Structure–function relationships of two paralogous single-stranded DNA-binding proteins from Streptomyces coelicolor: implication of SsbB in chromosome segregation during sporulation

The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, ∼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal medium, gene expression was prolonged for both genes and significantly upregulated for ssbB. The ssbA gene is transcribed as part of a polycistronic mRNA from two initiation sites, 163 bp and 75 bp upstream of the rpsF translational start codon. The ssbB gene is transcribed as a monocistronic mRNA, from an unusual promoter region, 73 bp upstream of the AUG codon. Distinctive DNA-binding affinities of single-stranded DNA-binding proteins monitored by tryptophan fluorescent quenching and electrophoretic mobility shift were observed. The crystal structure of SsbB at 1.7 Å resolution revealed a common OB-fold, lack of the clamp-like structure conserved in SsbA and previously unpublished S-S bridges between the A/B and C/D subunits. This is the first report of the determination of paralogous single-stranded DNA-binding protein structures from the same organism. Phylogenetic analysis revealed frequent duplication of ssb genes in Actinobacteria, whereas their strong retention suggests that they are involved in important cellular functions.     

Infection process of Colletotrichum dematium on mulberry leaves: An unusual method of sporulation

Abstract

The pre-penetration and infection process of Colletotrichum dematium on mulberry leaf was investigated by scanning electron microscope. Conidia produced on germination appressoria directly or at the end of short germ tubes. Appressoria were formed mostly over cuticle, but sometimes over stomata also. At 72 h post-inoculation, an extensive network of sub-cuticular runner hyphae (RH) was produced. The RH were traceable by the cuticular bulgings on leaf surface. The RH emerged to leaf surface through ruptured cuticle to form secondary infection hyphae (SIH). The SIH re-entered the leaf tissue by sending penetration branches through stomata. Conidia were formed singly on short conidiophores from the RH and SIH, at short intervals. The conidia developed on RH were exposed to leaf surface through ruptured cuticle. Some times conidia were released through stomata also. The RH and SIH had thick knots from which hyphal branches and conidia were developed. Definite acervuli were not developed.     

Nonsteroidal Anti-inflammatory Drugs Diclofenac and Celecoxib Attenuates Wnt/β-Catenin/Tcf Signaling Pathway in Human Glioblastoma Cells

Glioblastoma, the most common and aggressive primary brain tumors, carry a bleak prognosis and often recur even after standard treatment modalities. Emerging evidence suggests that deregulation of the Wnt/β-catenin/Tcf signaling pathway contributes to glioblastoma progression. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit tumor cell proliferation by suppressing Wnt/β-catenin/Tcf signaling in various human malignancies. In this study, we sought to inhibit Wnt/β-catenin/Tcf signaling in glioblastoma cells by the NSAIDs diclofenac and celecoxib. Both diclofenac and celecoxib significantly reduced the proliferation, colony formation and migration of human glioblastoma cells. Diclofenac and celecoxib downregulated β-catenin/Tcf reporter activity. Western and qRT-PCR analysis showed that diclofenac and celecoxib reduced the expression of β-catenin target genes Axin2, cyclin D1 and c-Myc. In addition, the cytoplasmic accumulation and nuclear translocation of β-catenin was significantly reduced following diclofenac and celecoxib treatment. Furthermore, diclofenac and celecoxib significantly increased phosphorylation of β-catenin and reduced the phosphorylation of GSK3β. These results clearly indicated that diclofenac and celecoxib are potential therapeutic agents against glioblastoma cells that act by suppressing the activation of Wnt/β-catenin/Tcf signaling.     

Parthenogenetic development of dwarf surf dam,Mulinia lateralis,oocytes treated with polar body suppressing agents

Summary

Parthenogenesis following oocyte activation has been observed in a number of marine invertebrates, but the fate of parthenogenesis in bivalve mollusc embryos is unclear. We used the dwarf surf clam, Mulinia lateralis, to examine parthenogenetic development of KC1-activated oocytes using the polar body suppressing agents caffeine and heat or cytochalasin B. Development was followed by epifluorescence microscopy and flow-cytometric analysis using the DNA-specific fluorochrome DAPI. All agents suppressed polar body formation to some degree, putatively increasing the ploidy level and retaining a meiotic centrosome in the zygote; but the zygotes failed to develop normally. Failure of the zygotes to develop suggests that the meiotic centrosome is incapable of participating in mitosis in bivalves.     

Preparation and characterization of mucilage polysaccharide for biomedical applications

In the present investigation, the polysaccharide/mucilage from waste of Abelmoscus esculentus by modification in hot extraction using two different solvents (Acetone, Methanol) were extracted, characterized and further compared with seaweed polysaccharide for their potential applications. The percentage yield, emulsifying capacity and swelling index of this mucilage were determined. The macro algae and okra waste, gave high % yield (22.2% and 8.6% respectively) and good emulsifying capacity (EC% = 52.38% and 54.76% respectively) with acetone, compared to methanol (11.3% and 0.28%; EC% = 50%) (PH = 7) while swelling index was greater with methanol than acetone extracts respectively. The infrared (I.R.) spectrum of the samples was recorded to investigate the chemical structure of mucilage. Thermal analysis of the mucilage was done with TGA (Thermal Gravimetric Analyzer) and DSC (Differential Scanning Calorimeter) which showed both okra and algal polysaccharide were thermostable hydrogels.     

β-Glucosidase 2 (GBA2) Activity and Imino Sugar Pharmacology

β-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher K_I) and a lower rate of enzyme inactivation (k_inact). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the β-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5–6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated β-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.     

