Floristic turnover in Cecropia stands showed strong and consistent negative frequency dependence. In contrast, Vismia stands exhibited little or no frequency dependence, likely due to local competitive interactions or priority effects. In these stands, species of the genera Vismia and Bellucia remained dominant throughout the monitoring period, whereas species initially of low abundance and frequency remained so. Differences in recruitment were the major driver of these alternative states. As species colonization proceeds, we expect dominance in the Vismia stands to diminish, albeit slowly. Our approach proved to be a useful tool for comparing species turnover in systems presenting alternative states.     

Galvanic vestibular stimulation improves the results of vestibular rehabilitation

Here, we present findings from a three-step investigation of the effect of galvanic vestibular stimulation (GVS) in normal subjects and in subjects undergoing vestibular rehabilitation (VR). In an initial study, we examined the body sway of 10 normal subjects after one minute of 2 mA GVS. The effect of the stimulation lasted for at least 20 minutes in all subjects and up to two hours in 70% of the subjects. We then compared a group of patients who received conventional VR (40 patients) with a group that received a combination of VR and GVS. Results suggest a significant improvement in the second group. Finally, we attempted to establish the optimal number of GVS sessions and to rule out a placebo effect. Fifteen patients received "systematic" GVS: five sessions, once a week. Five patients received "nonsystematic" galvanic stimulation in a sham protocol, which included two stimulations of the clavicle. These data were analyzed with Fisher's exact test and indicated that the best results were obtained after three sessions of GVS and no placebo effect was observed.     

The genetic diversity and phenotypic characterisation of Streptococcus agalactiae isolates from Rio de Janeiro, Brazil

Streptococcus agalactiae isolates are more common among pregnant women, neonates and nonpregnant adults with underlying diseases compared to other demographic groups. In this study, we evaluate the genetic and phenotypic diversity in S. agalactiae strains from Rio de Janeiro (RJ) that were isolated from asymptomatic carriers. We analysed these S. agalactiae strains using pulsed-field gel electrophoresis (PFGE), serotyping and antimicrobial susceptibility testing, as well as by determining the macrolide resistance phenotype, and detecting the presence of the ermA/B, mefA/E and lnuB genes. The serotypes Ia, II, III and V were the most prevalent serotypes observed. The 60 strains analysed were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampin and tetracycline was observed. Among the erythromycin and/or clindamycin resistant strains, the ermA, ermB and mefA/E genes were detected and the constitutive macrolides, lincosamides and streptogramin B-type resistance was the most prevalent phenotype observed. The lnuB gene was not detected in any of the strains studied. We found 56 PFGE electrophoretic profiles and only 22 of them were allocated in polymorphism patterns. This work presents data on the genetic diversity and prevalent capsular serotypes among RJ isolates. Approximately 85% of these strains came from pregnant women; therefore, these data may be helpful in developing future prophylaxis and treatment strategies for neonatal syndromes in RJ.     

Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival

The neurotrophins NGF and NT3 collaborate to support development of sympathetic neurons. Although both promote axonal extension via the TrkA receptor, only NGF activates retrograde transport of TrkA endosomes to support neuronal survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that?a Rac1-cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes. These actin-regulatory endosomal components are absent from NT3/TrkA endosomes, explaining the failure of NT3 to support retrograde TrkA transport and survival. The inability of NT3 to activate Rac1-GTP-cofilin signaling is likely due to the labile nature of NT3/TrkA complexes within the acidic environment of TrkA early endosomes. Thus, TrkA endosomes associate with actin-modulatory proteins to promote F-actin disassembly, enabling their maturation into transport-competent signaling endosomes. Differential control of this process explains how NGF but not NT3 supports retrograde survival of sympathetic neurons.     

Frequent loss of heterozygosity in the β2-microglobulin region of chromosome 15 in primary human tumors

Downregulation or total loss of HLA class I expression on tumor cells is known as a mechanism of cancer immune escape. Alterations of the HLA phenotype are frequently due to mutations affecting genes encoding the HLA class I heavy chains located on chromosome 6p21 or the β2-microglobulin (β2m) gene encoding the light chain of the HLA complex located on chromosome 15q21. Frequently irreversible total loss of HLA class I molecules is due to the coincidence of two molecular events, the mutation of one β2m gene and the loss of the second copy. The latter is detectable as loss of heterozygosity (LOH) of microsatellite markers in the β2m region on chromosome 15q21 (LOH-15q21). Thus, LOH-15q21 might be an important event in the processes of HLA class I downregulation and total loss. Here we studied the frequency of LOH-15q21 in tumor tissues of different entities. By determining the status of heterozygosity of two microsatellite markers we detected LOH-15q21 in 44% of bladder carcinomas (n = 69), in 35% of colon carcinomas (n = 95), in 16% of melanomas (n = 70) but only in 7% of renal cancers (n = 45). Moreover, we observed a frequent coincidence of LOH-15q21 and LOH-6p21 in colorectal carcinoma, bladder carcinoma and melanoma, but not for renal carcinoma. We believe that the high incidence of LOH-15q21 in some malignancies and especially the coincidence of LOH-15q21 and LOH-6p21 might have a strong impact on tumor immunogenicity and on the efficiency of cancer immunotherapy.