Liquid chromatography-mass spectrometric assay for quantitation of the short-chain fatty acid, 2,2-dimethylbutyrate (NSC 741804), in rat plasma

2,2-Dimethylbutyrate (DMB) is a potential treatment for thalassemia and hemoglobinopathies. To facilitate pharmacokinetic evaluation of DMB, we developed an LC-MS assay and quantitated DMB in plasma of rats after an oral dose of 500mg/kg. After acetonitrile protein precipitation, DMB and dimethylvaleric acid (DMV) internal standard were derivatized to benzylamides, chromatographed on a Hydro-RP column with acetonitrile, water, and 0.1% formic acid, and detected by electrospray positive-mode ionization mass spectrometry. The assay was accurate (97-107%) and precise (3.4-6.2%) between 100 and 10,000ng/mL. Recovery from plasma was >62%. Plasma freeze-thaw and room temperature stability were acceptable.     

Cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC(50) = 4.6 +/- 0.4 ng/ml) and crustaceans (Artemia nauplii LD(50) = 10 +/- 2 mug/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-A resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.     

The spinocerebellar ataxia 12 gene product and protein phosphatase 2A regulatory subunit Bbeta2 antagonizes neuronal survival by promoting mitochondrial fission

The neurodegenerative disorder spinocerebellar ataxia 12 (SCA12) is caused by CAG repeat expansion in the non-coding region of the PPP2R2B gene. PPP2R2B encodes Bbeta1 and Bbeta2, alternatively spliced and neuron-specific regulatory subunits of the protein phosphatase 2A (PP2A) holoenzyme. We show here that in PC12 cells and hippocampal neurons, cell stressors induced a rapid translocation of PP2A/Bbeta2 to mitochondria to promote apoptosis. Conversely, silencing of PP2A/Bbeta2 protected hippocampal neurons against free radical-mediated, excitotoxic, and ischemic insults. Evidence is accumulating that the mitochondrial fission/fusion equilibrium is an important determinant of cell survival. Accordingly, we found that Bbeta2 expression induces mitochondrial fragmentation, whereas Bbeta2 silencing or inhibition resulted in mitochondrial elongation. Based on epistasis experiments involving Bcl2 and core components of the mitochondrial fission machinery (Fis1 and dynamin-related protein 1), mitochondrial fragmentation occurs upstream of apoptosis and is both necessary and sufficient for hippocampal neuron death. Our data provide the first example of a proapoptotic phosphatase that predisposes to neuronal death by promoting mitochondrial division and point to a possible imbalance of the mitochondrial morphogenetic equilibrium in the pathogenesis of SCA12.     

Reproduction and development of Russian wheat aphid biotype 2 on crested wheatgrass, intermediate wheatgrass, and susceptible and resistant wheat

The Russian wheat aphid, Diuraphis noxia (Kurdjumov), is an economically important pest of small grains. Since its introduction into North America in 2003, Russian wheat aphid Biotype 2 has been found to be virulent to all commercially available winter wheat, Triticum aestivum L., cultivars. Our goal was to examine differences in Russian wheat aphid reproduction and development on a variety of plant hosts to gain information about 1) potential alternate host refuges, 2) selective host pressures on Russian wheat aphid genetic variation, and 3) general population dynamics of Russian wheat aphid Biotype 2. We studied host quality of two wheatgrasses (crested wheatgrass, Agropyron cristatum [L.] Gaertn., and intermediate wheatgrass, Agropyron intermedium [Host] Beauvoir) and two types of winter wheat (T. aestivum, one Biotype 2 susceptible wheat, 'Custer' and one biotype 2 resistant wheat, STARS02RWA2414-11). The susceptible wheat had the highest intrinsic rate of increase, greatest longevity and greatest fecundity of the four host studied. Crested wheatgrass and the resistant wheat showed similar growth rates. Intermediate wheatgrass had the lowest intrinsic rate of increase and lowest fecundity of all tested hosts.     

