Photocatalysis-dependent inactivation of Lactobacillus phage PL-1 by a ceramics preparation

A ceramics preparation (Cleansand-205), which was coated with a mixture of the oxides of Si, Al, Ti, and Ag, was found to inactivate Lactobacillus phage PL-1 suspended in a buffer solution. The inactivation of phage was dependent on the amounts of Cleansand-205 added, and the reaction obeyed almost first-order reaction kinetics. The phage inactivation was considerably accelerated by the presence of light.     

The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase (18.3 kDa, 162 amino acid residues) formed with the anticancer drug methotrexate using 2531 distance, 361 dihedral angle and 48 hydrogen bond restraints obtained from analysis of multidimensional NMR spectra. Simulated annealing calculations produced a family of 21 structures fully consistent with the constraints. The structure has four alpha-helices and eight beta-strands with two other regions, comprising residues 11 to 14 and 126 to 127, also interacting with each other in a beta-sheet manner. The methotrexate binding site is very well defined and the structure around its glutamate moiety was improved by including restraints reflecting the previously determined specific interactions between the glutamate alpha-carboxylate group with Arg57 and the gamma-carboxylate group with His28. The overall fold of the binary complex in solution is very similar to that observed in the X-ray studies of the ternary complex of L. casei dihydrofolate reductase formed with methotrexate and NADPH (the structures of the binary and ternary complexes have a root-mean-square difference over the backbone atoms of 0.97 A). Thus no major conformational change takes place when NADPH binds to the binary complex. In the binary complex, the loop comprising residues 9 to 23 which forms part of the active site has been shown to be in the "closed" conformation as defined by M. R. Sawaya & J. Kraut, who considered the corresponding loops in crystal structures of complexes of dihydrofolate reductases from several organisms. Thus the absence of the NADPH does not result in the "occluded" form of the loop as seen in crystal studies of some other dihydrofolate reductases in the absence of coenzyme. Some regions of the structure in the binary complex which form interaction sites for NADPH are less well defined than other regions. However, in general terms, the NADPH binding site appears to be essentially pre-formed in the binary complex. This may contribute to the tighter binding of coenzyme in the presence of methotrexate.     

Effects of some chelating agents on urinary copper excretion by the rat

In order to estimate the potential advantages of new chelating agents which can enhance copper excretion in the chronic copper intoxication arising in Wilson's disease, the relative ability of none chelating agents to induce the urinary excretion of copper was compared with that of D-penicillamine (DPA) and triethylenetetramine.2HCl (TRIEN), all given ip at 1 mmol/kg to male Sprague-Dawley rats. The compounds examined were as follows: tris(2-aminoethyl)-amine.3HCl (TREN), tetraethylenepentamine.5HCl (TETREN), pentaethylenehexamine.6HCl (PENTEN), 1,4,7,11-tetraazaundecane.4HCl (TAUD), 1,5,8,12-tetraazadodecane.4HCl (TADD), 1-N-benzyltriethylenetetramine.4HCl (BzTT), 4,7,10,13-tetraazatridecanoic acid.2H2SO4 (TTPA), 1,10-bis(2-pyridylmethyl)-1,4,7,10-tetraazadecane.4HCl (BPTETA), and N,N-bis(2-pyridylmethyl)-4-(aminomethyl)benzoic acid (4ABA). Of these, BzTT, TTPA, and 4ABA are new chelating agents not previously reported. The factors by which these chelating agents enhanced copper excretion over control (untreated) levels were as follows: DPA, 7.2; TREN, 1.6; TRIEN, 4.0; TETREN, 10.1; PENTEN, 7.8; TAUD, 7.8; TADD, 2.6; TTPA, 5.6; BzTT, 1.8; and 4ABA, 5.5. The results indicate that it may well be possible to develop additional chelating agents which are equal or superior to those now used in the treatment of Wilson's disease, as well as structural types whose immunological properties may be significantly different from DPA or TRIEN, the compounds currently used in the clinic for this disorder.     

