Genome-Wide Association Study in BRCA1 Mutation Carriers Identifies Novel Loci Associated with Breast and Ovarian Cancer Risk

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7×10-8, HR = 1.14, 95% CI: 1.09-1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4×10-8, HR = 1.27, 95% CI: 1.17-1.38) and 4q32.3 (rs4691139, P = 3.4×10-8, HR = 1.20, 95% CI: 1.17-1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific associat

Regulation of coenzyme utilization by bovine liver glutamate dehydrogenase: Investigations using thionicotinamide analogues of NAD and NADP in a dual wavelength assay

1. 1. The coenzyme preference of bovine liver glutamate dehydrogenase (GDH) was probed using dual wavelength spectroscopy and pairing the thionicotinamide analogues, S-NAD or S-NADP (which have absorbance maxima at 400 nm), with the natural coenzymes, NADP or NAD. 2. 2. S-NAD and S-NADP were found to be good alternate substrates for GDH : the apparent Kinm's for the thioderivatives were similar to those of the corresponding natural coenzymes, the apparent Kinm's for glutamate were unaltered by the substitution of the thioderivatives, and the effects of inhibitors and activators on S-NAD or S-NADP kinetics were qualitatively the same as those found for NAD or NADP, respectively. 3. 3. Dual wavelength assays paired NAD and S-NADP or S-NAD and NADP to study the simultaneous reduction of the two coenzymes. Conditions of increasing glutamate concentrations produced differential effects on the rates of the NAD vs NADP reactions, the result, with either nucleotide pair, promoting the NADP linked reaction. 4. 4. Activators and inhibitors of the GDH reaction also showed differential effects upon the NAD vs NADP linked reaction rates in the dual wavelength assay. ADP and leucine, which activate both the NAD and the NADP linked reactions in single coenzyme assays, preferentially activate the NADP or S-NADP linked reactions in the dual nucleotide assays. GTP produced greater inhibition of the NAD or S-NAD linked reactions than of the NADP or S-NADP reactions while ATP inhibited NAD or S-NAD reactions and activated NADP or S-NADP reactions. The net effect of all metabolite modulators was to promote the NADP linked reaction by decreasing the activity ratios, ν(Nad)/ν(S-Nadp) or ν(S-Nad)/ν(Nadp). 5. 5. The results are consistent with the suggestion that NADP is the preferred coenzyme for the oxidative deamination of glutamate by GDH even though the enzyme is capable of utilizing either coenzyme in vitro

Enzyme activities and isozyme composition of triglyceride, diglyceride and monoglyceride lipases in Periplaneta americana, Locusta migratoria and Polia adjuncta

1. 1. Measurements of maximal enzyme activities were combined with an electrophoretic study of isozyme make-up in an examination of triglyceride, diglyceride and monoglyceride lipases from the flight muscle, fat body and gut of the cockroach, Periplaneta americana and the locust, Locusta migratoria and from the flight muscle and fat body of the moth, Polia adjuncta. 2. 2. Tri-, di- and mono-glyceride lipases were present in all tissues of the insects with diglyceride lipase ≥ triglyceride lipase activity in all cases and monoglyceride lipase ≥ diglyceride lipase activity in locust and moth. 3. 3. In the flight muscle, a strong correlation was found between the activities of lipases and the known use of lipid as a fuel for flight in these insects. Lipase activities were lowest in the cockroach (a carbohydrate-based flight metabolism), intermediate in the locust (both carbohydrate and lipid-fueled flight), and highest in the moth (a non-feeding, lipid-catabolizing adult) flight muscle. 4. 4. Polyacrylamide gel electrophoresis, using substrate-impregnated gels and stained for fatty acids released by lipase action, demonstrated the presence of tissue specific isozymes of tri-, di- and mono-glyceride lipases in the three insects. In addition, some, but not all, tissues showed multiple molecular forms of one or more of the lipases. 5. 5. Diglyceride and monoglyceride lipase activities in both flight muscle and fat body of the insects coelectrophoresed suggesting the possibility that these two lipase activities might be catalyzed by a single enzyme protein

Tissue specific isozymes of glutamate dehydrogenase from the Japanese beetle, Popillia japonica: Catabolic vs anabolic GDH's

1. Glutamate dehydrogenase (GDH) from the Japanese beetle, Popillia japonica, occurs in tissue specific isozymic forms. Two forms, specific for flight muscle and fat body, were identified and were separable by starch gel electrophoresis and by differential elution from NAD-agarose. 2. The isozymes utilized both NAD(H) and NADP(H) as coenzymes with activity ratios NADH:NADPH of 6:1 for flight muscle and 8:1 for fat body. pH optima for both enzymes were similar. 3. GDH from the two tissues differed kinetically. Affinity for α-ketoglutarate was much higher for the fat body enzyme, S0.5 for the NADH and NADPH linked reactions being 0.81±0.09 and 0.26±0.03 mM for fat body and 2.4±0.03 and 2.3±0.4 mM for flight muscle GDH, respectively. 4. Flight muscle GDH was much more strongly regulated by nucleotides than was the fat body isozyme. The apparent activation constant, Ka, for ADP was 2-3 fold lower for the flight muscle enzyme for both forward and reverse reactions and ADP had a greater effect in lowering S0.5 for NH4 + for flight muscle GDH. GTP was a strong inhibitor of flight muscle GDH with apparent inhibitor constants, I50, of 15.5±3.0, 4.0±0.9 and 6.5±0.9 μM for the NADH, NADPH and NAD linked reactions, respectively. Fat body GDH, however, was only weakly affected by GTP with an I50 of 60±6μM for the NAD reaction and I50's of greater than 500 μM for the NADH and NADPH linked reactions. 5. The kinetic properties of the two GDH isozymes suit the probable roles of the enzyme in vivo. Flight muscle GDH has a major role in the oxidation of proline as a fuel for flight. Nucleotide control of GDH would allow enzyme activity to respond to the energy status of the cell and would achieve a rapid activation of GDH at the initiation of flight. Fat body GDH, however, has a major role in the biosynthesis of proline and other amino acids. Enzyme activity is probably regulated by substrate availability, the absence of strong nucleotide regul

Kinetic characterization of NADP-specific glutamate dehydrogenase from the sea anemone, anthopleura xanthogrammica: Control of amino acid biosynthesis during osmotic stress

1. 1. Glutamate dehydrogenase (GDH) from the sea anemone, Anthopleura xanthogrammica, was purified 430-fold to a final specific activity of 61.1 units/mg protein. 2. 2. The enzyme was a hexamer of molecular weight 325,000±5000 and subunit size 49,000. 3. 3. The enzyme was NADP(H) specific and utilized L-glutamine as an alternative substrate for ammonium ion; activity ratios, NH4 + vs glutamine, were 11:1 at pH 7 and 2:1 at pH 8. 4. 4. Kinetic constants, S0.5, for the reverse reaction were 61±4, 0.59±0.06 and 0.0091±0.0011 mM for NH4 -, α-ketoglutarate and NADPH for the NH4 + linked reaction and 5.1±1.0, 1.2±0.2 and 0.0027±0.004 mM for glutamine, α-ketoglutarate and NADPH for the glutamine linked reaction. 5. 5. The enzyme was not affected by allosteric modifiers, ADP, ATP, GTP or leucine but was affected by inorganic ions; salts (KCl, NaCl, K2SO4 or potassium phosphate) activated the reverse direction and had an inhibitory effect on the forward direction. 6. 6. Effects of salts on GDH would promote the de novo synthesis of glutamate during hyperosmotic stress giving GDH an important role in the synthesis of amino acids as intracellular osmolytes during adaptation to changes in external salinity