Aeromonas molluscorum Av27 is a potential tributyltin (TBT) bioremediator: phenotypic and genotypic characterization indicates its safe application

Aeromonas molluscorum Av27 is an estuarine bacterium highly resistant to tributyltin (TBT). Also, the strain is able to degrade TBT into the less toxic compounds dibutyltin and monobutyltin. Therefore, this bacterium has potential to be employed in bioremediation processes. In this context, defining its biological safety is crucial. With that purpose a number of intrinsic characteristics, usually present/associated with virulent strains, were investigated. Few virulence factors were detected in strain Av27. For instance, a DNase gene is present, but it is not apparently expressed in vitro. Motility, adherence factor and phospholipase activity were also detected. Additionally, cytotoxicity to Vero cells was negative. Resistance to penicillin (10 μg ml^?1), amoxicillin/clavulanic acid (30 μg ml^?1) and cephalothin (30 μg ml^?1) and also to the vibriostatic agent O/129 was observed. Five plasmids (4, 7, 10, 100 kb and one greater than 100 kb) were identified. No Class I and II integrons were detected. Study of the optimal growth conditions showed that Av27 easily adapts to different environmental conditions. Overall, the results suggest that A. molluscorum Av27 can be considered safe to use to bioremediate TBT in contaminated environments.     

Discovery and structure–activity relationship of thienopyridine derivatives as bone anabolic agents

A cell-based assay was performed for the discovery of novel bone anabolic agents. Alkaline phosphatase (ALPase) activity of ST2 cells was utilized as an indicator of osteoblastic differentiation, and thienopyridine derivative 1 was identified as a hit compound. 3-Aminothieno[2,3-b]pyridine-2-carboxamide was confirmed to be a necessary core structure for the enhancement of ALPase activity, and then optimization of the C4-substituent on the thienopyridine ring was carried out. Introduction of cyclic amino groups to the C4-position of the thienopyridine ring improved the activity. Especially, N-phenyl-homopiperazine derivatives were found to be strong enhancers of ALPase among this new series. Furthermore, 3-amino-4-(4-phenyl-1,4-diazepan-1-yl)thieno[2,3-b]pyridine-2-carboxamide (15k) was orally administered to ovariectomized (OVX) rats over 6 weeks for evaluating the effects on areal bone mineral density (aBMD), and statistically significant improvements in aBMD were observed from the dosage of 10 mg/kg/day.     

Studies on Transfer of Antiseptics to Microbes and their Toxic Effect

The adsorption of phenols and esters of acid antiseptics by the bacterial cell (Escherichia coli and Staphylococcus aureus) was investigated in relatoin to their toxic effect, and it has been observed that the definite quantity of antiseptics must be adsorbed on the solid phase of the bacterial cell in order to give the definite toxic effect, and the toxic effect is independent of the quantity dissolved in the inner cell fluid or in the lipid phase of the cell. The result shows that the toxic effect of these antiseptics on either bacteria and yeast, is exclusively limited by the adsorbed quantity.

The adsorbed quantity required for the definite toxic effect was nearly the same as that previously observed in the case of the yeast, and the mechanism of the toxic action of these antiseptics was assumed to be same with each other in any case of microbes.     

Permeability and Selective Toxicity of Nitrofurane Compounds

The bacterial growth is inhibited by nitrofurane compounds, although the yeast growth is hardly affected. In relation to the selective toxicity of nitrofuranes for bacteria, the interaction between microbes (Escherichia coli, Staphylococcus aureus and bakers– yeast) and nitrofurane compounds (5-nitro-2-furfural semicarbazone and 5-nitro-2-furylacryl amide) was examined.

Apparently, in the bacterial suspension containing energy substrate, nitrofuranes are continuously reduced to corresponding aminofuranes, respectively. The velocity of the bacterial reduction at the growth inhibiting condition was evaluated as great as above 30 per cent of the limit of supplying velocity of coenzymes in the cell, the reduction velocity of such value is enough to inhibit the bacterial growth, because the electron transfer in the cell metabolism is disordered.

On the other hand, in the yeast suspension, the reduction velocity was negligibly small. The difference of the reduction ability between bacteria and yeast was seemingly owing to the fact that the permeability of the nitrofuranes differs by the kind of microbe so that it was concluded that the antimicrobial effect of nitrofuranes is limited by the permeability for the microbe cell.     

Comparison of Mineral Balances in Germfree and Conventional Mice When Sodium Phytate is Added to Purified Diet

A strain of Alcaligenes isolated from soil was a good producer of β-glucuronidase, and the enzyme was purified from the cell-free extract by sequential column chromatography on DEAE-Toyopearl, Toyopearl HW-55F, and Phenyl-Sepharose CL-4B. By these procedures, two β-glucuronidases designated as β-glucuronidases I and II were purified 240- and 508-fold, respectively. β-Glucuronidase I, with a molecular weight of 75,000, had an optimum pH at 7.5 and the enzyme II, with a molecular weight of 300,000, had maximum activity at pH 6.0. Both enzymes were strongly inhibited by saccharo-1,4-lactone, glucaro-δ-lactam, p-chloromercuribenzoate, Hg²⁺, and N-bromosuccinimide. β-Glucuronidase I was active toward estrogen-3-β-glucuronides and inert toward β-glucuronide conjugates of menthol, estrogen-17β-, estrogen-16α-, androsterone-3α-, testosterone-17β-, cortisol-17α-. β-Glucuronidase II hydrolyzed all of these substrates. β-Glucuronidase I was inhibited by phenolphthalein and its glucuronide.     

