Evidence for a Further Dark Repair Process in Bacteria

Resistant (repair proficient) strains of Escherichia coli stop DNA synthesis almost immediately after low doses of ultraviolet (UV). Synthesis ceases for a period that is a function of the UV dose and is resumed at the same rate as unirradiated bacteria^1–4. The time at which synthesis restarts correlates well with the time at which excision-repair is completed. Billen and his collaborators showed that when DNA synthesis resumes, a new replication point is initiated preferentially at the chromosomal origin, the existing replication point remaining inactive^5–7. In contrast, excision-deficient (Uvr^?) or recombination-deficient (RecA^?) bacteria show decreased rates of synthesis as the dose is increased until (when the surviving fraction is very low) synthesis ceases to be measurable^1,3,8. Moreover in Uvr^? bacteria, DNA synthesis after UV continues largely at the existing replication point⁹. Thus in repair-proficient bacteria there exists after irradiation a “stalled” replication-complex, which must be repaired if the new replication point is to traverse the same region. At the least this must involve the elimination of one replicated arm by some breakage and rejoining process and may also involve the repair of radiation-induced lesions in that region.     

Further Evidence for a Growth-Promoting and Flower-Inhibiting Hormone in Strawberry

Further experiments with runner plants of the cultivated strawberryjoined in pairs as donor/receptor units are reported. Stolonproduction was promoted in receptor plants in short days bylong-day treatment of donor plants. This was a photoperiodiceffect induced by low intensity light. Again, as previously,petiole length was increased and flower induction inhibitedby these, or similar treatments. An attempt to demonstrate thetranslocation and activity of a vegetative-growth-inhibiting,flower-promoting hormone gave negative results. It is argued that the photoperiodic control of the growth habitis mediated mainly by a transmissible vegetative-growth-promotingand flower-inhibiting hormone, which is produced in leaves andacts at the growing regions.     

Evidence against a laminin receptor role for calsequestrin

Calsequestrin, a muscle calcium binding protein, has been shown to bind the extracellular matrix protein laminin and evidence has been presented that CAL (initially called aspartactin) is on the cell surface, consistent with a role as a laminin receptor (1). In this report, we present evidence that does not support a laminin receptor function for CAL. We found that CAL immunoreactivity could not be detected on live cultured chick myotubes unless they were permeabilized with detergent. Furthermore, polyclonal anti-CAL antibodies did not perturb myotube adhesion to laminin or the rate of myoblast fusion on laminin. Expression of the CAL cDNA in a melanoma cell line that was poorly adherent to laminin did not increase adhesion to laminin. In these cells, CAL could not be detected on the cell surface, and the majority of CAL was found to be secreted into the media.     

Further Evidence to Support a Nutritional Interpretation of Sclerophylly

A statistical analysis is made of published data on the leafcomposition of eighty-nine species, covering both sclerophyllousand mesophytic types of leaves, from North and Central America,Africa, and India. Expressing degree of sclerophylly by theratio crude fibre dry weight x 100/crude protein dry weight,the analysis shows: (1) that in terms of organic matter an increasein sclerophylly is associated with a decrease in percentageprotein content and with an increase in percentage fibre content,and (2) that in terms of mineral matter a decrease in phosphoruscontent below about 0.3 per cent. results in a proportionalincrease in degree of sclerophylly. These two results are consideredcomplementary. Since phosphorus is essential for protein synthesis,the observed protein deficiency of sclerophyllous leaves canbe correlated with their low phosphorus content. It is alsoreasonable to expect the observed reciprocal relationship betweenprotein content and fibre content on the basis of a common poolof intermediate metabolites. The above conclusions support the author's hypothesis, suggestedin a previous paper, that a sclerophyllous leaf is the expressionof a metabolism found in plants that can tolerate low levelsof phosphate. Strong circumstantial evidence is also providedby the fact that phosphate deficiency is characteristic of thesoils, either wet or dry, which carry sclerophyllous vegetation.The hypothesis thus provides a ready answer to the apparentparadox of why the same specialized type of leaf should occurin both wet and dry habitats.     

Further Evidence Supporting a Role for Gs Signal Transduction in Severe Malaria Pathogenesis

With the functional demonstration of a role in erythrocyte invasion by Plasmodium falciparum parasites, implications in the aetiology of common conditions that prevail in individuals of African origin, and a wealth of pharmacological knowledge, the stimulatory G protein (Gs) signal transduction pathway presents an exciting target for anti-malarial drug intervention. Having previously demonstrated a role for the G-alpha-s gene, GNAS, in severe malaria disease, we sought to identify other important components of the Gs pathway. Using meta-analysis across case-control and family trio (affected child and parental controls) studies of severe malaria from The Gambia and Malawi, we sought evidence of association in six Gs pathway candidate genes: adenosine receptor 2A (ADORA2A) and 2B (ADORA2B), beta-adrenergic receptor kinase 1 (ADRBK1), adenylyl cyclase 9 (ADCY9), G protein beta subunit 3 (GNB3), and regulator of G protein signalling 2 (RGS2). Our study amassed a total of 2278 cases and 2364 controls. Allele-based models of association were investigated in all genes, and genotype and haplotype-based models were investigated where significant allelic associations were identified. Although no significant associations were observed in the other genes, several were identified in ADORA2A. The most significant association was observed at the rs9624472 locus, where the G allele (∼20% frequency) appeared to confer enhanced risk to severe malaria [OR = 1.22 (1.09–1.37); P = 0.001]. Further investigation of the ADORA2A gene region is required to validate the associations identified here, and to identify and functionally characterize the responsible causal variant(s). Our results provide further evidence supporting a role of the Gs signal transduction pathway in the regulation of severe malaria, and request further exploration of this pathway in future studies.     

Further Evidence for an Intrinsic Neuraminidase in CNS Myelin

An intrinsic neuraminidase activity in rat brain CNS myelin has been demonstrated and compared with the neuraminidase activity in rat brain microsomes. With use of ganglioside GM3 as a substrate, the myelin-associated neuraminidase exhibited a shallow pH curve with an optimum at pH 4.8 whereas the microsomal activity had a marked optimum at pH 4-4.3. Neuraminidase activity in both fractions was optimized in 0.3% Triton CF-54 but activation was much greater in the microsomes. When the neuraminidase activities were examined at 60 degrees C, the myelin neuraminidase activity was more than sevenfold of that observed at 37 degrees C and was linear for at least 2 h; the microsomal activity increased only fivefold initially and exhibited a continual loss in activity. Addition of excess microsomes to the total homogenate prior to myelin isolation resulted in no change in myelin neuraminidase activity. When the two membrane fractions were examined at equivalent protein concentrations in the presence of additional cations or EDTA (1 mM), similar but not identical effects on neuraminidase activity were seen. The microsomal neuraminidase was considerably more susceptible to inhibition by divalent copper ion. Activity in both fractions was markedly inhibited by Hg2+ and Ag+ whereas EDTA had no effect on either activity. The myelin-associated neuraminidase activity was the highest in cerebral hemispheres, followed by brainstem, cerebellum, and spinal cord and was extremely low in sciatic nerve. In fact, the myelin neuraminidase activity was higher than the microsomal enzyme activity in the cerebral hemispheres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further Evidence for Selective Differences between Isoalleles in Drosophila

A number of separate strains of Drosophila pseudoobscura were inbred for 38 generations of brother-sister mating with forced heterozygosity for two alleles of either the octanol dehydrogenase or esterase-5 locus. Crosses were set up within each of these inbred lines such that simple mendelian ratios were expected, and eggs from these crosses were placed on media with additions of simple chemicals likely to interact with alleles of the two loci—octanol and ethanol for the ODH locus and tributyrin and triacetin for the E-5 locus. Similar crosses were set up involving parental flies with normally heterozygous genetic background as a control.—Significant deviations from mendelian expectation were observed in inbred E-5 flies grown on tributyrin, inbred ODH males grown on octanol, and inbred ODH females grown on ethanol. There was also a strong effect of octanol medium on males of one of the inbred E-5 lines, and a weak effect of tributyrin medium on ODH inbred females.—The probability that these results reflect interactions between these loci and the environment is assessed in the light of differences between the present results and those obtained at earlier stages of inbreeding.     

Evidence against a regulatory role for heme oxygenase in hepatic heme synthesis

The role of a microsomal enzyme system, heme oxygenase, in the regulation of intracellular heme concentration and of the heme biosynthetic pathway was investigated. It was determined that alterations in heme oxygenase activity were not consistent with the observed alterations of heme biosynthesis produced by the administration of various drugs. It is concluded that heme oxygenase does not play a role in the regulation of heme biosynthesis under these circumstances.     

