Kinetic study of the enzymatic cycling reaction conducted with 3alpha-hydroxysteroid dehydrogenase in the presence of excessive thio-NAD(+) and NADH

We have established a simple kinetic model applicable to the enzyme cycling reaction for the determination of 3alpha-hydroxysteroids. This reaction was conducted under the reversible catalytic function of a single 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) with nucleotide cofactors, thio-NAD(+) (one of the NAD(+) analogues) for the oxidation of 3alpha-hydroxysteroids and NADH for the reduction of 3-oxosteroids. This model was constructed based on the reaction mechanism of 3alpha-HSD, following an ordered bi-bi mechanism with cofactor binding first, under the assumption that the respective enzyme-cofactor complexes were distributed according to the initial ratio of thio-NAD(+) to NADH by the rapid equilibrium of both enzyme-cofactor complexes. The cycling rate in the new kinetic model could be expressed with the dissociation constants of enzyme-cofactor complexes and the initial concentrations of cofactors and enzyme. The cycling rate was verified by a comparison with the experimental data using 3alpha-HSD from Pseudomonas sp. B-0831. The results showed that the experimental data corresponded well with the results obtained from the kinetic model.     

The recognition and modification sites for the bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI

Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT).     

Construction of YAC Contigs at Human Chromosome 11q22.3-q23.1 Region Covering the Ataxia Telangiectasia Locus

Ataxia telangiectasia (AT) is an autosomal recessive diseaseof unknown etiology associated with cerebellar ataxia, telangiectasia,immune dysfunction, higher cancer risk, genomic instabilityand hypersensitivity to ionizing radiation. The major AT loci,AT-A and AT-C, are shown to be closely linked at chromosome11q22–q23. The most recent genetic linkage mapping andlinkage disequilibrium analysis have localized the major ATloci to a sequence of approximately 850 kb between the markersD11S1819 and D11S1818. The isolation of yeast artificial chromosomesspanning the AT region is an essential step to identify thegene or genes responsible for the mutation(s). We isolated atotal of 20 YAC clones from three independent YAC libraries,using sequence tagged sites mapped in the AT region as primersfor PCR-based YAC screening. The PCR assay for the presenceor absence of 16 different DNA markers allowed us to constructand to order four YAC contigs at the AT region. One of the contigswhich consists of the 10 YAC clones, covers about 2 Mb of DNAat the boundary between Giemsa-positive band 11q22.3 and Giemsa-negativeband 11q23.1 and includes the entire region of the major ATlocus between D11S1819 and D11S1818. Thus, the YAC contigs willfacilitate the positional cloning approach for searching transcribedsequences from the defined genomic region.     

Determination of Amino Acid Sequence in Surfactin,a Crystalline Peptidelipid Surfactant Produced by Bacillus subtilis

Some conditions of autolysis in cultured tobacco cells were examined for temperature, cell culture age and aeration. Cells autolyzed readily at 45°C. Seventy percent of the dry matter, almost 100% of the soluble sugar, 40% of the insoluble sugar and 60% of the total nitrogen in the initial cells were excreted within 5 hr of incubation in water. At lower physiological temperatures, excreted substances were reabsorbed into cells during the early period of incubation under aerobic conditions.

Rapidly growing cells excreted larger amounts of sugar, nitrogen and solid matter than did non-growing cells during autolysis at 30°C.

Plasmolysis was observed in autolyzed cells.

Autolysis was makedly stimulated by anaerobic conditions.     

Determination of the Location of Lactone Ring in Surfactin

A single form of exo-type cellulase (Exo I; M_w, 65,000), purified from a Trichoderma viride protease-depressed mutant, HK-75, digested Avicel to cellobiose exowise, and hydrolyzed cellotriose, cellotetraose, and cellopentaose in the strict manner of splitting off by cellobiose units. Exo I, however, hydrolyzed cellohexaose by both cellobiose and cellotriose units.

