Production of 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) and Its Derivatives by Vibrio tyrosinaticus

Six strains of bacteria belonging to Vibrio and Pseudomonas were selected as good producers of L-DOPA from L-tyrosine out of various bacteria. The condition for the formation of L-DOPA by Vibrio tyrosinaticus ATCC 19378 was examined and the following results were obtained. (1) Intermittent addition of L-tyrosine in small portions gave higher titer of L-DOPA than single addition of L-tyrosine. (2) Higher amount of L-DOPA was produced in stationary phase of growth than in logarithmic phase. (3) Addition of antioxidant, chelating agent or reductant such as L-ascorbic acid, araboascorbic acid, hydrazine, citric acid and 5-ketofructose increased the amount of L-DOPA formed. (4) L-Tyrosine derivatives such as N-acetyl-L-tyrosine amide, N-acetyl-L-tyrosine, L-tyrosine amide, L-tyrosine methyl ester and L-tyrosine benzyl ester were converted to the corresponding L-DOPA derivatives.

In the selected condition about 4 mg/ml of L-DOPA was produced from 4.3 mg/ml of L-tyrosine.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.     

Novel application of Shochu distillery by-products to prophylaxis against mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthracene in rats

The effect of the heat-dried product of Shochu distillery by-products (HSDB) derived from sweet potato on mammary carcinogenesis in rats was investigated. HSDB was fed at 2.5% or 5% of the total feed weight. Dietary HSDB at the 5% level suppressed the incidence and number of tumors, and delayed the latency of mammary tumor development relative to the control diet. Experiments were conducted to determine the relative polarity of the anticarcinogenic constituent(s). The number of tumors per tumor-bearing rat was lower in the diet group fed with an ethyl acetate extract of HSDB than in the control group. The tumor incidence evaluated at both palpation and autopsy was slightly lower in the group fed with a methanol extract than in the control group. These results suggest that HSDB contained at least two constituents of differing polarity that counteracted mammary carcinogenesis.     

Regulation of apolipoprotein E synthesis and mRNA by diet and hormones

Rats were fasted or fasted and refed simple purified diets so the effects of individual carbohydrates or fats could be studied. Freshly isolated hepatocytes from these animals were used to measure both apoE synthesis and mRNA levels so any changes in apoE synthesis that might occur without changes in its mRNA could be detected. Some of these experiments were done with both sexes. Both fasting and fasting and refeeding a 60% glucose fat-free diet significantly increased spoE synthesis. However, cyclic AMP is not likely to rapidly mediate the effect of fasting since dibutyryl cAMP slightly lowered (rather than increased) apoE synthesis and mRNA when injected into rats for 4.5 h. Dietary fat had no effect either in the absence of carbohydrate or when consumption of carbohydrate was constant in pair-fed rats. ApoE mRNA levels remained normal for 4 days in primary hepatocytes cultured in medium that had only amino acids as an energy source. Added hormones or fructose had no significant effect. Thus, only fasting and fasting and refeeding glucose were able to significantly change apoE synthesis or mRNA levels. Synthesis of apoE may be regulated to increase when apoE is secreted with very low density lipoprotein or when apoE in secreted high density lipoprotein is needed to acquire cholesteryl esters for the synthesis of bile salts and acids by liver.     

The clinicopathological significance of angiogenesis in hindgut neuroendocrine tumors obtained via an endoscopic procedure

Background

As the World Health Organization grading system for gastroenteropancreatic-neuroendocrine tumors (GEP-NETs) may not always correlate with tumor progression, it is imperative that other independent predictors of tumor progression be established. To identify such predictors, we conducted a retrospective histopathological study of hindgut NETs, obtained from endoscopic procedures, and used statistical analyses to evaluate predictive factors.

Methods

We first obtained clinicopathological data of cases of hindgut NETs. Tissue sections from tumor samples were prepared and subjected to pathological examination. In particular, we calculated the microvessel density (MVD) and lymphatic microvessel density (LMVD) values, and performed appropriate statistical analyses.

Results

A total of 42 cases of hindgut NETs were selected for the study, 41 from the rectum and 1 from the sigmoid colon. Based on the Ki-67 labeling index, 34 cases were classified as NET G1 tumors and 8 as NET G2 tumors. MVD values ranged from 1.4/mm² to 73.9/mm² and LMVD values from 0/mm² to 22.9/mm². MVD and LMVD were identified as risk factors for venous and lymphatic invasion of hindgut NETs. Moreover, MVD positively correlated with the maximum diameter of the tumor.

Conclusions

Tumor progression of NETs may cause angiogenesis and lymphangiogenesis, via an unknown mechanism, as well as lymphovascular invasion. Angiogenesis likely plays an important role in occurrence and progression in the initial phase of hindgut NETs.

     

Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea; therefore, the T9SS may be involved in biofilm formation by C. ochracea. Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae). Gliding motility was lost in each mutant, suggesting that, in C. ochracea, the proteins encoded by sprB, gldK, and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea.     

Involvement of chalcone reductase in the soybean isoflavone metabolon: identification of GmCHR5, which interacts with 2‐hydroxyisoflavanone synthase

Soybean (Glycine max) 5‐deoxyisoflavonoids (daidzein and its conjugates) are precursors of glyceollin phytoalexins. They are also converted to equol by microbes in the human intestine, resulting in health benefits. 5‐Deoxyisoflavonoids accumulate in the roots (93% mol/mol of the total root isoflavonoids) and seeds of unstressed soybean plants. Chalcone reductase (CHR) is a key enzyme mediating 5‐deoxyisoflavonoid biosynthesis because it catalyzes the production of 6′‐deoxychalcone through its effects on the chalcone synthase (CHS)‐catalyzed reaction. The soybean genome encodes at least 11 CHR‐related homologs, but it is unclear which ones are functionally important for daidzein accumulation in unstressed plants. Among the CHR homologs, the temporal and spatial expression patterns of GmCHR5 were the most correlated with the distribution patterns of 5‐deoxyisoflavonoids. The CHR activity of GmCHR5 was confirmed in vitro and in planta. In the in vitro assays, the ratio of CHR products (6′‐deoxychalcone) to total CHS products (R value) was dependent on GmCHR5 and CHS concentrations, with higher concentrations resulting in higher R values (i.e. approaching 90%). Subcellular localization analyses revealed that GmCHR5 was present in the cytoplasm and nucleus. Protein–protein interaction assays indicated that GmCHR5, but not GmCHR1 and GmCHR6, interacted with 2‐hydroxyisoflavanone synthase (IFS) isozymes. The CHS isozymes also interacted with IFS isozymes but not with GmCHR5. The proposed micro‐compartmentalization of isoflavone biosynthesis through the formation of an IFS‐mediated metabolon is probably involved in positioning GmCHR5 close to CHS, resulting in an R value that is high enough for the accumulation of abundant 5‐deoxyisoflavonoids in soybean roots.