Binding of the transition metal ion [(H20)(NH3)5Ru(II)]2+ to nucleosomal core and internucleosomal DNA

The goal of this study is to establish the nature of pentammineruthenium(III) binding to DNA in intact mouse liver nuclei. Also, we wish to determine whether the nucleosomal organization of mouse chromatin has a substantial effect on the relative Ru(III) binding levels of internucleosomal and nucleosomal core DNA. These questions are important because ammineruthenium compounds share chemical and biological properties with the cis-dichlorodiammineplatinum(II) or cisplatin chemotherapeutic agent. Therefore, they represent a potential class of new chemotherapeutic agents. We find that in intact nuclei the predominant DNA binding site for pentammineruthenium(II), followed by air oxidation to pentammineruthenium(III), is N-7 guanine, as is the case with cisplatin. Also, the Ru(III) distribution between internucleosomal and nucleosomal core DNA was found to be nearly identical as probed with three non-specific deoxyribonucleases.     

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

Metabolic correlates to glycerol biosynthesis in a freeze-avoiding insect,Epiblema scudderiana

Summary The course of glycerol biosynthesis, initiated by exposure to –4°C, was monitored in larvae of the goldenrod gall moth,Epiblema scudderiana, and accompanying changes in the levels of intermediates of glycolysis, adenylates, glycogen, glucose, fructose-2,6-bisphosphate, and fermentative end products were characterized. Production of cryoprotectant was initiated within 6 h after a switch from +16° to –4°C, with halfmaximal levels reached in 30 h and maximal content, 450–500 mol/g wet weight, achieved after 4 days. Changes in the levels of intermediates of the synthetic pathway within 2 h at –4°C indicated that the regulatory sites involved glycogen phosphorylase, phosphofructokinase, and glycerol-3-phosphatase. A rapid increase in fructose-2,6-bisphosphate, an activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase, appeared to have a role in maintaining flux in the direction of glycerol biosynthesis. Analysis of metabolite changes as glycerol production slowed suggested that the inhibitory restriction of the regulatory enzymes was slightly out of phase. Inhibition at the glycerol-3-phosphatase locus apparently occurred first and resulted in a build-up of glycolytic intermediates and an overflow accumulation of glucose. Glucose inhibition of phosphorylase, stimulating the conversion of the activea to the inactiveb forms, appears to be the mechanism that shuts off phosphorylase function, counteracting the effects of low temperature that are the basis of the initial enzyme activation. Equivalent experiments carried out under a nitrogen gas atmosphere suggested that the metabolic make-up of the larvae in autumn is one that obligately routes carbohydrate flux through the hexose monophosphate shunt. The consequence of this is that fermentative ATP production during anoxia is linked to the accumulation of large amounts of glycerol as the only means of maintaining redox balance.Abbreviations G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1, 6P fructose-1,6-bisphosphate - F2,6P ₂ fructose-2,6-bisphosphate - G3P grycerol-3-phosphate - DHAP dinydroxyacetonephosphate - GAP glyceraldehyde-3-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase - FBPase fructose-1,6-bisphosphatase - PK pyruvate kinase     

An assessment of short-term depletion of stream macroinvertebrate benthos by drift

A study was conducted to determine if emigration of drifting macroinvertebrates from a stream riffle which was blocked for one week from immigration by upstream colonists significantly reduced the abundance of drift collected from the tail of the riffle. The head of a 9 m long riffle of a 2nd order stream in Maryland (USA) was blocked from incoming drift by a 250 m mesh weir. Upstream immigration of invertebrates into the riffle was largely prevented by a partition placed at the tail of the riffle which held the drift nets. Benthos and drift samples were collected from the riffle prior to weir placement and following its removal, and drift was collected at dusk on each day. No difference in drift or in benthic abundance between the beginning and end of the study was observed. This is largely attributed to recruitment of immature insects (primarily hatching of eggs present at the outset), particularly of Dolophilodes distinctus and species of Tanytarsini, from within the riffle. Results suggest that recruitment of riffle species is of sufficient magnitude to more than compensate for short-term riffle depletion due to drift. Samples of drifting and non-drifting (benthic) animals were held without food for 12 h after collection and mortality within each group was determined. The mortality of drifting animals was three-fold that of benthic animals.     

