Reaction conditions for laccase catalyzed polymerization of catechol

Poly(catechol) was synthesized in batch runs with laccase from Trametes versicolor (ATCC 200801). The polymerization reaction was conducted in a closed, temperature controlled system containing acetone and sodium acetate buffer for pH control. The effects of the solvent mixture, monomer (catechol), enzyme, medium pH and temperature on the polymerization rate were investigated with respect to initial reaction conditions and depletion rate of dissolved oxygen in the medium. Maximum initial reaction rate was attained with 10% (v/v) acetone-sodium acetate buffer at pH 5.0, 25 degrees C, 0.02 U/ml enzyme and 250 mg/l initial catechol and 10 mg/l dissolved oxygen. A general saturation enzyme kinetics response was observed for catechol substrate. Temperature rise supported the rate increase up to 45 degrees C, after which the rate tended to be stable due to a drop in dissolved oxygen concentration as well as enzyme instability.     

Synthesis and antimalarial activity of 2-methoxyprop-2-yl peroxides derivatives

2-Methoxyprop-2-yl peroxides were synthesized and evaluated in vitro against Plasmodium falciparum. These acyclic artemisinin-related peroxides revealed moderate to good activity but were devoid of alkylating property towards the synthetic model of heme Mn(II)-TPP.     

Biodegradation of ethyl t-butyl ether (ETBE), methyl t-butyl ether (MTBE) and t-amyl methyl ether (TAME) by Gordonia terrae

Gordonia terrae strain IFP 2001 was selected from activated sludge for its capacity to grow on ethyl t-butyl ether (ETBE) as sole carbon and energy source. ETBE was stoichiometrically degraded to t-butyl alcohol (TBA) and the activity was inducible. A constitutive strain, G. terrae IFP 2007, derived from strain IFP 2001, was also selected. Methyl t-butyl ether (MTBE) and t-amyl methyl ether (TAME) were not used as carbon and energy sources by the two strains, but cometabolic degradation of MTBE and TAME was demonstrated, to TBA and t-amyl alcohol (TAA) respectively, in the presence of a carbon source such as ethanol. No two-carbon compound was detected during growth on ETBE, but formate was produced during cometabolic degradation of MTBE or TAME. A monooxygenase was involved in the degradation of ethers, because no degradation of ETBE was observed under anaerobic conditions and the presence of a cytochrome P-450 was demonstrated in G. terrae IFP 2001 after induction by cultivation on ETBE.     

Gender influence on jejunal migrating motor complex

The role of gender and the menstrual cycle in small bowel motility has not been clearly elucidated. Jejunal motility was recorded with a nasojejunal catheter incorporating five solid-state pressure transducers in ambulatory menstruating women and men of comparable age over 24 h. All women were studied twice, in the early follicular (early-F) and midluteal (mid-L) phases of the menstrual cycle, verified by determining serum levels of gonadal steroids and gonadotropins. The propagation velocity of phase III was slow and the contraction amplitude was high in both menstrual cycle phases compared with men, and these parameters were correlated with serum estrogen levels in the mid-L phase. In the early-F phase, migrating motor complex (MMC) cycle duration during sleep was long compared with other groups and positively correlated with estrogen concentrations, whereas in the mid-L phase MMC cycle duration during sleep was negatively correlated with serum progesterone levels. In all groups, the frequency of phase III contractions was low and the intercontractile interval measured from pressure peak to peak was long during sleep compared with the awake state. Postprandial motility did not display gender difference in any parameter examined. The results demonstrate that the majority of patterns of motility are similar in menstruating women and men, whereas certain aspects of the MMC, most conspicuously propagation velocity and phase III contraction amplitude, differ. We have also documented circadian variation of phase III contraction frequency in both women and men.     

