TRP64ARG β3-adrenergic receptor and obesity in Mexican Americans

The β3-adrenergic receptor (β3AR) is expressed in visceral fat and is a regulator of resting metabolic rate, thermogenesis, and lipolysis. We genotyped 61 unrelated Mexican Americans for a variant in the β3AR gene (codon 64 TGG^Trp→CGG^Arg; TRP64ARG). The allele frequency was 0.13. The TRP64ARG variant was significantly associated with an earlier age of onset of non-insulin-dependent diabetes mellitus (41.3 ± 4.6 years vs 55.6 ± 2.6 years; P < 0.02) and in non-diabetics, with elevated 2-h insulin levels during an oral glucose tolerance test (810 ± 120 pmol/l vs 384 ± 6 pmol/l; P < 0.005). Non-diabetic subjects with the variant allele tended to have higher body mass indices (BMI), waist-to-hip ratios, and diastolic blood pressures. The study group was expanded to include 421 related subjects from 31 families in the San Antonio Family Diabetes Study. Using a measured genotype analysis approach to estimate genotype-specific means for each trait, those who were homozygous for the TRP64ARG variant had significantly higher 2-h insulin levels (P = 0.036) and trends towards higher BMI compared to the other two genotypes. We detected no associations of these traits in the TRP64ARG heterozygotes in the larger group. We conclude that the TRP64ARG β3AR variant is a susceptibility gene for several features of the insulin resistance syndrome in Mexican Americans. Since its effects are modest, study design (e.g., subject selection, genetic background, and statistical analyses) may influence which traits are associated with this variant and whether or not the effect is detectable in heterozygotes. Received: 7 April 1997 / Accepted: 22 July 1997     

The inhibition of protein synthesis in meiotic cells and its effect on chromosome behavior

Autoradiographs show that tritiated leucine is incorporated into protein continually at an almost linear rate during meiotic prophase of lily microsporocytes in in vitro culture. Although label is mostly in the cytoplasm for the first hour, it becomes almost evenly distributed throughout the cell after a few hours. The amount of label decreases slightly, if at all, during a chase period extending through the rest of the prophase — a period of 3 to 4 days. — The incorporation of label was blocked by 95% by the protein inhibitor, cycloheximide, at a concentration of 3.5 × 10^-6 M. In the presence of this inhibitor, meiosis was arrested at all stages through metaphase I and even later. After temporary inhibition, however, or in low drug concentrations, characteristic cytological abnormalities subsequently developed, depending on the meiotic stage at which the inhibition occurred. One important observation was that the formation of chiasmata between homologs could be blocked if the inhibition was applied during the late zygotene or early pachytene stages.This work was supported by a grant from the National Science Foundation (GB-5173 X).USPHS postdoctoral fellow.     

Role of EXO1 nuclease activity in genome maintenance,the immune response and tumor suppression in Exo1D173A mice

DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here, we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1^DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1^−/− and EXO1 wild type Exo1^+/+ mice. We show that Exo1^DA/DA and Exo1^–/– mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1^DA/DA mice to be intermediate between Exo1^+/+ and Exo1^–/– mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1^DA/DA and Exo1^–/– mice was comparably defective, switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1^−/− mice that are infertile, meiosis progressed normally in Exo1^DA/DA and Exo1^+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1^DA/DA and Exo1^–/– mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.     

Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase.

CO2-capture methods have been used for assaying many decarboxylating enzymes including hydroxylation-coupled decarboxylation reactions. The traditional CO2-capture method involves performing the reaction in capped tubes and radiometric measurement of trapped 14CO2 by scintillation counting. In this report, a 14CO2-capture method in a 96-well microtiter plate format has been developed and a phosphor imaging system has been employed for sample measurement. The new assay method has been used successfully to assay aspartyl-beta-hydroxylase activity in microtiter plate format. The results obtained here compare favorably with those obtained from the traditional tube method. The method is sensitive, suitable for high throughput, and generally applicable to many CO2-releasing enzyme assays.     

A mitochondrial cytochrome b mutation but no mutations of nuclearly encoded subunits in ubiquinol cytochrome c reductase (complex III) deficiency

Ubiquinol cytochrome c reductase (complex III) deficiency represents a clinically heterogeneous group of mitochondrial respiratory chain disorders that can theoretically be subject to either a nuclear or a mitochondrial mode of inheritance. In an attempt to elucidate the molecular bases of the disease, we first determined the nucleotide sequence of three unknown subunits (9.5 kDa, 7.2 kDa, 6.4 kDa) by cyberscreening of human expressed sequence tag data bases and sequenced the 11 cDNA subunits encoding complex III in five patients with isolated complex III deficiency. No mutation in the nuclearly encoded complex III subunits was observed, but a mutation in the cd2 helix of the mitochondrial (mt) cytochrome b gene was found to alter the conformation of the bc ₁ complex in one patient with severe hypertrophic cardiomyopathy. The present study is highly relevant to genetic counseling as the absence of mtDNA mutations in all but one patient in our series strongly supports autosomal rather than maternal inheritance in the majority of patients with complex III deficiency. Received: 15 January 1999 / Accepted: 31 March 1999     

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [³H]zeatin riboside ([³H]ZR), a water‐soluble blue dye or [³H]H₂O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within 1 h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [³H]ZR and of blue dye in the same bud position was increased 3‐ to 10‐fold relative to intact plants, whereas content of [³H]H₂O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [³H]H₂O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [³H]ZR injected at physiological concentrations. Within 1 h, 80–95% of [³H]ZR was converted to other active CKs (mainly zeatin riboside‐5′phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O‐acetylZR, O‐acetylZRMP and a compound correlated with sites of high CK‐concentrations) and inactive catabolites (adenosine, adenine, 5′AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.     

