Concerning the biosynthesis of prostaglandin I2

Two diastereoisomers, 5R,6R-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (7) and 5S,6S-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (10) were synthesized for evaluation as possible biosynthetic intermediates in the enzymatic transformation of PGH₂ or PGG₂ into PGI₂. The synthetic sequence entails the stereospecific reduction of the 9-keto function in PGE₂ methyl ester after protecting the C-11 and C-15 hydroxyls as tbutyldimethylsilyl ethers. The resulting PGF_2α derivative was epoxidized exclusively at the C-5 (6) double bond to yield a mixture of epoxides, which underwent facile rearrangement with SiO₂ to yield the 5S,6S and 5R,6R-5-hydroxy-6(9α)-oxido cyclic ethers. It was found that dog aortic microsomes were unable to transform radioactive 9β-5S,6S[³H] or 9β-5R,6R[³H]-5-hydroxy-6(9α)-oxido cyclic ethers into PGI₂. Also, when either diastereoisomer was included in the incubation mixture, neither isomer diluted the conversion of [1-¹⁴C]arachidonic acid into [1-¹⁴C]PGI₂.     

THE ASSOCIATION BETWEEN ACE GENE VARIATION AND AEROBIC CAPACITY IN WINTER ENDURANCE DISCIPLINES

The aim of the study was to examine the possible relationship between I/D polymorphism of ACE gene and selected indices of aerobic capacity among male and female athletes practising winter endurance sports. Sixty-six well-trained athletes (female n = 26, male n = 40), aged 18.4 ± 2.8 years, representing winter endurance sports (cross-country skiing, n = 48; biathlon, n = 8; Nordic combined, n = 10) participated in the study. Genotyping for ACE I/D polymorphism was performed using polymerase chain reaction. Maximal oxygen consumption (VO₂max), maximal running velocity (V_max) and running velocity at anaerobic threshold (V_AT4) were determined in an incremental test to volitional exhaustion on a motorized treadmill. The ACE genotype had no significant effect on absolute VO₂max, relative VO₂max (divided by body mass or fat free body mass), V_AT4 or V_max. No interaction effect of gender x ACE genotype was found for each of the examined aerobic capacity indices. ACE gene variation was not found to be a determinant of aerobic capacity in either female or male Polish, well-trained endurance athletes participating in winter sports.     

Synthesis and biological activity of some known and putative duloxetine metabolites

Several putative phase I duloxetine metabolites, 4-hydroxy-, 5-hydroxy-, 6-hydroxy-, 5-hydroxy-6-methoxy-, 6-hydroxy-5-methoxy-, 5,6-dihydroxy-, and 4,6-dihydroxyduloxetine were synthesized, and their phase II metabolite as glucuronide or sulfate conjugates were also synthesized. Their in vitro binding activities were compared to that of parent compound duloxetine.     

AMP-activated protein kinase enhances the phagocytic ability of macrophages and neutrophils

Although AMPK plays well-established roles in the modulation of energy balance, recent studies have shown that AMPK activation has potent anti-inflammatory effects. In the present experiments, we examined the role of AMPK in phagocytosis. We found that ingestion of Escherichia coli or apoptotic cells by macrophages increased AMPK activity. AMPK activation increased the ability of neutrophils or macrophages to ingest bacteria (by 46 ± 7.8 or 85 ± 26%, respectively, compared to control, P<0.05) and the ability of macrophages to ingest apoptotic cells (by 21 ± 1.4%, P<0.05 compared to control). AMPK activation resulted in cytoskeletal reorganization, including enhanced formation of actin and microtubule networks. Activation of PAK1/2 and WAVE2, which are downstream effectors of Rac1, accompanied AMPK activation. AMPK activation also induced phosphorylation of CLIP-170, a protein that participates in microtubule synthesis. The increase in phagocytosis was reversible by the specific AMPK inhibitor compound C, siRNA to AMPKα1, Rac1 inhibitors, or agents that disrupt actin or microtubule networks. In vivo, AMPK activation resulted in enhanced phagocytosis of bacteria in the lungs by 75 ± 5% vs. control (P<0.05). These results demonstrate a novel function for AMPK in enhancing the phagocytic activity of neutrophils and macrophages.     

