Studies on the Corrinoids and Porphyrins in Streptomycetes

This paper deals with the confirmation of the existence of a cobalamin-dependent terminal step in the methionine synthesis, that is, the methyl transfer from N⁵-CH₃-H₄-folate to homocysteine to form methionine, in the cell-free extracts of Streptomyces olivaceus 605.

This transmethylation reaction required a reducing system and S-adenosylmethionine (SAM) as cofactors.

Methionine formation from serine was observed in the cell-free system and thus this reaction is considered to participate in a series of one-carbon metabolism from serine.     

Induction of hepatic microsomal propranolol N-desisopropylase activity by 3-methylcholanthrene and sudan III

Metabolism of propranolol in liver microsomes was markedly induced in rats and $\text{C57BL}\text{6J}$ mice treated with 3-methylcholanthrene (3-MC) or sudan III, inducers of cytochrome P-448. 7,8 Benzoflavone inhibited propranolol metabolism in microsomes from treated rats. 3-MC did not induce propranolol metabolism in genetically nonresponsive $\text{DBA}\text{2}$ mice. High-performance liquid chromatographical analysis of propranolol metabolites revealed a 6-fold increase in propranolol N-desisopropylase activities in liver microsomes from sudan III- or 3-methylcholanthrene-treated rats. It is concluded that propranolol N-desisopropylation is predominantly catalyzed by cytochrome P-448.     

Alternative splicing of PDLIM3/ALP, for α-actinin-associated LIM protein 3, is aberrant in persons with myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder of muscular dystrophy characterized by muscle weakness and wasting. DM1 is caused by expansion of CTG repeats in the 3′-untranslated region (3′-UTR) of DM protein kinase (DMPK) gene. Since CUG-repeat RNA transcribed from the expansion of CTG repeats traps RNA-binding proteins that regulate alternative splicing, several abnormalities of alternative splicing are detected in DM1, and the abnormal splicing of important genes results in the appearance of symptoms. In this study, we identify two abnormal splicing events for actinin-associated LIM protein 3 (PDLIM3/ALP) and fibronectin 1 (FN1) in the skeletal muscles of DM1 patients. From the analysis of the abnormal PDLIM3 splicing, we propose that ZASP-like motif-deficient PDLIM3 causes the muscular symptoms in DM. PDLIM3 binds α-actinin 2 in the Z-discs of muscle, and the ZASP-like motif is needed for this interaction. Moreover, in adult humans, PDLIM3 expression is highest in skeletal muscles, and PDLIM3 splicing in skeletal muscles is regulated during human development.     

The modulation of corticospinal excitability during motor imagery of actions with objects

We investigated whether corticospinal excitability during motor imagery of actions (the power or the pincer grip) with objects was influenced by actually touching objects (tactile input) and by the congruency of posture with the imagined action (proprioceptive input). Corticospinal excitability was assessed by monitoring motor evoked potentials (MEPs) in the first dorsal interosseous following transcranial magnetic stimulation over the motor cortex. MEPs were recorded during imagery of the power grip of a larger-sized ball (7 cm) or the pincer grip of a smaller-sized ball (3 cm)--with or without passively holding the larger-sized ball with the holding posture or the smaller-sized ball with the pinching posture. During imagery of the power grip, MEPs amplitude was increased only while the actual posture was the same as the imagined action (the holding posture). On the other hand, during imagery of the pincer grip while touching the ball, MEPs amplitude was enhanced in both postures. To examine the pure effect of touching (tactile input), we recorded MEPs during imagery of the power and pincer grip while touching various areas of an open palm with a flat foam pad. The MEPs amplitude was not affected by the palmer touching. These findings suggest that corticospinal excitability during imagery with an object is modulated by actually touching an object through the combination of tactile and proprioceptive inputs.     

