Zinc regulates the activity of kinase-phosphatase pair (BasPrkC/BasPrpC) in Bacillus anthracis

Bacillus anthracis Ser/Thr protein kinase PrkC (BasPrkC) is important for virulence of the bacterium within the host. Homologs of PrkC and its cognate phosphatase PrpC (BasPrpC) are the most conserved mediators of signaling events in diverse bacteria. BasPrkC homolog in Bacillus subtilis regulates critical processes like spore germination and BasPrpC modulates the activity of BasPrkC by dephosphorylation. So far, biochemical and genetic studies have provided important insights into the roles of BasPrkC and BasPrpC; however, regulation of their activities is not known. We studied the regulation of BasPrkC/BasPrpC pair and observed that Zn²⁺ metal ions can alter their activities. Zn²⁺ promotes BasPrkC kinase activity while inhibits the BasPrpC phosphatase activity. Concentration of Zn²⁺ in growing B. anthracis cells was found to vary with growth phase. Zn²⁺ was found to be lowest in log phase cells while it was highest in spores. This variation in Zn²⁺ concentration is significant for understanding the antagonistic activities of BasPrkC/BasPrpC pair. Our results also show that BasPrkC activity is modulated by temperature changes and kinase inhibitors. Additionally, we identified Elongation Factor Tu (BasEf-Tu) as a substrate of BasPrkC/BasPrpC pair and assessed the impact of their regulation on BasEf-Tu phosphorylation. Based on these results, we propose Zn²⁺ as an important regulator of BasPrkC/BasPrpC mediated phosphorylation cascades. Thus, this study reveals additional means by which BasPrkC can be activated leading to autophosphorylation and substrate phosphorylation.     

Effect of organic carbon sources and environmental factors on cell growth and lipid content of Pavlova lutheri

The present study aimed to investigate the effects of organic carbon sources, cultivation methods, and environmental factors on growth and lipid content of Pavlova lutheri for biodiesel production. In the 250-mL flask bioreactors, P. lutheri was cultivated in the modified artificial seawater (ASW) medium containing glucose, glycerol, sodium acetate, or sucrose as an organic carbon substrate. The effects of different growth conditions (phototrophic, mixotrophic, and heterotrophic) and environmental factors such as photoperiod, light intensity, and salinity were evaluated. Growth of P. lutheri was inhibited under heterotrophy but was enhanced in mixotrophy as compared to phototrophy. Biomass and lipid content of P. lutheri were significantly (p < 0.05) affected by changing photoperiod, light intensity, and salinity. Higher biomass concentration and lipid content were observed at a light intensity of 100 ± 2 μmol photons m−2 s−1, 18 h photoperiod, and 30% salinity, in a modified ASW medium supplemented with 10 mmol sucrose. An increase in biomass concentration from 320 ± 25.53 to 1106 ± 18.52 mg L−1 and high lipid content of 31.11 ± 1.65% (w/w) were observed with the optimized culture conditions, demonstrating a significant (p < 0.05) enhancement in biomass and lipid content due to the improved culture conditions. The present study emphasizes the possible use of sucrose for biomass and lipid production with P. lutheri under the optimized culture conditions. Using low-cost and relatively easy accessible feedstock such as sucrose would be a valuable alternative for growing microalgae with enhanced lipid content.     

Comparative analysis of the methods used for finding surface energy to investigate protein interaction behavior on chromatographic supports

Hydrophobic interaction chromatography, an important and effective purification strategy, is generally used for the purification of variety of biomolecules. A basic understanding of the protein interaction behavior is required to effectively separate these biomolecules. A colloidal type extended Derjaguin, Landau, Verwey, and Overbeek calculations were utilized to study the interactions behavior of model proteins to commercially available hydrophobic chromatographic materials that is, Toyopearl Phenyl 650C and Toyopearl Butyl 650C. Physicochemical properties of selected model proteins were achieved by contact angle and zeta potential measurements. The contact angle of chromatographic materials used was achieved through sessile drop method on disrupted beads and capillary penetration method (CPM) on intact beads. The surface properties were further used to calculate the interactions of the proteins to chromatographic supports. The calculated secondary energy minimum of the proteins with the chromatographic materials (from the contact angle values determined through both methods can be correlated with the retention volumes from the real chromatography. The secondary energy minimum values are higher for each protein to the chromatographic materials calculated from the inputs derived through sessile drop method compared to CPM. For instance, immunoglobulin G has secondary energy minimum value of 0.17 kT compared to 0.11 kT, obtained through sessile drop method and CPM, respectively. Average relative values of the energy minimum calculated for all proteins are as 1.51 kT and 1.29 kT for Toyopearl Butyl 650C and Toyopearl Phenyl 650C, respectively, as a conversion factor for estimation of secondary energy minimum for both methods.     

