Long-chain acyl-coenzyme A synthetase from rat brain microsomes. Kinetic studies using [1-14C]docosahexaenoic acid substrate

The activation of docosahexaenoic acid by rat brain microsomes was studied using an assay method based on the extraction of unreacted [1-14C]docosahexaenoic acid and the insolubility of [1-14C]docosahexaenoyl-CoA in heptane. This reaction showed a requirement for ATP, CoA, and MgCl2 and exhibited optimal activity at pH 8.0 in the presence of dithiothreitol and when incubated at 45 degrees C. The apparent Km values for ATP (185 microM), CoA (4.88 microM), MgCl2 (555 microM) and [1-14C]docosahexaenoic acid (26 microM) were determined. The presence of bovine serum albumin or Triton X-100 in the incubation medium caused a significant decrease in the Km and Vm values for [1-14C]docosahexaenoic acid. The enzyme was labile at 45 degrees C (t1/2:3.3 min) and 37 degrees C (t1/2:26.5 min) and lost 36% of its activity after freezing and thawing. The transition temperature (Tc) obtained from Arrhenius plot was 27 degrees C with the activation energies of 74 kJ/mol between 0 degrees C and 27 degrees C and 30 kJ/mol between 27 degrees C and 45 degrees C. [1-14C]Palmitic acid activation in rat brain and liver microsomes showed apparent Km values of 25 microM and 29 microM respectively, with V values of 13 and 46 nmol X min-1 X mg protein-1. The presence of Triton X-100 (0.05%) in the incubation medium enhanced the V value of the liver enzyme fourfold without affecting the Km value. Brain palmitoyl-CoA synthetase, on the other hand, showed a decreased Km value in the presence of Triton X-100 with unchanged V. The Tc obtained were 25 degrees C and 28 degrees C for brain and liver enzyme with an apparent activation energy of 109 and 24 kJ/mol below and above Tc for brain enzyme and 86 and 3.3 kJ/mol for liver enzyme. The similar results obtained for the activation of docosahexaenoate and palmitate in brain microsomes suggest the possible existence of a single long-chain acyl-CoA synthetase. The differences observed in the activation of palmitate between brain and liver microsomes may be due to organ differences. Fatty acid competition studies showed a greater inhibition of labeled docosahexaenoic and palmitic acid activation in the presence of unlabeled unsaturated fatty acids. The Ki values for unlabeled docosahexaenoate and arachidonate were 38 microM and 19 microM respectively for the activation of [1-14C]docosahexaenoate. In contrast, the competition of unlabeled saturated fatty acids for activation of labeled docosahexaenoate is much less than that for activation of labeled palmitate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution, endogamy, and isolation of Malas of Chittoor district, Andhra Pradesh, India

This study investigates the distribution of Malas, a scheduled caste population of Andhra Pradesh, their isolates in different eco-cultural zones, and their endogamy and isolation by marriage district. The Malas, formerly "untouchables," occupy the lowest status in the Hindu hierarchy. The sample consists of 10% of Malas from 10% of the villages in 2 taluks of Chitoor district of Andhra Pradesh. 6 Mala populations--Tangala, Maladasari, Pakanati, Rampala, Murikinati, and Bommanati--live in the area. These populations show a regionality in their distribution, with very little overlapping even when 2 populations inhabit the same village. Of 885 marriages in the 6 endogamous populations, all but 3 have been contracted between individuals belonging to the same Mala group. The 3 exogamous marriages took place between Mala men and women from another caste. Such small exceptions to the general rule do not mean that the Mala populations are not breeding isolates; these 6 populations satisfy Wright's island model. The high incidence of matings between closely related populations also contributes to their genetic and breeding isolation. Consanguineous marriages range from 26.76 to 38.75%. The distance between the birth place of spouses in miles, called marriage distance, shows a range from 7.72 to 15.71 miles. Lower values mean higher population densities. Groups within each population are isolated by distance and form small overlapping Mendelian populations, approaching a stepping stone model with continuous variation of genetic traits between adjacent groups of people.     

In vitro flowering and pod formation from cotyledons of groundnut (Arachis hypogaea L.)

Summary The response of groundnut cotyledons to the presence of various growth regulators in concentrations from 0.1 to 5 mg/l has been studied in detail using several genotypes of groundnut on two different media. Cotyledons with embryo axis, cultured on Blaydes' medium with cytokinins, produced shoots, in the axils of which 2–7 flower buds could be seen. The frequency of flower bud induction in general increased with increasing concentrations of cytokinins, the optimal levels being 3 mg/l of KN or 4 mg/l of BAP. Cotyledons without embryo axis, cultured on Blaydes' medium with BAP (0.5 mg/l), produced a cluster of flower buds directly, ranging in number from 8–28, without any vegetative growth. Excised embryo axes cultured on the same medium gave plantlets without flower buds. The growth regulators IAA, NAA, GA₃ and ABA failed to induce flower buds in independent treatments. However, lower concentrations of IAA and NAA in combination with cytokinins exerted a positive influence on flowering. The blooming of the flower buds was facilitated on media supplemented with low concentrations of cytokinins. Six percent of the induced flowers resulted in gynophore development and ultimately formed pods when cultured under complete dark conditions in modified MS medium supplemented with kinetin.     

