Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site.     

Protein kinase A inhibitors enhance radiation-induced apoptosis

In addition to a role for de novo protein synthesis in apoptosis we have previously shown that activation of a protein phosphatase or loss of activity of a kinase is also important in radiation-induced apoptosis in human cells [Baxter, and Lavin (1992): J Immunol 148:149–1954]. We show here that some inhibitors of protein kinases exacerbate radiation-induced apoptosis in the human cell line BM13674. The specific protein kinase A inhibitor isoquinoline sulfonamide (20 μM) gave rise to significantly increased levels of apoptosis at 2–6 h postirradiation compared to values after radiation exposure only. The same concentration of isoquinolinesulfonamide, which was effective in increasing apoptosis, reduced activity markedly. A 66% inhibition of cyclic AMP-dependent protein kinase A activity occurred in unirradiated cells at this concentration of H89 and activity was reduced to 58% in irradiated cells. Calphostin C, a specific inhibitor of protein kinase C, at a concentration of 0.1 μM, which caused 68% inhibition of enzyme activity in irradiated cells, failed to enhance the level of radiation-induced apoptosis. Other kinase inhibitors did not lead to an additional increase in apoptosis over and above that observed after irradiation. The results obtained here provide further support for an important role for modification of existing proteins during radiation-induced apoptosis.     

The Effect of Chronic Long-Term Intermittent Hypobaric Hypoxia on Bone Mineral Density in Rats: Role of Nitric Oxide

Intermittent hypoxia is the most common pattern of hypoxic exposure in humans. The effect of chronic long-term intermittent hypobaric hypoxia (CLTIHH) on bone metabolism is not investigated. We examined the effect of CLTIHH on bone metabolism and the role of nitric oxide (NO) in this process. The rats were divided into three groups in this study. The animals in groups I and II have been exposed to CLTIHH. The animals in group II were also treated with nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester. To obtain CLTIHH, rats were placed in a hypobaric chamber (430 mm Hg; 5 h/day, 5 days/week, 5 weeks). The group III (control) rats breathed room air in the same environment. At the begining of the experiments, bone mineral density (BMD) of the animals were measured, and blood samples were collected from the tail vein. After the 5-week CLTIHH period, the same measurements were repeated. Parathyroid hormone, calcium, phosphate, bone alkaline phosphatase (b-ALP), NO, interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha levels were determined. The cytokines, NO levels, and BMD in CLTIHH-induced rats were higher compared with baseline and control values. The cytokines, b-ALP, and BMD increased while NO levels decreased in the group II compared with baseline values. BMD values of group II were lower than group I but higher than control group. Our results suggested that CLTIHH has positive effects on bone density. Intermittent hypoxia protocols may be developed for treatment and prevention of osteopenia and osteoporosis.     

Production of human lysozyme in biofilm reactor and optimization of growth parameters of Kluyveromyces lactis K7

Lysozyme (1,4-β-N-acetylmuramidase) is a lytic enzyme, which degrades the bacterial cell wall. Lysozyme has been of interest in medicine, cosmetics, and food industries because of its anti-bactericidal effect. Kluyveromyces lactis K7 is a genetically modified organism that expresses human lysozyme. There is a need to improve the human lysozyme production by K. lactis K7 to make the human lysozyme more affordable. Biofilm reactor provides high biomass by including a solid support, which microorganisms grow around and within. Therefore, the aim of this study was to produce the human lysozyme in biofilm reactor and optimize the growth conditions of K. lactis K7 for the human lysozyme production in biofilm reactor with plastic composite support (PCS). The PCS, which includes polypropylene, soybean hull, soybean flour, bovine albumin, and salts, was selected based on biofilm formation on PCS (CFU/g), human lysozyme production (U/ml), and absorption of lysozyme inside the support. To find the optimum combination of growth parameters, a three-factor Box–Behnken design of response surface method was used. The results suggested that the optimum conditions for biomass and lysozyme productions were different (27 °C, pH 6, 1.33 vvm for biomass production; 25 °C, pH 4, no aeration for lysozyme production). Then, different pH and aeration shift strategies were tested to increase the biomass at the first step and then secrete the lysozyme after the shift. As a result, the lysozyme production amount (141 U/ml) at 25 °C without pH and aeration control was significantly higher than the lysozyme amount at evaluated pH and aeration shift conditions (p?<?0.05).     

Biocontrol of wilt disease complex of pea using Pseudomonas fluorescens and Rhizobium sp.

