Structural analysis of dextrans containing 4-O-α-d-glucosylated α-d-glucopyranosyl residues at the branch points,by use of 13c-nuclear magnetic resonance spectroscopy and gas-liquid chromatography-mass spectrometry

Dextran fractions from NRRL strain Streptococcus sp. B-1526 and the native, structurally homogeneous dextrans from Acetobacter capsulatum B-1225, Leuconostoc mesenteroides B-1307, and L. dextranicum B-1420 were examined by ¹³C-n.m.r. spectroscopy at 90°. Dextran B-1526 fraction I and dextran B-1420 were also examined by g.l.c:-m.s., methylation-structural analysis. All of these dextrans and dextran fractions branch, either primarily or exclusively, through α-d-(1→4)-glucopyranosyl linkages; however, their degrees of branching differ. Several ¹³C-n.m.r. resonances that are diagnostic for 4,6-di-O-substituted α-d-glucopyranosyl residues have been identified. Comparison was made with dextrans from L. mesenteroides B-742 fraction L and Streptobacterium dextranicum B-1254 fraction S[L], for which previously published, methylation-structural analyses had established the presence of 4,6-di-O-substituted α-d-glucopyranosyl residues at the branch points. These fermentation culture, and in a sedimented gum-phase (fraction I). The product from the soluble phase is designated here as fraction S in order to simplify the terminology. Originally⁷, this product was not designated a fraction, because it was, by definition⁸, the main dextran product. The same distinction also applies to the pairs of products from strains B-1380, B-1420, and b-1394 (see ref. 7). The attempts thus made to establish the significance of the phase separation were indeterminant.Methods.— Methods previously described were used for the mythylation⁹ of the dextrans and for structural analysis^6.38 by combined g.l.c-electron-impact mass spectrometry of the aldononitriles. For each permethylation, three successive Hakomori³⁹ methylations were employed on an initial, 40-mg sample, with ～80% (final weight) recovery of each permethylated dextran. Successive formolysis and acetic acid hydrolysis were employed, and, after each step, the resulting solutions were clear, colorless, and free from suspended material. All mass spectra were recorded with a Hewlett-Packard 5980A GC/MS integrated g.l.c.-m.s.-computer system. The g.l.c. peak-integrals reported in Table II were obtained with a Barber-Coleman Series 5000 g.l.c. instrument equipped with hydrogen-flame detectors. On-column injection with glass columns (2mmi.d. x 1.23m) was employed for all chromatograhy.The ¹³C-n.m.r. conditions and the methods for the preparation of dextran samples have been described⁴. In general, a Varian XL-100-15 spectrometer equipped with a Nicolet TT-100 system was employed in the Fourier-transform mode. The dextran samples, ～0.3g/4 mL of deuterium oxide, were maintained at 90°. Chemical shifts are expressed in p.p.m. relative to external tetramethylsilane, but were actually calculated by reference to the solvent lock-signal. The convolution-difference resolution-enhancement (c.d.r.e.) technique has been described⁴⁰.     

Donut-shaped “miniparticles” in nuclei of human and rat cells

Donut-shaped “miniparticles” were extracted from nuclei of various types of human and rat cells. Electron-microscopic investigations showed these particles were predominantly in sucrose density gradient fractions that had an approximate sedimentation coefficient of 21S. These particles were 113±8_A^o in diameter and had an electron dense center of 29±6_A^o. They appeared to be composed of 8 subunits. Quantitative analysis of the number of these particles by electron-micrographic field counting showed nuclei of tumor samples had a larger amount of the particles than the cytosol. However, normal cell cytosol had a larger number of particles than the nuclei. A group of proteins in the 25, 000–33, 000 molecular weight range was shown to be the main protein component by two dimensional gel electrophoresis.     

Hemagglutination Inhibition of Passively Sensitized Red Blood Cells

It has been shown that sheep red blood cells sensitized with the polysaccharides of Haemophilus influenzae type B and pneumococcal types I, III, IV, VII, VIII, XII, and XIV respond readily in hemagglutination-inhibition testing and exhibit antigen inhibition at levels of 0.09 to 4.0 ng.     

Systematic implications of sesquiterpene lactones in the subtribe melampodiinae

Within the tribe Heliantheae of the Asteraceae, the genetic boundaries of the subtribe Melampodinae have recently been drastically revised by Stuessy. The number of genera within the subtribe has been reduced and new generic groupings have been established. The present study correlates the distribution of sesquiterpene lactones found in these genera with the newly revised subtribal boundaries. The genera Acanthospermum, Melampodium, Polymnia and Sigesbeckia produce predominantly melampolide-type sequiterpene lactones. Limited chemical data support Stuessy's removal of the genera Desmanthodium, Clibadium and Ichthyothere from the subtribe Melampodiinae. The occurrence of melampolide-type sesquiterpene lactones in members of the genera Tetragonotheca (Helianthinae) and Enhydra (Ecliptinae) indicate a possible position of these genera in the Melampodiinae.     

