Purification and Properties of a Pectate Lyase in Erwinia aroideae

A strain of Erwinia aroideae produced an extracellular pectolytic enzyme under growth conditions with pectin or pectic acid as the inducer. This strain also produced a pectin lyase when nalidixic acid is added to a culture medium. The pectolytic enzyme produced under the growth conditions was purified approximately 40-fold from the culture fluid by carboxy- methyl cellulose and Sephadex G-75 gel column chromatographies. The purified enzyme was almost homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, having a molecular weight of about 36,000 to 38,000. This enzyme, with optimal activity at pH 9.0 to 9.2, produced reaction products which had a strong absorption at 230 nm indicating a lyase type of the reaction. The enzyme activity was markedly stimulated by calcium ion and completely inhibited by cobalt and mercuric ions and by ethylenediaminetetraacetate. Pectic acid or pectin with lower methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectin with higher methoxyl content was used as a substrate. It was concluded that the enzyme produced under the normal growth conditions is an endo-pectate lyase and differs from the pectin lyase induced by nalidixic acid.     

Anti‐barnacle Activity of Isocyanides Derived from Amino Acids

Creation of new potent antifouling active compounds is important for the development of environmentally friendly antifouling agents. Fifteen isocyanide congeners derived from proteinogenic amino acids were synthesized, and the antifouling activity and toxicity of these compounds against cypris larvae of the barnacle Balanus amphitrite were investigated. All synthesized amino acid‐isocyanides exhibited potent anti‐barnacle activity with EC₅₀ values of 0.07 – 10.34 μg/ml after 120 h exposure without significant toxicity. In addition, seven compounds showed more than 95% settlement inhibition of the cypris larvae at 10 μg/ml after 120 h exposure without any mortality observed. Considering their structure, these amino acid‐isocyanides would eventually be biodegraded to their original nontoxic amino acids. These should be useful for further research focused on the development of environmentally friendly antifoulants.     

Anti-Tumor Effects of Second Generation β-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression

Cholangiocarcinoma (CCA) has a poor prognosis due to widespread intrahepatic spread. Aspartate β-hydroxylase (ASPH) is a transmembrane protein and catalyzes the hydroxylation of aspartyl and asparaginyl residues in calcium binding epidermal growth factor (cbEGF)-like domains of various proteins, including Notch receptors and ligands. ASPH is highly overexpressed (>95%) in human CCA tumors. We explored the molecular mechanisms by which ASPH mediated the CCA malignant phenotype and evaluated the potential of ASPH as a therapeutic target for CCA. The importance of expression and enzymatic activity of ASPH for CCA growth and progression was examined using shRNA “knockdown” and a mutant construct that reduced its catalytic activity. Second generation small molecule inhibitors (SMIs) of β-hydroxylase activity were developed and used to target ASPH in vitro and in vivo. Subcutaneous and intrahepatic xenograft rodent models were employed to determine anti-tumor effects on CCA growth and development. It was found that the enzymatic activity of ASPH was critical for mediating CCA progression, as well as inhibiting apoptosis. Mechanistically, ASPH overexpression promoted Notch activation and modulated CCA progression through a Notch1-dependent cyclin D1 pathway. Targeting ASPH with shRNAs or a SMI significantly suppressed CCA growth in vivo.     

4-Vinylphenol production from glucose using recombinant <Emphasis Type="Italic">Streptomyces mobaraense</Emphasis> expressing a tyrosine ammonia lyase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Objectives

To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species.

Results

The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme’s k_cat/K_m value was 0.54 mM ^?1 s^?1. Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l^?1 from 10 g glucose l^?1.

Conclusion

A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.

     

Vitamin B12 deficiency-induced increase of osteoclastic bone resorption caused by abnormal renal resorption of inorganic phosphorus via Napi2a

Vitamin B₁₂ deficiency is a risk factor for bone disorders via mechanisms not fully understood. In this study, an increase in serum inorganic phosphorus (Pi) concentrations was associated with a vitamin B₁₂ deficiency. Napi2a, a renal cotransporter for Pi reabsorption, accumulated on plasma membranes in a vitamin B₁₂ deficiency suggests that vitamin B₁₂ plays an important role in Pi homeostasis.     

Prion infection correlates with hypersensitivity of P2X7 nucleotide receptor in a mouse microglial cell line

We recently established mouse microglial cells persistently infected with mouse-adapted scrapie ME7 (ScMG20/ME7) for in vitro study of prion pathogenesis. Here, we found that ScMG20/ME7 cells were hypersensitive to P2X7 receptor agonists, as demonstrated by sustained Ca(2+) influx, membrane pore formation, cell death, and interleukin-1beta release. P2X7 mRNA expression was upregulated in these cells, and also in scrapie-infected mice brains. Treatment with pentosan polysulfate eliminated the infectivity and disease-related forms of prion protein from ScMG20/ME7 cell cultures, however, hypersensitivity of P2X7 receptors remained. These results suggest that prion infections may strongly affect the P2X7 receptor system in mouse microglial cells.     

Generation and maintenance of suspension cultures from cotyledons and their organogenic potential of two mangrove species, Sonneratia alba and S. caseolaris

Liquid cultures were successfully generated from cotyledons of two Sonneratia species, S. alba and S. caseolaris in Murashige and Skoog (MS) medium containing 0.1 μmol L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious roots differentiated from cotyledons of S. alba. Proliferated cells were subcultured and a large volume of suspension cells was subsequently established in 100-mL flasks. All the cytokinins tested inhibited cell proliferation. After three years of culture, the potential to differentiate was tested as indicated by greening of the cells. Greening occurred when suspension cells were transferred to solid MS medium with and without 0.1 μmol L⁻¹ 2,4-D. Greening was stimulated by low concentrations of the weak auxins indolebutyric acid (IBA) and naphthaleneacetic acid (NAA) while 2,4-D stimulated late-stage greening. Abscisic acid (ABA) inhibited greening. Gibberellic acid (GA₃) at 1.0 μmol L⁻¹ stimulated callus greening and was not inhibitory even when tested at high concentrations. Cytokinins were inhibitory in combination with 0.1 μmol L⁻¹ of either IBA or NAA. The cause of different effects of plant hormones on growth and differentiation was discussed. Small-scale liquid media and 24-well culture plates of solid media methods developed in this paper are suitable for the optimization of hormonal conditions for cell proliferation and differentiation.     

Physiological effects of Shinrin-yoku (taking in the atmosphere of the forest)--using salivary cortisol and cerebral activity as indicators

The purpose of this study is to examine the physiological effects of Shinrin-yoku (taking in the atmosphere of the forest). The subjects were 12 male students (22.8+/-1.4 yr). On the first day of the experiments, one group of 6 subjects was sent to a forest area, and the other group of 6 subjects was sent to a city area. On the second day, each group was sent to the opposite area for a cross check. In the forenoon, the subjects were asked to walk around their given area for 20 minutes. In the afternoon, they were asked to sit on chairs and watch the landscapes of their given area for 20 minutes. Cerebral activity in the prefrontal area and salivary cortisol were measured as physiological indices in the morning at the place of accommodation, before and after walking in the forest or city areas during the forenoon, and before and after watching the landscapes in the afternoon in the forest and city areas, and in the evening at the place of accommodation. The results indicated that cerebral activity in the prefrontal area of the forest area group was significantly lower than that of the group in the city area after walking; the concentration of salivary cortisol in the forest area group was significantly lower than that of the group in the city area before and after watching each landscape. The results of the physiological measurements show that Shinrin-yoku can effectively relax both people's body and spirit.