Population dynamics of Triatoma vitticeps (Stål, 1859) in Itanhomi, Minas Gerais, Brazil

Taxonomic markers (head structure morphometry, isoenzymes and randon amplified polymorphism of DNA - RAPD) were used to understand the population dynamics of Triatoma vitticeps, predominant triatomine species in Itanhomi district, using samples obtained from domestic, peridomiciliary and sylvatic habitats. Morphometric analysis revealed sexual dimorphism within the three samples although specimens could not be separated according to the habitat in which they were captured. Forty-two bands were analyzed from RAPD profiles generated using four primers. A dendrogram constructed from Dice's similarity coefficient values showed that migration of the insects between the habitats has occurred, without structuring of populations. Moreover, the dendrogram obtained from the genetic distance values showed an important gene flow between the sylvatic and domestic habitats. No polymorphism was found in the electrophoretic mobility of proteins for the ten enzymes studied. Our results revealed movement of triatomines between the three habitats, suggesting that the presence of T. vitticeps in houses should not be ignored. As invasion of houses by sylvatic insects is frequent and the natural infection indices of this species are among the highest known, epidemiological vigilance studies may reveal possible changes in T. vitticeps behaviour which could present future risks to public health.     

Production of cachexia mediators by Walker 256 cells from ascitic tumors

In neoplasic cachexia, chemical mediators seem to act as initiators or perpetuators of this process. Walker 256 cells, whose metabolic properties have so far been little studied with respect to cancer cachexia, are used as a model for the study of this syndrome. The main objective of this research was to pinpoint the substances secreted by these cells that may contribute to the progression of the cachectic state. Since inflammatory mediators seem to be involved in the manifestation of this syndrome, the in vitro production of nitric oxide (NO), cytokines (tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6)), and prostaglandin E2 (PGE2) was evaluated in Walker 256 cells isolated from ascitic tumors. After 4 or 5 h, a significant increase in NO production was observed (2.55 +/- 1.56 and 4.05 +/- 1.99 nmol NO per 10(7) cells, respectively). When isolated from a 6-day-old tumor, a significantly lower production of IL-6 and higher production of TNF-alpha than in cells from a 4-day-old tumor were observed, indicating a relationship between the production of cytokines and the time of tumor development after implantation. Considerable production of PGE(2) by Walker 256 cells isolated from the 6-day-old tumor was also observed. Polyamines were also determined in Walker 256 cells. Levels of putrescine, spermidine, and spermine did not show significant differences in tumors developed during 4 or 6 days. Direct evidence of the release of proinflammatory cytokines and PGE2 by Walker 256 cells suggests that these mediators can drive the cachectic syndrome in the host, the effect being dependent on tumor development time.     

Combating protein misfolding and aggregation by intracellular antibodies

Conformational or misfolding diseases are a large class of devastating human disorders associated with protein misfolding and aggregation. Most conformational diseases are caused by a combination of genetic and environmental factors, suggesting that spontaneous events can destabilize the protein involved in the pathology or impair the clearance mechanisms of misfolded aggregates. Aging is one of the risk factors associa-ted to these events, and the clinical relevance of conformational disorders is growing dramatically, as they begin to reach epidemic proportions due to increases in mean lifespan. Currently, there are no effective strategies to slow or prevent these diseases. Intrabodies are promising therapeutic agents for the treatment of misfolding diseases, because of their virtually infinite ability to specifically recognize the different conformations of a protein, including pathological isoforms, and because they can be targeted to the potential sites of aggregation (both intra- and extracellular sites). These molecules can work as neutralizing agents against amylo-idogenic proteins by preventing their aggregation, and/or as molecular shunters of intracellular traffic by re-routing the protein from its potential aggregation site. The fast-developing field of recombinant antibody technology provides intrabodies with enhanced binding specificity and stability, together with lower immunogenicity, for use in a clinical setting. This review provides an update on the applications of intrabodies in misfolding diseases, with particular emphasis on an evaluation of their multiple and feasible modes of action.     

A decreased mitochondrial DNA content is related to insulin resistance in adolescents

The aim of this study was to investigate whether mitochondrial DNA (mtDNA) content is associated with insulin resistance (IR) in a sample of adolescents with features of metabolic syndrome. We further studied the link between polymorphisms in three genes involved in mitochondrial biogenesis and the presence of deleted mtDNA and mtDNA content. Data and blood samples were collected from 175 adolescents out of a cross-sectional, population-based study of 934 high school students. On the basis of the median value of homeostasis model assessment of IR (HOMA-IR) of the whole sample (2.2), the population was divided into two groups: noninsulin resistance (NIR) and IR. mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out using a real-time quantitative PCR method. Genotyping for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) (pro12Ala), PPAR- gamma coactivator-1alpha (PGC-1alpha) (Gly482Ser), and Tfam (rs1937 and rs12247015) polymorphisms was performed by PCR-based restriction fragment length polymorphism. Long-extension PCR was performed to amplify the whole mitochondrial genome. The mtDNA/nDNA ratio was significantly lower in the IR group (median: 9.08, range: 68.94) in comparison with the NIR group (12.24, 71.92) (P<0.03). Besides, the mtDNA/nDNA ratio was inversely correlated with HOMA (R: -0.18, P<0.02), glucose (R: -0.21, P<0.008), and uric acid (R: -0.18, P<0.03). Genotypes for the PPAR- gamma, PGC-1alpha, and Tfam variants were not associated with the mtDNA/nDNA ratio. Long-extension PCR did not show significant levels of mtDNA deletions. In conclusion, our findings indicate that reduced mtDNA content in peripheral leukocytes is associated with IR. This result seems not to be related with the previously mentioned variants in genes involved in the regulation of mitochondrial biogenesis.     

