Biological Activity and Mode of Action of a Specific Antiphage Substance,AFS

Purified AFS (anti-filamentous phage substance) produced by Streptomyces lavendulae AM–7a showed specific antiphage activity against the male specific, deoxyribonucleic acid-containing filamentous phages of Escherichia coli without any activity against other DNA-phages nor the male-specific ribonucleic acid-containing phages of E. coli. AFS brought about no inactivation of free particles of filamentous phage, fl, nor the receptor of the host cells for the phage, while it showed strong killing effect against the fl-infected host cells at the concentration below 0.01 μg/ml. Antiphage activity of AFS might be due to its highly specific killing effect only on the E. coli cells infected with the filamentous DNA phages, while it exerted no effect on the growth of the unifected E. coli nor other microorganisms. Killing by AFS seemed to require the energy metabolism of the phage-infected host cells. Macro-molecular synthesis and respiration of the infected host cells were inhibited soon after the addition of small amounts of AFS without any cell lysis.     

A Metaphosphate-dependent Nicotinamide Adenine Dinucleotide Kinase from Brevibacterium ammoniagenes

The enzyme utilizing metaphosphate for nicotinamide adenine dinucleotide phosphorylation was purified 500-fold from B. ammoniagenes and its properties were studied. The isolated enzyme appeared homogeneous on disc gel electrophoresis; its molecular weight was determined to be 9.0 × 10⁴ by gel filtration. This enzyme specifically phosphorylated nicotinamide adenine dinucleotide at the optimum pH at 6.0. Of phosphoryl donors tested, metaphosphate was most effective for the reaction, and adenosine-5′-triphosphate was less effective. The activity was inhibited by adenosine-5′-monophosphate, adenosine-5′-diphosphate or reduced pyridine nucleotides. The enzyme did not exhibit catalytic activity in the absence of a divalent cation. We concluded that the enzyme phosphorylating nicotinamide adenine dinucleotide in the presence of metaphosphate is distinct from adenosine-5′-triphosphate-dependent nicotinamide adenine dinucleotide kinase, and tentatively designated it metaphosphate-dependent nicotinamide adenine dinucleotide kinase.     

Stimulation of Methionine Sulfoxide Reduction and Ethylene Production by Isoprothiolane in Relation to Its Activity against Non-parasitic Damping-off (MURENAE Disease) of Rice Seedlings

A rice blast controlling agent, isoprothiolane (diisopropyl 1,3-dithiolan-2-ylidenemalonate), stimulated the reduction of methionine sulfoxide to methionine by the rice plant. In the presence of isoprothiolane, the methionine/(methionine + its sulfoxide) ratio was increased to 129~208% of the control. The ethylene production by the plant was also enhanced by isoprothiolane, probably because methionine is an important precursor of ethylene. The non-parasitic damping-off caused by chilling stress on rice seedlings was effectively prevented with the application of isoprothiolane as well as ethephon, which easily decomposes to ethylene and acids. Therefore, the ethylene level modified by isoprothiolane and ethephon can contribute to their protective activity against the non-parasitic damping-off of rice seedlings. Indeed, a close relationship between the ethylene level and the protective activity against damping-off was obtained with isoprothiolane, but not with ethephon. Endogenous ethylene seems to be more effective in controlling the damping-off than exogenous ethylene from ethephon.     

Production of 5′-dRiboflavin-α-d-glucopyranoside in Growing Cultures by the Mold Mucor

During the investigations on riboflavin glycoside formation by Aspergillus, Mucor, Penicillium and Rhizopus, a remarkable production of 5′-d-riboflavin-α-d-glucopyranoside was observed in several strains belonging to the genus Mucor when grown on a, medium containing maltose and riboflavin. Several conditions on 5′-d-riboflavin-α-d-glucopyranoside formation were also investigated with washed mycellium of M. javanicus. Maltosyl compounds such as maltose, dextrin, amylose and soluble starch were the effective glucosyl donor, whereas glucose, fructose, sucrose, lactose and dextran were inactive.     

Improved Microbial Production of Colominic Acid,a Homopolymer of N-Acetylneuraminic Acid

A culture medium has been devised for producing colominic acid in improved yields. Major improvements were obtained by using sorbitol as a source of carbon, by adding phosphate in high concentrations, and by supplementing a limited amount of yeast extract. E. coli O 16: Kl: HNM produced approximately 3000 µg/ml of colominic acid on cultivation at 37°C for 46 hr with a liquid medium consisting of sorbitol (2.0%), (NH₄)₂SO₄ (0.5%), K₂HPO₄ (1.4%), MgSO₄·7H₂O (0.05%), and yeast extract (0.05%).

Isolation and purification by deproteinization with ammonium sulfate, precipitation with ethanol, and by column chromatography on anion exchange resins resulted in a pure colominic acid preparation devoid of internal ester linkages.

In producing colominic acid, strains forming S-type colonies were more active than those forming R-type colonies.     

Ameloblastin in Hertwig’s Epithelial Root Sheath Regulates Tooth Root Formation and Development

Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig’s epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-2′-deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21^Cip1 and p27^Kip1 was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.     

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.     

Soluble Isoform of the Receptor for Advanced Glycation End Products as a Biomarker for Postoperative Respiratory Failure after Cardiac Surgery

Purpose

Postoperative respiratory failure is a major problem which can prolong the stay in the intensive care unit in patients undergoing cardiac surgery. We measured the serum levels of the soluble isoform of the receptor for advanced glycation end products (sRAGE), and we studied its association with postoperative respiratory failure.

Methods

Eighty-seven patients undergoing elective cardiac surgery were enrolled in this multicenter observational study in three university hospitals. Serum biomarker levels were measured perioperatively, and clinical data were collected for 7 days postoperatively. The duration of mechanical ventilation was studied for 28 days.

Results

Serum levels of sRAGE elevated immediately after surgery (median, 1751 pg/mL; interquartile range (IQR) 1080–3034 pg/mL) compared with the level after anesthetic induction (median, 884 pg/mL; IQR, 568–1462 pg/mL). Postoperative sRAGE levels in patients undergoing off-pump coronary artery bypass grafting (median, 1193 pg/mL; IQR 737–1869 pg/mL) were significantly lower than in patients undergoing aortic surgery (median, 1883 pg/mL; IQR, 1406–4456 pg/mL; p = 0.0024) and valve surgery (median, 2302 pg/mL; IQR, 1447–3585 pg/mL; p = 0.0005), and postoperative sRAGE correlated moderately with duration of cardiopulmonary bypass (r_s = 0.44, p<0.0001). Receiver operating characteristic curve analysis demonstrated that postoperative sRAGE had a predictive performance with area under the curve of 0.81 (95% confidence interval 0.71–0.88) for postoperative respiratory failure, defined as prolonged mechanical ventilation >3 days. The optimum cutoff value for prediction of respiratory failure was 3656 pg/mL, with sensitivity and specificity of 62% and 91%, respectively.

Conclusions

Serum sRAGE levels elevated immediately after cardiac surgery, and the range of elevation was associated with the morbidity of postoperative respiratory failure. Early postoperative sRAGE levels appear to be linked to cardiopulmonary bypass, and may have predictive performance for postoperative respiratory failure; however, large-scale validation studies are needed.