DNA reassociation kinetic analysis of the brine shrimp, Artemia salina

DNA reassociation kinetics have been partly elucidated for the brine shrimp $\text{Artemia salina}$, using calf thymus DNA as a standard. The $\text{Artemia}$ single-copy DNA sequences comprise 45% of the genome; sequences having a repetition frequency of about 2–90 are not detectable. The average repetition frequency of the intermediately redundant DNA component is about 5,000 copies. Reassociation kinetic data are consistent with a unit genome size of 1.5 pg.     

Chloroplast sulfhydryl groups and the light activation of fructose-1,6-bisphosphatase

Studies of isolated intact spinach (Spinacia oleracea L.) chloroplasts reveal that most of the available sulfhydryl groups are associated with stromal protein as opposed to a thylakoid membrane fraction under non-denaturing conditions. Increases in sulfhydryl content of approximately 50% occurred with illumination and could be correlated kinetically with a reductive activation of fructose-1,6-bisphosphatase during CO₂-assimilation. Inhibition of linear electron flow with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prevented light driven increases in both fructose-1,6-bisphosphatase activity and the relative sulfhydryl number. These results provide evidence for the operation of a reductive enzyme activating system in vivo.     

Food Deprivation Suppresses a Preference for the Top-Scent Mark of an Over-Mark in Meadow Voles (Microtus pennsylvanicus)

Food availability affects whether mammals communicate their interest in interacting with opposite‐sex conspecifics. This study examined the responses of voles to over‐marks, and factors that influence the formation and maintenance of a preference for the top‐scent in an over‐mark. Specifically, we investigated how food deprivation affected the amount of time male and female voles exposed to an over‐mark, later responded to the marks of the top‐ and bottom‐scent donors when subsequently presented with the two scents side by side. Males and females that were not food deprived and males that were food deprived 6 h before exposure to an over‐mark later maintained a preference for the donor of the top‐scent mark compared with the donor of the bottom‐scent mark of the over‐mark. Females that were food deprived for 6 h before or after exposure of the over‐mark and males food deprived 6 h after the exposure to the over‐mark showed no preference for the top‐scent mark donor. Re‐feeding females that were food deprived for 6 h before exposure to an over‐mark was sufficient to restore their preference for the mark of the top‐scent male over that of the bottom‐scent male. The different responses of food‐deprived male and female voles to over‐marks of opposite‐sex conspecifics may be associated with differences in their tactics for interacting with potential mates and the higher energetic costs of reproduction in female voles than in male voles.     

Erythrocytes possess an intrinsic barrier to nitric oxide consumption

It has been reported that free hemoglobin (Hb) reacts with NO at an extremely high rate (K(Hb) approximately 10(7) M(-1) s(-1)) and that the red blood cell (RBC) membrane is highly permeable to NO. RBCs, however, react with NO 500-1000 times slower. This reduction of NO reaction rate by RBCs has been attributed to the extracellular diffusion limitation. To test whether additional limitations are also important, we designed a competition test, which allows the extracellular diffusion limitation to be distinguished from transmembrane or intracellular resistance. This test exploited the competition between free Hb and RBCs for NO generated in a homogenous phase by an NO donor. If the extracellular diffusion resistance is negligible, then the results would follow a kinetic model that assumes homogenous reaction without extracellular diffusion limitation. In this case, the measured effective reaction rate constant, K(RBC), would remain invariant of the hematocrit, extracellular-free Hb concentration, and NO donor concentration. Results show that the K(RBC) approaches a constant only when the hematocrit is greater than 10%, suggesting that at higher hematocrit, the extracellular diffusion resistance is negligible. Under such a condition, the NO consumption by RBCs is still 500-1000 times slower than that by free Hb. This result suggests that intrinsic RBC factors, such as transmembrane diffusion limitation or intracellular mechanisms, exist to reduce the NO consumption by RBCs.     

Stability of the topoisomerase II closed clamp conformation may influence DNA-stimulated ATP hydrolysis

Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II alpha with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr28 --> Phe) was introduced in yeast Top2. The ATPase activities of human TOP2alpha(Tyr50 --> Phe) and yeast Top2(Tyr28 --> Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.     

The development of potent and selective bisarylmaleimide GSK3 inhibitors

Many 3-aryl-4-(1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indol-7-yl)maleimides exhibit potent GSK3 inhibitory activity (<100 nM IC(50)), although few show significant selectivity (>100x) versus CDK2, CDK4, or PKCbetaII. However, combining 3-(imidazo[1,2-a]pyridin-3-yl), 3-(pyrazolo[1,5-a]pyridin-3-yl) or aza-analogs with a 4-(2-acyl-(1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indol-7-yl)) group on the maleimide resulted in very potent inhibitors of GSK3 (160 to >10,000-fold selectivity versus CDK2/4 and PKCbetaII. These compounds also inhibited tau phosphorylation in cells and were effective in lowering plasma glucose in a rat model of type 2 diabetes (ZDF rat).     