Subunit‐dependent inhibition and potentiation of 5‐HT3 receptor by the anticancer drug,topotecan

The 5‐hydroxytryptamine (serotonin, 5‐HT) type 3 (5‐HT3) receptor belongs to the superfamily of Cys‐loop ligand‐gated ion channels, and can be either homopentameric (5‐HT3A) or heteropentameric (5‐HT3AB) receptor. Several modulators are known, which either inhibit or potentiate this channel, but few have any appreciable selectivity between the two subtypes or can modulate one receptor differently to the other. In this study, we show that the anticancer drug, topotecan, bidirectionally modulates the 5‐HT3 receptor using a two‐electrode voltage clamp technique. Topotecan inhibited 5‐HT‐gated current through homomeric 5‐HT3A receptors. Interestingly, however, additional expression of the 5‐HT3B subunit changed the response to topotecan dramatically from an inhibitory to a potentiatory one. This effect was dependent on the level of 5‐HT3B subunit expression. Moreover, the effect was reduced in the receptors containing the 5‐HT3B(Y129S) polymorphic variant. These finding could explain individual differences in the sensitivity to topotecan‐induced nausea and vomiting.     

Immobilization of β-Galactosidase by Polyacrylamide in the Presence of Protective Agents

β-Galactosidase from Aspergillus oryzae was immobilized in crosslinked polyacrylamide gel beads. The presence of the enzyme inhibitor, such as glucono-δ-lactone or galactono-γ-lactone, during polymerization procedure enhanced the residual enzymatic activity in the polymer beads, and activity yield attained up to 45%. Such enhancement effect was also observed when bovine serum albumin, dithiothreitol or glutathione was added during polymerization. Temperature and pH optima were not affected by the immobilization. The Michaelis constants for free and immobilized β-galactosidase were comparable. Lyophilized beads exhibited good stability without loss of enzymatic activity when stored at 4°C for 47 days.     

Purification and Substrate Specificity of Glucoamylase of Paecilomyces varioti AHU 9417

A glucoamylase was purified from the culture broth of Paecilomyces varioti AHU 9417 by precipitation with ethanol, chromatography on DEAE-Sepharose CL-6B, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was estimated to be 6.9×10⁴ by SDS-disc electrophoresis, and the optimum pH was 4.5. The enzyme preferentially hydrolyzes α-1,4-glucosidic linkages in a series of maltooligosaccharides, and is capable to slowly hydrolyzing nigerose, isomaltose, and panose, but is not active on kojibriose. Soluble starch, amylopectin, glycogen, and β-limit dextrin are rapidly degraded. Activity toward raw starches is very low, but rice and waxy corn raw starches are relatively subject to attack. The subsite affinities (Aⁱ) of each subsite (i) in the active site of the enzyme were evaluated from the Michaelis constants (Km) and the molecular activities (k₀) for a series of maltooligosaccharides according to the subsite theory. The active site was considered to be made up of about four subsites: A₁?0.46 kcal/mol, A₂?4.78 kcal/mol, A₃?1.76 kcal/mol and A₄?0.67 kcal/mol.     

Riboflavin Biosynthesis by Candida robusta

During the study on the oxidative sugar metabolism of yeasts, it was found that six strains of Candida robusta, isolated from fruits, produced large amounts of yellow pigment in shaking culture and this pigment was identified as riboflavin. Riboflavin production by C. robusta has never been reported.

Some notable characteristics of C. robusta in riboflavin production were found. As nitrogen sources, ammonium salts and urea were favorable, but nitrate and organic nitrogen sources such as glycine, asparagine and peptone were not utilized for riboflavin production. Riboflavin was not produced in still culture; a highly aerobic condition, as may be obtained by shaking culture, wa, essential. The addition of excess CaCO₃ was also necessary. Acetic acid, added as the Ca salt in its production as a sole carbon source, was more effective than sugars and optimum concentration of this acid was 7%. Riboflavin were obtained in yields as high as 32 to 34 mg % from the acetate medium after 8 days.