Direct association of RhoA with specific domains of PKC-alpha

Previous studies performed at our laboratory have shown that agonist-induced contraction of smooth muscle is associated with translocation of protein kinase C (PKC)- and RhoA to the membrane and that this interaction is due to a direct protein-protein interaction. To determine the domains of PKC- involved in direct interaction with RhoA, His-tagged PKC- proteins of individual domains and different combinations of PKC- domains were used to perform in vitro binding assays with the fusion protein glutathione-S-transferase (GST)-RhoA. Coimmunoprecipitation was also performed using smooth muscle cells transfected with truncated forms of PKC- in this study. The data indicate that RhoA directly bound to full-length PKC-, both in vitro (82.57 ± 15.26% above control) and in transfected cells. RhoA bound in vitro to the C1 domain of PKC- [PKC- (C1)] (70.48 ± 20.78% above control), PKC- (C2) (72.26 ± 29.96% above control), and PKC- (C4) (90.58 ± 26.79% above control), but not to PKC- (C3) (0.64 ± 5.18% above control). RhoA bound in vitro and in transfected cells to truncated forms of PKC-, PKC- (C2, C3, and C4), and PKC- (C3 and C4) (94.09 ± 12.13% and 85.10 ± 16.16% above control, respectively), but not to PKC- (C1, C2, and C3) or to PKC- (C2 and C3) (0.47 ± 1.26% and 7.45 ± 10.76% above control, respectively). RhoA bound to PKC- (C1 and C2) (60.78 ± 13.78% above control) only in vitro, but not in transfected cells, and PKC- (C2, C3, and C4) and PKC- (C3 and C4) bound well to RhoA. These data suggest that RhoA bound to fragments that may mimic the active form of PKC-. The studies using cells transfected with truncated forms of PKC- indicate that PKC- (C1 and C2), PKC- (C1, C2, and C3), and PKC- (C2 and C3) did not associate with RhoA. Only full-length PKC-, PKC- (C2, C3, and C4), and PKC- (C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC- with RhoA may require the C4 domain. domains; histidine; fusion proteins     

PON1 Paraoxonase Activity is Reduced During HDL Oxidation and is an Indicator of HDL Antioxidant Capacity

The aim of this study was to investigate the effect of HDL oxidation on PON1 paraoxonase activity. Also, we were interested in investigating the mechanism by which PON1 could be inactivated and the correlation between its enzymatic activity and the antioxidant properties of HDL. Three different oxidation systems were used for the HDL oxidation: (1) oxidation induced by THP1 cells, (2) oxidation induced by copper ions at a concentration 10 &#119 M, and (3) oxidation induced by &#148 OH and O 2 &#148 &#109 oxygen free radicals produced by &#110 -radiolysis. HDL oxidation was followed by the measurement of lipid peroxide formation, and PON1 activity was determined by measuring the rate of paraoxon hydrolysis. Our results show that HDL oxidation is accompanied by a reduction in the PON1 paraoxonase activity. The extent of PON1 inactivation depends both on the extent of HDL oxidation and on the oxidation system used. The rates of HDL oxidation and PON1 inactivation were significantly correlated ( r =0.93, p <0.0054). Our results show that oxidized HDL loses its protective effect toward LDL oxidation. The antioxidant action of HDL towards LDL oxidation and the degradation of PON1 paraoxonase activity were significantly correlated ( r =0.95, p <0.04).     

Synthesis of Novel α-l-Arabinopyranosides of β-Lactams with Potential Antimicrobial Activity

Synthetic routes toward the synthesis of some novel 1-(2,3,4-tri-O-acetyl-α-l-arabinopyranosyl)-azetidin-2-ones are described. Antimicrobial screening of three selected compounds revealed their activity against Bacillus subtilis and Escherichia coli.     

Transient permeability induced by alkyl derivatives of amphotericin B in lipid membranes

Individual ionic channels were shown to be formed in the brain cholesterol containing phospholipid membranes by two-sided addition of the amphotericin B alkyl derivatives. At concentrations between 10(-8) and 10(-7) M, the resulting conductance appeared to be transient. Existence of different antibiotic assemblies was justified by the kinetic analysis of the membrane conductance decline following the antibiotic washing out. In order to account for the transient characteristics of the induced conductance, it was proposed that the antibiotic oligomers incorporate into the membrane from the aqueous phase, form channels aggregating with cholesterol, and then dissociate in the bilayer into non-active degraded oligomeric or monomeric forms.     

Production of ethanol by alginate-entrapped Saccharomyces cerevisiae strain "14-12"

Cells of S. cerevisiae strain "14-12" of different ages were immobilized in sodium alginate and used for conversion of glucose to ethanol. Immobilized cells of 48 hr old were the most potential. Employment of high counts of alginate-entrapped cells shortened the period required for production of the maximal alcohol yield. However, the percentage surviving cells decreased with increasing initial cell counts. Maximal accumulation of ethanol (4.18 g/100 ml) was obtained after 4 days of static fermentation with 1.8 X 10(8) immobilized yeast cells. The residual viable cell count was found to represent 3-fold the surviving percentage in a control experiment using an inoculum of the free yeast cells. Immobilized yeast cells could convert about 85% of the available sugars to ethanol over 28 days of the repeated-batch fermentation. The immobilized cells retained 50% of their viability for 16 days. After 48 days of repeated fermentation only 6% of the yeast cells were viable, and on the 52nd day no viable cells could be detected.     

Changes in physical,chemical and microbial parameters during the composting of municipal sewage sludge

Changes in physical, chemical and microbial parameters were investigated during the composting of municipal sewage sludge. Raw sewage sludge (30% dry matter) was mixed with compost from sewage sludge (85% dry matter) in 3:1 ratio (v/v). The mixture was divided into 4 windrows which were composted under the same conditions except the turning factor. The turning was every 7, 10, 15 days and according to the temperature which must be (55–65°C) for windrow 1 (W1), windrow 2 (W2), windrow 3 (W3) and windrow 4 (W4), respectively. Water was added to adjust the moisture content (40–60%). The composting process consisted of 2 periods; fermentation (12 weeks) and maturation (4 weeks). The results showed that the temperature reached the maximum after 12 weeks for W1 and 11 weeks for W2, W3 and W4 and then decreased. The final compost was nearly odourless and black, especially in case of W4. The general trend indicates a decrease in organic matter, organic carbon and nitrogen (N), whereas ash, potassium (K) and phosphorus (P) increased and consequently C/K and C/P ratios decreased. There was a slight increase in C/N ratio. The pH increased and then decreased to near neutrality at the end. The mesophilic bacteria increased during the fermentation period and decreased after that, whereas the thermophilic ones increased with increasing of temperature, decreased after 2 weeks and increased again during the fermentation period and then decreased. The mesophilic and thermophilic fungi were present during the first week and disappeared after that. The final compost was pathogens-free as indicated by the counts of coliforms and Salmonella.