Biochemical characterization of hepatic microsomal leukotriene B4 hydroxylases

omega-Hydroxylation of leukotriene B4 (LTB4) has been reported in human and rodent polymorphonuclear leukocytes; preliminary information indicates that this metabolism is cytochrome P-450 dependent. Therefore, these studies were initiated to characterize the cytochrome P-450-dependent metabolism of LTB4 in other tissues. LTB4 was metabolized by rat hepatic microsomes to two products, 20-hydroxy(omega)-LTB4 and 19-hydroxy(omega-1)-LTB4. The formation of these metabolites was both oxygen and NADPH dependent indicating that a monooxygenase(s) was responsible for these reactions. The apparent Km and Vmax for LTB4 omega-hydroxylase were 40.28 microM and 1202 pmol/min/mg of protein, respectively. In contrast, the apparent Km and Vmax for LTB4 (omega-1)-hydroxylase were 61.52 microM and 73.50 pmol/min/mg of protein, respectively. Both LTB4 omega- and (omega-1)-hydroxylases were inhibited by metyrapone in a concentration-dependent fashion. However, SK&F 525A inhibited LTB4 (omega-1)- but not omega-hydroxylase. In contrast, alpha-naphthoflavone decreased LTB4 omega- but not (omega-1)-hydroxylase activities. The differences in the Km apparent for substrate as well as the differential inhibition by inhibitors of cytochrome P-450 suggest that the omega- and (omega-1)-hydroxylations of LTB4 in hepatic microsomes are mediated by different isozymes of P-450. Furthermore, several additional characteristics of LTB4 hydroxylases indicate that these isozymes of P-450 may be different from those which catalyze similar reactions on medium-chain fatty acids, such as laurate and prostaglandins.     

Structural and functional characterization of major platelet membrane components derived by limited proteolysis of glycoprotein IIIa

The authors isolated a product of proteolytic degradation of glycoprotein IIIa (GPIIIa) which is formed on the surface of human platelets during incubation with chymotrypsin and which was previously described as the 66 kDa platelet membrane component. This component migrated with an apparent Mr 62,400 in a non-reduced system of sodium dodecyl sulfate polyacrylamide gel electrophoresis. In a reduced system it yielded two major subunits migrating with apparent Mr 14,000-17,000 and 65,000. The low-molecular weight component began with the NH2-terminal sequence of GPIIIa (GPNICTTR...) and the larger component with residue 348 of GPIIIa (GKIRSKKA...) as deduced from a cDNA clone of this glycoprotein. The two subunits appeared to be linked by one or more S-S bridges supporting the contention that GPIIIa is a highly folded molecule on the platelet membrane. In contrast to GPIIIa, the '66 kDa component' did not bind to GRGDSPK-agarose, to fibrinogen-agarose nor to insolubilized monoclonal antibody recognizing the GPIIb/IIIa complex. The exposure of fibrinogen receptors during the course of incubation of platelets with chymotrypsin preceded the formation of the '66 kDa component' characterized in this study. An intermediate product of GPIIIa proteolysis migrating with an apparent Mr 120,000 in a non-reduced system and Mr 80,000 in a reduced system was identified as a precursor of the '66 kDa component'. The '120 kDa component' was not retained on GRGDSPK-agarose or on fibrinogen-agarose but it was retained on insolubilized antibody recognizing the GPIIb/IIIa complex. Incubation of platelets with porcine pancreatic elastase or human granulocytic elastase resulted in the formation of similar proteolytic degradation fragments.     

Rate of erythropoietin formation in humans in response to acute hypobaric hypoxia

This study was carried out to investigate the early changes in erythropoietin (EPO) formation in humans in response to hypoxia. Six volunteers were exposed to simulated altitudes of 3,000 and 4,000 m in a decompression chamber for 5.5 h. EPO was measured by radioimmunoassay in serum samples withdrawn every 30 min during altitude exposure and also in two subjects after termination of hypoxia (4,000 m). EPO levels during hypoxia were significantly elevated after 114 and 84 min (3,000 and 4,000 m), rising thereafter continuously for the period investigated. Mean values increased from 16.0 to 22.5 mU/ml (3,000 m) and from 16.7 to 28.0 mU/ml (4,000 m). This rise in EPO levels corresponds to 1.8-fold (3,000 m) and 3.0-fold (4,000 m) increases in the calculated production rate of the hormone. After termination of hypoxia, EPO levels continued to rise for approximately 1.5 h and after 3 h declined exponentially with an average half-life time of 5.2 h.     

Advances in bioconversion of anthracycline antibiotics

During the last decade new anthracycline-type structures with potential usefulness in cancer treatment have been supplied both by new microbial strains and by bioconversions of precursor molecules employing cells or enzymes. We highlight recent advances in bioconversion of anthracycline structures with the main focus on late transformations such as are carried out by oxidoreductases.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。