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.     

MAJOR CLADES OF THE ANGIOSPERMS

Abstract— Our knowledge of fundamental angiosperm interrelationships is still very incomplete. The absence of a narrowly circumscribed gymnosperm outgroup, ideally the sister group, makes character evaluation, necessary for a cladistic analysis, difficult. According to current views the superorder Magnoliiflorae with a number of other groups, for example the monocotyledons, may represent a complex of families near the base of the angiosperms. Interrelationships of groups within the monocotyledons are much better understood than those between groups within the dicotyledons. A cladogram of monocotyledon orders based on earlier work by R. Dahlgren, H. T. Clifford, and F. N. Rasmussen is presented. A data matrix for a sample of the angiosperms with 61 characters for 49 taxa, mostly magnoliifloran and related families, is presented. The characters are polarized mainly according to the current view that the primitive angiosperm morphotype is a woody dicotyledon with strobiloid flowers. As an alternative the matrix is adjusted following W. C. Burger's conjecture that the primitive angiosperm was a herbaceous monocotyledon with trimerous flowers. Both matrices were run in a computerized parsimony analysis, resulting in numerous equally parsimonious solutions. This result is illustrative of the great homoplasy in the available character information, and also of how little actually is known about fundamental angiosperm interrelationships or phylogeny.     

Enhanced fibrin formation in high-altitude pulmonary edema

Blood coagulation, fibrinolysis, and arterial blood gases were examined in 66 nonacclimatized mountaineers at 4,557 m. Subjects were classified according to a clinical score as healthy (n = 25), having mild acute mountain sickness (AMS) (n = 24), showing severe AMS (n = 13), and suffering from high-altitude pulmonary edema (HAPE) (n = 4). Coagulation times, euglobulin lysis time, and fibrin(ogen) fragment E were normal in all groups without significant changes. Fibrinopeptide A (FPA), a molecular marker of in vivo fibrin formation, was elevated in HAPE to 4.2 +/- 2.7 ng/ml (P less than 0.0001) compared with the other groups showing mean values between 1.6 +/- 0.4 and 1.8 +/- 0.7 ng/ml. FPA was normal in one patient with HAPE, however. Severe AMS was accompanied by a significant decrease in arterial PO2 due to an increase in alveolar-arterial O2 difference, whereas arterial PCO2 did not change significantly. We conclude that activation of blood coagulation is not involved in the pathogenesis of AMS and the impairment of gas exchange in this disease. Fibrin generation occurring in HAPE is probably an epiphenomenon of edema formation.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Isolation and Characterization of a Virus Inhibitor from Spinach (Spinacia oleracea L.)

A virus inhibiting protein (VI) was isolated from spinach (Spinacia oleracea L.). The VI inhibited infections of test plants with plus- and minus-strand RNA viruses. Inoculation of both local lesion and systemic hosts with TMV in the presence of varying amounts of the VI resulted in typical dose response curves for the number of local lesions or the amount of virus respectively. The lowest concentration of VI leading to a significant reduction in the number of local lesions was 0.06 μg/ml. The VI was found to inhibit local lesion formation only when applied within 2–3 h p.i. but still reduced the number of local lesions when applied up to 9 h prior to virus inoculation. The antiviral activity could be attributed to a protein of molecular weight 29,000 dalton with an isoelectric point of 10.3. Its activity was destroyed by heating for 30 min to 70°C. These characteristics resemble those of other virus inhibiting proteins described for members of the order Caryophyllales such as the Phytolacca inhibitor against which a serological relationship was obtained.     

Coagulation and fibrinolysis in acute mountain sickness and beginning pulmonary edema

To examine whether intravascular coagulation and/or decreased fibrinolysis precedes high-altitude pulmonary edema (HAPE) we examined 25 male mountaineers (median age 40 yr) at low altitude (550 m) and after 6, 18, and 42 h at an altitude of 4,559 m, which was climbed in 24 h. In 14 subjects, 2 of whom showed radiological evidence of HAPE after 42 h, symptoms of acute mountain sickness (AMS) were mild or absent. Eleven subjects suffered from AMS, six of whom developed radiologically documented HAPE after 18 or 42 h. In the absence of AMS there were no significant changes at high altitude, with the exception of a decrease in bleeding time from 246 +/- 18 to 212 +/- 13 (SE) (P less than 0.05). In AMS, partial thromboplastine time decreased from 34.2 +/- 0.8 to 31.1 +/- 0.5 s (P less than 0.001) and factor VIII procoagulant activity and von Willebrand factor antigen were increased by 57 +/- 12 and 70 +/- 13%, respectively (P less than 0.001), whereas there were no significant changes in beta-thromboglobulin (BTG), fibrinopeptide A (FPA), and fibrin fragment B beta 15-42. In subjects with HAPE, BTG, FPA, and B beta 15-42 were normal before and in beginning HAPE. Preceding HAPE, euglobulin clot lysis time declined at high compared with low altitude from 289 +/- 48 to 201 +/- 42 min without venous occlusion (VO) and from 107 +/- 36 to 86 +/- 31 min after VO (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

CD4+ T cell response to the human acetylcholine receptor alpha subunit in myasthenia gravis. A study with synthetic peptides

The production of antinicotinic acetylcholine receptor (AcChR) antibodies in myasthenia gravis (MG) is modulated by specific Th (CD4+) lymphocytes that can recognize epitopes on the denatured AcChR alpha subunit. Thirty-two overlapping synthetic peptides corresponding to the complete sequence of human AcChR alpha subunit were used to investigate the anti-alpha subunit response of unselected lymphocytes and of CD8(+)-depleted, CD4(+)-enriched lymphocytes from the blood of nine MG patients and from four healthy controls. One subject was a newly diagnosed MG patient that was tested three times after the development of the disease. An anti-AcChR response of the CD4(+)-enriched cells was present that could be detected only after removal of the CD8+ population and that seems to be related to the clinical conditions of the patient. The high basal rate of the cell proliferation of the unselected unstimulated blood lymphocytes and the normal basal rate observed for the CD8(+)-depleted population suggested the presence of activated CD8+ cells. The study of surface markers of the T cells confirmed the existence of activated CD8+ and CD4+ cells in numbers correlated with the severity of the disease and the results of the in vitro response of the T cells. The anti-AcChR activity of the CD4+ cells in MG may be a useful marker of the activity of the disease and it seems to be influenced by activated CD8+ cells present in the patients' blood.