Exo I was proteolyzed by papain into two fragments; GPExo (M_w, 9,000) and Exo I′ (M_w, 56,000). The GPExo intensively adsorbed onto Avicel but did not hydrolyze it. Exo I′ had nearly identical activity to that of intact Exo I toward cellooligosaccharides but was almost inert to Avicel in digestion and adsorption. Sequence analysis of N-terminal and C-terminal amino acids showed that GPExo was between Gly⁴³⁵ and Leu⁴⁹⁶ and Exo I′ between Glu¹ and Gly⁴³⁴ in Exo I. Exo I therefore consists of two domains, one for adsorption to Avicel, as demonstrated by the Avicel-affinity site, GPExo and the other for the cleavage of glycosidic linkages as demonstrated in Exo 1′.     

2-(2-Phenylmorpholin-4-yl)pyrimidin-4(3H)-ones; A new class of potent,selective and orally active glycogen synthase kinase-3β inhibitors

A series of 2-(2-phenylmorpholin-4-yl)pyrimidin-4(3H)-ones was synthesized and examined for their inhibitory activity against glycogen synthase kinase-3β (GSK-3β). We found 21, 29 and 30 to possess potent in vitro GSK-3β inhibitory activity with good in vitro PK profiles. 21 demonstrated significant decrease of tau phosphorylation after oral administration in mice and excellent PK profiles.     

Additional effects of silver nanoparticles on bactericidal efficiency depend on calcination temperature and dip-coating speed

There is an increasing interest in the application of photocatalytic properties for disinfection of surfaces, air, and water. Titanium dioxide is widely used as a photocatalyst, and the addition of silver reportedly enhances its bactericidal action. However, the synergy of silver nanoparticles and TiO(2) is not well understood. The photocatalytic elimination of Bacillus atrophaeus was examined under different calcination temperatures, dip-coating speeds, and ratios of TiO(2), SiO(2), and Ag to identify optimal production conditions for the production of TiO(2)- and/or TiO(2)/Ag-coated glass for surface disinfection. Photocatalytic disinfection of pure TiO(2) or TiO(2) plus Ag nanoparticles was dependent primarily on the calcination temperature. The antibacterial activity of TiO(2) films was optimal with a high dip-coating speed and high calcination temperature (600°C). Maximal bacterial inactivation using TiO(2)/Ag-coated glass was also observed following high-speed dip coating but with a low calcination temperature (250°C). Scanning electron microscopy (SEM) showed that the Ag nanoparticles combined together at a high calcination temperature, leading to decreased antibacterial activity of TiO(2)/Ag films due to a smaller surface area of Ag nanoparticles. The presence of Ag enhanced the photocatalytic inactivation rate of TiO(2), producing a more pronounced effect with increasing levels of catalyst loading.     

Effects of dietary methylmercury on the zebrafish brain: histological, mitochondrial, and gene transcription analyses

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for wildlife and human health through fish consumption. To study the neurotoxic impact of MeHg on piscivorous fish, we contaminated the model fish species Danio rerio for 25 and 50 days with food containing 13.5 μg/g dry weight (dw) of MeHg (0.6 μg MeHg/fish/day), an environmentally relevant dose leading to brain mercury concentrations of 30 ± 4 μg of Hg g⁻¹ (dw) after 25 days of exposure and 46 ± 7 μg of Hg g⁻¹ (dw) after 50 days. Brain mitochondrial respiration was not modified by exposure to MeHg, contrary to what happens in skeletal muscles. A 6-fold increase in the expression of the sdh gene encoding the succinate dehydrogenase Fe/S protein subunit was detected in the contaminated brain after 50 days of exposure. An up regulation of 3 genes, atp2b3a, atp2b3b, and slc8a2b, encoding for calcium transporters was noticed after 25 days of exposure but the atp2b3a and atp2b3b were repressed and the slc8a2b gene expression returned to its basal level after 50 days, suggesting a perturbation of calcium homeostasis. After 50 days, we detected the up regulation of glial fibrillary acidic protein and glutathione S-transferase genes (gfap and gst), along with a repression of the glutathione peroxidase gene gpx1. These results match well with a MeHg-induced onset of oxidative stress and inflammation. A transmission electron microscopic observation confirmed an impairment of the optical tectum integrity, with a decrease of the nucleal area in contaminated granular cells compared to control cells, and a lower density of cells in the contaminated tissue. A potential functional significance of such changes observed in optical tectum when considering wild fish contaminated in their natural habitat might be an impaired vision and therefore a lowered adaptability to their environment.