Cryosurgery of the monkey (macaque) prostate. II. Apparent immunopathologic alterations following cryostimulation.

Previous investigation of the development of humoral immunologic responsiveness following cryostimulation of the monkey prostate up to 30 days postoperatively disclosed low to modest titres of antibodies to prostatic tissue antigens. In the present study, the possible occurrence and predominance of a cellular response and its ensuing immunopathologic effects on the prostate and other accessory sexual glands of these animals, not initially examined, together with further serologic evaluation and correlation of this cellular response with the presence of antiprostatic antibodies, has been made 41 to 90 days following freezing. A reduction in the size of the prostate observed following freezing was accompanied by what were suggestive of immunopathologic alterations. Alterations occurred principally in the caudal lobe, and were characterized by what appeared to be specific periacinar foci of lymphocytic infiltrates. These lymphocytes were observed to infiltrate onto the acinar epithelial cells with subsequent separation of epithelial cells from the basal lamina and epithelial cell destruction. Other observed alterations in the prostate consisting of stromal fibrosis, periodic presence of inflammatory cells, proliferation of regenerating glands, and squamous metaplasia were in consonance with previous histologic studies of the prostate following cryosurgery by others. Antiprostatic antibodies were present in only one of the seven animals evaluated at the time which these observations were made. The present preliminary observations provide evidence suggestive of the development of a specific cellular immunologic response following cryosurgery of the prostate. Fending confirmation of the study of a larger series of animals, these observations may be of potential significance in providing an explanation of reported cases of eradication of human prostatic carcinomas following cryotherapy.     

Further Studies on Bacteriophage T4 DNA Synthesis in Sucrose-Plasmolyzed Cells

This paper describes several technical improvements in the sucrose-plasmolyzed cell system used in earlier experiments on DNA synthesis in situ with Escherichia coli infected by DNA-defective mutants of bacteriophage T4 (W. L. Collinsworth and C. K. Mathews, J. Virol. 13:908-915, 1974). Using this system, which is based primarily on that of M. G. Wovcha et al. (Proc. Natl. Acad. Sci. U.S.A. 70:2196-2200, 1973), we reinvestigated the properties of mutants bearing lesions in genes 1, 41, and 62, and we resolved some disagreements with data reported from that laboratory. We also asked whether the DNA-delay phenotype of T4 mutants is related to possible early leakage of DNA precursors from infected cells. Such cells display defective DNA synthesis in situ, even when ample DNA precursors are made available. Thus, the lesions associated with these mutations seem to manifest themselves at the level of macromolecular metabolism. Similarly, we examined an E. coli mutant defective in its ability to support T4 production, apparently because of a lesion affecting DNA synthesis (L. Simon et al., Nature [London] 252:451-455). In the plasmolyzed cell system, reduced nucleotide incorporation is seen, indicating also that the genetic defect does not involve DNA precursor synthesis. The plasmolyzed cell system incorporates deoxynucleotide 5'-monophosphates into DNA severalfold more rapidly than the corresponding 5'-triphosphates. This is consistent with the idea that DNA precursor-synthesizing enzymes are functionally organized to shuttle substrates to their sites of utilization.     

Coupled ribonucleoside diphosphate reduction, channeling, and incorporation into DNA of mammalian cells

There is rapid and specific channeling of ribonucleoside diphosphates into DNA through reactions beginning with ribonucleotide reductase and terminating with DNA polymerase. Lysolecithin-permeabilized Chinese hamster embryo fibroblasts in culture rapidly reduced ribonucleoside diphosphates by ribonucleotide reductase action when dithiothreitol was provided as a reducing agent and incorporated these deoxynucleotides into DNA. The radioactive label provided in ribo-CDP was not diluted by added deoxyribo-CTP during its incorporation into DNA, showing that the ribo-CDP does not pass through a deoxy-CTP pool. Under the conditions that permitted rapid incorporation of ribonucleoside diphosphates, deoxynucleoside triphosphates were very poorly incorporated. Ribonucleotide reductase with the rate-limiting enzyme for the overall process. The Km values for the reductase reaction and the overall process were similar and low enough for saturation by in vivo pools. Natural feedback inhibitors dATP or dTTP inhibited incorporation of labeled ribo-CDP into deoxyribonucleotides and into DNA to the same extent. Ribonucleotide reductase behaved like other enzymes that are associated in a rapidly sedimenting form. It was concentrated in the nucleus during S phase, and most of the enzyme activity in these nuclear extracts was co-sedimented with DNA polymerase on sucrose density gradients. These data support the hypotheses that a physically associated complex of enzymes (replitase) catalyzes the production of deoxynucleotides and their incorporation into DNA in S phase cells.     

