Human StarD5, a cytosolic StAR-related lipid binding protein

Recently identified StarD5 belongs to the StarD4 subfamily, a subfamily of steroidogenic acute regulatory related lipid transfer (START) domain proteins that includes StarD4 and StarD6, proteins whose functions remain unknown. The objective of this study was to confirm StarD5's protein localization and sterol binding capabilities as measures to pursue function. Using rabbit polyclonal antibody against newly purified human histidine-tagged/StarD5 protein, StarD5 was detected in human liver. In parallel studies, increased expression of StarD5 in primary hepatocytes led to a marked increase in microsomal free cholesterol. Cell fractionation studies demonstrated StarD5 protein in liver cytosolic fractions only, suggesting StarD5 as a directional cytosolic sterol carrier. Supportive in vitro binding assays demonstrated a concentration-dependent binding of cholesterol by StarD5 similar to that of the cholesterol binding START domain protein StarD1. In contrast to selective cholesterol binding by StarD1, StarD5 bound the potent regulatory oxysterol, 25-hydroxycholesterol, in a concentration-dependent manner. StarD5 binding appeared selective for cholesterol and 25-hydroxycholesterol, as no binding was observed for other tested sterols. The ability of StarD5 to bind not only cholesterol but also 25-hydroxycholesterol, a potent inflammatory mediator and regulatory oxysterol, raises basic fundamental questions about StarD5's role in the maintenance of cellular cholesterol homeostasis.     

Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

We studied the reaction between vinyl butyrate and 2-phenyl-1-propanol in acetonitrile catalyzed by Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY and on Accurel PA-6. The choice of 2-phenyl-1-propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. With all the supports, initial rates of transesterification were higher at a water activity (a(w)) of 0.2 than at a(w) = 0.7, and the reverse was true for initial rates of hydrolysis. By providing acid-base control in the medium through the use of solid-state buffers that control the parameter pH-pNa, which we monitored using an organo-soluble chromoionophoric indicator, we were able, in some cases, to completely eliminate dissolved butyric acid. However, none of the buffers used were able to improve the rates of transesterification relative to the blanks (no added buffer) when the enzyme was immobilized at an optimum pH of 8.5. When the enzyme was immobilized at pH 5 and exhibited only marginal activity, however, even a relatively acidic buffer with a pK(a) of 4.3 was able to restore catalytic activity to about 20% of that displayed for a pH of immobilization of 8.5, at otherwise identical conditions. As a(w) was increased from 0.2 to 0.7, rates of transesterification first increased slightly and then decreased. Rates of hydrolysis showed a steady increase in that a(w) range, and so did total initial reaction rates. The presence or absence of the buffers did not impact on the competition between transesterification and hydrolysis, regardless of whether the butyric acid formed remained as such in the reaction medium or was eliminated from the microenvironment of the enzyme through conversion into an insoluble salt. Cutinase enantioselectivity towards 2-phenyl-1-propanol was indeed low and was not affected by differences in immobilization support, enzyme protonation state, or a(w).     

Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.     

Reduced L-selectin (CD62LLow) expression identifies tumor-specific type 1 T cells from lymph nodes draining an autologous tumor cell vaccine

Reduced expression of CD62L can identify tumor-specific T cells in lymph nodes draining murine tumors. Here, we examined whether this strategy could isolate tumor-specific T cells from vaccinated patients. Tumor vaccine-draining lymph node (TVDLN) T cells of seven patients were separated into populations with reduced (CD62LLow) or high levels of CD62L (CD62LHigh). Effector T cells generated from CD62LLow cells maintained or enriched the autologous tumor-specific type 1 cytokine response compared to unseparated TVDLN T cells in four of four patients showing tumor-specific cytokine secretion. Interestingly, effector T cells generated from CD62LLow or CD62LHigh TVDLN were polarized towards a dominant type 1 or type 2 cytokine profile, respectively. For CD62LLow T cells the type 1 cytokine profile appeared determined prior to culture. Since a tumor-specific type 1 cytokine profile appears critical for mediating anti-tumor activity in vivo, this approach might be used to isolate T cells for adoptive immunotherapy.     

Signaling through Gs alpha is required for the growth and function of neuromuscular synapses in Drosophila

Although synapses are assembled in a highly regulated fashion, synapses once formed are not static structures but continue to expand and retract throughout the life of an organism. One second messenger that has been demonstrated to play a critical role in synaptic growth and function is cAMP. Here, we have tested the idea that signaling through the heterotrimeric G protein, Gs, plays a coincident role with increases in intracellular Ca(+2) in the regulation of adenylyl cyclases (ACs) during synaptic growth and in the function of synapses. In larvae containing a hypomorphic mutation in the dgs gene encoding the Drosophila Gs alpha protein, there is a significant decrease in the number of synaptic boutons and extent of synaptic arborization, as well as defects in the facilitation of synaptic transmission. Microscopic analysis confirmed that Gs alpha is localized at synapses both pre- and postsynaptically. Restricted expression of wild-type Gs alpha either pre- or postsynaptically rescued the mutational defects in bouton formation and defects in the facilitation of synaptic transmission, indicating that pathways activated by Gs alpha are likely to be involved in the reciprocal interactions between pre- and postsynaptic cells required for the development of mature synapses. In addition, this Gs alpha mutation interacted with fasII, dnc, and hyperexcitability mutants in a manner that revealed a coincident role for Gs alpha in the regulation of cAMP and FASII levels required during growth of these synapses. Our results demonstrate that Gs alpha-dependent signaling plays a role in the dynamic cellular reorganization that underlies synaptic growth.     

Habitat fragmentation and biodiversity: testing for the evolutionary effects of refugia

Concordant areas of endemism among taxa have important implications both for understanding mechanisms of speciation and for framing conservation priorities. Here we discuss the need for careful testing of phylogeographic data for evidence of such concordance, with particular reference to the Indonesian island of Sulawesi. This is because there are good reasons to question whether concordance between taxa is likely to be a common pattern, and because of the serious implications of incorrectly concluding that the biodiversity of a given area can be partitioned in this way.     

A Novel ZZ/ZW Sex Chromosome System for the Genus Leporinus (Pisces, Anostomidae, Characiformes)

A wide range of sex chromosome mechanisms, including simple and multiple chromosome systems is characteristic of fishes. The Leporinus genus represent a good model to study sex chromosome mechanisms, because an unambiguous ZZ/ZW sex chromosome system was previously described for seven species, while the remaining studied species of the genus do not show differentiated sex chromosomes. The occurrence of sex chromosomes in Leporinus trifasciatus and Leporinus sp2 from the Araguaia river, Amazon basin, Brazil, was here investigated. ZZ/ZW sex chromosomes were detected for both species. The Z and W chromosome morphology of L. trifasciatus is the same as described for other species of the genus Leporinus. However, the Z and W chromosomes of L. sp2 were quite different in their morphology and banding pattern suggesting that the ZW system of this species have originated independently from the ZW system previously described for other Leporinus.     

The Physical Gene Hsp70 Map on Polytene Chromosomes of Anopheles darlingi from the Brazilian Amazon

In situ hybridization was used to determine the physical location of the Hsp70 genes in salivary polytene chromosomes of Anopheles darlingi from Manaus and Macapá, Brazil, and to assess the usefulness of the Hsp70 locus as a genetic marker in A. darlingi populations. In both populations, the double markings corresponding to the Hsp70-12A and Hsp70-14A genes were located on the right arm of chromosome 2. The Hsp70 locus was considered to be an excellent marker for studying chromosomal evolution and relationships among A. darlingi populations.     

Experimentally increased testosterone affects social rank and primary sex ratio in the spotless starling

It has been suggested that the amount of maternal testosterone allocated into the eggs might be implicated in the process of sex determination. However, recent findings on the effect that female social rank has on the level of egg testosterone suggest that reported associations between male-biased sex ratios and yolk testosterone may represent an indirect hormonal effect mediated by the interdependence among maternal hormones, female social rank, and sex ratio. Here, we report the results of a field experiment in which we manipulated the circulating levels of testosterone in female spotless starlings (Sturnus unicolor) before egg formation. Focal females were controlled in subsequent years to explore possible delayed effects of hormone manipulation on primary sex ratio and social status that could persist because of permanent hormonal change or through hormone-dominance interactions. The results indicate that testosterone-implanted females (T-females) produced significantly more sons than control females (C-females) in the year in which they were manipulated. These differences in offspring sex ratio between T- and C-females persisted in the next 3 years, although no additional hormone treatments were given. These results were not mediated by an eventual effect of testosterone treatment on the quality of the females' mates. A similar proportion of T- and C-females acquired a nest box and bred either in the manipulation year or in Year 1 after manipulation, but T-females tended to be more successful in acquiring a nest box than C-females in Years 2 and 3 after manipulation. These results suggest that added testosterone had a direct role on the acquisition and maintenance of high social rank. Delayed effects of testosterone on primary sex ratio might have been caused by altered endogenous production of T-females. Alternatively, the maintenance of sex ratio differences between T- and C-females long after having being implanted might be attributed to the positive effect that enhanced social rank of T-females has on their circulating testosterone levels.