Mutations Affecting Acetylcholine Levels in the Nematode Caenorhabditis elegans

Gene cha-1.unc-17 of the nematode Caenorhabditis elegans is a complex gene, consisting of at least two complementation groups. One part (cha-1 region) of the gene encodes the enzyme choline acetyltransferase (ChAT), but the function of the other part (unc-17 region) is still unclear. We measured the ChAT activity and ACh levels of the cha-1 and unc-17 complex gene mutants. We show here that alterations in ACh levels, rather than the ChAT activity, reflect abnormal phenotypes accompanying cha-1.unc-17 mutations, that is, the decreased ACh levels in cha-1 mutations and abnormal accumulation in unc-17 mutations. Our results suggest that the unc-17 region may encode functions necessary for storage and/or release of ACh at the presynaptic level.     

N1-Methyl-2-125I-Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors

Methylation of 2-125I-lysergic acid diethylamide (125I-LSD) at the N1 position produces a new derivative, N1-methyl-2-125I-lysergic acid diethylamide (125I-MIL), with improved selectivity and higher affinity for serotonin 5-HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I-MIL represents 80-90% of total binding, and the apparent dissociation constant (KD) for serotonin 5-HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I-MIL also displays a high affinity for serotonin 5-HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I-MIL exhibits at least 60-fold higher affinity for serotonin 5-HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I-MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0 degree C. Autoradiographic experiments confirm the improved specificity of 125I-MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I-MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I-MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.     

Polymorphism of the vitamin D binding protein (DBP) among primates: an evolutionary analysis

The distribution of the DBP (vitamin D binding protein) polymorphism is now well characterized among human populations but for primates only limited results are known. The aim of this paper is to describe the electrophoretic polymorphism of this protein among various species. Using three different electrophoretic methods, we are able to detect an unknown polymorphism and to classify the different alleles observed. These results may be used to set an international nomenclature for further comparisons. The different electrophoretic mobilities between Old and New World Monkeys show that: 1) the Cercopithecoïdea are presenting the largest genetic heterogeneity; 2) the DBP among the Galago corresponds to the lowest isoelectric points observed among Primates; 3) during the evolution from nonhuman Primates to Man, the DBP is able to keep its affinity for vitamin D derivatives despite the occurrence of significant molecular modifications; 4) among Anthropoïdea, the electrophoretic patterns of DBP are very close to the human Gc 1 proteins. These results show that evolution at the DBP level can be considered as a continous mechanism of structural modifications. A significant transition occurs during the differentiation between Cercopithecoïdea and Anthropoïdea. It is not too speculative to consider that some electrophoretic forms detected among Gorilla, Pongo, or Pan may be identical to rare variants observed among humans.     

紫胶虫四种寄主树次生韧皮部与周皮的解剖学观察

紫胶虫的4种寄主树钝叶黄檀、南岭黄檀、木豆和苏门答腊金合欢的次生韧皮部轴向系统均由筛管、伴胞、韧皮薄壁细胞、纤维组成,径向系统由射线组成。木豆、苏门答腊金合欢中尚有少量石细胞。苏门答腊金合欢筛管分子端壁较倾斜,具复筛板,无明显的 P-蛋白质。其余3种筛管分子端壁均为横向,具单筛板,具 P-蛋白质。根据观察结果分析,4种寄主树2—3年生枝条或具表皮,或木栓层很薄;次生韧皮部的筛管看来均具相对较长的寿命;4种寄主中有3种筛管与伴胞均属特化程度高的类型。     

Naturally occurring monoterpenoids in needles of Picea abies (L.) Karst

Summary An analysis was made of the effects of different sampling and extraction techniques on the amounts and pattern of monoterpenoids isolated from needles of Norway spruce. The following isolation and analysis procedure was finally adopted: liquid nitrogen-cooled needles were pulverized by a microdismembrator, extracted with pentane overnight at 2°–3°C and concentrated to a volume not less than 3 ml/g fresh weight on a Vigreux column. The crude extract was injected splitless (with solvent split) onto a cold programmed temperature vaporized (PTV) precolumn of a gas chromatograph and the vaporizable compounds heated to a capillary column. This method was tested for production of artefacts and quantitative extraction and applied to needles of eleven 80-year-old spruce trees.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Rhodobacter capsulatus MT113: A single mutation results in the absence of c-type cytochromes and in the absence of the cytochrome bc1 complex

MT113, a nonphotosynthetic mutant of Rhodobacter capsulatus previously characterized as lacking cytochrome c₂ is shown to lack also cytochrome c₁, the Rieske iron-sulfur cluster and the antimycin sensitive semiquinone Q_c, all components of the cytochrome bc₁ complex. Although MT113 contained b-type cytochromes and other iron-sulfur clusters at nearly wild-type level, it lacks c-type cytochromes. Based on antibody detection, c₂ apoprotein was absent in MT113, however the apoproteins corresponding to the cytochromes b and c₁ and the Rieske iron-sulfur cluster were present in reduced amounts. Genetic analysis indicated that the lesion appears to be due to a single mutation which is not localized in the structural genes of cytochrome c₂ or the bc₁ complex. These data taken together suggest that the pleiotropic mutation in MT113 might be related to the biosynthesis of c-type cytochromes.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae

The ATP synthase complex of Klebsiella pneumoniae (KF₁F₀) has been purified and characterized. SDS-gel electrophoresis of the purified F₁F₀ complexes revealed an identical subunit pattern for E. coli (EF₁F₀) and K. pneumoniae. Antibodies raised against EF₁ complex and purified EF₀ subunits recognized the corresponding polypeptides of EF₁F₀ and KF₁F₀ in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA 9-amino-6-chloro-2-methoxyacridine - DCCD N,N-dicyclohexylcarbodiimide - FITC fluorescein isothiocyanate - SDS sodium dodecyl sulfate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole