Function of MoaB proteins in the biosynthesis of the molybdenum and tungsten cofactors

Molybdenum (Mo) and tungsten (W) enzymes catalyze important redox reactions in the global carbon, nitrogen, and sulfur cycles. Except in nitrogenases both metals are exclusively associated with a unique metal-binding pterin (MPT) that is synthesized by a conserved multistep biosynthetic pathway, which ends with the insertion and thereby biological activation of the respective element. Although the biosynthesis of Mo cofactors has been intensively studied in various systems, the biogenesis of W-containing enzymes, mostly found in archaea, is poorly understood. Here, we describe the function of the Pyrococcus furiosus MoaB protein that is homologous to bacterial (such as MogA) and eukaryotic proteins (such as Cnx1) involved in the final steps of Mo cofactor synthesis. MoaB reconstituted the function of the homologous Escherichia coli MogA protein and catalyzes the adenylylation of MPT in a Mg2+ and ATP-dependent way. At room temperature reaction velocity was similar to that of the previously described plant Cnx1G domain, but it was increased up to 20-fold at 80 degrees C. Metal and nucleotide specificity for MPT adenylylation is well conserved between W and Mo cofactor synthesis. Thermostability of MoaB is believed to rely on its hexameric structure, whereas homologous mesophilic MogA-related proteins form trimers. Comparison of P. furiosus MoaB to E. coli MoaB and MogA revealed that only MogA is able to catalyze MPT adenylylation, whereas E. coli MoaB is inactive. In summary, MogA, Cnx1G, and MoaB proteins exhibit the same adenylyl transfer activity essential for metal insertion in W or Mo cofactor maturation.     

The Adipose Tissue Phenotype of Hormone‐Sensitive Lipase Deficiency in Mice

Objective: To directly ascertain the physiological roles in adipocytes of hormone‐sensitive lipase (HSL; E.C. 3.1.1.3), a multifunctional hydrolase that can mediate triacylglycerol cleavage in adipocytes. Research Methods and Procedures: We performed constitutive gene targeting of the mouse HSL gene (Lipe), subsequently studied the adipose tissue phenotype clinically and histologically, and measured lipolysis in isolated adipocytes. Results: Homozygous HSL^?/? mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue, and heterozygous mice have intermediate levels with respect to wild‐type and deficient littermates. HSL‐deficient mice have normal body weight but reduced abdominal fat mass compared with normal littermates. Histologically, both white and brown adipose tissues in HSL^?/? mice show marked heterogeneity in cell size, with markedly enlarged adipocytes juxtaposed to cells of normal morphology. In isolated HSL^?/? adipocytes, lipolysis is not significantly increased by β₃‐adrenergic stimulation, but under basal conditions in the absence of added catecholamines, the lipolytic rate of isolated HSL^?/? adipocytes is at least as high as that of cells from normal controls. Cold tolerance during a 48‐hour period at 4 °C was similar in HSL^?/? mice and controls. Overnight fasting was well‐tolerated clinically by HSL^?/? mice, but after fasting, liver triglyceride content was significantly lower in HSL^?/? mice compared with wild‐type controls. Conclusions: In isolated fat cells, the lipolytic rate after β‐adrenergic stimulation is mainly dependent on HSL. However, the observation of a normal rate of lipolysis in unstimulated HSL^?/? adipocytes suggests that HSL‐independent lipolytic pathway(s) exist in fat. Physiologically, HSL deficiency in mice has a modest effect under normal fed conditions and is compatible with normal maintenance of core body temperature during cold stress. However, the lipolytic response to overnight fasting is subnormal.     