Roles of proteins in cation/membrane interactions of isolated rat cardiac sarcolemmal vesicles

To ascertain the roles of the membrane proteins in cation/sarcolemmal membrane binding, isolated rat cardiac sarcolemmal vesicles were extensively treated with Protease (S. aureus strain V.8). SDS-gel electrophoresis, protein and phosphate analysis confirmed that at least 20–22% of the protein, but none of the phospholipid, was solubilized by this procedure, and that the remaining membrane proteins were extensively hydrolyzed into small fragments. The cation binding properties of the treated vesicles were then examined by analyzing their aggregation behavior. The results demonstrate that this procedure had no effect on the selectivity series for di- and trivalent cation binding, or the divalent cation-induced aggregation behavior of the sarcolemmal vesicles at different pHs, indicating that proteins are probably not involved in these interactions and cannot be the low affinity cation binding sites previously observed [21, 22]. It did, however, change the pH at which protons induced sarcolemmal vesicle aggregation, suggesting a possible role for proteins in these processes. Protease treatment also modified the effects of fluorescamine labelling on divalent cation-induced vesicle aggregation, indicating that the NH, groups being labelled with fluorescamine are located on the sarcolemmal proteins. Together, these results support the hypothesis that di- and trivalent cation binding to the sarcolemmal membrane is largely determined by lipid/lipid and/or lipid/carbohydrate interactions within the plane of the sarcolemmal membrane, and that membrane proteins may exert an influence on these interactions, but only under very specialized conditions.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino) propanesulfonic acid - HEPES N-2-Hydroxyethylpiperizine-N-2- ethanesulfonic acid - CHES 2(N-Cyclohexylamino) ethanesulfonic acid - DTT DL-Dithiothreitol - PMSF Phenylmethyl-sulfonyl fluoride     

Comparative studies of hepatotoxicity and fumonisin B1 and B2 content of water and chloroform/methanol extracts of Fusarium moniliforme strain MRC 826 culture material

Fusarium moniliforme has been associated with several diseases including equine leukoencephalomalacia, human esophageal cancer and hepatotoxicity/hepatocarcinogenicity in laboratory animals. The potential health risks to animals and humans posed by F. moniliforme contaminated grains cannot be assessed until the toxins are identified and toxicologically evaluated. As part of a systematic approach to identifying the hepatotoxins produced by F. moniliforme, diets containing aqueous and chloroform/methanol (11) extracts of F. moniliforme strain MRC 826 culture material (CM) and/or the extracted CM residues were fed to male Sprague-Dawley rats for four weeks. Serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities were increased after two and four weeks and microscopic liver lesions were found in those animals fed aqueous CM extract and the CM residue after chloroform/ methanol extraction. Fumonisins B₁ and B₂ were extracted from the CM by water, but not chloroform/ methanol, and were present in the toxic diets at concentrations of 93–139 and 82–147 ppm, respectively. Nontoxic diets contained 22 ppm fumonisin B₁ and 65 ppm fumonisin B₂.Abbreviations CM culture material - ELEM equine leukoencephalomalacia Mention of a trademark, proprietory name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.     

The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro

Summary Semigranular cells from the crayfish, Pacifastacus leniusculus, were separated by Percoll gradient centrifugation and were used to study the encapsulation of foreign particles. The semigranular cells were found strongly to encapsulate glass beads coated with haemocyte lysate in which the prophenoloxidase-activating system had been activated with laminarin or with a low concentration of calcium ions. The granular cells only weakly encapsulated these particles. The encapsulationpromoting factor was purified from haemocyte lysates and found to be a 76 kD protein which was recognized by an antiserum to the previously described 76 kD cell-adhesion factor. After the last step in purification (Con A-Sepharose chromatography), the flowthrough consisted of several proteins, which had some, but less, encapsulation-promoting activity and contained a 30 kD band that was also recognized by the antiserum to the 76 kD cell-adhesion factor. If the haemocyte lysate prepared in low [Ca²⁺] was incubated with a -1,3-glucan prior to purification, no 76 kD protein could be isolated but only a 30 kD protein. The 30 kD protein thus seems to be a degradation product of the 76 kD cell-adhesion factor. We conclude that the 76 kD protein which is released from degranulating haemocytes, and to a lesser extent its 30 kD fragment, can promote encapsulation. Phenoloxidase did not have any encapsulation-promoting activity.     

Solubilization and characterization of a lactogenic receptor from human placental chorion membranes

Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.     

The human metallothionein gene cluster is not disrupted in myelomonocytic leukemia

The human metallothionein gene complex on chromosome 16 has been remapped to 16q13 using high-resolution in situ hybridization. The complex is not disrupted by the rearrangement breakpoint on the long arm of chromosome 16 in patients with myelomonocytic leukemia with abnormal eosinophils, as had been previously reported. The locus order on 16q is cen-MT-FRA16B-D16S4-inversion breakpoint-HP-tel.