Development of fertile fruit bodies in a single-strain culture ofNeurospora crassa

Fertile fruit-bodies (perithecia) were formed in the single-strain culture (EmA) ofNeurospora crassa in the presence of the sex and fertility-inducing substances isolated from the cross EmA x Ema. Analysis of the progeny showed high mortality and an abnormal segregation ratio.     

Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways

Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5′-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5′-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations.     

<Emphasis Type="Italic">Vibrio cholerae</Emphasis> in waters of the Sunderban mangrove: relationship with biogeochemical parameters and chitin in seston size fractions

Wetland dynamics are probably linked to cholera endemicity in South Asia. We focus on links between Vibrio cholerae abundance, chitin content and suspended particle load in size fractions of suspended particulate matter (SPM) along the salinity gradient of Sunderban mangrove waters. SPM decreased downstream, while salinity increased from 0.2 to 4. Particulate organic carbon (90 ± 25 μM) and nitrogen (9.1 ± 3.3 μM) highly correlated with SPM and turbidity, suggesting a significant contribution of fine particles to organic matter. Total chitin ranged 1–2 mg/l and decreased downstream. The distribution among size fractions of SPM, chitin and V. cholerae O1 (the bacterial serogroup mainly associated with cholera epidemics) was similar, with ~98% of the total in the fraction <20 μm. In comparison, the number of V. cholerae O1 attached to zooplankton and microplankton size classes >20 μm was almost negligible, in contrast to usual assumptions. Thus, microdetritus, nanoplankton and fungal cells in size classes <20 μm represent a chitinaceous substrate on which V. cholerae can grow and survive. Total bacteria, cultivable vibrios and V. cholera O1 increased 5–10 times downstream, together with salinity and nitrite concentration. Overall, nitrate and silicate concentrations were relatively constant (>22 μM N and 100 μM Si). However, nitrite increased ~9 times in the outer sector, reaching ~1.2 μM N, probably as a result of increased abundance of nitrate-reducing vibrios. A characterization of Vibrio habitats that takes account of the presence of nitrate-reducing bacteria could improve the understanding of both mangrove nitrogen cycling and cholera seasonality.     

A new lectin from the tuberous rhizome of Kaempferia rotunda: isolation, characterization, antibacterial and antiproliferative activities

A lectin (designated as KRL) was purified from the extracts of Kaempferia rotunda Linn. tuberous rhizome by glucose-sepharose affinity chromatography. KRL was determined to be a 29.0 ± 1.0 kDa polypeptide by SDS-PAGE under both reducing and non-reducing conditions. KRL was a divalent ion dependent glycoprotein with 4% neutral sugar which agglutinated different groups of human blood cells. Methyl-α-D-mannopyranoside, D-mannose and methyl-α-D-glucopyranoside were the most potent inhibitors. N-terminal sequence of KRL showed similarity to some mannose/ glucose specific lectins but the main differences with their molecular masses and sugar content. KRL lost its activity markedly in the presence of denaturants and exhibited high agglutination activity from pH 6.0 to 8.2 and temperature 30 to 60° C. The lectin showed toxicity against brine shrimp nauplii with the LC50 value of 18 ± 6 μg/ml and strong agglutination activity against seven pathogenic bacteria. KRL inhibited the growth of six bacteria partially and did not show antifungal activity. In addition, antiproliferative activity against Ehrlich ascites carcinoma (EAC) cells showed 51% and 67% inhibition in vivo in mice administered 1.25 mg/kg/day and 2.5 mg/kg/day of KRL respectively by injection for five days.     

Facile synthesis and stereochemical investigation of Mannich base derivatives: evaluation of antioxidant property and antituberculostic potency

