Purification and Partial Characterization of a Prolyl-Dipeptidyl Aminopeptidase from Lactobacillus helveticus CNRZ 32

X-prolyl-dipeptidyl aminopeptidase, which hydrolyzed Gly-Pro-p-nitroanilide (relative activity [RA] = 100%) and Arg-Pro-p-nitroanilide (RA, 130%), was purified to homogeneity from the cell extract of Lactobacillus helveticus CNRZ 32. The enzyme also hydrolyzed Ala-Pro-Gly (RA, 11%) and Ala-Ala-p-nitroanilide (RA, 2%) but was not active on Ala-Leu-Ala, dipeptides, and endopeptidase and carboxypeptidase substrates. The enzyme was purified 145-fold by streptomycin sulfate precipitation, ammonium sulfate fractionation, and a series of column chromatographies on DEAE-cellulose, arginine-Sepharose 4B, and glycyl-prolyl-AH-Sepharose 4B. The purified enzyme appeared as a single band on native polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses and had a molecular weight of 72,000. Optima for activity by the purified enzyme were pH 7.0 and 40°C. The enzyme was incubated at 40°C for 15 min with various metal ions. It was activated by Mg²⁺ (2.5 mM), Ca²⁺ (0.1 to 2.5 mM), Na⁺ (10 to 50 mM), and K⁺ (10 to 50 mM) and was inhibited by Hg²⁺ (0.1 to 2.5 mM), Cu²⁺ (0.1 to 2.5 mM), and Zn²⁺ (0.1 to 2.5 mM). Enzyme activity was partially inhibited by EDTA (1.0 mM, 20 h at 40°C), 1,10-phenanthroline (1.0 mM, 15 min at 40°C), phenylmethylsulfonyl fluoride (1.0 mM), N-ethylmaleimide (1.0 mM), and iodoacetate (1.0 mM). It was completely inhibited by diisopropyl fluorophosphate (1.0 mM, 2 h at 40°C) and p-chloromercuribenzoate (1.0 mM, 15 min at 40°C). The enzyme was not affected by dithioerythritol (1.0 to 10 mM).     

Calcium activated proteolysis of fibrous proteins in central nervous system

A Ca²⁺ activated protease(s) capable of hydrolyzing several polypeptides at neutral pH including cytoskeletal proteins, actin, myosin, tubulin and neurofilament triplet was identified in calf brain cortex. The enzyme activity precipitates at 75 mM KCl, pH 6.5 – 7.0 and is inhibited by the sulfhydryl inhibitors, N-ethylmaleimide and para-chloromercuribenzoate and the protease inhibitors, antipain, pepstatin and leupeptin, leupeptin being the most effective.     

Improved sensitivity of glutamine synthetase assay and of acyl hydroxamates

Sensitivity of glutamine synthetase assay by the hydroxamate method can be doubled by replacing ferric chloride with ferric nitrate or ferric perchlorate, each with the appropriate acid.     

Light and electron microscopical study of Nosema eurytremae

A nuclear-polyhedrosis virus (NPV) of the silkworm, Bombyx mori, which forms an icosahedral inclusion body, was transmitted to larvae of the rice stem borer, Chilo suppressalis. Serial passages of Bombyx NPV in the alternate host by injecting the supernatant of diseased hemolymph produced inclusion bodies with cuboidal and other shapes that differed from the original shape formed in Bombyx. These different shapes increased with times of passages, and after the twelfth passage, only cuboidal inclusion bodies were formed. The icosahedral inclusion bodies in B. mori and the cuboidal inclusion bodies in C. suppressalis occluded singly enveloped virions of the same size (350 × 75 nm), but the cuboidal inclusion bodies contained only a few virions and a large number of membraneous spherical structures. The formation process of the cuboidal inclusion body differed from that of the icosahedral. At first, irregularly branched inclusion bodies containing “vacant” spaces appeared in the infected nuclei. The bodies grew larger with the deposition of protein in the spaces between the branches, and this was accompanied with the occlusion of a large number of membraneous structures formed in the vicinity of the inclusion bodies, which became cuboidal in shape.     

Acetophenone and stilbene derivatives from Gnetum ula

The isolation of bergenin, 2-hydroxy-4-benzyloxyacetophenone and the related dimer and stilbene from Gnetum ula is reported.     

Inter-rater reliability of physiotherapists using the Action Research Arm Test in chronic stroke

Objectives:The purpose of this study is to establish whether physiotherapists’ ratings are consistent, when using the Action Research Arm Test (ARAT) to score a chronic stroke patient.Methods:This was part of a large project establishing the reliability in chronic stroke. This study used a correlational design comparing the association between physiotherapist scores of the same patient, to establish the ARAT’s inter-rater reliability. The COSMIN checklist was followed to enhance the methodology of the study.Results:Twenty physiotherapists (8 female and 12 male) aged between 25 and 53 years were selected. There were no participant dropouts or withdrawals. The sample size was normally distributed. The physiotherapists appeared representative of the UK physiotherapy population, with the exception of gender. The distribution of scores showed a normal distribution with standard deviation of score of 1.9. The Kendall’s W test showed 0.711 of agreement between the raters. The scores achieved statistical significance showing consistency between physiotherapists’ scores with chronic stroke. Limitations of the study were the use of a small single center convenience sample that may reduce the generalizability of the findings.Conclusions:The ARAT is consistent when scored by physiotherapists in a chronic stroke population. The inter-rater reliability range was (0.70 to 0.90) which is categorized as good.     

Molecular docking analysis of HER-2 inhibitor from the ZINC database as anticancer agents

The human epidermal growth factor (HER2) is a transmembrane receptor that is highly expressed in breast cancer and in different other cancers. Therefore, it is of interest to identify the new HER2 inhibitors from a selected 300 compounds in the ZINC database. The top two hit compounds (ZINC000014780728 (-11.0 kcal/mol) and ZINC000014762512 (-10.8 kcal/mol)) showed a high affinity with HER2 relative to the reference compound (lapatinib (-10.2 kcal/mol)) for further consideration.