Baseline susceptibility of western corn rootworm (Coleoptera: Crysomelidae) to Cry3Bb1 Bacillus thuringiensis toxin

Susceptibility to Cry3Bb1 toxin from Bacillus thuringiensis (Bt) was determined for western corn rootworm, Diabrotica virgifera virgifera LeConte, neonates from both laboratory and field populations collected from across the Corn Belt. Rootworm larvae were exposed to artificial diet treated with increasing Cry3Bb1 concentrations, and mortality and growth inhibition were evaluated after 4-7 d. The range of variation in Bt susceptibility indicated by growth inhibition was similar to that indicated by mortality. Although interpopulation variation in susceptibility was observed, the magnitude of the differences was comparable with the variability observed between generations of the same population. In general, the toxin was not highly toxic to larvae and estimated LC50 and EC50 values were several times higher than those reported for lepidopteran-specific Cry toxins by using similar bioassay techniques. These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among rootworm populations and provide a baseline for estimating potential shifts in susceptibility that might result from selection and exposure to Cry3Bb1-expressing corn hybrids.     

Bph1p, the Saccharomyces cerevisiae homologue of CHS1/beige, functions in cell wall formation and protein sorting

Mutations in the Chediak-Higashi syndrome gene (CHS1) and its murine homologue Beige result in the formation of enlarged lysosomes. BPH1 (Beige Protein Homologue 1) encodes the Saccharomyces cerevisiae homologue of CHS1/Beige. BPH1 is not essential and the encoded protein was found to be both cytosolic and peripherally bound to a membrane. Neither disruption nor overexpression of BPH1 affected vacuole morphology as assessed by fluorescence microscopy. The deltabph1 strain showed an impaired growth on defined synthetic media containing potassium acetate buffered below pH 4.25, increased sensitivity to calcofluor white, and increased agglutination in response to low pH. A library screen identified VPS9, FLO1, FLO9, BTS1 and OKP1 as high copy suppressors of the growth defect of deltabph1 on both low pH potassium acetate and calcofluor white. The deltabph1 strain demonstrated a mild defect in sorting vacuolar components, including increased secretion of carboxypeptidase Y and missorting of alkaline phosphatase. Overexpression of VPS9, BTS1 and OKP1 suppressed the carboxypeptidase Y secretion defect of deltabph1. Overexpression of BPH1 was found to suppress the calcofluor white sensitivity of a class E VPS deletion strain, deltavta1. Together, these data suggest that Bph1p associates with a membrane and is involved in protein sorting and cell wall formation.     

Contribution of maternal effect QTL to genetic architecture of early growth in mice

Existing approaches to characterizing quantitative trait loci (QTL) utilize a paradigm explicitly focused on the direct effects of genes, where phenotypic variation among individuals is mapped onto genetic variation of those individuals. For many characters, however, the genotype of the mother via its maternal effect accounts for a considerable portion of the genetically based variation in progeny phenotypes. Thus the focus on direct effect QTL may result in an insufficient or misleading characterization of genetic architecture due to the omission of the potentially important source of genetic variance contributed by maternal effects. We analyze the relative contribution of direct and maternal effect (ME) QTL to early growth in mice using a three-generation intercross of the Small (SM/J) and Large (LG/J) inbred mouse lineages. Using interval mapping and composite interval mapping, direct effect (DE) QTL for early growth (change in body mass during the interval from week 1 to 2) were detected in the F(2) generation of the intercross (n = 510), where no maternal genetic effect variance is present (all individuals are progeny of genetically identical F(1) mothers). ME QTL were detected by treating the phenotypes of cross-fostered F(3) pups as a characteristic of their nurse-dam (n = 168 dams with cross-fostered progeny). Five DE QTL, significant at a chromosome wide level (alpha = 0.05), were detected, with two significant at a genome wide level. FourME QTL significant at the chromosome wide level were detected, with three significant at the genome wide level. A model containing only DE QTL accounted for 11.8% of phenotypic variance, while a model containing only ME QTL accounted for 31.5% of the among litter variance in growth. There was no evidence for pleiotropy of DE and ME loci since there was no overlap between loci detected in these two analyses. Epistasis between all pairs of loci was analyzed for both DEs and MEs. Ten pairs of loci showed significant epistasis for MEs (alpha = 0.05 corrected for multiple comparisons) while four pairs showed significant epistasis for DEs on early growth.