Abnormally spliced messenger RNA in erythroid cells from patients with β+ thalassemia and monkey cells expressing a cloned β+-thalassemic gene

The reduced β-globin synthesis characterizing the β⁺ thalassemia phenotype has been shown to be caused by anomalous processing within the small Intervening sequence (IVS1) of the β-globin mRNA precursor. The β-globin gene from such patients contains a single base substitution within IVS1, located 22 bp from the 3′ junction between IVS1 and exon 2, creating an alternative splice site within IVS1 and resulting in retention of the 3′-terminal 19 bases of IVS1. We have identified this abnormally spliced mRNA in the reticulocyte RNA of two patients with β⁺ thalassemia, by S1 nuclease mapping and primer-extension analysis. Moreover, a cloned β⁺-thalassemic gene preferentially generated the anomalously spliced RNA when expressed In monkey kidney cells. The anomalously spliced RNA constituted approximately 80%–90%, and normal β RNA approximately 10%–20%, of the total β mRNA. In contrast, the small amount of β mRNA present in reticulocytes from such patients consisted predominantly of normal β mRNA. These results suggest that the reduced amount of normally functioning β mRNA present in such patients results from preferential processing at the alternative splice site, with subsequent Instability, reduced nuclear processing and/or inadequate cytoplasmic transport of the abnormal RNA species.     

Effect of prostaglandins on ornithine decarboxylase activity in the testis of immature rat

Intratesticular injection of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rats. PGE2 at a dose of 10 microgram per testis was maximally effective 2 hours after the injection. Dibutyryl cyclic AMP (cAMP) and 1 methyl, 3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor, also stimulated ODC activity. Simultaneous injection of PGE2 and FSH or LH caused additional stimulation of ODC activity. Similarly injection of PGE2 in addition to cAMP or MIX also caused increased stimulation of ODC. Indomethacin (IM, 60 microgram/testis) inhibited LH, FSH or cAMP induced ODC activity. However, IM at the same dose inhibited the synthesis of total proteins. These results suggest that PGE2 and PGF2 alpha stimulate the activity of ODC. The action of prostaglandins may be independent of the action of gonadotropic hormones. cAMP appears to mediate the action of prostaglandins in the testis of rat.     

Partial purification and immunoreactivity of an 80000-molecular-weight polypeptide associated with peroxisome proliferation in rat liver

Hypolipidaemic drugs and industrial plasticizers such as di-(2-ethylhexyl) phthalate, which cause proliferation of hepatic peroxisomes, also cause an increase in an 80000-mol.wt. polypeptide in the liver of rats and mice. This polypeptide has been designated as PPA-80 (PPA, for peroxisome-proliferation-associated; 80 for 80000mol.wt.). The polypeptide PPA-80 was purified to over 90% purity from livers of rats treated with the peroxisome proliferators Wy-14,643, nafenopin, tibric acid and clofibrate by a single-step preparative sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic procedure. The antibodies raised against the PPA-80 polypeptide isolated from livers of rats treated with Wy-14,643 cross-reacted with polypeptide PPA-80 purified from the livers of rats treated with Wy-14,643, as well as from the livers of rats treated with nafenopin, tibric acid and clofibrate. The anti-(polypeptide PPA-80) antibodies did not cross-react with catalase, a marker enzyme for peroxisomes, or with NADPH–cytochrome P-450 reductase, which has the same approximate mol.wt., 80000. The intensity of immunoprecipitin bands formed with microsomal, large-particle and postnuclear fractions from livers of animals pretreated with peroxisome proliferators was significantly greater compared with equal amounts of protein from corresponding fractions obtained from control animals, suggesting that these agents all enhance the synthesis of the same 80000-mol.wt. polypeptide. Although the polypeptide PPA-80 was increased in the postnuclear, large-particle and microsomal fractions of livers of rats pretreated with peroxisome proliferators, the relative abundance of this peptide in the peroxisome-rich light-mitochondrial fraction and its lack in highly purified mitochondrial fractions suggest the localization of this polypeptide in peroxisomes and/or microsomal fraction. Additional studies are needed to establish unequivocally the subcellular localization of the polypeptide PPA-80 and to ascertain if this polypeptide is identical with the multi-functional protein displaying enoyl-CoA hydratase and β-hydroxyacyl-CoA dehydrogenase activities that was purified by Osumi & Hashimoto [(1979) Biochem. Biophys. Res. Commun. 89, 580–584].