Gravel dredging alters diversity and structure of riverine fish assemblages

1. Human activities affect fish assemblages in a variety of ways. Large‐scale and long‐term disturbances such as in‐stream dredging and mining alter habitat and hydrodynamic characteristics within rivers which can, in turn, alter fish distribution. Habitat heterogeneity is decreased as the natural riffle–pool–run sequences are lost to continuous pools and, as a consequence, lotic species are displaced by lentic species, while generalist and invasive species displace native habitat specialists. Sediment and organic detritus accumulate in deep, dredged reaches and behind dams, disrupting nutrient flow and destroying critical habitat for habitat specialist species. 2. We used standard ecological metrics such as species richness and diversity, as well as stable isotope analysis of δ¹³C and δ¹⁵N, to quantify the differences in fish assemblages sampled by benthic trawls among dredged and undredged sites in the Allegheny River, Pennsylvania, U.S.A. 3. Using mixed‐effects models, we found that total catch, species richness and diversity were negatively correlated with depth (P < 0.05), while species richness, diversity and proportion of species in lithophilic (‘rock‐loving’) reproductive guilds were lower at dredged than at undredged sites (P < 0.05). 4. Principal components analysis and manova revealed that taxa such as darters in brood hider and substratum chooser reproductive guilds were predominantly associated with undredged sites along principal component axis 1 (PC1 and manova P < 0.05), while nest spawners such as catfish and open substratum spawners including suckers were more associated with dredged sites along PC2 (P < 0.05). 5. Stable isotope analysis of δ¹³C and δ¹⁵N revealed shifts from reliance on shallow water and benthic‐derived nutrients at undredged sites to reliance on phytoplankton and terrestrial detritus at deep‐water dredged sites. Relative trophic positions were also lower at dredged sites for many species; loss of benthic nutrient pathways associated with depth and dredging history is hypothesised. 6. The combination of ecological metrics and stable isotope analysis thus shows how anthropogenic habitat loss caused by gravel dredging can decrease benthic fish abundance and diversity, and that species in substratum‐specific reproductive guilds are at particular risk. The effects of dredging also manifest by altering resource use and nutrient pathways within food webs. Management and conservation decisions should therefore consider the protection of relatively shallow areas with suitable substratum for spawning for the protection of native fishes.     

The field-dependence of the solid-state photo-CIDNP effect in two states of heliobacterial reaction centers

The solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect is studied in photosynthetic reaction centers of Heliobacillus mobilis at different magnetic fields by ¹³C MAS (magic-angle spinning) NMR spectroscopy. Two active states of heliobacterial reaction centers are probed: an anaerobic preparation of heliochromatophores (“Braunstoff”, German for “brown substance”) as well as a preparation of cells after exposure to oxygen (“Grünstoff”, “green substance”). Braunstoff shows significant increase of enhanced absorptive (positive) signals toward lower magnetic fields, which is interpreted in terms of an enhanced differential relaxation (DR) mechanism. In Grünstoff, the signals remain emissive (negative) at two fields, confirming that the influence of the DR mechanism is comparably low.     

Synthetic Lethal Targeting of Superoxide Dismutase 1 Selectively Kills RAD54B-Deficient Colorectal Cancer Cells

Synthetic lethality is a rational approach to identify candidate drug targets for selective killing of cancer cells harboring somatic mutations that cause chromosome instability (CIN). To identify a set of the most highly connected synthetic lethal partner genes in yeast for subsequent testing in mammalian cells, we used the entire set of 692 yeast CIN genes to query the genome-wide synthetic lethal datasets. Hierarchical clustering revealed a highly connected set of synthetic lethal partners of yeast genes whose human orthologs are somatically mutated in colorectal cancer. Testing of a small matrix of synthetic lethal gene pairs in mammalian cells suggested that members of a pathway that remove reactive oxygen species that cause DNA damage would be excellent candidates for further testing. We show that the synthetic lethal interaction between budding yeast rad54 and sod1 is conserved within a human colorectal cancer context. Specifically, we demonstrate RAD54B-deficient cells are selectively killed relative to controls via siRNA-based silencing and chemical inhibition and further demonstrate that this interaction is conserved in an unrelated cell type. We further show that the DNA double strand breaks, resulting from increased reactive oxygen species following SOD1 inhibition, persist within the RAD54B-deficient cells and result in apoptosis. Collectively, these data identify SOD1 as a novel candidate cancer drug target and suggest that SOD1 inhibition may have broad-spectrum applicability in a variety of tumor types exhibiting RAD54B deficiencies.     

Evolvosides C–E,flavonol-4-O-triglycosides from Evolvulus alsinoides and their anti-stress activity

Phytochemical investigation of the n-butanol fraction of Evolvulus alsinoides (Linn.) led to the isolation of three new phenolic glycosides, evolvosides C, D and E (1–3) along with six known compounds (4–9). The structures of the compounds were elucidated on the basis of spectroscopic analysis, viz. 1D and 2D NMR experiments, chemical study, and comparison with literature data. Evolvoside C (1) was characterized as kaempferol 4′-O-β-d-glucopyranosyl-(1→2)-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside, whereas evolvosides D and E (2–3) were found to be mono and di-O-methyl derivatives of 1. The new compounds (1–3) represent rare triglycoside derivatives of flavonol at C-4′. The isolated compounds (1–6) were screened for acute stress-induced biochemical changes in male Sprague–Dawley rats at a dose of 40 mg/kg body weight. Compounds 1 and 2 displayed anti-stress effects by normalizing hyperglycemia, plasma corticosterone, plasma creatine kinase, and adrenal hypertrophy. Compounds 3 and 6 were also found to be effective in normalizing most of these stress parameters, whereas compounds 4 and 5 were ineffective in normalizing most of these effects.