Detection and kinetic studies of triplex formation by oligodeoxynucleotides using real-time biomolecular interaction analysis (BIA).

Real-time biomolecular interaction analysis (BIA) has been applied to triplex formation between oligodeoxynucleotides. 5'-Biotinylated oligonucleotides were immobilised on the streptavidin-coated surface of a biosensor chip and subsequently hybridised to their complementary strand. Sequence-specific triplex formation was observed when a suitable third-strand oligopyrimidine was injected over the surface-bound duplex. In addition, a single-stranded oligonucleotide immobilised on the chip surface was able to capture a DNA duplex by triplex recognition. The presence of spermine increases the rate of association between the third strand and immobilised duplex, but at elevated spermine concentrations non-specific association is observed. A preliminary kinetic analysis of triplex formation at pH 5.2 by an 11mer third strand containing thymine, cytosine and uracil is reported. Values for the association and dissociation rate constants were determined to be (1.9 +/- 0.2) x 10(3) M-1 s-1 and (8.1 +/- 1.9) x 10(-5) s-1, respectively.     

Divergent effects of extracellular oxygen on the growth, morphology, and function of human skin microvascular endothelial cells

Partial pressure of extracellular oxygen influences a number of major cellular functions. The purpose of this study was to determine if the proliferation, morphology, and synthesis of proteins important in the function of skin microvascular endothelial cells were significantly altered by an extracellular oxygen tension used to culture endothelial cells. Microvascular endothelial cells were isolated from the dermis of neonatal foreskins and were studied at a venous capillary oxygen level (5% O(2), 38 mm Hg) and at an atmospheric oxygen level (20.8% O(2,) 158 mm Hg). At all time points studied and at all passage numbers, a significant inhibition of proliferation was observed at 20.8% O(2) compared to identical cultures grown and subcultured at 5% O(2). Two morphologically distinct endothelial cell populations were observed at 5% O(2). When mediators of angiogenesis and inflammation-such as basic fibroblast growth factor (bFGF), phorbol myristate acetate (PMA), and interleukin-1beta (IL-1beta)-were studied, additional differences in proliferation were observed. Atmospheric O(2) inhibited the synthesis of a major basement membrane protein (Type IV collagen), a major surface protein (PECAM-1), and increased the synthesis of von Willebrand factor (vWf). The rate of vascular channel formation induced by collagen gels was decreased at 5% O(2). These results demonstrate that an increase in extracellular oxygen tension from 5 to 20.8% can significantly alter the cellular physiology of human skin microvascular endothelial cells.     

Proteomic analysis of survival of Rhodococcus jostii RHA1 during carbon starvation

Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ～250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO(2) utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ΔsigG deletion mutant showed only minor changes in cell survival. Stationary-phase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.