A mini-library of diversely substituted 2,4-diaryl-3-azabicyco[3.3.1]nonan-9-one O-methyloximes and their N-methyl analogs were synthesized by a non-laborious, modified and an optimized Mannich condensation in good yields. Both the ring N-methylation and oxime O-methylation were employed by various methods; of them, the usage of ^tBuOK was found to be the superior in terms of good yield in short time. Stereochemistry of all the synthesized compounds was unambiguously established by their NMR spectral (¹H, ¹³C, ¹H-¹H COSY, ¹H-¹³C one and multiple bond COSY and NOESY) as well as single-crystal XRD studies. Irrespective of the nature and position of the substituents, all the synthesized oxime ethers of the bicyclic Mannich bases as well as their N-methyl analogs adopted the twin-chair conformation with equatorial orientations of all the substituents. All the synthesized oxime ethers were evaluated for their antioxidant property by DPPH radical scavenging method. According to the structure-activity correlations, compound 4y was found to be a lead molecule with the IC₅₀ of 0.187 mg/mL. Thus, the present study exploits the scope of finding more active analogs by further optimization with the incorporation of more electron enriched alkoxy/amino and/or phenolic groups on the heterocycle as well as oxime ether pharmacophore. Most of the synthesized molecules were screened for their antituberculostic potency against Mycobacterium tuberculosis H₃₇Rv by zone of inhibition method. Of them, 4w/5d and 4x showed very promising inhibition zones of 21 and 23 mm, respectively, which leads to the optimization of 4x by introducing various substituents on the O-benzyl moiety to enhance the antituberculostic potency.     

2,4-Diacetylphloroglucinol suppresses zoosporogenesis and impairs motility of Peronosporomycete zoospores

2,4-Diacetylphloroglucinol (DAPG) produced by Pseudomonas fluorescens, shows toxicity to many microorganisms including fungi, bacteria, and peronosporomycetes. Zoosporogenesis and motility of zoospores are critical for a complete disease cycle and pathogenicity of the peronosporomycete phytopathogens. The aim of this study was to test the effects of DAPG and its derivatives on zoosporogenesis and motility of zoospores of a downy mildew pathogen, Plasmopara viticola, and a damping-off pathogen, Aphanomyces cochlioides. In both cases, DAPG inhibited zoosporogenesis (5 μg/ml) and the motility of zoospores (10 μg/ml) in a dose-dependent manner. Generally, zoospores became immotile shortly after exposure to DAPG followed by lysis. However, a fraction of DAPG treated A. cochlioides zoospores formed round cystospores instead of lysis and then germinated with excessively-branched germ tubes. All derivatives of DAPG had similar inhibitory activities but at varying doses. Among them, 2,4-dipropylphloroglucinol exerted the highest inhibitory activity against both zoosporogenesis and motility of zoospores. This revealed that the degree of hydrogen atoms substitution in the benzene ring by acyl groups and the length of substituted acyl groups were related to the level of bioactivity. This is the first report of inhibitory activities of DAPG and its derivatives against zoosporogenesis and motility of zoospores of two important peronosporomycete phytopathogens.     

What Point-of-Use Water Treatment Products Do Consumers Use? Evidence from a Randomized Controlled Trial among the Urban Poor in Bangladesh

Background

There is evidence that household point-of-use (POU) water treatment products can reduce the enormous burden of water-borne illness. Nevertheless, adoption among the global poor is very low, and little evidence exists on why.

Methods

We gave 600 households in poor communities in Dhaka, Bangladesh randomly-ordered two-month free trials of four water treatment products: dilute liquid chlorine (sodium hypochlorite solution, marketed locally as Water Guard), sodium dichloroisocyanurate tablets (branded as Aquatabs), a combined flocculant-disinfectant powdered mixture (the PUR Purifier of Water), and a silver-coated ceramic siphon filter. Consumers also received education on the dangers of untreated drinking water. We measured which products consumers used with self-reports, observation (for the filter), and chlorine tests (for the other products). We also measured drinking water''s contamination with E. coli (compared to 200 control households).

Findings

Households reported highest usage of the filter, although no product had even 30% usage. E. coli concentrations in stored drinking water were generally lowest when households had Water Guard. Households that self-reported product usage had large reductions in E. coli concentrations with any product as compared to controls.

Conclusion

Traditional arguments for the low adoption of POU products focus on affordability, consumers'' lack of information about germs and the dangers of unsafe water, and specific products not meshing with a household''s preferences. In this study we provided free trials, repeated informational messages explaining the dangers of untreated water, and a variety of product designs. The low usage of all products despite such efforts makes clear that important barriers exist beyond cost, information, and variation among these four product designs. Without a better understanding of the choices and aspirations of the target end-users, household-based water treatment is unlikely to reduce morbidity and mortality substantially in urban Bangladesh and similar populations.     

