Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Monocarbonyl Analogs of Curcumin with Potential to Treat Colorectal Cancer

Curcumin has a plethora of biological properties, making this compound potentially effective in the treatment of several diseases, including cancer. However, curcumin clinical use is compromised by its poor pharmacokinetics, being crucial to find novel analogs with better pharmacokinetic and pharmacological properties. Here, we aimed to evaluate the stability, bioavailability and pharmacokinetic profiles of monocarbonyl analogs of curcumin. A small library of monocarbonyl analogs of curcumin 1a–q was synthesized. Lipophilicity and stability in physiological conditions were both assessed by HPLC-UV, while two different methods assessed the electrophilic character of each compound monitored by NMR and by UV-spectroscopy. The potential therapeutic effect of the analogs 1a–q was evaluated in human colon carcinoma cells and toxicity in immortalized hepatocytes. Our results showed that the curcumin analog 1e is a promising agent against colorectal cancer, with improved stability and efficacy/safety profile.     

Sampling properties of DNA sequence data in phylogenetic analysis

We inferred phylogenetic trees from individual genes and random samples of nucleotides from the mitochondrial genomes of 10 vertebrates and compared the results to those obtained by analyzing the whole genomes. Individual genes are poor samples in that they infrequently lead to the whole-genome tree. A large number of nucleotide sites is needed to exactly determine the whole-genome tree. A relatively small number of sites, however, often results in a tree close to the whole-genome tree. We found that blocks of contiguous sites were less likely to lead to the whole-genome tree than samples composed of sites drawn individually from throughout the genome. Samples of contiguous sites are not representative of the entire genome, a condition that violates a basic assumption of the bootstrap method as it is applied in phylogenetic studies.      

Economic evaluation of water loss saving due to the biological control of water hyacinth at New Year’s Dam,Eastern Cape province,South Africa

Water hyacinth Eichhornia crassipes is considered the most damaging aquatic weed in the world. However, few studies have quantified the impact of this weed economically and ecologically, and even fewer studies have quantified the benefits of its control. This paper focuses on water loss saving as the benefit derived from biological control of this plant between 1990 and 2013 at New Year’s Dam, Alicedale, Eastern Cape, South Africa. Estimates of water loss due to evapotranspiration from water hyacinth vary significantly; therefore, the study used three different rates, high, medium and low. A conservative raw agriculture value of R 0.26 per m³ was used to calculate the benefits derived by the water saved. The present benefit and cost values were determined using 10% and 5% discount rates. The benefit/cost ratio at the low evapotranspiration rate was less than one, implying that biological control was not economically viable but, at the higher evapotranspiration rates, the return justified the costs of biological control. However, at the marginal value product of water, the inclusion of the costs of damage to infrastructure, or the adverse effects of water hyacinth on biodiversity, would justify the use of biological control, even at the low transpiration rate.     

Is the association between optimistic cardiovascular risk perceptions and lower rates of cardiovascular disease mortality explained by biomarkers of systemic inflammation or endothelial function? A case-cohort study

Background  

More optimistic perceptions of cardiovascular disease risk are associated with substantively lower rates of cardiovascular death among men. It remains unknown whether this association represents causality (i.e. perception leads to actions/conditions that influence cardiovascular disease occurrence) or residual confounding by unmeasured factors that associate with risk perceptions and with physiological processes that promote cardiovascular disease (i.e. inflammation or endothelial dysfunction).     

Treatment with apolipoprotein A-1 mimetic peptide reduces lupus-like manifestations in a murine lupus model of accelerated atherosclerosis

Introduction

The purpose of this study was to evaluate the effects of L-4F, an apolipoprotein A-1 mimetic peptide, alone or with pravastatin, in apoE^-/-Fas^-/-C57BL/6 mice that spontaneously develop immunoglobulin G (IgG) autoantibodies, glomerulonephritis, osteopenia, and atherosclerotic lesions on a normal chow diet.

Methods

Female mice, starting at eight to nine weeks of age, were treated for 27 weeks with 1) pravastatin, 2) L-4F, 3) L-4F plus pravastatin, or 4) vehicle control, followed by disease phenotype assessment.

Results

In preliminary studies, dysfunctional, proinflammatory high-density lipoproteins (piHDL) were decreased six hours after a single L-4F, but not scrambled L-4F, injection in eight- to nine-week old mice. After 35 weeks, L-4F-treated mice, in the absence/presence of pravastatin, had significantly smaller lymph nodes and glomerular tufts (P_{L, LP} < 0.05), lower serum levels of IgG antibodies to double stranded DNA (dsDNA) (P_L < 0.05) and oxidized phospholipids (oxPLs) (P_{L, LP} < 0.005), and elevated total and vertebral bone mineral density (P_{L, LP} < 0.01) compared to vehicle controls. Although all treatment groups presented larger aortic root lesions compared to vehicle controls, enlarged atheromas in combination treatment mice had significantly less infiltrated CD68⁺ macrophages (P_LP < 0.01), significantly increased mean α-actin stained area (P_LP < 0.05), and significantly lower levels of circulating markers for atherosclerosis progression, CCL19 (P_{L, LP} < 0.0005) and VCAM-1 (P_L < 0.0002).

Conclusions

L-4F treatment, alone or with pravastatin, significantly reduced IgG anti-dsDNA and IgG anti-oxPLs, proteinuria, glomerulonephritis, and osteopenia in a murine lupus model of accelerated atherosclerosis. Despite enlarged aortic lesions, increased smooth muscle content, decreased macrophage infiltration, and decreased pro-atherogenic chemokines in L-4F plus pravastatin treated mice suggest protective mechanisms not only on lupus-like disease, but also on potential plaque remodeling in a murine model of systemic lupus erythematosus (SLE) and accelerated atherosclerosis.     

A conserved genetic mechanism specifies deutocerebral appendage identity in insects and arachnids

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). Here we show that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.