Solution structure of a brodimoprim analogue in its complex with Lactobacillus casei dihydrofolate reductase

Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating-frame NOESY (ROESY) spectra were used to assign essentially all the protons in a 1:1 complex of Lactobacillus casei dihydrofolate reductase formed with an analogue of the antibacterial drug brodimoprim [2,4-diamino-5-(3',5'-dimethoxy-4'-bromobenzyl)pyrimidine]. The analogue has a 4,6-dicarboxylic acid side chain substituted on the 3'-O position designed to interact with the Arg 57 and His 28 residues in L. casei dihydrofolate reductase; it binds a factor of 10(3) more tightly to the enzyme than does the parent compound. Thirty-eight intermolecular and 11 intramolecular NOEs were measured involving the bound brodimoprim-4,6-dicarboxylic acid analogue. These provided the distance constraints used in conjunction with an energy minimization and simulated annealing protocol (using Discover from Biosym Ltd.) to dock the brodimoprim analogue into dihydrofolate reductase. In calculations where side chains and backbone fragments for binding-site residues were allowed flexibility, 90% of the 40 calculated structures had reasonable covalent geometry and none of them had NOE distance violations of greater than 0.36 A. The conformations of the aromatic rings in the bound ligand were well-defined in all the structures, with torsion angles tau 1 = -153 degrees +/- 4 degrees (C4-C5-C7-C1') and tau 2 = 53 degrees +/- 4 degrees (C5-C7-C1'-C2'): the aromatic rings of the ligand occupied essentially the same space in all the calculated structures (root mean square deviation value 1.83 A). Inclusion of the electrostatic interactions into the energy minimizations indicated that structures in which the 4,6-dicarboxylate group of the ligand interacts with the side chains of Arg 57 and His 28 are of low energy. Significant differences in side-chain and backbone conformations were detected between binding-site residues in the enzyme complexes with the brodimorpim analogue and methotrexate.     

Peculiarities of primary and secondary structure of phage FI-1 DNA

The composition of nitrous bases of phage FI-1 DNA was studied. As is evidenced from the values of buoyant density in CsCl (p=1,7093 g/cm(3)), melting temperature (T degrees m=86,05 degrees), spectral parameters and direct chromatographic determination, the DNA analysed contains 41,5 mole% pairs of guanine-cystosine. 5-hydroxymethylcytosine and other anomalous bases were not found. Chemical identification and jaxtposition of data of buoyant density in CsCl and Cs2SO4 (p=1,4466 g/cm(3)) and T degrees m. showed the presence of the extra-sugar component in DNA, most likely in the form of hentibiose. Spectral character of thermal denaturation of DNA in different solvents is indicative of the double helixity of its structure. DNA is characterized by enthalpies of conformational transitions "helix coil" (deltaH=12,3 kcal/g) and (deltaH=10 kcal/g) for the solvents, 1 x SSC, and 0,1 x SSC, correspondingly. The presence of extra-sugar in DNA with standard set of nitrous bases is discussed.     

Infectivity of the DNA of PM-2 phage

The infectivity of PM-2 phage free DNA was studied versus competent cells of marine bacteria Pseudomonas Bal-31 depending on the time of incubation with the bacterial cells, on the DNA and calcium concentration in the solution. Optimal conditions for the infection are the following: DNA concentration about 3 microgram/ml, CaCl2 concentration 0.1 M, incubation time 20 min.     