Attributes of Individual Flowers of Brassica napus L. are Affected by Defoliation but not by Intraspecific Competition

To investigate the effect of intraspecific competition on floweringin Brassica napus‘Westar’, a series of 30 pots wasestablished that spanned a range of one–96 plants perpot. In each pot, the following attributes of individual flowerswere quantified: petal length; petal width; stamen length; pistillength; pollen production; diameter of pollen grains; and nectarproduction. Certain plants contained a transgenic marker thatenabled the vigour of their pollen to be quantified by screeningthe progeny from post-pollination trials against conventionalmale competitors. Plant size was strongly affected by plantdensity; plants in the lowest density pots comprised ten-timesmore dry biomass than individuals in the highest density pots.However, none of the attributes of individual flowers variedwith density. In contrast, the number of flowers per plant declinedrapidly with density. In the face of resource scarcity, theplants apparently conserved flower size rather than flower number.There was no difference in the response to density between transgenicand conventional plants, but transgenic plants obtained morefertilizations than expected in post-pollination competitionagainst conventional competitors. A separate experiment demonstratedthat B. napus is, nevertheless, capable of plasticity in certainflower attributes (petal size, stamen length) in response todefoliation. Explanations for the stability of flower size relativeto flower number are discussed. Copyright 2001 Annals of BotanyCompany Brassica napus, defoliation, density effects, flowers, flower size, intraspecific competition, nectar production, oilseed rape, pollination, transgenic plants     

Analysis of short stature homeobox-containing gene (<Emphasis Type="Italic">SHOX</Emphasis>) and auxological phenotype in dyschondrosteosis and isolated Madelung deformity

Dyschondrosteosis (DCO; also called Léri-Weill syndrome) is a skeletal dysplasia characterised by disproportionate short stature because of mesomelic shortening of the limbs. Madelung deformity is a feature of DCO that is distinctive, variable in expressivity and frequently observed. Mutations of the SHOX (short stature homeobox-containing) gene have been previously described as causative in DCO. Isolated Madelung deformity (IMD) without the clinical characteristics of DCO has also been described in sporadic and a few familial cases but the genetic defect underlying IMD is unknown. In this study, we have examined 28 probands with DCO and seven probands with IMD for mutations in the SHOX gene by using polymorphic CA-repeat analysis, fluorescence in situ hybridisation (FISH), Southern blotting, direct sequencing and fibre-FISH analyses. This was combined with auxological examination of the probands and their family members. Evaluation of the auxological data showed a wide intra- and interfamilial phenotype variability in DCO. Out of 28 DCO probands, 22 (79%) were shown to have mutations in the SHOX gene. Sixteen unrelated DCO families had SHOX gene deletions. Four novel DCO-associated mutations were found in different families. In two additional DCO families, the previously described nonsense mutation (Arg195Stop) was detected. We conclude that mutations in the SHOX gene are the major factor in the pathogenesis of DCO. In a female proband with severe IMD and her unaffected sister, we detected an intrachromosomal duplication of the SHOX gene.     

General Synthesis of 2′(3′)-O-Aminoacyl Oligoribonucleotides Related to the 3′-Terminus of aa-tRNA

Abstract

A general method is presented for the synthesis of the title compounds using phosphotriester methodology and employing a unique combination of protecting groups, including the double protection of the guanosine aglycon.     

The arctic-alpine polyploids Cerastium alpinum and C. nigrescens (Caryophyllaceae) in a sympatric situation: breakdown of species integrity?

 The high polyploids Cerastium alpinum (8x) and C. nigrescens (12x) were investigated in a mixed population in central Norway to evaluate whether hybridisation has resulted in continuous variation in morphology and genetic markers, a hypothesis previously proposed to account for the extensive taxonomic confusion in this species group. Isozyme, fertility, and morphological (37 characters) variation were examined among 347, 265, and 237 plants, respectively. A PCO analysis based on 23 quantitative morphological characters identified two main groups, corresponding to C. alpinum and C. nigrescens. The groups were also clearly separated in isozyme markers, several qualitative morphological characters, and chromosome numbers. Only 20 plants (8.4%) were more or less intermediate in the PCO analysis. These plants had a parental or hybrid isozyme multilocus phenotype and typically few well-developed anthers, low pollen stainability, and no seed set. Several plants within the C. nigrescens group also had reduced pollen stainability. These results indicate that later-generation hybrids and/or backcrosses towards C. nigrescens are formed. Thus, interspecific, interploidal gene flow probably occurs, but at rates that are insufficient to break down species integrity. Received July 5, 2000 Accepted August 2, 2001