Recent Findings in the Chemistry of Odorants from Four Baccharis Species and Their Impact as Chemical Markers

Baccharis is a widespread genus belonging to the Asteraceae family that includes almost 400 species exclusively from the Americas. Even when studied in detail, the taxonomic classification among species from this genus is not yet fully defined. Within the framework of our study of the volatile composition of the Baccharis genus, four species (B. trimera, B. milleflora, B. tridentata, and B. uncinella) were collected from the ‘Campos de Cima da Serra’ highlands of the Brazilian state of Rio Grande do Sul. The aerial parts were dried and extracted by the simultaneous distillation extraction (SDE) procedure. This is the first time that SDE has been applied to obtain and compare the volatile‐extract composition in the Baccharis genus. Characterization of the volatile extracts allowed the identification of 180 peaks with many coeluting components; these latter being detailed for the first time for this genus. The multivariate statistical analyses allowed separating the volatile extracts of the four populations of Baccharis into two separate groups. The first one included the B. milleflora, B. trimera, and B. uncinella volatile extracts. The three species showed a high degree of similarity in their volatile composition, which was characterized by the presence of high contents of sesquiterpene compounds, in particular of spathulenol. The second group comprised the extract of B. tridentata, which contained α‐pinene, β‐pinene, limonene, and (E)‐β‐ocimene in high amounts.     

ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.     

Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L(-1) at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined.     

Novel series of substituted biphenylmethyl urea derivatives as MCH-R1 antagonists for the treatment of obesity

We have designed and synthesized two novel series of MCH-R1 antagonists based on a substituted biphenylmethyl urea core. SAR was explored, suggesting that optimal binding with the receptor was achieved when the biphenylmethyl group and the linker were substituted on the same nitrogen of the urea moiety. Compound 1-(3'-cyano-4-biphenylmethyl)-3-(2-hydroxy-1,1-dimethylethyl)-1-{2-[1-(4-methylbenzyl)-4-piperidinyl]ethyl}urea 2t showed the best antagonist binding activity to the MCH-R1 with a 43 nM K(i).     

Preferential Targeting of Nav1.6 Voltage-Gated Na+ Channels to the Axon Initial Segment during Development

During axonal maturation, voltage-gated sodium (Na_v) channels accumulate at the axon initial segment (AIS) at high concentrations. This localization is necessary for the efficient initiation of action potentials. The mechanisms underlying channel trafficking to the AIS during axonal development have remained elusive due to a lack of Na_v reagents suitable for high resolution imaging of channels located specifically on the cell surface. Using an optical pulse-chase approach in combination with a novel Na_v1.6 construct containing an extracellular biotinylation domain we demonstrate that Na_v1.6 channels are preferentially inserted into the AIS membrane during neuronal development via direct vesicular trafficking. Single-molecule tracking illustrates that axonal channels are immediately immobilized following delivery, while channels delivered to the soma are often mobile. Neither a Na_v1.6 channel lacking the ankyrin-binding motif nor a chimeric K_v2.1 channel containing the Na_v ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Na_v1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS.     

Molecular-based estimate of species number,phylogenetic relationships and divergence times for the genus Stenotaenia (Chilopoda,Geophilomorpha) in the Italian region

Stenotaenia is one of the largest and most widespread genera of geophilid centipedes in the Western Palearctic, with a very uniform morphology and about fifteen species provisionally recognized. For a better understanding of Stenotaenia species-level taxonomy, we have explored the possibility of using molecular data. As a preliminary assay, we sampled twelve populations, mainly from the Italian region, and analyzed partial sequences of the two genes COI and 28S. We employed a DNA-barcoding approach, complemented by a phylogenetic analysis coupled with divergence time estimation. Assuming a barcoding gap of 10–16% K2P pairwise distances, we found evidence for the presence of at least six Stenotaenia species in the Italian region, which started diverging about 50 million years ago, only partially matching with previously recognized species. We found that small-sized oligopodous species belong to a single clade that originated about 33 million years ago, and obtained some preliminary evidence of the related genus Tuoba being nested within Stenotaenia.     

Esterase-6 polymorphism inDrosophila melanogaster:Effects of temperature and methyl malonate on genotypic trajectories in polymorphic populations set up with highly inbred lines

It is generally difficult to identify possible effects of selection at a specific locus because of the heterogeneity of the genetic background. Geographical patterns ofEst-6 gene frequencies suggest that there is selection at this locus but selection on loci closely linked to it cannot be excluded. Differences in catalytic properties between allozymes have been shownin vitro; further, several laboratory studies have shown apparent fitness differences between allozymes. Our study used inbred lines highly homogeneous in the genetic background. Four populations were set up fromEst-6s andEst-6F homozygous females inseminated by males of the same genotype at each combination of three factors: temperature (18 and 25°C); methyl malonate (presence or absence); input gene frequencies [p(S) = 0.2 and 0.8]. The populations were sampled periodically for about 28 generations. Methyl malonate was chosen to exert pressure in the enzymatic function of esterase-6. Statistical analyses show that: there are no sex differences; gene frequencies change from input values to those of the first sampling, when only individuals of the first generation are present at 18^oC or individuals of the second generation just begin to appear at 25°C; gene frequencies do not change thereafter and Hardy-Weinberg equilibrium is established. The changes in gene frequencies observed in the first generations suggest thatEst-6 can under certain conditions be a target of selection. Such conditions may not, however, occur in natural populations.