Deregulation of hamster fibroblast proliferation by mutated ras oncogenes is not mediated by constitutive activation of phosphoinositide-specific phospholipase C

Stable expression of high levels of activated forms of Haras (T24) or v-Ki-ras by transfection of Chinese hamster lung fibroblasts (CCL39) yielded cells highly tumorigenic in nude mice. Two classes of transformed cells were distinguished, one with moderate p21 expression (10-fold increased) had retained growth factor dependency, the second with higher level of p21 (greater than 50-fold) appeared autonomous for growth. Neither class of transformants expressing Ki-ras or Ha-ras displayed a significant basal activity of polyphosphoinositide-specific phospholipase C, measured either in serum-starved cells or during exponential growth in the presence of growth factors of the tyrosine kinase family (EGF, FGF, insulin). In the growth-factor-dependent class of T24-Ha-ras-transfected cells (clone 39THaB), phospholipase C could be stimulated normally by serum, thrombin and AlF-4. In the more growth autonomous class (clones 39THaC and 39Ki9), release of inositol phosphates after stimulation with thrombin or serum was drastically reduced. This desensitization, apparently at the receptor level since the response to AlF-4 persisted, is, however, not specific to ras expression. We observed it to the same degree in polyoma virus-transformed CCL39 cells. Finally, expression of mutated forms of p21 ras did not abrogate the sensitivity of phospholipase C activation to pertussis toxin. We conclude that the transforming potential of activated forms of p21ras does not result from persistent activation of phospholipase C and that ras GTP-binding proteins cannot substitute for Gp.     

Monohydroxylated fatty acid substrate specificity of human leukocyte 5-lipoxygenase and omega-hydroxylase.

Various monohydroxylated fatty acids were synthesized from eicosapolyenoic acids, namely arachidonic (20:4 omega-6), timnodonic (20:5 omega-3), dihomogammalinolenic (20:3 omega-6) and mead (20:3 omega-9) acids. 12-Hydroxy derivatives, as well as 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), were produced with platelets as the enzyme source, and 15-hydroxy derivatives were produced by soya bean lipoxygenase treatment. Each monohydroxylated fatty acid was incubated with human leukocytes in the presence or absence of the calcium ionophore A23187, and dihydroxylated products were analysed by h.p.l.c. 12-Hydroxy derivatives of 20:4 omega-6, 20:5 omega-3 and 20:3 omega-9 were similarly oxygenated by both the 5-lipoxygenase and the omega-hydroxylase. As expected, the 12-hydroxy derivative of 20:3 omega-6 was not a substrate for 5-lipoxygenase, but surprisingly, omega-6 oxygenated products, like 15-OH-20:4 or HHT, were not converted by the enzyme, although being potential substrates because of the presence of two double bonds at C-5 and C-8. omega-6 oxygenated derivatives were also poorly converted by leukotriene B4 omega-hydroxylase, a cytochrome P-450-dependent enzyme. It is concluded that both leukocyte 5-lipoxygenase and omega-hydroxylase exhibit a substrate specificity towards monohydroxylated fatty acids with respect to their double bonds and/or the carbon position of the alcohol function.     

New chromosome 21 DNA markers isolated by pulsed field gel electrophoresis from an ETS2-containing Down syndrome chromosomal region

To generate new chromosome 21 markers in a region that is critical for the pathogenesis of Down syndrome (D21S55-MX1), we used pulsed field gel electrophoresis (PFGE) to isolate a 600-kb NruI DNA fragment from the WA17 hybrid cell line, which has retained chromosome 21 as the only human material. This fragment, which contains the oncogene ETS2, was used to construct a partial genomic library. Among the 14 unique sequences that were isolated, 3 were polymorphic markers and contained sequences that are conserved in mammals. Five of these markers mapped on the ETS2-containing NruI fragment and allowed us to define an 800-kb high-resolution PFGE map.     

