The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.     

Volatile profiles of human skin cell cultures in different degrees of senescence

It is known that skin releases volatile organic compounds to the environment, and also that its emission pattern changes with aging of the skin. It could be considered, that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, a simple and non-destructive method consisting of SPME sampling and GC/MS analysis was developed to identify volatile organic emanations from cell cultures. This technique, applied to skin cells culture, indicates that the cells or cell metabolism produce several skin emissions. Chemometric analysis was performed in order to explore the relationship between a volatile profile and the senescence of cell cultures. Volatile profiles were different for cell cultures in different degrees of senescence, indicating that volatile compound patterns could be used to provide information about the age of skin cells.     

Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Physical microhabitat requirements of freshwater pearl mussels,Margaritifera margaritifera (L.)

Surface sediment diatoms from the east coast of Lake Tanganyika were analysed using ordination and classification techniques, and compared with assemblages previously described from the northern part of the lake. Grain-size analyses were performed on subsamples. Four groups of diatom assemblages were recognised. The first group clusters samples taken in the north, far from the Rusizi river mouth. The second group comprises samples taken on silty sediment along the Tanzanian coast, including one sample taken near the mouth of the Malagarazi river and those from the northernmost part of the lake. The third group comprises surface sediments along the Burundian coast (near Ramba and Magara), and the fourth is characterised by epipsammic taxa. A sample taken near the central arm of the Malagarazi river is included in the latter group. The impact of small rivers on the diatom assemblages in the surface sediments is restricted to the mouth area.     

Products of mitochondrial protein synthesis in Tetrahymena.

The products of mitochondrial protein synthesis have been investigated in Tetrahymena after labelling with [35S]methionine in the presence of cycloheximide. The labelled proteins were analyzed by sodium dodecyl sulfate slab polyacrylamide gel electrophoresis. We have identified 13 electrophoretically discrete bands as well as 4 other bands with a more variable occurrence. These proteins ranged in apparent molecular weight from 8100 to 57,500. The cycloheximide-resistant incorporation could be blocked with chloramphenicol. The mitochondrial proteins appeared to be in a disaggregated state and were stable to agents such as trichloroacetic acid (hot or cold) and chloroform-methanol. The pattern of proteins was similar following labelling times ranging from 30 min to 3 h.