Further Evidence for a Role of Carbohydrates in Insulin Binding : Studies in Lectin-Purified Receptors

Abstract

Purification of liver membrane insulin receptors on concanavalin A-and ricin I-lectin columns gave a 15-fold enrichment in the insulin binding capacity per milligramm of protein. Final receptor and protein recoveries were 53 and 3.8 % respectively. Lectin-purification increased the receptor affinity for insulin, as indicated by a left-ward shift in the binding competition curve and a steeper slope in the Scatchard plot. Lectin-purification increased the receptor sensitivity towards the glycosidic probes. The maximal effects of β-galactosidase, ricin I (galactose-binding lectin) and α-mannosidase were markedly amplified : 80, 90 and 60 % inhibition, versus 45, 40 and 15 % with particulate membranes. The limulus polyphemus (LPA) and wheat germ (WGA) agglutinins (sialic acid- and N-acetyl-glucosaminyl-binding lectins) became effective in modifying the insulin binding : 45 and 80 % inhibition, respectively. The effects were dose-dependent, reversed by the monosaccharide competitors (lectin effects) and unrelated to the state of receptor occupancy. These findings indicate that, within the hormone recognition area, peptide chains containing galactose, mannose and N-acetyl-glucosamine are strictly required for insulin-receptor interaction and suggest that change in the receptor affinity is related to the role of carbohydrate in insulin binding.     

Further evidence against an IAA-tRNA complex

Phenol extraction of pea stems treated with indoleacetic acid-³Hresults in the presence of label in water soluble/ethanol insolublematerial. This label is largely not in RNA as detected by MAKchromatography or polyribosome density gradient centrifugation.IAA can be recovered from the material non adsorbed to MAK. (Received April 23, 1971; )     

Evidence for specificity in symbiont-conferred protection against parasitoids

Many insects harbour facultative symbiotic bacteria, some of which have been shown to provide resistance against natural enemies. One of the best-known protective symbionts is Hamiltonella defensa, which in pea aphid (Acyrthosiphon pisum) confers resistance against attack by parasitoid wasps in the genus Aphidius (Braconidae). We asked (i) whether this symbiont also confers protection against a phylogenetically distant group of parasitoids (Aphelinidae) and (ii) whether there are consistent differences in the effects of bacteria found in pea aphid biotypes adapted to different host plants. We found that some H. defensa strains do provide protection against an aphelinid parasitoid Aphelinus abdominalis. Hamiltonella defensa from the Lotus biotype provided high resistance to A. abdominalis and moderate to low resistance to Aphidius ervi, while the reverse was seen from Medicago biotype isolates. Aphids from Ononis showed no evidence of symbiont-mediated protection against either wasp species and were relatively vulnerable to both. Our results may reflect the different selection pressures exerted by the parasitoid community on aphids feeding on different host plants, and could help explain the maintenance of genetic diversity in bacterial symbionts.     

Further Evidence for Lack of Gene Expression in the Tetrahymena Micronucleus

Certain galA mutations in the ciliated protozoan Tetrahymena thermophila confer an almost total loss of galactokinase activity in homozygotes. Heterokaryons have been constructed that are homogeneous for the galA1 mutation in the (45n) macronucleus, but which contain a galA⁺ (2n) micronucleus. Soluble cell extracts prepared from these heterokaryons have been assayed for galactokinase activity, using a radiometric assay for the conversion of galactose to galactose-1-phosphate (gal-1-P). No galactokinase activity attributable to the micronuclear genes is observed in such heterokaryons. These results, obtained with the galA1 marker, provide the first direct, quantitative evidence for the lack of micronuclear (germ line) gene expression in Tetrahymena during vegetative growth, and substantiate the predictions of previous phenotypic observations on heterokaryons and autoradiographic studies of micronuclear RNA synthesis. The generality of this conclusion will be established in the future when other enzymically assayable mutations become available for similar studies.     

Thioureas react with superoxide radicals to yield a sulfhydryl compound. Explanation for protective effect against paraquat

Thiourea and superoxide dismutase were effective antidotes to paraquat toxicity in an HL60 cell culture system, whereas other hydroxyl scavengers were ineffective. The efficacy of thioureas was not due to blockage of intracellular paraquat uptake, inhibition of NADPH-P-450 reductase, or reaction with the paraquat radical. Thiourea also competitively inhibited the reduction of cytochrome c by the xanthine/xanthine oxidase superoxide-generating system, and the release of iron from ferritin by superoxide radicals. The reaction of superoxide with thiourea produced a sulfhydryl compound distinct from products formed by hydrogen peroxide or hydroxyl radicals. Spectrophotometric and chromatographic studies indicated the carbon-sulfide double bond was converted to a sulfhydryl group which reacted with Ellman's reagent. Additional confirmatory evidence for the sulfhydryl compound was obtained with carbon-13 NMR and mass spectroscopies. Thus, thioureas are direct scavengers of superoxide radicals as well as hydroxyl radicals and hydrogen peroxide. The rate constant for the reduction of thiourea by superoxide was estimated at 1.1 x 10(3) M-1 s-1. The implication of this finding on free radical studies, the mechanism of paraquat toxicity, and the metabolism of thioureas is discussed.