Fluorescent Amplified Fragment Length Polymorphism Analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

The Eating Inventory and Body Adiposity from Leanness to Massive Obesity: a Study of 2509 Adults

Objective: To examine how chronic dietary restraint, disinhibition, and hunger, as assessed by the Eating Inventory, vary over a broad range of BMI values from leanness to massive obesity, in subjects with family obesity. Eating Inventory factors were also studied as a function of personal weight history. Research Methods and Procedures: Subjects were 2509 participants in a genetic study of obesity. BMIs ranged from 15 to 87 kg/m². Six BMI groups were formed (<27, 27 to 30, 30 to 35, 35 to 40, 40 to 45, and >45). Results: Multivariate analyses showed that restraint and disinhibition were significantly associated with BMI in men, whereas only disinhibition was in women. Disinhibition scores correlated strongly with hunger scores in both genders in all BMI categories; dietary restraint tended to correlate with the other two factors positively in leaner subjects and negatively in the highest BMI categories. Highly restrained normal‐weight subjects were likely to exhibit disinhibition and hunger, whereas massively obese persons with very high disinhibition scores showed high hunger but little restraint. The highest restraint scores were observed in nonobese adult women with previous obesity in childhood and/or adolescence. Discussion: The factor most strongly associated with BMI in this large population was disinhibition, suggesting that obesity treatment should target behaviors associated with disinhibition, especially in individuals showing a low level of dietary restraint. High restraint scores in formerly obese normal‐weight women suggest that dietary restraint may exert a beneficial influence on body weight control under conditions that deserve further investigation.     

Chronic glutamine supplementation increases nasal but not salivary IgA during 9 days of interval training.

Oral glutamine supplementation during and after exercise abolishes exercise-induced decreases in plasma glutamine concentration but does not affect secretory IgA (sIgA) salivary output. Whether chronic glutamine supplementation during high-intensity interval training influences salivary and nasal sIgA concentration is unknown. The purpose of this study was examine the effects of chronic glutamine supplementation on sIgA during intense running training. Runners (n = 13, body mass 69.9 +/- 2.8 kg, peak whole body oxygen uptake 55.5 +/- 2 ml.kg(-1).min(-1), age 29.1 +/- 2.8 yr) participated in twice-daily interval training for 9-9.5 days, followed by recovery (5-7 days). Oral glutamine supplement (0.1 g/kg) or placebo was given four times daily for the first 14 days. After an overnight fast, venous blood, nasal washes, and stimulated saliva were collected at baseline (T1), midtraining (T2), posttraining (T3), and after recovery (T4). Mood states were assessed by using Profile of Mood States (POMS) inventories. We found that glutamine concentration in resting subjects decreased from T1 to T4 (P < 0.05) and was not altered by supplementation. Salivary IgA concentration and output were unchanged by training or supplementation. Mean nasal IgA across the study period was greater in runners receiving glutamine (264.7 +/- 35.0 microg/mg protein) vs. placebo (172.4 +/- 33.7 microg/mg protein; P < 0.05). POMS analyses indicated that vigor was lower at T3 vs. T1 (P < 0.05) and fatigue was higher at T2 vs. T1 and T4 (P < 0.05). We conclude that chronic glutamine supplementation during interval training results in higher nasal IgA than placebo but does not affect salivary IgA concentration or output.     

Total evidence phylogeny of Gasterosteidae: combining molecular, morphological and behavioral data

Genealogical relationships among Gasterosteidae (Teleostei: Gasterosteiformes) were tested with 84 morphological, 48 behavioral, and 2879 molecular characters. Phylogenetic analysis of the combined data set identified a single (CI = 0.735) best‐supported hypothesis (Spinachia (Apeltes ((Pungitius + Culaea)(Gasterosteus aculeatus + G. wheatlandi)))). This hypothesis is identical to previous phylogenetic propositions proposed on the basis of behavioral, and behavioral plus morphological data. Our hypothesis, however, differed from a molecular‐based phylogeny in the placement of Apeltes. This analysis highlights the importance of combining all available evidence in order to produce the best‐supported proposition of genealogical relationships.     