Contribution of HIF-P4H isoenzyme inhibition to metabolism indicates major beneficial effects being conveyed by HIF-P4H-2 antagonism

Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs 1–3) are druggable targets in renal anemia, where pan-HIF-P4H inhibitors induce an erythropoietic response. Preclinical data suggest that HIF-P4Hs could also be therapeutic targets for treating metabolic dysfunction, although the contributions of HIF-P4H isoenzymes in various tissues to the metabolic phenotype are inadequately understood. Here, we used mouse lines that were gene-deficient for HIF-P4Hs 1 to 3 and two preclinical pan-HIF-P4H inhibitors to study the contributions of these isoenzymes to the anthropometric and metabolic outcome and HIF response. We show both inhibitors induced a HIF response in wildtype white adipose tissue (WAT), liver, and skeletal muscle and alleviated metabolic dysfunction during a 6-week treatment period, but they did not alter healthy metabolism. Our data indicate that HIF-P4H-1 contributed especially to skeletal muscle and WAT metabolism and that its loss lowered body weight and serum cholesterol levels upon aging. In addition, we found HIF-P4H-3 had effects on the liver and WAT and its loss increased body weight, adiposity, liver weight and triglyceride levels, WAT inflammation, and cholesterol levels and resulted in hyperglycemia and insulin resistance, especially during aging. Finally, we demonstrate HIF-P4H-2 affected all tissues studied; its inhibition lowered body and liver weight and serum cholesterol levels and improved glucose tolerance. We found very few HIF target metabolic mRNAs were regulated by the inhibition of three isoenzymes, thus suggesting a potential for selective therapeutic tractability. Altogether, these data provide specifications for the future development of HIF-P4H inhibitors for the treatment of metabolic diseases.     

Identification of small peptides mimicking the R2 C‐terminus of Mycobacterium tuberculosis ribonucleotide reductase

Ribonucleotide reductase (RNR) is a viable target for new drugs against the causative agent of tuberculosis, Mycobacterium tuberculosis. Previous work has shown that an N‐acetylated heptapeptide based on the C‐terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. Here the synthesis and binding affinity, evaluated by competitive fluorescence polarization, of several truncated and N‐protected peptides are described. The protected single‐amino acid Fmoc‐Trp shows binding affinity comparable to the N‐acetylated heptapeptide, making it an attractive candidate for further development of non‐peptidic RNR inhibitors. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Arachidonic acid is a physiological activator of the ryanodine receptor in pancreatic beta-cells

Pancreatic beta-cells have ryanodine receptors but little is known about their physiological regulation. Previous studies have shown that arachidonic acid releases Ca(2+) from intracellular stores in beta-cells but the identity of the channels involved in the Ca(2+) release has not been elucidated. We studied the mechanism by which arachidonic acid induces Ca(2+) concentration changes in pancreatic beta-cells. Cytosolic free Ca(2+) concentration was measured in fura-2-loaded INS-1E cells and in primary beta-cells from Wistar rats. The increase of cytosolic Ca(2+) concentration induced by arachidonic acid (150microM) was due to both Ca(2+) release from intracellular stores and influx of Ca(2+) from extracellular medium. 5,8,11,14-Eicosatetraynoic acid, a non-metabolizable analogue of arachidonic acid, mimicked the effect of arachidonic acid, indicating that arachidonic acid itself mediated Ca(2+) increase. The Ca(2+) release induced by arachidonic acid was from the endoplasmic reticulum since it was blocked by thapsigargin. 2-Aminoethyl diphenylborinate (50microM), which is known to inhibit 1,4,5-inositol-triphosphate-receptors, did not block Ca(2+) release by arachidonic acid. However, ryanodine (100microM), a blocker of ryanodine receptors, abolished the effect of arachidonic acid on Ca(2+) release in both types of cells. These observations indicate that arachidonic acid is a physiological activator of ryanodine receptors in beta-cells.     

mlo‐based powdery mildew resistance in hexaploid bread wheat generated by a non‐transgenic TILLING approach

Wheat is one of the most widely grown cereal crops in the world and is an important food grain source for humans. However, wheat yields can be reduced by many abiotic and biotic stress factors, including powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt). Generating resistant varieties is thus a major effort in plant breeding. Here, we took advantage of the non‐transgenic Targeting Induced Lesions IN Genomes (TILLING) technology to select partial loss‐of‐function alleles of TaMlo, the orthologue of the barley Mlo (Mildew resistance locus o) gene. Natural and induced loss‐of‐function alleles (mlo) of barley Mlo are known to confer durable broad‐spectrum powdery mildew resistance, typically at the expense of pleiotropic phenotypes such as premature leaf senescence. We identified 16 missense mutations in the three wheat TaMlo homoeologues, TaMlo‐A1, TaMlo‐B1 and TaMlo‐D1 that each lead to single amino acid exchanges. Using transient gene expression assays in barley single cells, we functionally analysed the different missense mutants and identified the most promising candidates affecting powdery mildew susceptibility. By stacking of selected mutant alleles we generated four independent lines with non‐conservative mutations in each of the three TaMlo homoeologues. Homozygous triple mutant lines and surprisingly also some of the homozygous double mutant lines showed enhanced, yet incomplete, Bgt resistance without the occurrence of discernible pleiotropic phenotypes. These lines thus represent an important step towards the production of commercial non‐transgenic, powdery mildew‐resistant bread wheat varieties.