Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.     

Chitosan-iron oxide nano-composite platform for mismatch-discriminating DNA hybridization for Neisseria gonorrhoeae detection causing sexually transmitted disease

Electrochemically fabricated nano-composite film of chitosan (CH)-iron oxide (Fe(3)O(4)) has been used to detect gonorrhoea, a sexually transmitted disease (STD) via immobilization of biotinylated probe DNA (BDNA) using avidin-biotin coupling for rapid and specific (mismatch-discriminating) DNA hybridization. The presence of Fe(3)O(4) nanoparticles (～18nm) increases the electro-active surface area of the nano-biocomposite that provides desirable environment for loading of DNA with better conformation leading to increased electron transfer kinetics between the medium and electrode. The differential pulse voltammetric (DPV) studies have been conducted using BDNA/avidin/CH-Fe(3)O(4)/ITO electrode owing to the reduction of the methylene blue (MB) indicator and investigate electron transfer between MB moieties and electrode for one and two-bases mismatch. This STD biosensor is found to have a detection limit (1 × 10(-15)M) and a wide dynamic range (from 1 × 10(-16)M to 1 × 10(-6)M) using the complementary target DNA. In addition, the sensing system can be utilized to accurately discriminate complementary sequence from mismatch sequences.     

Crystallizing membrane proteins using lipidic bicelles

Crystallization of membrane proteins remains a significant challenge. For proteins resistant to the traditional approach of directly crystallizing from detergents, lipidic phase crystallization can be a powerful tool. Bicelles are an excellent medium for crystallizing membrane proteins in a lipidic environment. They can be described as bilayer discs formed by the mixture of a long-chain phospholipid and an amphiphile in an aqueous medium. Membrane proteins can be readily reconstituted into bicelles, where they are maintained in a native-like bilayer environment. Importantly, membrane proteins have been shown to be fully functional in bicelles under physiological conditions. Protein-bicelle mixtures can be manipulated with almost the same ease as detergent-solubilized membrane proteins, making bicelles compatible with standard equipment including high-throughput crystallization robots. A number of membrane proteins have now been successfully crystallized using the bicelle method, including bacteriorhodopsin, β2 adrenergic receptor, voltage-dependent anion channel, xanthorhodopsin and rhomboid protease. Because of the success with a variety of membrane proteins and the ease of implementation, bicelles should be a part of every membrane protein crystallographer's arsenal.     

Conditional stimulation of type V and VI adenylyl cyclases by G protein betagamma subunits

In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein beta2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the Gbeta(1) subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that Gbetagamma subunits enhance the activity of these enzymes provided either Galpha(s) or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of Gbetagamma subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by Gbetagamma subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous Gbetagamma subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G(i) was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, Gbetagamma subunits both in vitro and in intact cells. Moreover, Gbetagamma subunits derived from a source(s) other than G(i) are necessary for the full activation of hACVI by isoproterenol in intact cells.     

Identification and characterization of chickpea genotypes for early flowering and higher seed germination through molecular markers

Molecular Biology Reports - Chickpea is the fourth most important legume crop contributing 15.42% to the total legume production and a rich source of proteins, minerals, and vitamins. Determination...     

Effect of Eucalyptus and Mentha leaves and Curcuma rhizomes on Tyrophagus putrescentiae (Schrank) (Acarina: Acaridae) in wheat

Varying concentrations of powdered leaves of Eucalyptus and Mentha and rhizomes of Curcuma were evaluated in controlling Tyrophagus putrescentiae in wheat flour. At higher concentrations, all these plant materials significantly decreased the population build-up of the mite. Eucalyptus and Mentha leaf powders were effective at concentrations ranging from 100 to 5% in bringing about a decrease in the fecundity of the mite and reducing the egg numbers to 51.66 and 25.49 per female, respectively, as compared to 98.16 eggs per female in the controls. Rhizomes of Curcuma were effective even at a concentration of 0.1% reducing the egg laying to 7.66 eggs per female in wheat flour. These materials were more deleterious to immature stages (ova and larvae) than to the mature stages of T. putrescentiae. When tested on whole grains of wheat these plant materials showed more pronounced effects in controlling the mite population compared to that in wheat flour. Curcuma rhizomes were the most promising for possible use against T. putrescentiae.     

Nitric oxide in dengue and dengue haemorrhagic fever: necessity or nuisance?

Advances in free radical research show that reactive oxygen and nitrogen oxide species, for example superoxide, nitric oxide (NO) and peroxynitrite, play an important role in the pathogenesis of different viral infections, including dengue virus. The pathogenic mechanism of dengue haemorrhagic fever (DHF) is complicated and is not clearly understood. The hallmarks of the dengue disease, the antibody-dependent enhancement, the shift from T-helper type 1 (Th1) to Th2 cytokine response and the cytokine tsunami resulting in vascular leakage can now be explained much better with the knowledge gained about NO and peroxynitrite. This paper makes an effort to present a synthesis of the current opinions to explain the pathogenesis of DHF/shock syndrome with NO on centre stage.     

Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division.

A eukaryotic-type protein serine/threonine kinase, PknA, was cloned from Mycobacterium tuberculosis strain H37Ra. Sequencing of the clone indicated 100% identity with the published pknA sequence of M. tuberculosis strain H37Rv. PknA fused to maltose-binding protein was expressed in Escherichia coli; it exhibited a molecular mass of approximately 97 kDa. The fusion protein was purified from the soluble fraction by affinity chromatography using amylose resin. In vitro kinase assays showed that the autophosphorylating ability of PknA is strictly magnesium/manganese-dependent, and sodium orthovanadate can inhibit this activity. Phosphoamino-acid analysis indicated that PknA phosphorylates at serine and threonine residues. PknA was also able to phosphorylate exogenous substrates, such as myelin basic protein and histone. A comparison of the nucleotide-derived amino-acid sequence of PknA with that of functionally characterized prokaryotic serine/threonine kinases indicated its possible involvement in cell division/differentiation. Protein--protein interaction studies revealed that PknA is capable of phosphorylating at least a approximately 56-kDa soluble protein from E. coli. Scanning electron microscopy showed that constitutive expression of this kinase resulted in elongation of E. coli cells, supporting its regulatory role in cell division.     

Subcellular localization of LGN during mitosis: evidence for its cortical localization in mitotic cell culture systems and its requirement for normal cell cycle progression

Mammalian LGN/AGS3 proteins and their Drosophila Pins orthologue are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in their asymmetric division. Using overexpression studies in different cell line systems, we demonstrate here that, like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. We find that in WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression.     

Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity

In this study we extend tetramerization technology to T-cell receptors (TCRs). We identified TCR alpha beta pairs in the absence of accessory molecules, ensuring isolation of high-affinity TCRs that maintain stable binding characteristics after tetramerization. Subtle changes in cognate peptide levels bound to the class I molecule were accurately reflected by parallel changes in the mean fluorescence intensity of cells that bound TCR tetramers, allowing us to accurately assess the binding affinity of a panel of peptides to major histocompatibility complex (MHC) class I. Using a TCR tetramer specific for the Mamu-A(*)01 allele, we identified animals expressing this restricting class I allele from a large cohort of outbred rhesus macaques. TCR tetramers should facilitate analysis of the MHC-peptide interface and, more generally, the design of immunotherapeutics and vaccines.