Phosphorus and intraspecific density alter plant responses to arbuscular mycorrhizas

We investigated how phosphorus availability, intraspecific density, and their interaction affect plant responses to arbuscular mycorrhizas. Four facultatively mycotrophic species: chile, cilantro, tomato, and corn were examined separately in pot experiments that employed a tropical phosphorus-immobilizing soil. Each experiment comprised nine soluble phosphorus additions, two levels of intraspecific plant density, and inoculation with arbuscular mycorrhizal fungi or not. High phosphorus signi- ficantly diminished mycorrhizal colonization of corn, cilantro, and tomato, but not chile, which was highly variably colonized. Corn roots were colonized by other root-inhabiting fungi, and mycorrhizas significantly reduced colonization by these potential root parasites. High phosphorus significantly increased relative growth rates (RGR) of all species, and high density significantly reduced RGR of cilantro, tomato, and corn. Chile showed little growth at any but the highest phosphorus additions, and consequently had no RGR response to density or mycorrhizas. Mycorrhiza inoculation caused transient depression of corn growth during the first month, but mycorrhizas increased corn RGR during the second month of growth. Both RGR and dry weights at harvest, cilantro, tomato, and corn benefited from mycorrhizas at low phosphorus availability, but this benefit diminished or changed to disadvantage as phosphorus availability increased. At low phosphorus availability, high density increased the dry weight of mycorrhizal cilantro and thereby amplified the benefit of mycorrhizas. At high phosphorus availability, increased density diminished the effects of mycorrhizas on dry weight, reducing mycorrhiza benefit to tomato and chile and reducing mycorrhiza detriment to cilantro. This study demonstrates that for three of the four plant species examined, phosphorus availability, intraspecific density, and their interaction significantly modify plant responses to arbuscular mycorrhizas.     

Drug recognition of a DNA single strand break: nogalamycin intercalation between coaxially stacked hairpins.

Two DNA hairpin motifs (5'-GCGAAGC-3' and 5'-ACGA AGT-3'), both stabilized by a 5'-GAA loop, have been used to design novel intramolecular double hairpin structures (5'-GCGAAGCACGAAGT-3' and 5'-ACGAAGTGCG AAGC-3') in which coaxial stacking of the two hairpin components generates a double-stranded stem region effectively with a single-strand break in the middle of the sequence at either the TG or CA step between unconnected 3' and 5' terminal bases. We have investigated by NMR the conformation and dynamics of the DNA at the strand break site. We show that mutual stacking significantly enhances the stability of each hairpin. Further, the anthracycline antibiotic nogalamycin binds cleanly to the 5'-TG (5'-CA) site formed by the mutually stacked hairpins despite the break in the sugar-phosphate backbone on one strand. The complex resembles the structure of nogalamycin-DNA complexes with the drug bound at 5'-TG sites in intact duplex sequences, with pi-stacking interactions probably the single dominant stabilizing interaction.     

Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme.

The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling of extracellular stimulation to nitric oxide production. Because eNOS is activated by Akt-dependent phosphorylation to produce nitric oxide (NO), we determined the subcellular distribution of eNOS phosphorylated on serine 1179 using a variety of methodologies. Based on sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection of eNOS with Akt and stimulation of endothelial cells with vascular endothelial growth factor (VEGF) increased the ratio of P-eNOS to total eNOS but did not change the relative intracellular distribution between these domains. The proper localization of eNOS to intracellular membranes was required for agonist-dependent phosphorylation on serine 1179, since VEGF did not increase eNOS phosphorylation in cells transfected with a non-acylated, mistargeted form of eNOS. Confocal imaging of P-eNOS and total eNOS pools demonstrated co-localization in the Golgi region and plasmalemma of transfected cells and native endothelial cells. Finally, VEGF stimulated a large increase in NO localized in both the perinuclear region and the plasma membrane of endothelial cells. Thus, activated, phosphorylated eNOS resides in two cellular compartments and both pools are VEGF-regulated to produce NO.     

Local and global cerebral blood flow and glucose utilization in the α-galactosidase A knockout mouse model of Fabry disease

Fabry disease is an X-linked lysosomal disorder characterized by deficient alpha-galactosidase A activity and intracellular accumulations of glycosphingolipids, mainly globotriaosylceramide (Gb3). Clinically, patients occasionally present CNS dysfunction. To examine the pathophysiology underlying brain dysfunction, we examined glucose utilization (CMR(glc)) and cerebral blood flow (CBF) globally and locally in 18 brain structures in the alpha-galactosidase A gene knockout mouse. Global CMR(glc) was statistically significantly reduced by 22% in Fabry mice (p < 0.01). All 18 structures showed decreases in local CMR(glc) ranging from 14% to 33%. The decreases in all structures of the diencephalon, caudate-putamen, brain stem, and cerebellar cortex were statistically significant (p < 0.05). Global cerebral blood flow (CBF) and local CBF measured in the same 18 structures were lower in Fabry mice than in control mice, but none statistically significantly. Histological examination of brain revealed no cerebral infarcts but abundant Gb3 deposits in the walls of the cerebral vessels with neuronal deposits localized to the medulla oblongata. These results indicate an impairment in cerebral energy metabolism in the Fabry mice, but one not necessarily due to circulatory insufficiency.