Swelling of articular cartilage depends on the integrity of adjacent cartilage and bone

Articular cartilage swells when its collagen network is degraded, both in osteoarthritis (OA) and following mechanical trauma. However, most of the experimental evidence actually shows that it is small excised samples of cartilage that swell, implying that the cartilage was not greatly swollen in-situ before it was excised. We hypothesise that degraded cartilage can be prevented from swelling in-situ by restraint from adjacent normal cartilage and subchondral bone. Four adjacent osteochondral specimens, 20 x 20 mm, were obtained from regions of the humeral heads of each of 11 skeletally-mature cows. The central region of each specimen was injured by compressive loading using a 9 mm-diameter flat metal indenter, and cartilage surface damage was confirmed using Indian ink. Damaged cartilage was allowed to swell in physiological saline for 1 h under one of four conditions of restraint: (A) normal in-situ restraint from subchondral bone and surrounding cartilage, (B) restraint from bone only, (C) restraint from cartilage only, (D) no restraint (excised specimen). Cartilage hydration was assessed by freeze-drying to constant weight. Proteoglycan loss from damaged cartilage was quantified by analyzing the GAG content of the surrounding bath using the DMB assay. Hydration of damaged cartilage after swelling depended on restraint (p < 0.001), averaging: (A) 76.8%, (B) 78.2%, (C) 78.0%, (D) 81.3%. GAG loss following cartilage surface damage was insufficient to explain observed differences in hydration. The 6% increase in hydration between (A) and (D) can be attributed to swelling which is prohibited when the cartilage remains in-situ. Swelling of degraded cartilage can be largely prevented if it remains in-situ, supported by adjacent healthy bone and cartilage. Adverse physico-chemical consequences of cartilage degradation and swelling may become apparent only when this support is diminished, either because the affected region is large, or following deterioration of adjacent bone or cartilage.     

Retinoic acid-induced alveolar cellular growth does not improve function after right pneumonectomy.

To determine whether all-trans retinoic acid (RA) treatment enhances lung function during compensatory lung growth in fully mature animals, adult male dogs (n = 4) received 2 mg x kg(-1) x day(-1) po RA 4 days/wk beginning the day after right pneumonectomy (R-PNX, 55-58% resection). Litter-matched male R-PNX controls (n = 4) received placebo. After 3 mo, transpulmonary pressure (TPP)-lung volume relationship, diffusing capacities for carbon monoxide and nitric oxide, cardiac output, and septal volume (V(tiss-RB)) were measured under anesthesia by a rebreathing technique at two lung volumes. Lung air and tissue volumes (V(air-CT) and V(tiss-CT)) were also measured from high-resolution computerized tomographic (CT) scans at a constant TPP. In RA-treated dogs compared with controls, TPP-lung volume relationships were similar. Diffusing capacities for carbon monoxide and nitric oxide were significantly impaired at a lower lung volume but similar at a high lung volume. Whereas V(tiss-RB) was significantly lower at both lung volumes in RA-treated animals, V(air-CT) and V(tiss-CT) were not different between groups; results suggest uneven distribution of ventilation consistent with distortion of alveolar geometry and/or altered small airway function induced by RA. We conclude that RA does not improve resting pulmonary function during the early months after R-PNX despite histological evidence of its action in enhancing alveolar cellular growth in the remaining lung.     

An exchange of botanical information in the early contact situation: Wisakon of the Southeastern Algonquians

The termwisakon, recorded among Southeastern Algonquian Indians in the seventeenth and eighteenth centuries, frequently has been iden tified as one of several plant species. However, it appears thatwisakon referred not to any particular plant species but to a general category of substances that included both plant and nonplant materials. The misunderstanding illustrates some of the problems and procedures involved in the exchange and integration of botanical information by Europeans and Indians in the early culture contact situation.     

Effects of exogenous fatty acids on calcium uptake by brush-border membrane vesicles from rabbit small intestine

The uptake of a variety of fatty acids by isolated brush-border membranes from rabbit small intestine was studied. This uptake increased with acyl chain-length and was not diminished by washing of the lipid-treated membranes with 0.25 M CsBr. The binding of fatty acid was not accompanied by a decrease in endogenous acyl groups or of cholesterol and therefore corresponded to a net uptake accountable qualitatively and quantitatively by the fatty acid added to the membranes. The uptake of Ca2+ was stimulated by treatment of the membranes with low concentrations of unsaturated fatty acids (0.05 mM) as well as with various concentrations of caprylic acid (0.10-3.00 mM) and inhibited by treatment with higher concentrations of unsaturated fatty acids (0.20-0.60 mM). Saturated fatty acids had no marked effects on Ca2+ uptake. The stimulatory concentrations of unsaturated fatty acids did not change the Ca2+-binding characteristics of the membranes, whereas the higher concentrations decreased equilibrium binding of Ca2+ and very probably the number of high-affinity binding sites. The results of this study are assessed in terms of the effects of normal fatty acids found in the diet on the absorptive properties of the brush-border membranes.