Biocontrol of wilt disease complex of pea caused by the root-knot nematode Meloidogyne incognita and Fusarium oxysporum f. sp. pisi was studied on pea (Pisum sativum L.) using plant growth-promoting rhizobacterium Pseudomonas fluorescens and root nodule bacterium Rhizobium sp. Inoculation of M. incognita and F.oxysporum alone caused significant reductions in plant growth over un-inoculated control. Reduction in plant growth caused by M. incognita was statistically equal to that caused by F. oxysporum. Inoculation of M. incognita plus F. oxysporum together caused a greater reduction in plant growth than the sum of damage caused by these pathogens singly. Inoculation of P. fluorescens and Rhizobium sp. individually or both together increased plant growth in pathogen inoculated and un-inoculated plants. Inoculation of P. fluorescens to pathogen-inoculated plants caused a greater increase in plant growth than caused by Rhizobium sp. Application of Rhizobium plus P. fluorescens caused a greater increase in plant growth than caused by each of them singly. Inoculation of P.fluorescens caused higher reduction in galling and nematode multiplication than caused by Rhizobium sp. Use of Rhizobium plus P. fluorescens caused higher reduction in galling and nematode multiplication than their individual inoculation. Plants inoculated with both pathogens plus Rhizobium showed less nodulation than plants inoculated with single pathogen plus Rhizobium. Inoculation of Rhizobium plus P. fluorescens resulted in higher root-nodulation than inoculated only with Rhizobium. Wilting indices were 4 and 5, respectively, when plants were inoculated with F. oxysporum and F. oxysporum plus M. incognita. Wilting indices were reduced maximum to 1 and 2, respectively, when plants inoculated with F.oxysporum and plants with both pathogens were treated with P. fluorescens plus Rhizobium.     

A novel homozygous 10 nucleotide deletion in BBS10 causes Bardet–Biedl syndrome in a Pakistani family

Bardet–Biedl Syndrome is a multisystem autosomal recessive disorder characterized by central obesity, polydactyly, hypogonadism, learning difficulties, rod-cone dystrophy and renal dysplasia. Bardet–Biedl Syndrome has a prevalence rate ranging from 1 in 100,000 to 1 in 160,000 births although there are communities where Bardet–Biedl Syndrome is found at a higher frequency due to consanguinity. We report here a Pakistani consanguineous family with two affected sons with typical clinical features of Bardet–Biedl Syndrome, in addition to abnormal liver functioning and bilateral basal ganglia calcification, the latter feature being typical of Fahr's disease. Homozygous regions obtained from SNP array depicted three known genes BBS10, BBS14 and BBS2. Bidirectional sequencing of all coding exons by traditional sequencing of all these three genes showed a homozygous deletion of 10 nucleotides (c.1958_1967del), in BBS10 in both affected brothers. The segregation analysis revealed that the parents, paternal grandfather, maternal grandmother and an unaffected sister were heterozygous for the deletion. Such a large deletion in BBS10 has not been reported previously in any population and is likely to be contributing to the phenotype of Bardet–Biedl Syndrome in this family.     

New binary copper(II) complexes containing intercalating ligands: DNA interactions,an unusual static quenching mechanism of BSA and cytotoxic activities

New binary copper(II) complexes – [Cu(4-mphen)₂(NO₃)]NO₃·H₂O (1), [Cu(5-mphen)₂ (NO₃)]NO₃·H₂O (2), the known complex [Cu(dmphen)₂(NO₃)]NO₃ (3) and [Cu(tmphen)₂ (NO₃)]NO₃·H₂O (4) - (4-mphen: 4-methyl-1,10-phenanthroline, 5-mphen: 5-methyl-1,10-phenanthroline, dmphen: 4,7-dimethyl-1,10-phenanthroline, tmphen: 3,4,7,8-tetramethyl-1,10-phenanthroline), have been synthesized and characterized by CHN analysis, ESI-MS, FTIR and single-crystal X-ray diffraction techniques. Interaction of these complexes with calf thymus DNA (CT-DNA) has been investigated by absorption spectral titration, ethidium bromide (EB) and Hoechst 33,258 displacement assay and thermal denaturation measurement. These complexes cleaved pUC19 plasmid DNA in the absence and presence of an external agent. Notably, in the presence of H₂O₂ as an activator, the cleavage abilities of these complexes are obviously enhanced at low concentration. Addition of hydroxyl radical scavengers like DMSO shows significant inhibition of the DNA cleavage activity of these complexes. BSA quenching mechanism was investigated with regard to the type of quenching, binding constant, number of binding locations and the thermodynamic parameters. The experimental results suggested that the probable quenching mechanism was an unusual static process and hydrophobic forces play a dominant role. The CT-DNA and BSA binding efficiencies of these complexes follow the order: 4 > 3 > 1 > 2. Furthermore, in vitro cytotoxicities of these complexes on tumor cells lines (Caco-2, MCF-7 and A549) and healthy cell line (BEAS-2B) showed that these complexes exhibited anticancer activity with low IC₅₀ values. The effect of hydrophobicity of the methyl-substituted phenanthrolines on DNA and protein binding activities of these complexes is discussed.