Drug Resistance of Staphylococci VI. Genetic Determinant for Chloramphenicol Resistance

Naturally occurring strains of staphylococci which are resistant to chloramphenicol (CM) inactivate this antibiotic. One of the inactivation products of CM showed the chromatographic behavior of 3-acetoxychloramphenicol. Induction of resistance occurred after prior exposure to subinhibitory concentrations of the antibiotic. The resistance of induced populations, as well as CM-inactivation ability, was decreased when they were grown in CM-free medium. The CM-inactivation property was transduced together with CM resistance. Transductional analysis and CM-resistance elimination experiments indicated that CM resistance in naturally occurring strains of staphylococci is mainly accounted for by inactivation of the drug.     

Effect of the structure of dietary epoxylignan on its cytotoxic activity: relationship between the structure and the activity of 7,7′-epoxylignan and the introduction of apoptosis by caspase 3/7

We compared the cytotoxic activities of dietary epoxylignans and their stereoisomers and found (?)-verrucosin, which is (7S,7′R,8R,8′R)-7,7′-epoxylignan, to be the most cytotoxic epoxylignan against HeLa cells (IC₅₀ = 6.6 μM). On the other hand, the activity was about a factor of 10 less against HL-60. In this research on the relationship between the structure and cytotoxic activity of (?)-verrucosin 13, the 7-(4-methoxyphenyl)-7′-(3,4-dimethoxyphenyl) derivative 60, for which the activity (IC₅₀ = 2.4 μM) is three times greater than (?)-verrucosin 13, was discovered. The induction of apoptosis by caspase 3/7 was observed upon treatment with the (?)-verrucosin derivative.     

Effect of inoculation of symbiotic fungi on the growth and antioxidant enzymes’ activities in the presence of <Emphasis Type="Italic">Fusarium subglutinans</Emphasis> f. sp. <Emphasis Type="Italic">ananas</Emphasis> in pineapple plantlets

The inoculation with symbiotic fungi, Arbuscular mycorrhizal fungi (AMF) and/or Piriformospora indica on the growth, nutrient absorption, and induction of antioxidant enzyme activities in plantlets of pineapple ‘Imperial’ (fusariosis-resistant) and ‘Pérola’ (fusariosis-susceptible) in the presence of Fusarium subglutinans f. sp. ananas was investigated. The experiment was comprised by two cultivars, with or without fungal inoculation (Claroideoglomus etunicatum, Rhizophagus clarus, and P. indica, a mixture of all the fungi, and the control—absence of fungal inoculation), with or without applying Fusarium conidia, and with four replicates. In both cultivars, nutrient absorption was higher in the AMF plantlets compared to those inoculated with P. indica or the control ones, although it was more efficient in ‘Imperial’ than in ‘Pérola’. Inoculation with AMF and/or P. indica as well as the pathogen influenced differently the activities of superoxide dismutase, catalase, glutathione reductase, peroxidase, and polyphenol oxidase, in the shoots or roots of pineapple plantlets in both cultivars. Inoculated plantlets with mixture of all the fungi also exhibited a better growth and nutrient absorption, and generally, the ‘Imperial’ responded better than ‘Pérola’. In addition, these plantlets developed better than the control even in the presence of pathogen, indicating that inoculation with AMF and/or P. indica may contribute to the production of more resistant propagative material. Increased antioxidant enzyme activity is a potential strategy for managing this plant for explore biological control as an alternative to reduce environmental and health impacts by reducing the use of fungicides.     

Structural and functional analysis of tomato β‐galactosidase 4: insight into the substrate specificity of the fruit softening‐related enzyme

Plant β‐galactosidases hydrolyze cell wall β‐(1,4)‐galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato β‐galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of β‐(1,4)‐galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1–7 that belongs to the β‐galactosidase/exo‐β‐(1,4)‐galactanase subfamily. The enzyme can hydrolyze a wide range of plant‐derived (1,4)‐ or 4‐linked polysaccharides, and shows a strong ability to attack β‐(1,4)‐galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with β‐d ‐galactose. TBG4 comprises a catalytic TIM barrel domain followed by three β‐sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 β‐galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards β‐(1,6)‐galactobiose, and ~0.6‐fold activity towards β‐(1,4)‐galactobiose, compared with wild‐type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant β‐galactosidases towards β‐1,4 and β‐1,6 linkages.     

A real-time PCR-based quantitative assay for 3-methylcytosine demethylase activity of ALKBH3

Human AlkB homolog 3 (ALKBH3), a homolog of the Escherichia coli protein AlkB, demethylates 1-methyladenine and 3-methylcytosine (3-meC) in single-stranded DNA and RNA by oxidative demethylation. Immunohistochemical analyses on clinical cancer specimens and knockdown experiments using RNA interference in vitro and in vivo indicate that ALKBH3 is a promising molecular target for the treatment of prostate, pancreatic, and non-small cell lung cancer. Therefore, an inhibitor for ALKBH3 demethylase is expected to be a first-in-class molecular-targeted drug for cancer treatment. Here, we report the development of a novel, quantitative real-time PCR-based assay for ALKBH3 demethylase activity against 3-meC by highly active recombinant ALKBH3 protein using a silkworm expression system. This assay enables us to screen for inhibitors of ALKBH3 demethylase, which may result in the development of a novel molecular-targeted drug for cancer therapy.     

Theoretical approaches for dynamical ordering of biomolecular systems

Background

Living systems are characterized by the dynamic assembly and disassembly of biomolecules. The dynamical ordering mechanism of these biomolecules has been investigated both experimentally and theoretically. The main theoretical approaches include quantum mechanical (QM) calculation, all-atom (AA) modeling, and coarse-grained (CG) modeling. The selected approach depends on the size of the target system (which differs among electrons, atoms, molecules, and molecular assemblies). These hierarchal approaches can be combined with molecular dynamics (MD) simulation and/or integral equation theories for liquids, which cover all size hierarchies.

Degradation of amyloid β peptide by neprilysin expressed from Borna disease virus vector

Accumulation of amyloid β (Aβ40 and Aβ42) in the brain is a characteristic of Alzheimer disease (AD). Because neprilysin (NEP) is a major Aβ‐degrading enzyme, NEP delivery in the brain is a promising gene therapy for AD. Borna disease virus (BoDV) vector enables long‐term transduction of foreign genes in the central nerve system. Here, the proteolytic ability of NEP transduced by the BoDV vector was evaluated and it was found that the amounts of Aβ40 and Aβ42 decreased significantly, suggesting that NEP expressed from the BoDV vector is functional in that it degrades Aβ.

     

Further Characterization of a Myelin-Associated Neuraminidase: Properties and Substrate Specificity

A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.     

No evidence for linkage between the loci for coagulation factor XIII-A and HLA

Summary The linkage analysis between the locus for coagulation factor XIII-A (F13A) and HLA region genes (HLA-A,-C,-B) was performed. In males, the maximum of lod scores between F13A and HLA was 0.33 at =0.30, and in females lod scores were negative at all values of . The results provided no evidence for close linkage between F13A and HLA genes.