Cockroaches that lack Blattabacterium endosymbionts: the phylogenetically divergent genus Nocticola

Phylogenetic relationships among termites, mantids and the five traditionally recognized cockroach families have been the subject of several studies during the last half-century. One cockroach lineage that has remained notably absent from such studies is the Nocticolidae. This group of small, elusive surface- and cave-dwelling species from the Old World Tropics has been proposed to represent an additional family. Using molecular sequences, we performed an initial phylogenetic examination of Nocticola spp. The hypothesis that they are phylogenetically divergent was confirmed from the analyses of three genes and a combined dataset. To supplement our phylogenetic analyses, we attempted to amplify 16S rRNA from the obligate mutualistic endosymbiont Blattabacterium cuenoti, present in all cockroaches studied to date. Unexpectedly, amplification was unsuccessful in all Nocticola spp. examined. This result was confirmed by microscopic examinations of fat body tissue. These Nocticola spp. are the first cockroaches found to be uninfected by B. cuenoti, which raise questions about when the bacterium first infected cockroaches.     

The effect of Tookad-mediated photodynamic ablation of the prostate gland on adjacent tissues--in vivo study in a canine model.

Photodynamic therapy (PDT) mediated with vascular acting photosensitizer Tookad (Pd-bacteriopheophorbide) was investigated as an alternative modality for treating prostate cancer. Photodynamic effects on the prostate gland and its adjacent tissues were evaluated in a canine model. Interstitial prostate PDT was performed by irradiating individual lobes with a cylindrical diffuser fiber at various drug/light doses. The sensitivity of the adjacent tissues to Tookad PDT was determined by directly irradiating the surface of the bladder, colon, abdominal muscle and pelvic plexus with a microlens fiber at various drug/light doses. The prostate and adjacent tissues were harvested one-week after the treatment and subjected to histopathological examination. PDT-induced prostate lesions were characterized by marked hemorrhagic necrosis. The bladder, colon, abdominal muscle and pelvic plexus appeared to be sensitive to PDT although the Tookad PDT-induced responses in these tissues were minimal compared to that of the prostate gland at the same dose levels. Nevertheless, the protection of the adjacent tissues should be taken into consideration during the total prostate ablation process due to their sensitivity to PDT. The sensitivity of the prostatic urethra is worth further investigation. Direct intraurethral irradiation might provide an ideal means to determine the sensitivity of the prostatic urethra and might lead to transurethral PDT protocols for the management of benign prostatic hyperplasia (BHP).     

Simulating association studies: a data-based resampling method for candidate regions or whole genome scans

MOTIVATION: Reductions in genotyping costs have heightened interest in performing whole genome association scans and in the fine mapping of candidate regions. Improvements in study design and analytic techniques will require the simulation of datasets with realistic patterns of linkage disequilibrium and allele frequencies for typed SNPs. METHODS: We describe a general approach to simulate genotyped datasets for standard case-control or affected child trio data, by resampling from existing phased datasets. The approach allows for considerable flexibility in disease models, potentially involving a large number of interacting loci. The method is most applicable for diseases caused by common variants that have not been under strong selection, a class specifically targeted by the International HapMap project. RESULTS: Using the three population Phase I/II HapMap data as a testbed for our approach, we have implemented the approach in HAP-SAMPLE, a web-based simulation tool.     

Endotoxin priming of neutrophils requires endocytosis and NADPH oxidase-dependent endosomal reactive oxygen species

NADPH oxidase 2 (Nox2)-generated reactive oxygen species (ROS) are critical for neutrophil (polymorphonuclear leukocyte (PMN)) microbicidal function. Nox2 also plays a role in intracellular signaling, but the site of oxidase assembly is unknown. It has been proposed to occur on secondary granules. We previously demonstrated that intracellular NADPH oxidase-derived ROS production is required for endotoxin priming. We hypothesized that endotoxin drives Nox2 assembly on endosomes. Endotoxin induced ROS generation within an endosomal compartment as quantified by flow cytometry (dihydrorhodamine 123 and Oxyburst Green). Inhibition of endocytosis by the dynamin-II inhibitor Dynasore blocked endocytosis of dextran, intracellular generation of ROS, and priming of PMN by endotoxin. Confocal microscopy demonstrated a ROS-containing endosomal compartment that co-labeled with gp91(phox), p40(phox), p67(phox), and Rab5, but not with the secondary granule marker CD66b. To further characterize this compartment, PMNs were fractionated by nitrogen cavitation and differential centrifugation, followed by free flow electrophoresis. Specific subfractions made superoxide in the presence of NADPH by cell-free assay (cytochrome c). Subfraction content of membrane and cytosolic subunits of Nox2 correlated with ROS production. Following priming, there was a shift in the light membrane subfractions where ROS production was highest. CD66b was not mobilized from the secondary granule compartment. These data demonstrate a novel, nonphagosomal intracellular site for Nox2 assembly. This compartment is endocytic in origin and is required for PMN priming by endotoxin.