High frequency of copy number variations and sequence variants at CYP21A2 locus: implication for the genetic diagnosis of 21-hydroxylase deficiency

Background

The systematic study of the human genome indicates that the inter-individual variability is greater than expected and it is not only related to sequence polymorphisms but also to gene copy number variants (CNVs). Congenital Adrenal Hyperplasia due to 21-hydroxylase deficiency (21OHD) is the most common autosomal recessive disorder with a carrier frequency of 1∶25 to 1∶10. The gene that encodes 21-hydroxylase enzyme, CYP21A2, is considered to be one of the most polymorphic human genes. Copy number variations, such as deletions, which are severe mutations common in 21OHD patients, or gene duplications, which have been reported as rare events, have also been described. The correct characterization of 21OHD alleles is important for disease carrier detection and genetic counselling

Methodology and Findings

CYP21A2 genotyping by sequencing has been performed in a random sample of the Spanish population, where 144 individuals recruited from university students and employees of the hospital were studied. The frequency of CYP21A2 mutated alleles in our sample was 15.3% (77.3% were mild mutations, 9% were severe mutations and 13.6% were novel variants). Gene dosage assessment was also performed when CYP21A2 gene duplication was suspected. This analysis showed that 7% of individuals bore a chromosome with a duplicated CYP21A2 gene, where one of the copies was mutated.

Conclusions

As far as we know, the present study has shown the highest frequency of 21OHD carriers reported by a genotyping analysis. In addition, a high frequency of alleles with CYP21A2 duplications, which could be misinterpreted as 21OHD alleles, was found. Moreover, a high frequency of novel genetic variations with an unknown effect on 21-hydroxylase activity was also found. The high frequency of gene duplications, as well as novel variations, should be considered since they have an important involvement in carrier testing and genetic counseling.     

BicaudalD Actively Regulates Microtubule Motor Activity in Lipid Droplet Transport

Background

A great deal of sub-cellular organelle positioning, and essentially all minus-ended organelle transport, depends on cytoplasmic dynein, but how dynein''s function is regulated is not well understood. BicD is established to play a critical role in mediating dynein function—loss of BicD results in improperly localized nuclei, mRNA particles, and a dispersed Golgi apparatus—however exactly what BicD''s role is remains unknown. Nonetheless, it is widely believed that BicD may act to tether dynein to cargos. Here we use a combination of biophysical and biochemical studies to investigate BicD''s role in lipid droplet transport during Drosophila embryogenesis.

Methodology/Principal Findings

Functional loss of BicD impairs the embryo''s ability to control the net direction of droplet transport; the developmentally controlled reversal in transport is eliminated. We find that minimal BicD expression (near-BicD^null) decreases the average run length of both plus and minus end directed microtubule (MT) based transport. A point mutation affecting the BicD N-terminus has very similar effects on transport during cellularization (phase II), but in phase III (gastrulation) motion actually appears better than in the wild-type.

Conclusions/Significance

In contrast to a simple static tethering model of BicD function, or a role only in initial dynein recruitment to the cargo, our data uncovers a new dynamic role for BicD in actively regulating transport. Lipid droplets move bi-directionally, and our investigations demonstrate that BicD plays a critical—and temporally changing—role in balancing the relative contributions of plus-end and minus-end motors to control the net direction of transport. Our results suggest that while BicD might contribute to recruitment of dynein to the cargo it is not absolutely required for such dynein localization, and it clearly contributes to regulation, helping activation/inactivation of the motors.     

Extra-nuclear signaling of progesterone receptor to breast cancer cell movement and invasion through the actin cytoskeleton

Progesterone plays a role in breast cancer development and progression but the effects on breast cancer cell movement or invasion have not been fully explored. In this study, we investigate the actions of natural progesterone and of the synthetic progestin medroxyprogesterone acetate (MPA) on actin cytoskeleton remodeling and on breast cancer cell movement and invasion. In particular, we characterize the nongenomic signaling cascades implicated in these actions. T47-D breast cancer cells display enhanced horizontal migration and invasion of three-dimensional matrices in the presence of both progestins. Exposure to the hormones triggers a rapid remodeling of the actin cytoskeleton and the formation of membrane ruffles required for cell movement, which are dependent on the rapid phosphorylation of the actin-regulatory protein moesin. The extra-cellular small GTPase RhoA/Rho-associated kinase (ROCK-2) cascade plays central role in progesterone- and MPA-induced moesin activation, cell migration and invasion. In the presence of progesterone, progesterone receptor A (PRA) interacts with the G protein G alpha(13), while MPA drives PR to interact with tyrosine kinase c-Src and to activate phosphatidylinositol-3 kinase, leading to the activation of RhoA/ROCK-2. In conclusion, our findings manifest that progesterone and MPA promote breast cancer cell movement via rapid actin cytoskeleton remodeling, which are mediated by moesin activation. These events are triggered by RhoA/ROCK-2 cascade through partially differing pathways by the two compounds. These results provide original mechanistic explanations for the effects of progestins on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.     

Tissue inhibitor of metalloproteinases-2 binding to membrane-type 1 matrix metalloproteinase induces MAPK activation and cell growth by a non-proteolytic mechanism

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.