Overrepresentation of the COL3A1 AA genotype in Polish skiers with anterior cruciate ligament injury

Although various intrinsic and extrinsic risk factors for anterior cruciate ligament (ACL) rupture have been identified, the exact aetiology of the injury is not yet fully understood. Type III collagen is an important factor in the repair of connective tissue, and certain gene polymorphisms may impair the tensile strength. The aim of this study was to examine the association of the COL3A1 rs1800255 polymorphism with ACL rupture in Polish male recreational skiers. A total of 321 male Polish recreational skiers were recruited for this study; 138 had surgically diagnosed primary ACL ruptures (ACL-injured group) and 183 were apparently healthy male skiers (control group – CON) who had no self-reported history of ligament or tendon injury. Both groups had a comparable level of exposure to ACL injury. Genomic DNA was extracted from the oral epithelial cells. All samples were genotyped on a real-time polymerase chain reaction instrument. The genotype distribution in the ACL-injured group was significantly different than in CON (respectively: AA=10.1 vs 2.2%, AG=22.5 vs 36.1, GG=67.4 vs 61.8%; p=0.0087). The AA vs AG+GG genotype of COL3A1 (odds ratio (OR)=5.05; 95% confidence interval (CI), 1.62-15.71, p=0.003) was significantly overrepresented in the ACL-injured group compared with CON. The frequency of the A allele was higher in the ACL-injured group (21.4%) compared with CON (20.2%), but the difference was not statistically significant (p=0.72). This study revealed an association between the COL3A1 rs1800255 polymorphism and ACL ruptures in Polish skiers.     

Induction of HLA class II molecules on human T cells: relationship to immunoregulation and the pathogenesis of AIDS.

Human T cells express HLA class II molecules upon activation. The factors that regulate the induction of expression of these molecules are for the most part unknown. Here we report preliminary results indicating that tumor necrosis factor-alpha (TNF-alpha) regulates the induction of cell-surface HLA-DR, DO, and DP molecules in human T cells stimulated with PHA. In contrast, recombinant interferon-gamma (rIFN-gamma), recombinant interleukin-1 alpha (rIL-1 alpha), or rIL-4 appear to have no effect on class II expression. The role of class II molecules on activated T cells is discussed in relationship to immunoregulation and the progression of HIV infection. Three non-mutually exclusive hypotheses are discussed. In the first hypothesis, we consider the role of these class II molecules in antigen presentation of endogenously synthesized HIV envelope by CD4+ cells. The second is a clonal inactivation of virus-specific helper T cells that might occur as a consequence of a direct T cell to T cell interaction and a bypass of the "accessory signal" normally delivered by antigen-presenting cells such as macrophages. The third is a molecular mimicry between HIV envelope proteins and HLA class II molecules, which may lead to the development of autoimmunity against CD4+ T-cell-expressing class II molecules.     

o-Phthalyl amidase in the synthesis of loracarbef: Process development using this novel biocatalyst

Summary A dephthalylation step utilizing a novel enzyme, o-phthalyl amidase, was developed. This step was part of a potentially new large scale synthetic route for a novel beta-lactam antibiotic Loracarbef. The enzyme was isolated from the organism Xanthobacter agilis. Purification of the enzyme to near homogeneity was accomplished by a 3-step procedure. Studies indicated that the phthalimido group can be opened chemically to generate the o-phthalyl derivative. This enzyme then can remove the phthalyl group from o-phthalylated amides. Optimization of the process was achieved by combining these two hydrolysis steps. Conversion yields of 85–97.8% (mol/mol) were obtained from reactions at substrate concentrations of 5–10% (w/v).