Structure and function of the repressor of bacteriophage lambda

Summary By mutagenizing a cIts (cI857) lysogen, a mutant has been isolated with a wild-type phenotype. This mutant phage lysogenizes with low efficiency and produces a low burst. Though the initial rates of repressor synthesis in Escherichia coli after infection with wild-type and mutant are the same, the maximum level of repressor that is synthesized in the latter case is only about 30% of that synthesized in the former. Virulent plates on the lysogen of mutant with slightly less efficiency producing very tiny plaques. Operator-binding studies made in vitro with purified mutant and wild-type repressors show that the binding curve of the former repressor is a rectangular hyperbola while that of the latter is sigmoid. The half-lives of the complexes of mutant and wild-type repressors with right operator are 133 and 27 min, respectively. All these results suggest that the mutant repressor possibly has a higher affinity for the operators. This mutant has been named cIha (ha=high affinity).     

Control of sulfidogenic bacteria in produced water from the Kathloni oilfield in northeast India

A full-scale study was conducted to evaluate microbiocide efficacy to control the indigenous sulfide-producing bacterial population in produced water at Kathloni, Oil India Limited (OIL), Assam (northeast India). The sulfide-producing culturable bacterial strains present in produced water of Kathloni oilfield were identified as Anaerobaculum mobile, Garciella nitratireducens, Clostridium sporogenes, Thermosediminibacter oceani, Coprothermobacter sp., Thermodesulfovibrio sp., Thermodesulfobacterium sp., Thermodesulfotobacterium sp., and Caldanaerobacter sp. These strains could produce sulfide in the range of 50–200 mg l^?1 and volatile fatty acids such as acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid. Out of 10 microbiocides screened, three (sodium hypochlorite, benzyl trimethyl ammonium chloride, and 2-bromo-2-nitropropane-1,3-diol) were selected to control the growth and sulfide production by the aforementioned mixed bacterial species. A strategy was designed in which these three microbiocides were sequentially applied in an oilfield for 132 days. There was no significant difference in the chemical composition of the produced water after the treatment.     

Antioxidant response and Lea genes expression under exogenous ABA and water deficit stress in wheat cultivars contrasting in drought tolerance

Two wheat (Triticum aestivum) cultivars, C306 (drought tolerant) and PBW343 (drought susceptible) were compared for their response to exogenous ABA, water stress (WS) and combined (ABA plus WS) during their seedlings growth. Their responses were studied in the form of seedlings growth, antioxidant potential of roots and shoots and expression levels of LEA genes in shoots. ABA treatment led to increase in levels of ascorbate, ascorbate to dehydroascorbate ratio, antioxidant enzymes and decreases in levels of dehydroascorbate, malondialdehyde (MDA). Decrease in biomass, ascorbate contents, ascorbate to dehydroascorbate ratios and antioxidant enzymes was more in PBW343 than in C306 under WS. Dehydroascorbate and MDA levels were higher in PBW343 than in C306 under WS. ABA plus WS improved some of these features from their levels under WS in PBW343. Proline contents were not increased significantly under ABA in both cultivars. Out of ten LEA genes studied, six LEA genes were induced more under WS than under ABA in C306 but equally induced in PBW343. Four LEA genes were induced earlier in PBW343 but later in C306. Wdhn13 was induced more under ABA than under WS in C306 while it was non-responsive to both stresses in PBW343.     

Catalytic phosphorylation of Na,K-ATPase drives the outward movement of its cation-binding H5-H6 hairpin