Variants of the anti-Müllerian hormone gene in a compound heterozygote with the persistent Müllerian duct syndrome and his family

Summary The human genome contains a large number of interspersed simple repeat sequences that are variable in length and can therefore serve as highly informative, polymorphic markers. Typing procedures include conventional multilocus and single locus probing, and polymerase chain reaction aided analysis. We have identified simple sequences in a cosmid clone stemming from the human Y chromosome and consisting of (gata)_n repeats. We have compared these with two equivalent simple repeat loci from chromosome 12. After amplifying the tandemly repeated motifs, we detected between four and eight different alleles at each of the three loci. Codominant inheritance of the alleles was established in family studies and the informativity of the simple repeat loci was determined by typing unrelated individuals. The polymorphisms are suitable for application in linkage studies, practical forensic case work, deficiency cases in paternity determination, and for studying ethnological questions. The mutational mechanisms that bring about changes in simple repeats located both on the autosomes and on the sex chromosomes, are discussed.Professor Dr. Otto Prokop (Humboldt-Universität Berlin) on the occasion of his 70th birthday     

Distribution of tritium labeled 12(S) hydroxy-eicosatetraenoic acid (12-HETE) in the rat.

The in vivo metabolism of 12-(S)-Hydroxy-eicosatetraenoic acid (12-HETE), the end-lipoxygenase product of arachidonic acid in platelets, has been investigated in the rat. Fifty microcuries of 5,6-[3H]-12-HETE (50 Ci/mmol) were injected to anesthetized rats and the radioactivity was followed in plasma. At the end of the experiment, various organs of the animal were removed and the radioactivity attached to them was determined. The label of the plasma plateaued to approximately one third of the initial radioactivity ten minutes after the injection. Among the various organs tested (brain, heart, intestine, kidney, liver, lungs, spleen, testis/uterus) the kidney was far the most active to accumulate 12-HETE and/or its labeled metabolites, and no radioactivity could be detected in urine during the course of the experiment. The analysis of lipid extracts from the various tissues revealed that 12-HETE was not accumulating in its unesterified form but was likely bound to phospholipids. We conclude that, although the label providing from the initial 12-HETE did not completely disappear from plasma, circulating 12-HETE cannot be considered as a circulating marker of cell activation.     

Hydroperoxides produced by n-6 lipoxygenation of arachidonic and linoleic acids potentiate synthesis of prostacyclin related compounds

In a previous paper we reported that arachidonic acid (20:4(n-6] strongly enhances the endothelial cell synthesis of prostaglandin I3 (PGI3) from eicosapentaenoic acid (20:5(n-3], in stimulating the cyclooxygenase rather than the prostacyclin synthase (Bordet et al. (1986) Biochem. Biophys. Res. Commun. 135, 403-410). In the present study, endothelial cell monolayers were co-incubated with exogenous 20:5(n-3) or docosatetraenoic acid (22:4(n-6], and n-6 lipoxygenase products of 20:4(n-6) or linoleic acid (18:2(n-6], namely 15-HPETE and 13-HPOD, respectively. Prostaglandins or dihomoprostaglandins were then measured by gas chromatography-mass spectrometry. Both hydroperoxides, up to 20 microM, stimulated the cyclooxygenation of 20:5(n-3) and 22:4(n-6), in particular the formation of PGI3 and dihomo-PGI2, respectively. Higher concentrations inhibited prostacyclin synthetase. In contrast, the reduced products of hydroperoxides, 15-HETE and 13-HOD, failed to stimulate these cyclooxygenations, 13-HPOD appeared more potent than 15-HPETE and the cyclooxygenation of 22:4(n-6) seemed to require higher amounts of hydroperoxides to be efficiently metabolized than 20:5(n-3). These data suggest that prostacyclin potential of endothelium might be enhanced by raising the peroxide tone.     

