Isolation and Identification of Microorganism,Producing Microbial Alkaline Proteinase Inhibitor (MAPI)

In the screening of actinomycetes’ culture filtrate for inhibitor of subtilisin and various microbial alkaline proteinases, a novel inhibitor was found in a cultured broth of strain WT-27. This inhibitor was named as MAPI, abbreviation of microbial alkaline proteinase inhibitor.Judging from the morphological and physiological properties of the actinomycetes which produced MAPI, this strain was identified as Streptomyces nigrescens.

For the production of MAPI, this strain was aerobically cultured at 25 ~ 27%C in a jar fermentor which contained an optimum medium consisting of polypepton 3 %, meat extract 1%, glucose 1%, NaCl 0.1%, K₂HPO₄ 0.1% and MnSO₄·nH₂O 0.0001%, pH 7.0. The production of MAPI reached its maximum after 21 ~ 24 hr cultivation.

MAPI had an inhibitory activity against various microbial alkaline proteinases, α-chymotrypsin and papain but not against trypsin, kallikrein, thermolysin, or pepsin.     

Isolation and Characterization of Nα-Benzyloxycarbonyl Amino Acid Urethane Hydrolase II from Lactobacillus fermenti 36 ATCC 9338

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase II, was purified from a cell-free extract of Lactobacillus fermenti 36 ATCC 9338. The enzyme catalyzed the stoichiometric hydrolysis of N^α-benzyloxycarbonyl arginine to form benzyl alcohol and arginine. The enzyme was purified 106-fold with an activity yield of 3%. The purified enzyme was homogeneous by disc gel electrophoresis. The molecular weight of the native enzyme is about 200,000 by gel filtration, and a molecular weight of 27,000 was found for the reduced and denaturated enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the enzyme was 5.0, it was inhibited by disodium ethylenediamine tetraacetate and p-chloromercuribenzoate, and the presence of a divalent cation, i.e. Co²⁺, is essential for its activity.     

Peptide hydrogen exchange rates in Streptomyces subtilisin inhibitor.

The exchange reaction of the peptide NH protons of a microbial protease inhibitor (Streptomyces subtilisin inhibitor) with deuterium atoms in 2H2O (p2H 6.8) has been studied by proton magnetic resonance in the temperature range 56-71 degrees C. Both slowly and rapidly exchanging processes have been observed. The number of slowly exchanging protons is estimated to be 25 +/- 2 per subunit of the protein molecule. The decay of the slowly exchanging proton signals follows a single time-exponential function at each temperature. The observed first-order rate constants have been analyzed to give the denaturated fraction of the protein as a function of temperature with a consequent enthalpy (56 kcal/mol) and an entropy (137 cal/degree per mol) of denaturation. The results indicate the high conformational stability of this protein against heat denaturation.     

Design and synthesis of novel allosteric MEK inhibitor CH4987655 as an orally available anticancer agent

The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differentiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional optimization including metabolic stability, physicochemical properties and safety profiles were effectively performed and led to the identification of a clinical candidate for an orally available potent MEK inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benzamide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK profile and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.     

Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection

Background

Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue.

Methods and Findings

A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome.

Conclusions

Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.     

Intake of green tea inhibited increase of salivary chromogranin A after mental task stress loads

Background

Green tea has become renowned for its health benefits. In this study, we investigated the anti-stress effect of two kinds of green tea against a mental stress task load.

Methods

Warm water, ordinary green tea (Sagara), and shaded white tea, which contains more amino acid components than Sagara, were used as test samples in a randomized cross-over design study. Eighteen students (nine male and nine female) participated in three experimental trials on different days at intervals of seven days. Saliva was collected before beverage intake and after performing the mental stress load tasks. Concentration of chromogranin A (CgA) in the saliva was used as an index of autonomic nervous system activity.

Results

CgA level increased after the mental tasks, but intake of green tea inhibited this increase; the anti-stress effect was even greater after consumption of shaded white tea. Intake of shaded white tea also lowered Total Mood Disturbance (TMD) score on the Profile of Mood States (POMS); subjects in this condition tended to perform more calculations in the arithmetic task than those in the warm water treatment condition.

Conclusions

Salivary CgA concentration levels increased after mental stress load tasks, but ingestion of green tea inhibited this increase. This anti-stress effect was larger after the consumption of shaded white tea than after Sagara. Shaded white tea intake also lowered TMD score (POMS) and tended to improve performance on an arithmetic task compared to warm water, suggesting that shaded white tea might also improve mood during and after mental stress load.     

Enzymatic Synthesis of Trehalose from Maltose

Synthesis of trehalose from maltose by a coupled enzyme system with trehalose Phosphorylase and maltose Phosphorylase has been studied. Trehalose Phosphorylase was partially purified from Euglena gracilis and maltose Phosphorylase was obtained from Lactobacillus brevis. The optimum pH of the reaction was 6.5~7.0 and the reaction rate was faster in the rection mixture containing a low concentration of phosphate. The final ratio of conversion (the ratio of trehalose to maltose) in the pH range between 6.0 and 8.0 was about 60%.

Immobilized maltose and trehalose Phosphorylase in κ-carageenan could be used without any appreciable loss of activity for batch reactions at least 10 times.     

Effect of Pepsin Inhibitor (S-PI) on the Growth of Rhodotorula glutinis No. K-24

A tissue-type transglutaminase (TGase) was purified from liver tissue of the red sea bream, Pagrus major, by ion-exchange chromatography and heparin-Sepharose affinity chromatography. Its activity was assessed using a fluorometric assay to measure the incorporation of monodansylcadaverine into N,N′-dimethyl casein. The molecular mass of purified TGase was estimated to be 78kDa by SDS–polyacrylamide gel electrophoresis. The enzyme required Ca²⁺ to express its activity, although 10 mM Sr²⁺ also activated the enzyme fully. TGase activity was maximal at pH 9.0–9.5, and the enzyme was strongly inhibited by sulfhydryl reagents. The purified enzyme catalyzed the cross-linking of myosin heavy chain obtained from Alaska pollack, resulting in gelation of an actomyosin solution. The partial amino acid sequence of this fish TGase showed divisionally significant similarity to TGase from guinea pig liver.