The Na,K-ATPase undergoes conformational transitions during its catalytic cycle that mediate energy transduction between the phosphorylation and cation-binding sites. Structure-function studies have shown that transmembrane segments H5 and H6 in the alpha subunit of the enzyme participate in cation binding and transport. The Ca-ATPase crystal structure indicates that the H5 helix extends into the cytoplasmic ATP binding domain, finishing 4-5 A from the phosphorylation site. Here, we test whether the phosphorylation of the Na,K-ATPase leads to conformational changes in the cation-binding H5-H6 hairpin. Using as background an enzyme where all wild-type Cys in the transmembrane region were replaced, Cys were introduced in the joining loop and extracellular ends of H5 and H6. Mutated proteins were expressed in COS cells and probed with Hg(2+), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and biotin-maleimide, applied to the extracellular media while placing the cells in two different media (K-medium and Na-medium). We assumed that under these treatment conditions most of the enzyme would be in one of two predominant conformations: E1 (K-medium) and E2P (Na-medium). The extent of enzyme inactivation by Hg(2+) or MTSET treatment was dependent on the targeted position; i.e., proteins carrying Cys in the outermost positions were more affected by treatment. Moreover, in the case of proteins carrying Cys at positions 785, 787, and 797, driving the enzyme to phosphorylated conformations (Na-media) led to a larger inactivation. Similarly, biotinylation of introduced Cys was also influenced by the enzyme conformation, with a larger extent of modification after treatment of cells in the Na-medium (E2P form). These results can be explained by the enzyme phosphorylation driving the outward movement of the H5 helix. Thus, they provide experimental evidence for a structure-function mechanism where, via H5, enzyme phosphorylation leads to a conformational change at the cation-binding site and the consequent cation translocation.     

A study of the hydration and thermodynamics of warm-water and cold-water fish collagens.

The hydrated volumes, Vh, of collagens extracted from various fish species were calculated by using the Simha-Einstein equation, and it was found that the hydration of warm-water fish collagen is greater than that of cold-water fish collagen (halibut). Although the intrinsic viscosities of warm-water fish (bigeye-tuna, carp and catfish) collagens are almost the same, the hydrated volume of bigeye-tuna collagen is approx. 1.5 and 3 times those of carp and catfish collagens respectively. The extent of hydration at 20 degrees C is in the following order: bigeye tuna greater than carp greater than catfish greater than halibut. The various thermodynamic activation parameters (delta G*, delta H* and delta S*) were calculated and it was found that they are useful for determining the exact denaturation temperature. It was calculated that the denaturation temperatures of halibut, bigeye-tuna, carp and catfish collagens are 17, 31, 32 and 26-30 degrees C respectively. The variations of hydration, intrinsic viscosity, denaturation temperature and the thermodynamic parameters with the variation of concentration of catfish collagen were also thoroughly examined. The change of thermodynamic parameters from coiled-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results.     

Activation of Wnt-β-catenin pathway in basal–parabasal layers of normal cervical epithelium comparable during development of uterine cervical carcinoma

In this study, importance of Wnt-β-catenin pathway in the development of uterine cervical carcinoma was evaluated. For this purpose, the profiles (expression/methylation/deletion) of β-catenin, p-β-catenin (Y654), Wnt3a, and APC were studied in disease free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of primary tumors (n = 70), CIN (n = 28), CACX (n = 102) samples, and two CACX cell lines (HeLa and SiHa). Immunohistochemical analysis revealed high/medium (74–95%) expression of β-catenin/p-β-catenin (Y654) and Wnt3a and low expression (23–26%) of APC in proliferating basal–parabasal layers contrary to differentiated spinous layer in normal cervix irrespective of HPV16 infection. The expression profile of the genes in the basal–parabasal layers did not change significantly during development of CACX. High (66%) promoter methylation of APC was seen in basal–parabasal layers and the cervical lesions (42–69%), unlike in spinous layers (25%). The promoter methylation status of APC was validated by in vitro demethylation experiments using 5-aza-dC in CACX cell lines. However, additional deletion of APC was significantly increased from CIN (12%) to stage I/II (40%) and became comparable in stage III/IV (48%) of the tumor. Patients with alterations (deletion/methylation) of APC and high/medium expression of Wnt3a/β-catenin/p-β-catenin (Y654) showed significantly poor survival. Thus our data indicate that cumulative effect of Wnt3a overexpression and APC inactivation are needed for overexpression of β-catenin during the development of CACX.