脂质体介导质粒DNA细胞转染的优化研究

Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years. The aim of this study was to achieve a better outcome of AAV-293 cells transfection by plasmid DNA.Methods:We studied the optimal condition for higher efficiency of cationic lipid-mediated cell transfection. Four experimental groups were set. Plasmid DNA and liposome were mixed in each groups at different ratios (μg :μL), 1:2.5,1:3.5,1:4.0 and 1:5.0, respectively. LacZ gene functioned as reporter gene, measuring the transfection efficiency of the four groups using the method of X-gal staining. Results:When the ratio was 1:3.5, the cell transfection rate was the highest. While the ratio of 1:2.5recommended by product manual achieve the lowest transfection rate. Their difference had statistical significance. Conclusion:In order to obtain a higher transfection efficiency, optimization on conditions of the ratio of plasmid DNA to liposome is necessary in cell transfection.     

Stability and DNA binding of the phd protein of the phage P1 plasmid addiction system

The plasmid addiction module of bacteriophage P1 encodes two proteins, Doc, a toxin that is stable to proteolytic degradation, and Phd, the toxin's antidote that is proteolytically unstable. Phd has been shown to autoregulate its expression by specific DNA binding. Here, we investigate the secondary structure and thermal stability of Phd, the effect of operator DNA binding on the structure and stability of Phd, and the stoichiometry, affinity, and cooperativity of Phd binding to operator subsites and intact operator DNA. Phd folds as a monomer at low temperatures or in the presence of osmolytes but exists predominantly in an unfolded conformation at 37 degreesC. The native state of Phd is stabilized by operator binding. Two Phd monomers bind to each operator subsite, and four monomers bind to the intact operator. The subsite binding reaction shows a second-order dependence on protein concentration and monomer-bound DNA species are unpopulated, suggesting that two Phd molecules bind cooperatively to each operator subsite. In intact operator binding experiments, both dimer-bound and tetramer-bound DNA species are populated, and binding occurs at protein concentrations similar to those required for subsite binding, suggesting that there is no significant dimer-dimer cooperativity.     

Purification and DNA-binding properties of the integrase protein Int encoded by Lactobacillus plantarum phage

The Lactobacillus plantarum temperate phage phi g1e (42,259 bp) encodes an integrase gene int linked to a phage attachment site attP (Kakikawa et al., 1997). To investigate phi g1e recombination, the integrase protein Int was overproduced in Escherichia coli under the T7 promoter, and purified. The Int protein had an apparent molecular mass of 42.0 kDa, corresponding well with that (45.5 kDa) predicted from the DNA sequence. Amino-acid sequencing revealed that the N-terminal 20 amino-acids of the purified Int protein completely coincided with those deduced from the DNA sequence, although deficient in the first methionine. Gel mobility-shift assays demonstrated that Int bound specifically to the attP region. In addition, footprinting analysis showed that Int protected about 35 bases, containing the 24-bp core domain at attP, from DNase I attack. These results are indicative of site-specific interaction of Int with the attP site, the reaction prerequisite for integration and excision of the phi g1e genome into and/or out of the host chromosome.     

The effect of consumption of milk fermented by Lactobacillus casei strain Shirota on the intestinal microflora and immune parameters in humans

OBJECTIVE: To determine the effect of consumption of milk fermented by Lactobacillus casei strain Shirota (L. casei Shirota) on the composition and metabolic activities of the intestinal microflora, and immune parameters in humans. SUBJECTS: Twenty healthy male subjects aged 40-65 years were selected. DESIGN: A placebo-controlled trial was performed in which 10 subjects were randomly assigned to a control and 10 to a treatment group. During the first and last two weeks of the 8-week study the subjects received a strictly controlled diet without fermented products. The same controlled diet was given during the intermediate 4-week test period but then the treatment group received three times daily 100 ml of fermented milk containing 10(9) CFU L. casei Shirota/ml, whereas the same amount of unfermented milk was given to the subjects in the control group. RESULTS: In comparison to the control group, the consumption of L. casei Shirota-fermented milk resulted in an increase of the Lactobacillus count in the faeces in which the administered L. casei Shirota was predominant at the level of 10(7) CFU/g wet faeces. This was associated with a significant increase in Bifidobacterium counts (P < 0.05). Some shifts in the other bacterial species were found, such as a decreased number of Clostridium; however the differences were not statistically different between the treatment and the control groups. The beta-glucuronidase and beta-glucosidase activities per 10(10) bacteria decreased significantly (P < 0.05) at the second week of the 4-week test period with the consumption of L. casei Shirota-fermented milk. Furthermore, the consumption of the fermented milk product resulted in a slight but significant increase in the moisture content of the faecal samples (P < 0.05). No treatment effects were observed for any of the immune parameters measured (including natural killer (NK) cell activity, phagocytosis and cytokine production). CONCLUSIONS: The results suggest that consumption of L. casei Shirota-fermented milk is able to modulate the composition and metabolic activity of the intestinal flora and indicate that L. casei Shirota-fermented milk does not influence the immune system of healthy immunocompetent males.     

Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage

We have used two selection techniques to study sequence-specific DNA recognition by the zinc finger, a small, modular DNA-binding minidomain. We have chosen zinc fingers because they bind as independent modules and so can be linked together in a peptide designed to bind a predetermined DNA site. In this paper, we describe how a library of zinc fingers displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets. The amino acid sequences of selected fingers which bind the same triplet are compared to examine how sequence-specific DNA recognition occurs. Our results can be rationalized in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few alpha-helical positions. In the paper following this one, we describe a complementary technique which confirms the identity of amino acids capable of DNA sequence discrimination from these positions.     

Inductive electron-withdrawal from ammonium ion headgroups of cationic lipids and the influence on DNA transfection

We have prepared a panel of lipidic ammonium tetrafluoroborate salts that contain trifluoromethyl, trichloromethyl, and methyl groups attached to the headgroup. 19F-NMR analyses of the cationic lipid panel revealed that the differences in electron-withdrawal from the ammonium ion headgroup accounted for differences in ion-pairing. Exchange of the tetrafluoroborate counterion by complexation to DNA-phosphate of a reporter gene enabled us to probe the influence of inductive electron-withdrawal in cationic lipid-mediated DNA transfection. We tested the lipid panel for transfection activity in two cell lines. The results indicate that the inductive effects of electron-withdrawing functionality diminish transfection activity in modest (2-4-fold) increments. The present study suggests that the mechanism whereby poly(alcohol)- or poly(ether)-substituted headgroups improve DNA transfection is not based on electronic activation of the ammonium ion.     

Action of intercalating agents on the activity of DNA polymerase I

The effect of intercalating compounds such as 9-aminoacridine, quinacrine (atebrin), proflavine and daunomycin on the activity of DNA polymerase I(EC 2.7.7.7) was studied in vitro and compared with the binding of these acridines to native DNA. The enzyme kinetics were followed at various concentrations of DNA 3'-OH primer end groups and constant concentrations of deoxynucleosidetriphosphates as well as under the opposite conditions. The Km values for the DNA 3'-OH end groups were 16--38 nM and for the deoxynucleosidetriphosphates 2--5 micrometer, depending on the buffer and pH used in the enzymatic assay. All acridine derivates inhibit the DNA polymerase; at variable DNA concentrations a competitive inhibition was observed, where the Ki values ranged between 0.87 and 8.5 micrometer. At variable concentrations of deoxynucleosidetriphosphates and constant DNA concentration a non-competitive inhibition was observed. On denatured 3'-OH DNA as well as on poly(dA) - (dT)10 as substrate no inhibition by 9-aminoacridine was observed. 5'--3' exonuclease activity of DNA polymerase is inhibited by 9-aminoacridine but 3'--5' exonuclease activity on denatured DNA is not influenced by this intercalating compound. The affinity of the acridines to DNA was determined spectrophotometrically under conditions similar to those in the enzymatic assay and the computed frequency of intercalation was related to the inhibition of enzymatic activity. The mechanism of inhibition is explained by a disturbance of the structure of the double helical DNA due to the interaction of the bound acridine derivates.