Insulin resistance in nonobese patients with polycystic ovary syndrome

OBJECTIVES: Most patients with polycystic ovary syndrome (PCOS) are obese and known to have insulin resistance. Obesity per se is a cause of insulin resistance. This study was performed to determine whether insulin resistance occurs in patients with PCOS in the absence of obesity and acanthosis nigricans. METHOD: For this purpose, an euglycemic hyperinsulinemic clamp study was performed in 12 nonobese patients with PCOS and in 10 healthy control subjects matched for age and weight. RESULTS: The mean serum testosterone and luteinizing hormone (LH) levels were significantly elevated (4.09 +/- 1.32 vs. 1.18 +/- 0.53 pg/ml, p < 0.001, and 11.63 +/- 5.37 vs. 4.98 +/- 2.73 mIU/ml, p < 0.001, respectively), and the serum sex hormone binding globulin level was significantly reduced (40.96 +/- 14.94 vs. 73.98 +/- 30.40 nmol/l, p < 0.001) in patients with PCOS as compared with the values in control subjects. The mean serum insulin level was also elevated in patients with PCOS as compared with control subjects (32.33 +/- 4.98 vs. 19.56 +/- 2.21 microU/ml, p < 0.05). The insulin sensitivity was lower in patients with PCOS as compared with the control subjects (200 +/- 27.8 vs. 427.8 +/- 88.9 micromol x kg(-1) x min(-1), p < 0.001). In patients with PCOS, the serum levels of free testosterone (r = -0.89, p < 0.001) and LH were inversely correlated with the insulin sensitivity (r = -0.63, p < 0.05). Serum follicle-stimulating hormone, prolactin, and dehydroepiandrosterone sulfate levels were similar in both groups. CONCLUSIONS: These results indicate that a significant degree of insulin resistance exists in nonobese patients with PCOS and that this insulin resistance is significantly related to serum LH and free testosterone levels. Thus, measures to decrease insulin resistance may have to be considered earlier to decrease the potential risks of developing diabetes mellitus and coronary artery disease at later ages of life in these patients.     

Secretin, glucagon, gastric inhibitory polypeptide, parathyroid hormone, and related peptides in the regulation of the hypothalamus- pituitary-adrenal axis

Secretin, glucagon, gastric inhibitory polypeptide (GIP), and parathyroid hormone (PTH) belong, together with vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase (AC)-activating polypeptide, to a family of peptides (the VIP-secretin-glucagon family), which also includes growth hormone-releasing hormone and exendins. All the members of this peptide family possess a remarkable amino-acid sequence homology, and bind to G-protein-coupled receptors, whose signaling mechanism primarily involves AC/protein kinase A and phospholipase C/protein kinase C cascades. VIP and pituitary AC-activating polypeptide play a role in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis, and in this review we survey findings that also other members of the VIP-secretin-glucagon family may have the same function. Secretin and secretin receptors are expressed in the hypothalamus and pituitary gland, and secretin inhibits adrenocorticotropic hormone (ACTH) release. No evidence is available for the presence of secretin receptors in adrenal glands, but secretin selectively depresses the glucocorticoid response to ACTH of dispersed zona fasciculata-reticularis (ZF/R) cells. Glucagon and glucagon-like peptide-1 are contained in the hypothalamus, and all the components of the HPA axis are provided with glucagon and glucagons-like-1 receptors. These peptides exert a short-term inhibitory effect on stress-induced pituitary ACTH release and depress the ZF/R cell response to ACTH by inhibiting the AC/protein kinase A cascade; they also stimulate hypothalamic arginine-vasopressin release. GIP receptors are present in the ZF/R of the normal adrenals, and are particularly abundant in some types of adrenocortical adenomas and hyperplasias. GIP, through the activation of the AC/protein kinase A cascade, evokes a sizeable glucocorticoid secretagogue effect, leading to the identification of a food/GIP-dependent Cushing's syndrome. PTH and PTH-related protein are expressed in the hypothalamus and pituitary gland, and PTH and PTH-related protein receptors in all the components of the HPA axis. Both peptides enhance ACTH and arginine-vasopressin release, as well as stimulate aldosterone and glucocorticoid secretion of dispersed zona glomerulosa and ZF/R cells, respectively. The involvement of growth hormone-releasing hormone and exendins in the functional regulation of the HPA axis has not yet been extensively investigated.     