A phenyl-beta-galactoside-specific monoclonal antibody reactive with murine and rat NK cells

The phenyl-beta-galactoside (phi-beta-gal)-specific monoclonal antibody (mAb) 49H.8 cross-reacts with the terminal disaccharide structure of the asialo GM1 (AGM1) molecule. It was found to react with phi-beta-gal determinants on murine and rat splenic natural killer (NK) cells, as measured by complement depletion studies. Flow cytometric analysis identified the antigen on two IL 2-dependent cloned murine NK cell lines and the rat large granular lymphocyte leukemia RNK. We have compared the 49H.8 reactivity to that of anti-AGM1 antisera (alpha-AGM1) on NK cells and a panel of NK related killer cells, including bone marrow-derived killer cells, lymphokine-activated killer cells (LAK), and anomalous killer cells (AK). We found that the 49H.8 specificity closely paralleled that of alpha-AGM1. When tested against Con A-reactive T cells, the 49H.8 mAb was less reactive than the alpha-AGM1, indicating that it may be a more specific marker for splenic NK populations than the alpha-AGM1.     

Studies on platelet lipoxygenase specificity towards icosapolyenoic and docosapolyenoic acids

Two docosapolyenoic acids (22:5(n-3) and 22:5(n-6)) were isolated from the liver of normal and 18:3(n-3)-deficient trout, respectively. They were prepared by combined thin-layer chromatography (TLC) and reversed-phase high performance liquid chromatography (HPLC). Their purity, checked by capillary gas liquid chromatography, was greater than 95%. Each fatty acid was oxygenated into monohydroxy derivatives by human platelets. The hydroxy compounds were purified by TLC and HPLC and then derivatized for gas chromatography-mass spectrometry analysis. Whereas 22:5(n-6) was only converted into 14-OH-22:5, three hydroxy derivatives (11, 13 and 14) were obtained from 22:5(n-3). However, 13-hydroxy was not formed in the presence of aspirin, indicating that platelet lipoxygenase catalyses the formation of both 11- and 14-hydroxy derivatives from 22:5(n-3), as described previously, from 22:6(n-3). Further studies showed that 22:4(n-6) and 20:5(n-3) were only converted into 14- and 12-hydroxy derivatives. We conclude then that, besides the well-known n-9 oxygenation, lipoxygenase of human platelets is able to catalyse an n-12 oxygenation on docosapolyenoic acids of the n-3 family.     

Myelin-associated glycoprotein and myelin basic protein are present in central and peripheral nerve myelin throughout phylogeny

This phylogenetic study of central and peripheral nervous system myelin proteins demonstrates that important changes occur in the composition of certain myelin proteins during evolution. Only two components, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) are present in all Gnathostomata representatives investigated. While MBP components varied considerably even among the representatives of a given order, the apparent molecular weight of MAG showed little variation indicating that the conservation of the molecular structure could be important for the function of MAG in glia axon interactions.     

Serology of Rubella—Comparison of Fluorescent Antibody,Complement Fixation and Neutralization Tests for Diagnosis of Current Infections and Determination of Sero-immunity

Neutralization, complement fixation (CF) and indirect fluorescent antibody (FA) assays for rubella virus were compared for sensitivity in the serologic diagnosis of infection, for demonstrating antibody in the sera of infants with suspected rubella syndrome, and in the detection of antibody elicited by past infection (determination of immunity status). The combination of CF and FA tests was shown to be the most useful for serologic diagnosis of infection, largely eliminating the need for the slower and more cumbersome interference neutralization test.Neutralizing antibodies were found to appear rapidly in the course of infection, antibodies demonstrable by immunofluorescent staining appeared slightly later, and CF antibodies were rarely demonstrable in sera collected earlier than 14 days after onset of illness. Antibodies detected by all three techniques showed good correlation in infants with clinical evidence of rubella syndrome and corresponding maternal sera. The indirect FA technique compared favorably with the neutralization test for the detection of antibody elicited by past infection (determination of immunity status) and offered distinct advantages in ease of technical performance and more rapid results. In both current and past infections, FA titers tended to be higher than neutralizing antibody titers.