Substructural specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N- acetylgalactosamine/galactose lectins

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc- derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.      

Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath)

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.     

Chimpanzee to chimpanzee American Sign Language

The current study examined how five chimpanzees combined the signs of American Sign Language with their nonverbal communication during high arousal interactions. Thirty-five hours of videotape were analyzed for the presence of high arousal interactions. Similar to deaf children, the chimpanzees signed to one another during high arousal interactions, and they emphatically modulated their signs by signing more vigorously, enlarging the sign’s movement, prolonging the sign, reiterating the sign, or by using a two-handed version of a sign regularly signed with one hand. The majority of the chimpanzees’ sign utterances were contextually consistent. The chimpanzees’ sign utterances were scored as contextually consistent if they were used in previous high arousal interactions which were not part of the current study. Individual chimpanzee differences in signing frequency, emphatic modulation, and recipient allocation were found. Similar to humans, the chimpanzees’ verbal communication is a robust phenomena which continues to occur even during high arousal interactions.     

Cytochrome P-450 monooxygenase,epoxide hydrolase and flavin monooxygenase activities in clara cells and alveolar type II cells isolated from rabbit

The activities of several enzymes which metabolize xenobiotics were measured and compared in freshly isolated rabbit Clara cells (50–70% purity) and alveolar type II cells (80–95% purity) or microsomal preparations from the isolated cell fractions. The presence of 1 mM nicotinamide in protease and cell isolation buffers increased significantly 7-ethoxycoumarin (7-EC) deethylase and epoxide hydrolase activities in the isolated Clara and type II cells. Isolated Clara cell fractions metabolized 7-EC to umbelliferone at a rate of 241 ± 27 pmoles/mg prot/min (mean ± S.E., N=5), while the 7-EC deethylation rate in type II cells was 111 ± 15 pmoles/mg prot/min. Coumarin hydroxylation activity, however, was more than ten times greater in the Clara cells than in the type II cells on a per mg cellular protein basis. N-oxidation of N,N-dimethylaniline, catalyzed by a flavin monooxygenase, was about 2 times as great in microsomes of Clara cells as in microsomes of type II cells. Epoxide hydrolase activity with benzo(a)pyrene 4,5-oxide as substrate was about 10 times higher in Clara cells than in type II cells. Because of the greater cellular, structural and functional heterogeneity in lung, differential distribution of enzymes responsible for xenobiotic metabolism in this tissue may contribute to cell selective chemical toxicity and carcinogenesis.Abbreviations 7-EC 7-ethoxycoumarin - DMA N,N-dimethylaniline     

Hepatic lipid droplets. Isolation, morphology and composition

The floating lipid layer isolated centrifugation of rat liver was examined for composition and ultrastructure. It was chiefly composed of triglycerides and cholesterol esters plus much smaller amounts of free cholesterol, diglycerides, phospholipid and protein. No free fatty acids were detected. The triglyceride and cholesterol ester fractions consisted mostly of esters of linoleic acid, oleic acid and palmitic acid. Electron micrographs of the floating lipid layer revealed numerous spherical osmiophilic droplets having a mean diameter of 0.5-2mum with a very-thin dense outer coat. Similar structures were observed as organelles in electron micrographs of the intact liver cell. The amount of triglyceride in the layer decreased in rats starved for 72h, but pellet triglyceride (homogenate minus the floating lipid layer) was unchanged. These results suggest that the floating lipid layer is the representative in vitro of lipid-rich organelles which probably function as a depot form of hepatic-cell neutral lipid.     

The effect of Hg2+ on rabbit hepatic and pulmonary solubilized,partially purified N,N-dimethylaniline N-oxidases

Solubilization and partial purification of the rabbit pulmonary and hepatic N,N-dimethylaniline N-oxidases were carried out in order to study the effect of Hg²⁺ in vitro observed previously in the microsomal enzymes. Rabbit lung microsomal N,N-dimethylaniline (DMA) N-oxidase activity was stimulated 1.5–2 times by 0.1 mM Hg²⁺ added in vitro. This concentration of mercury inhibited hepatic microsomal N-oxidase by 50%. Upon solubilization and partial purification of the lung N-oxidase enzyme, stimulation of the N-oxidase activity by 0.1 mM Hg²⁺ was lost. It was found that the concentration of Hg²⁺ that would stimulate the partially purified pulmonary N-oxidases was 25 μM or less. Stimulation by 0.1 mM Hg²⁺ of the partially purified N-oxidase from lung was restored by addition of flavins (FMN or FAD) or a heat-stable (NH₄)₂SO₄ precipitated fraction obtained during the purification of the N-oxidase from solubilized pulmonary or hepatic microsomes. However, addition of the flavins or the solubilized, heat-stable fraction from liver or lung microsomes did not reverse inhibition by 0.1 mM Hg²⁺ of the N-oxidase in hepatic microsomes or in partially purified preparations from these hepatic microsomes. Kinetic data suggest that flavins and the heatstable factor isolated from microsomes lower the concentration of free Hg²⁺.The determination of kinetics of Hg²⁺ inhibition (liver) and activation (lung) with the partially purified N-oxidases showed that the pulmonary and hepatic DMA N-oxidase enzymes are markedly different with respect to their in vitro response to Hg²⁺. This suggests that the N-oxidases from liver and lung may be different enzymes.     

Sequence arrangement of the 16S and 26S rRNA genes in the pathogenic haemoflagellate Leishmania donovani.

Kinetic and chemical analysis show that the haploid genome of Leishmania donovani has between 4.6 and 6.5 X 10(7) Kb pairs of DNA. Cot analysis shows that the genome contains 12% rapidly reassociating DNA, U3% middle repetitive DNA with an average reiteration frequency of 77 and 62% single copy DNA. Saturation hybridization experiments show that 0.82% of the nuclear DNA is occupied by rRNA coding sequences. The average repetition frequency of these sequences is determined to be 166. Sedimentation velocity studies indicate the two major rRNA species have sedimentation values of 26S and 16S, respectively. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA has been determined by the examination of the structure of rRNA:DNA hybrids prepared for electron microscopy by the gene 32-ethidium bromide technique. Long DNA strands are observed to contain several gene sets (16S + 26S). One repeat unit contains the following sequences in the order given: (a) A 16S gene of length 2.12 Kb, (b) An internal transcribed spacer (Spl) of length 1.23 Kb, which contains a short sequence that may code for a 5.8S rRNA, (C) 26S gene with a length of 4.31 Kb which contains an internal gap region of length 0.581 Ib, (d) An external spacer of average length 5.85 Kb.     

Sequence arrangement of the rRNA genes of the dipteran Sarcophaga bullata

Velocity sedimentation studies of RNA of Sarcophaga bullata show that the major rRNA species have sedimentation values of 26S and 18S. Analysis of the rRNA under denaturing conditions indicates that there is a hidden break centrally located in the 26S rRNA species. Saturation hybridization studies using total genomic DNA and rRNA show that 0.08% of the nuclear DNA is occupied by rRNA coding sequences and that the average repetition frequency of these coding sequences is approximately 144. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA was determined by the examination of the structure of rRNa:DNA hybrids in the electron microscope. Long DNA strands contain several gene sets (18S + 26S) with one repeat unit containing the following sequences in order given: (a) An 18S gene of length 2.12 kb, (b) an internal transcribed spacer of length 2.01 kb, which contains a short sequence that may code for a 5.8S rRNA, (c) A 26S gene of length 4.06 kb which, in 20% of the cases, contains an intron with an average length of 5.62 kb, and (d) an external spacer of average length of 9.23 kb.