Distinction between Ca(2+) pump and Ca(2+)/H(+) antiport activities in synaptic vesicles of sheep brain cortex

Synaptic vesicles, isolated from a sheep brain cortex, accumulate Ca(2+) in a manner that depends on the pH and pCa values. In the presence of 100 microM CaCl(2), most of the Ca(2+) taken up by the vesicles was vanadate-inhibited (86%) at pH 7.4, whereas at pH 8.5, part of the Ca(2+) accumulated (36%) was DeltapH-dependent (bafilomycin and CCCP inhibited) and part was insensitive to those drugs (31%). We also observed that both vanadate-sensitive and bafilomycin-sensitive Ca(2+) accumulations were completely released by the Ca(2+) ionophore, ionomycin, and that these processes work with high (K(0.5)=0.6 microM) and low (K(0.5)=217 microM) affinity for Ca(2+), respectively. The DeltapH-dependent Ca(2+) transport appears to be largely operative at Ca(2+) concentrations (>100 microM) which completely inhibited the vanadate-sensitive Ca(2+) uptake. These Ca(2+) effects on the Ca(2+) accumulation were well correlated with those observed on the vanadate-inhibited Ca(2+)-ATPase and bafilomycin-inhibited H(+)-ATPase, respectively. The Ca(2+)-ATPase activity reached a maximum at about 25 microM (pH 7.4) and sharply declined at higher Ca(2+) concentrations. In contrast, Ca(2+) had a significant stimulatory effect on the H(+)-ATPase between 250 and 500 microM Ca(2+) concentration. Furthermore, we found that DeltapH-sensitive Ca(2+) transport was associated with proton release from the vesicles. About 21% of the maximal proton gradient was dissipated by addition of 607.7 microM CaCl(2) to the reaction medium and, if CaCl(2) was present before the proton accumulation, lower pH gradients were reached. Both vanadate-inhibited and bafilomycin-inhibited systems transported Ca(2+) into the same vesicle pool of our preparation, suggesting that they belong to the same cellular compartment. These results indicate that synaptic vesicles of the sheep brain cortex contain two distinct mechanisms of Ca(2+) transport: a high Ca(2+) affinity, proton gradient-independent Ca(2+) pump that has an optimal activity at pH 7.4, and a low Ca(2+) affinity, proton gradient-dependent Ca(2+)/H(+) antiport that works maximally at pH 8.5.     

The protective effect of lisinopril on membrane-bound enzymes in myocardial preservation

A number of studies have reported that oxidant stress reduces the activity of isolated Na(+)-K(+) ATPase and Ca(2+) ATPase which are known to affect the cell membrane integrity. The aim of the study is to determine whether the administration of lisinopril is able to protect the membrane-bound enzyme levels in isolated guinea pig hearts and also ascertain whether or not a relationship exists between oxygen free radicals and membrane bound Na(+)-K(+) ATPase and Ca(2+) ATPase. Forty guinea pig hearts were studied in an isolated Krebs-Henseleit solution-perfused Langendorff cardiac model. In all groups cardioplegic arrest was achieved by administering St. Thomas' Hospital cardioplegic solution (STHCS). Group 1 (control, n=10) received only STHCS. Group 2 (n=10) were arrested with lisinopril (l micromol l(-1)) added STHCS. Group 3 (n=10) were pretreated with oral lisinopril (0.2 mg kg(-1) twice a day) for 10 days and then arrested with STHCS. Group 4 were also pretreated with oral lisinopril (0.2 mg kg(-1) twice a day for 10 days), arrested with STHCS and reperfused with lisinopril added to Krebs-Henseleit solution (l micromol l(-1)). Hearts were subjected to normothermic global ischaemia for 90 min and then reperfused at 37 degrees C. Pretreatment and addition of lisinopril in the reperfusion buffer improved the levels of membrane-bound enzymes. When the treated groups were compared with control hearts, the best results were achieved in group 4. The Na(+)-K(+) and Ca(2+) ATPase levels increased from 466.38+/-5.99 to 560.12+/-18.02 and 884.69+/-9.13 to 1287.71+/-13.01 nmolPi mg(-1) protein h(-1) respectively (p<0.05). These results suggest that lisinopril protects the cell membrane integrity and lessens free radical-induced oxidant stress.     

Divergent N-terminal sequences target an inducible testis deubiquitinating enzyme to distinct subcellular structures

Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-gamma-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action.