Purification and Some Properties of Arthrobacter globiformis Exo-l,6-α-glucosidase

A gram-positive and pleomorphic bacterium (strain I-42) isolated from soil as a producer of exo-l,6-α-glucosidase [EC 3.2.1.70] was identified as Arthrobacter globiformis. This Arthrobacter enzyme, inducible by dextran extracellularly, was partially purified from a cell-free culture supernatant. It was found most active at pH around 6.0 and most stable at pH 6.0~6.5. The enzyme was proved, by several experiments, to attack dextran in the exo-wise fashion to release only glucose leaving a macromolecular limit dextrandextrin. Transglucosylation from dextran to accumulating or added glucose was not observed.     

The Chromatographic Purification of Yeast Invertase by an Ion-Exchange Resin Method and Some Properties of the Enzyme Obtained

The purification of yeast invertase was attempted by application of the chromatographic method using Duolite C-10, a sulfonic acid cation exchange resin. This method was found to be extremely simple in process and significantly effective for the improvement of purity of the enzyme, compared with those other methods reported, hitherto. In the present paper, the procedure of the purification and some properties of the enzyme obtained thereby, are described, and some discussion of the implications is presented.     

Studies on γ Globulin of Rice Embryo

An improved method has been described for the isolation and purification of γ globulin from rice embryo. The method involves the extraction with phosphate buffer, pH 7.0 and ionic strength 0.1, the fractionation in saline solution of ionic strength 0.31, the removal of nucleic acids by precipitation with ammonium sulfate and the gel filtration chromatography on a Sephadex G-200 column. Although the preparations exhibited homogeneous patterns in sedimentation analysis, the electrophoretic patterns on polyacrylamide gels at pH 8.35 and ionic strength 0.11 exhibited at least two components. Three major components, γ₁, γ₂ and γ₃ globulins, were isolated by ion exchange chromatography on a DEAE Sephadex A-50 column. These components were revealed to be homogeneous in electrophoresis as well as sedimentation. N-Terminal amino acid compositions have also been described.

The molecular weight of γ₁ globulin was determined as 2.0 × 10⁵ by the Archibald method, and the intrinsic viscosity, [η], and the sedimentation coefficient, s_{20, w}^°, were found to be 0.0424 dl/g and 7.26S respectively. These values indicated the large asymmetry of the protein. The protein was composed of 18 residues of hexose, 3 residues of pentose, 6 residues of hexosamine and 1751 residues of amino acids: Lys₅₈, His₄₇, Arg₁₄₈, Asp₁₂₆, Glu₂₇₃, Gly₁₆₁, Ala₁₄₄, Val₁₂₁, Leu₁₀₆, Ile₇₂, Pro₈₃, Ser₁₃₆, Thr₄₈, Hyp₆₈, Cys₁₇, Met₁₆, Tyr₄₄, Trp₈, Phe₇₅ and amide ammonia₁₆₃. The N-terminal amino acid analysis suggested that the protein was composed of ten subunits. The properties and the composition were discussed in comparison with those of the 7S globulin of soybean cotyledon.     

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs’ program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.     

Relationship between G1287A of the NET Gene Polymorphisms and Brain Volume in Major Depressive Disorder: A Voxel-Based MRI Study

Background

Earlier studies implicated norepinephrine transporter (NET) gene (SLC6A2) polymorphisms in the etiology of major depressive disorder (MDD). Recently, two single nucleotide SLC6A2 polymorphisms, G1287A in exon 9 and T-182C in the promoter region, were found to be associated with MDD in different populations. We investigated the relationship between the brain volume and these two polymorphisms of the SLC6A2 in MDD patients.

Methods

We obtained 3D high-resolution T1-weighted images of 30 first-episode MDD patients and 48 age- and sex-matched healthy subjects (HS). All were divided into 4 groups based on polymorphism of either the G1287A or the T-182C genotype. VBM analysis examined the effects of diagnosis, genotype, and genotype-diagnosis interactions.

Results

Diagnosis effects on the brain morphology were found in the left superior temporal cortex. No significant genotype effects were found in the T-182C and the G1287A. A significant genotype (G1287A)–diagnosis interaction was found in the left dorsolateral prefrontal cortex. No significant genotype (T-182C)–diagnosis interaction effects were observed in any brain region.

Conclusions

In MDD patients there seems to be a relationship between the volume of the dorsolateral prefrontal cortex and polymorphism of the SLC6A2 G1287A gene.