Effect of amines as activators on the alcohol-oxidizing activity of pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenase

Pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenases (PQQ-ADH) require ammonia or primary amines as activators in in vitro assays with artificial electron acceptors. We found that PQQ-ADH from Pseudomonas putida KT2440 (PpADH) was activated by various primary amines, di-methylamine, and tri-methylamine. The alcohol oxidation activity of PpADH was strongly enhanced and the affinity for substrates was also improved by pentylamine as an activator.     

Fermentative Production of l-Leucine with Auxotrophic Mutants of Corynebaeterium glutamicum

An auxotrophic mutant of Corynebaeterium glutamicum was found to accumulate a large amount of l-leucine in the culture medium. The nutritional requirement of the mutant is rather complex but it’s growth was most remarkably stimulated by l-phenylalanine. Acetate (1.5~3.0%) or pyruvate (3%) stimulated the l-leucine production. By a further mutagenic treatment, 329 mutants earring some defect in addition to phenylalanine auxotrophy were derived from the mutant No. 190. Among them, a histidine auxotrophic derivative produced twice as much l-leucine as the parent strain, i.e., the level of l-leucine production by this derivative reached 16 mg/ml in a medium containing 12% glucose, 1 % (NH₄)₂SO₄ and 2.5% CH₃COONH₄ as carbon and nitrogen sources. Some other auxotrophic markers such as isoleucine- (or threonine-), threonine-, purine(s)-, homoserine-, or methionine- auxotrophy also improved the L-leucine production by No, 190.     

A Root Growth-promoting Factor,Capillarol, from Artemisia capillaris Thunb

A root growth-promoting factor was isolated from Artemisia capillaris Thunb. The chemical structure of this compound was elucidated as methyl 3-(3-methylbut-2-enoyl)-4-hydroxycinnamate (capillarol) on the basis of its spectroscopic analysis. At 5×10^–4m this compound promoted rice root growth to 180% of the control value.     

Studies on Menthol Derivatives

(–)-Menthyl carbinol (1-(R)-methyl-3-(R)-hydroxymethyl-4-(S)-isopropylcyclohexane) (4) was prepared stereospecifically in good yield by treatment of formaldehyde with the Grignard reagent from (–)-menthyl chloride (2), which was prepared from (–)-menthol-(1-(R)-methyl-3-(R)-hydroxy-4-(S)-isopropylcyclohexane) (1) by chlorination using the Lucas reagent (HCl+ZnCl₂). The configuration of 4 was assigned by the chemical method.     

Novel mutations of CDKN1C in Japanese patients with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an imprinting-related human disease that is characterized by macrosomia, macroglossia, abdominal wall defects, and variable minor features. BWS is caused by several genetic/epigenetic alterations, such as loss of methylation at KvDMR1, gain of methylation at H19-DMR, paternal uniparental disomy of chromosome 11, CDKN1C mutations, and structural abnormalities of chromosome 11. CDKN1C is an imprinted gene with maternal preferential expression, encoding for a cyclin-dependent kinase (CDK) inhibitor. Mutations in CDKN1C are found in 40 % of familial BWS cases with dominant maternal transmission and in ~5 % of sporadic cases. In this study, we searched for CDKN1C mutations in 37 BWS cases that had no evidence for other alterations. We found five mutations—four novel and one known—from a total of six patients. Four were maternally inherited and one was a de novo mutation. Two frame-shift mutations and one nonsense mutation abolished the QT domain, containing a PCNA-binding domain and a nuclear localization signal. Two missense mutations occurred in the CDK inhibitory domain, diminishing its inhibitory function. The above-mentioned mutations were predicted by in silico analysis to lead to loss of function; therefore, we strongly suspect that such anomalies are causative in the etiology of BWS.     

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.     

Inhibition of the Growth Factor MDK/Midkine by a Novel Small Molecule Compound to Treat Non-Small Cell Lung Cancer

Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.     

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.