Bioorganic synthesis of a recombinant HIV-1 fusion inhibitor,SC35EK,with an N-terminal pyroglutamate capping group

The bioorganic synthesis of an end-capped anti-HIV peptide from a recombinant protein was investigated. Cyanogen bromide-mediated cleavage of two Met-Gln sites across the target anti-HIV sequence generated an HIV-1 fusion inhibitor (SC35EK) analog bearing an N-terminal pyroglutamate (pGlu) residue and a C-terminal homoserine lactone (Hsl) residue. The end-capped peptide, pGlu-SC35EK-Hsl, had similar bioactivity and biophysical properties to the parent peptide, and an improved resistance to peptidase-mediated degradation was observed compared with the non-end-capped peptide obtained using standard recombinant technology.     

Functional Interactions between Sphingolipids and Sterols in Biological Membranes Regulating Cell Physiology

Sterols and sphingolipids are limited to eukaryotic cells, and their interaction has been proposed to favor formation of lipid microdomains. Although there is abundant biophysical evidence demonstrating their interaction in simple systems, convincing evidence is lacking to show that they function together in cells. Using lipid analysis by mass spectrometry and a genetic approach on mutants in sterol metabolism, we show that cells adjust their membrane composition in response to mutant sterol structures preferentially by changing their sphingolipid composition. Systematic combination of mutations in sterol biosynthesis with mutants in sphingolipid hydroxylation and head group turnover give a large number of synthetic and suppression phenotypes. Our unbiased approach provides compelling evidence that sterols and sphingolipids function together in cells. We were not able to correlate any cellular phenotype we measured with plasma membrane fluidity as measured using fluorescence anisotropy. This questions whether the increase in liquid order phases that can be induced by sterol–sphingolipid interactions plays an important role in cells. Our data revealing that cells have a mechanism to sense the quality of their membrane sterol composition has led us to suggest that proteins might recognize sterol–sphingolipid complexes and to hypothesize the coevolution of sterols and sphingolipids.     

Long-term Administration of Probiotics to Asymptomatic Pre-school Children for Either the Eradication or the Prevention of Helicobacter pylori Infection

Background:  The role of probiotics in the armamentarium remains to be defined. The aims of this study were to investigate whether the long-time administration of Lactobacillus gasseri OLL2716 (LG21) strain can eradicate H. pylori in asymptomatic pre-school children and/or prevent H. pylori infection.
Methods: A total of 440 children, from 5–7 years of age, attending a kindergarten in Thailand were screened by the Helicobacter pylori stool antigen (HpSA) test. Thereafter 132 H. pylori positive and 308 H. pylori negative children were recruited to eradication and randomized prevention arms, respectively. Children in the active and placebo treatment groups received Lactobacillus gasseri OLL2716 (LG21) containing cheese and ordinary cheese, respectively, for 12 months. Eradication was defined as reversion by HpSA at 12 months. Prevention was defined as persistently HpSA negative at 12 months.
Results:  Eighty-two of 132 H. pylori positive (62%) completed the eradication arm, of which 24 (29.3%) were negative at 12 months according to the HpSA test. In the randomized prevention arm, 123 of 156 (79%) and 99 of 122 (81%) completed active and placebo arms, respectively, of which 4.1% and 8.1%, respectively, were HpSA positive at 12 months based on a per-protocol analysis ( p  = .21).
Conclusion: Further trials are needed.     

Induction and isolation of pigmentation mutants of Porphyra yezoensis (Bangiales, Rhodophyta) by heavy-ion beam irradiation

The present study describes the isolation of pigmentation mutants of Porphyra yezoensis Ueda induced by heavy-ion beam irradiation for the first time. The gametophytic blades were irradiated with ¹²C⁺⁶ ion beams within a dose range of 25–400 Gy. From the survival rate and cell growth of the irradiated blades, it is suggested that a dose of 150 Gy or less is suitable to induce mutation for the isolation of mutants of P. yezoensis . After irradiation, red, green and deep reddish brown-colored gametophytic blades developed from archeospores that were released from each of the mutated cell clusters of the respective different colors, and the red mutant strain (IBY-R1) and green mutant strain (IBY-G1) were established as a conchocelis colony in culture. Blades of the mutants were characterized by their growth and photosynthetic pigment contents compared with those of the wild-type. From these results, it is clear that heavy-ion beam mutagenesis will be an effective tool for genetic and breeding studies of Porphyra , and also for other algal research.     

A simple method for isolation of nuclei from <Emphasis Type="Italic">Basidiobolus ranarum</Emphasis> (Zygomycota)

A simple method for isolating the nuclei from Basidiobolus ranarum was established. To improve the yield and purity of nuclei, we investigated maceration methods, buffer composition, and centrifugation conditions to establish an optimal procedure. Basidiobolus ranarum cultured for 5 days was enzymatically macerated and then homogenized and filtrated through stainless steel sieves. The crude cell homogenate was loaded on a layer of buffer containing 50% glycerol and centrifuged at 1500 g. The resultant pellet contained pure nuclei.     

Preliminary estimation of chum salmon stock composition in the Bering Sea and North Pacific Ocean using polymorphic microsatellite DNA markers

Variation at the three microsatellite (ms) DNA loci in chum salmon was applied to estimate preliminarily the stock composition using a conditional maximum likelihood method in more than 700 fish collected from 14 stations in the Bering Sea and adjacent North Pacific Ocean during September 2003. Regional stock assignment accuracy with these msDNA markers was nearly the same as the previous estimation with mitochondrial (mt) DNA for the Japanese and North American stocks, but decreased for Russian stocks. The temporal stock estimation with msDNA gave a nonrandom distribution pattern of chum stocks, in that the Japanese and Russian stocks increased in the western to central Bering Sea, and the North American stocks were abundant in the eastern Bering Sea and near the Aleutian Islands. However, predominance of the North American stocks in nearly all of the surveyed area was different from the previous mtDNA estimation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CERT-mediated trafficking of ceramide

The transport and sorting of lipids from the sites of their synthesis to their appropriate destinations are fundamental for membrane biogenesis. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER), and transported from the ER to the trans Golgi regions, where it is converted to sphingomyelin. CERT mediates the ER-to-Golgi trafficking of ceramide. CERT contains several functional domains and motifs including i) a START domain capable of catalyzing inter-membrane transfer of ceramide, ii) a pleckstrin homology domain, which serves to target the Golgi apparatus, iii) a FFAT motif which interacts with the ER-resident membrane protein VAP, and iv) a serine-repeat motif, of which hyperphosphorylation down-regulates CERT activity. It has been suggested that CERT extracts ceramide from the ER and carries it to the Golgi apparatus in a non-vesicular manner and that efficient CERT-mediated trafficking of ceramide occurs at membrane contact sites between the ER and the Golgi apparatus.