红火蚁病原真菌AUGM47早期发育超微结构研究

在前期筛选已获得对红火蚁Solenopsis invicta Buren高效致病真菌罗伯茨绿僵菌Metarhizium robertsii AUGM47的基础上,为进一步明确病原真菌对寄主昆虫的侵染机制。本试验在室内条件下,以红火蚁工蚁为侵染对象,利用荧光显微镜和透射电镜观察了罗伯茨绿僵菌AUGM47侵染单元分生孢子在体表附着萌发、穿透和体内增殖的早期发育过程。结果表明,菌株AUGM47分生孢子在红火蚁体表可萌发并形成附着胞侵入,接种后12 h观察到萌发,在36 h内普遍出现穿透结构穿透体壁。接种后48 h为菌体在血腔内的增殖阶段。菌丝体在穿透表皮和体腔内增殖过程中伴随着机械压力和酶的活动。接种后96 h,观察到自噬现象,菌体通过自噬降解并回收细胞器,为从体内穿出的晚期发育过程提供物质基础。本研究对罗伯茨绿僵菌AUGM47分生孢子在红火蚁体外至体内的发育进程研究证实了菌株的高致病性,为红火蚁生防真菌菌种改良和后续开发利用奠定理论基础。     

Enhancing crop growth, nutrients availability, economics and beneficial rhizosphere microflora through organic and biofertilizers

Field experiment was conducted on fodder maize to explore the potential of integrated use of chemical, organic and biofertilizers for improving maize growth, beneficial microflora in the rhizosphere and the economic returns. The treatments were designed to make comparison of NPK fertilizer with different combinations of half dose of NP with organic and biofertilizers viz. biological potassium fertilizer (BPF), Biopower, effective microorganisms (EM) and green force compost (GFC). Data reflected maximum crop growth in terms of plant height, leaf area and fresh biomass with the treatment of full NPK; and it was followed by BPF+full NP. The highest uptake of NPK nutrients by crop was recorded as: N under half NP+Biopower; P in BPF+full NP; and K from full NPK. The rhizosphere microflora enumeration revealed that Biopower+EM applied along with half dose of GFC soil conditioner (SC) or NP fertilizer gave the highest count of N-fixing bacteria (Azotobacter, Azospirillum, Azoarcus andZoogloea). Regarding the P-solubilizing bacteria,Bacillus was having maximum population with Biopower+BPF+half NP, andPseudomonas under Biopower+EM+half NP treatment. It was concluded that integration of half dose of NP fertilizer with Biopower+BPF / EM can give similar crop yield as with full rate of NP fertilizer; and through reduced use of fertilizers the production cost is minimized and the net return maximized. However, the integration of half dose of NP fertilizer with biofertilizers and compost did not give maize fodder growth and yield comparable to that from full dose of NPK fertilizers.     

Down-regulation of reduced folate carrier may result in folate malabsorption across intestinal brush border membrane during experimental alcoholism

Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The intestinal folate uptake is tightly and diversely regulated, and disturbances in folate homeostasis are observed in alcoholism, attributable, in part, to intestinal malabsorption of folate. The aim of this study was to delineate the regulatory mechanisms of folate transport in intestinal absorptive epithelia in order to obtain insights into folate malabsorption in a rat model of alcoholism. The rats were fed 1 g.kg(-1) body weight of ethanol daily for 3 months. A reduced uptake of [(3)H]folic acid in intestinal brush border membrane was observed over the course of ethanol administration for 3 months. Folate transport exhibited saturable kinetics and the decreased intestinal brush border membrane folate transport in chronic alcoholism was associated with an increased K(m) value and a low V(max) value. Importantly, the lower intestinal [(3)H]folic acid uptake in ethanol-fed rats was observed in all cell fractions corresponding to villus tip, mid-villus and crypt base. RT-PCR analysis for reduced folate carrier, the major folate transporter, revealed that reduced folate carrier mRNA levels were decreased in jejunal tissue derived from ethanol-fed rats. Parallel changes were observed in reduced folate carrier protein levels in brush border membrane along the entire crypt-villus axis. In addition, immunohistochemical staining for reduced folate carrier protein showed that, in alcoholic conditions, deranged reduced folate carrier localization was observed along the entire crypt-villus axis, with a more prominent effect in differentiating crypt base stem cells. These changes in functional activity of the membrane transport system were not caused by a general loss of intestinal architecture, and hence can be attributed to the specific effect of ethanol ingestion on the folate transport system. The low folate uptake activity observed in ethanol-fed rats was found to be associated with decreased serum and red blood cell folate levels, which might explain the observed jejunal genomic hypomethylation. These findings offer possible mechanistic insights into folate malabsorption during alcoholism.     

Fanconi anemia: contribution of molecular analyses to the identification of bone marrow graft donors and the study of chimerism in grafted patients

We report on the effectiveness of molecular studies regarding Fanconi anemia (FA) for a better selection of bone marrow graft donors and for post-transplant follow up. Ten unrelated FA patients and their families were analyzed by microsatellite markers. In 9 cases, the cytogenetic investigation of potential human leukocyte antigen (HLA)-identical related donors was normal, and the molecular analyses confirmed that they were also either normal or heterozygous carriers. For 1 patient, cytogenetic analysis of an HLA-identical sibling donor yielded ambiguous results with a relatively high number of chromosomal breakages using cross-linking agents. However, genotyping of this potential donor demonstrated his heterozygous state. Nine patients have received allogeneic bone marrow transplantation from HLA-matched related donors. Microsatellite analysis showed complete chimerism (CC) in all cases. The median follow up was 54 months (range 8-144 months). One patient out of 9 with CC rejected her graft without prior detection of a transitional mixed chimerism. Among these patients, 1 died 25 months after the transplantation of a chronic graft-versus-host-disease (GVHD). We conclude that, when the cytogenetic studies are not conclusive, molecular analyses are crucial to distinguish heterozygous carriers from asymptomatic FA Tunisian patients. Molecular analyses also allowed the evaluation of hematopoietic chimerism after allogeneic bone marrow transplantation and might be of value to identify patients with a high risk for graft rejection.     

Induction of micronuclei by Zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of Vitamin E

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin mainly produced by Fusarium graminaerum, found as a world-wide contaminant mainly of corn and wheat. Previous studies have demonstrated that among several other effects on animals and humans, ZEN also displays hepatotoxicity, immunotoxicity and nephrotoxicity. ZEN is mainly known as a hormonal disrupter due to its estrogenic activities and consequent toxicity for reproduction. Furthermore, mutagenic and genotoxic proprieties of ZEN were disclosed recently, the molecular mechanisms of which are not yet well understood. In the present study, the genotoxic potential of ZEN was evaluated using genotoxicity tests: the 'cytokinesis block micronucleus assay' in Vero monkey kidney cells and the 'in vivo mouse bone marrow micronucleus assay'. In cultured cells treated with 5, 10 and 20 microM ZEN, the frequency of binucleated micronucleated cells (BNMN) was assessed in 1000 binucleated cells and in mice given oral doses of 10, 20 and 40 mg/kg bw, the frequency of polychromatic erythrocytes micronucleated (PCEMN) in bone marrow cells was assessed in 2000 polychromatic erythrocytes (PCE). The potential prevention of ZEN-induced effects by 25 microM Vitamin E (Vit E) was also evaluated.In vivo, doses of 10, 20 and 40 mg/kg bw ZEN representing, respectively 2, 4 and 8% of the LD50 (LD50 of ZEN in mice is 500 mg/kg bw), were administered to animals either with or without pre-treatment with Vit E (216.6 mg/kg bw) in order to evaluate its preventive potential.ZEN was found to induce micronuclei (MN) in a dose-dependent manner in cultured Vero cells as well as in mouse bone marrow cells. The present data emphasise the likely clastogenic pathway among the molecular mechanisms that underlay the ZEN-induced genotoxicity. Vit E was found to prevent partially-from 30 to 50%-these toxic effects, most likely acting either as a structural analogue of ZEN or as an antioxidant.