Co-regulation of histone-modifying enzymes in cancer

Cancer is characterized by aberrant patterns of expression of multiple genes. These major shifts in gene expression are believed to be due to not only genetic but also epigenetic changes. The epigenetic changes are communicated through chemical modifications, including histone modifications. However, it is unclear whether the binding of histone-modifying proteins to genomic regions and the placing of histone modifications efficiently discriminates corresponding genes from the rest of the genes in the human genome. We performed gene expression analysis of histone demethylases (HDMs) and histone methyltransferases (HMTs), their target genes and genes with relevant histone modifications in normal and tumor tissues. Surprisingly, this analysis revealed the existence of correlations in the expression levels of different HDMs and HMTs. The observed HDM/HMT gene expression signature was specific to particular normal and cancer cell types and highly correlated with target gene expression and the expression of genes with histone modifications. Notably, we observed that trimethylation at lysine 4 and lysine 27 separated preferentially expressed and underexpressed genes, which was strikingly different in cancer cells compared to normal cells. We conclude that changes in coordinated regulation of enzymes executing histone modifications may underlie global epigenetic changes occurring in cancer.     

Global to community scale differences in the prevalence of convergent over divergent leaf trait distributions in plant assemblages

Aim The drivers of species assembly, by limiting the possible range of functional trait values, can lead to either convergent or divergent distributions of traits in realized assemblages. Here, to evaluate the strengths of these species assembly drivers, we partition trait variance across global, regional and community scales. We then test the hypothesis that, from global to community scales, the outcome of co‐occurring trait convergence and divergence is highly variable across biomes and communities. Location Global: nine biomes ranging from subarctic highland to tropical rain forest. Methods We analysed functional trait diversity at progressively finer spatial scales using a global, balanced, hierarchically structured dataset from 9 biomes, 58 communities and 652 species. Analyses were based on two key leaf traits (foliar nitrogen content and specific leaf area) that are known to drive biogeochemical cycling. Results While 35% of the global variance in these traits was between biomes, only 15% was between communities within biomes and as much as 50% occurred within communities. Despite this relatively high within‐community variance in trait values, we found that trait convergence dominated over divergence at both global and regional scales through comparisons of functional trait diversity in regional and community assemblages against random (null) models of species assembly. Main conclusions We demonstrate that the convergence of traits occurring from global to regional assemblages can be twice as strong as that from regional to community assemblages, and argue that large differences in the nature and strength of abiotic and biotic drivers of dominant species assembly can, at least partly, explain the variable outcome of simultaneous trait convergence and divergence across sites. Ultimately, these findings stress the urgent need to extend species assembly research to address those scales where trait variance is the highest, i.e. between biomes and within communities.     

Single wall carbon nanotubes enter cells by endocytosis and not membrane penetration

Background

Quantitative analysis of nanoparticle uptake at the cellular level is critical to nanomedicine procedures. In particular, it is required for a realistic evaluation of their effects. Unfortunately, quantitative measurements of nanoparticle uptake still pose a formidable technical challenge. We present here a method to tackle this problem and analyze the number of metal nanoparticles present in different types of cells. The method relies on high-lateral-resolution (better than 30 nm) transmission x-ray microimages with both absorption contrast and phase contrast -- including two-dimensional (2D) projection images and three-dimensional (3D) tomographic reconstructions that directly show the nanoparticles.

Results

Practical tests were successfully conducted on bare and polyethylene glycol (PEG) coated gold nanoparticles obtained by x-ray irradiation. Using two different cell lines, EMT and HeLa, we obtained the number of nanoparticle clusters uptaken by each cell and the cluster size. Furthermore, the analysis revealed interesting differences between 2D and 3D cultured cells as well as between 2D and 3D data for the same 3D specimen.

Conclusions

We demonstrated the feasibility and effectiveness of our method, proving that it is accurate enough to measure the nanoparticle uptake differences between cells as well as the sizes of the formed nanoparticle clusters. The differences between 2D and 3D cultures and 2D and 3D images stress the importance of the 3D analysis which is made possible by our approach.