Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.     

Two Amino-acid Carriers in Pea Chloroplasts

CHLOROPLASTS are more permeable to amino-acids^1,2 than are cell membranes³ and also discriminate between their optical isomers². This has led to the suggestion that the passage of amino-acids through the limiting membranes of chloroplasts is facilitated by carriers². Using the additional criteria of saturation and competition, we support this suggestion and show that at least two carriers are probably involved.     

The unpredictability of favourability: condition assessment and protected areas in England

The probability that protected areas will deliver their potential for maintaining or enhancing biodiversity is likely to be maximised if they are appropriately and effectively managed. As a result, governments and conservation agencies are devoting much attention to the management of protected areas. In the U.K., the demand for performance accountability has resulted in Public Service Agreements (PSA) that set out targets for government departments to deliver results in return for investments being made. One such target for England is to ensure that all nationally important wildlife sites are in favourable condition by 2010. Here, we tested the hypothesis, of potential strategic importance, that the ecological condition of these sites is predictable from relationships with a range of physical, environmental and demographic variables. We used binary logistic regression to investigate these relationships, using the results of English Nature’s 1997–2003 condition assessment exercise. Generally, sites in unfavourable condition tend to be larger in area, located at higher elevations, but with higher human population density and are more spatially isolated from units of the same habitat. However, despite the range of different parameters included in our models, the extent to which the condition of any given site could be predicted was low. Our results have implications for the delivery of PSA targets, funding allocation, and the location of new protected areas.     

A simple method using PCR for direct sequencing of genomic DNA from frozen tumor tissue embedded in optimal cutting temperature compound.

Described here is a three-day protocol that directly yields DNA sequence after isolating and PCR amplifying genomic DNA from a small sample of frozen nasopharyngeal carcinoma tissue embedded in optimal cutting temperature (OCT) compound. The method is consistently successful, reproducible and will facilitate the rapid analysis of DNA sequence from very small samples.     

Natural relationship between bacteroides and flavobacteria.

Comparisons among 16S rRNA sequences from various eubacteria reveal a natural relationship between the bacteroides (represented by the Bacteroides fragilis sequence) and a phylogenetic unit that comprises the flavobacteria, cytophagae, flexibacteria, and others (represented by the Flavobacterium heparinum sequence). Although the relationship is not a close one, it is, nevertheless, specific. rRNAs from these two organisms are not only closer to one another in overall sequence than they are to outgroup species (such as Bacillus subtilis, Escherichia coli, Desulfovibrio desulfuricans, and Agrobacterium tumefaciens), but they show common idiosyncrasies (i.e., derived characteristics) in both rRNA sequences and higher-order structures.     

Mise en evidence de l'existence de sensilles chemorecepteurs sur les antennes d'un crustace mysidace cavernicole et de l'intervention de ces sensilles dans la detection d'un extrait de nourriture

The cavernicolous crustacean Antromysis juberthiei showed unexpected behavior when presented with food extract : it avoids the area of odor inflow. This avoidance reaction is reduced by removal of various combinations of antennae and disappears almost with bilateral removal of the first antennae (the aesthetasc-bearing antennules).     

Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17-3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting [1,7,16-3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of 3H2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16-3H]progesterone and [4-14C]progesterone was employed.