Synthesis of N-[3-(2,3-dimethyl-1H-indol-1-yl)-4-hydroxyphenyl]arylsulfon(aroyl)amides

Indole derivatives are an essential elements of many natural and synthetic compounds having significant biological activity. New derivatives of 2,3-dimethylindole were synthesized in this work by the reaction of N-(4-oxocyclohexa-2,5-dien-1-ylidene)arylsulfon(aroyl)amides and 4-methyl-N-(4-oxonaphthalen-1(4H)-ylidene)benzene-1-sulfonamide with 2,3-dimethylindole. The possibility of these reactions is determined by the redox potential of the starting amides and steric factor. If there is a free C=C bond in the quinoid ring of the starting arylsulfonamides, the reaction proceeds under the 1,4-addition scheme with the formation of corresponding N-[3-(2,3-dimethyl-1H-indol-1-yl)-4-hydroxyphenyl(naphthyl-1)]arylsulfonamides which are potentially biologically active compounds. N-(4-Oxocyclohexa-2,5-diene-1-ylidene)aroylamides have a high redox potential as compared with similar arylsulfonamides and naphthalene derivatives; therefore, two processes proceed in their reaction with 2,3-dimethylindole: reduction and 1,4-addition. The 1,4-addition product was obtained only for the 2,3-dimethyl derivative, which has the lowest redox potential among the investigated aroylamides. Analysis of the potential biological activity of the synthesized compounds by using the PASS program showed that the synthesized products can exhibit the following activities: para amino benzoic acid antagonist, glutamyl endopeptidase II inhibitor, CYP3A2 substrate, insulysin inhibitor, membrane integrity agonist and phobic disorders treatment.

Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria

Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity- and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9- and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture.

Structural insights into the role of the N-terminus in the activation and function of extracellular serine protease from Staphylococcus epidermidis

Extracellular serine protease (Esp) from Staphylococcus epidermidis is a glutamyl endopeptidase that inhibits the growth and formation of S. aureus biofilms. Previously, crystal structures of the matured and active Esp have been determined. Interestingly, many of the staphylococcal glutamyl endopeptidase zymogens, including V8 from Staphylococcus aureus and Esp from S. epidermidis, contain unusually long pro-peptide segments; however, their function is not known. With the aim of elucidating the function of these pro-peptide segments, crystal structures of the Esp zymogen (Pro-Esp) and its variants were determined. It was observed that the N-terminus of the Pro-Esp crystal structure is flexible and is not associated with the main body of the enzyme, unlike in the known active Esp structure. In addition, the loops that border the putative substrate-binding pocket of Pro-Esp are flexible and disordered; the structural components that are responsible for enzyme specificity and efficiency in serine proteases are disordered in Pro-Esp. However, the N-terminal locked Pro-Esp variants exhibit a rigid substrate-binding pocket similar to the active Esp structure and regain activity. These structural studies highlight the role of the N-terminus in stabilizing the structural components responsible for the activity and specificity of staphylococcal glutamyl endopeptidases.

Effects ofBacillusSerine Proteases on the Bacterial Biofilms

Serratia marcescensis an emerging opportunistic pathogen responsible for many hospital-acquired infections including catheter-associated bacteremia and urinary tract and respiratory tract infections. Biofilm formation is one of the mechanisms employed byS. marcescensto increase its virulence and pathogenicity. Here, we have investigated the main steps of the biofilm formation byS. marcescensSR 41-8000. It was found that the biofilm growth is stimulated by the nutrient-rich environment. The time-course experiments showed thatS. marcescenscells adhere to the surface of the catheter and start to produce extracellular polymeric substances (EPS) within the first 2 days of growth. After 7 days,S. marcescensbiofilms maturate and consist of bacterial cells embedded in a self-produced matrix of hydrated EPS. In this study, the effect ofBacillus pumilus3-19 proteolytic enzymes on the structure of 7-day-oldS. marcescensbiofilms was examined. Using quantitative methods and scanning electron microscopy for the detection of biofilm, we demonstrated a high efficacy of subtilisin-like protease and glutamyl endopeptidase in biofilm removal. Enzymatic treatment resulted in the degradation of the EPS components and significant eradication of the biofilms.

A first case report of UDP-galactose-4′-epimerase deficiency in China: genotype and phenotype

The aim of the study was to investigate the incidence and genotype-phenotype characteristics of UDP-galactose-4′-epimerase (GALE) deficiency in newborn screening of Chinese population.Neonates were screened at the Newborn Screening Center of Zhejiang Province, China for GALE deficiency and their condition was confirmed by testing of theA total of 350,023 of newborns were screened; of which, the condition of one female neonate was diagnosed with GALE deficiency, accounting for an incidence rate of approximately 1:350,000 in our sample. The patient with GALE deficiency clinically manifested slight increase in levels of blood galactose (122–251 mg/L), glutamyl endopeptidase (61 U/L), total bile acid (17 μmol/L), and lactic acid (1.8 mmol/L). The neonate was fed with lactose-free powdered milk and followed-up to 1 year. Re-examination showed that all biochemical indicators recovered to normal range, whereas physical and mental development appeared normal without cataract change. The genotype of GALE deficiency was identified as compound heterozygous mutations: c.505C>T (p.R169W) and c.452G>A (p.G151D). The latter was a novel mutation. The GALE enzyme value was 42% of control.GALE deficiency is relatively rare in China. The genotype of compound heterozygous mutations at R169W and G151D clinically manifest as mild-type; it is recommended to limit galactose diet.

An Experimental Study on the Mechanisms of Ge Hua Jie Cheng Decoction for Rats with Alcoholic Liver Injuries

Objective: To explore the effects and underlying mechanisms of Ge Hua Jie Cheng Decoction on rats with alcoholic liver injuries. Methods: 60 Wistar rats were randomly assigned to six groups: normal group, model group, Yi Gan Ling group, and Ge Hua Jie Cheng Decoction groups in low, middle and high concentrations, 10 rats in each group. Except for the normal group, rats in other groups were administered white wine for eight weeks to establish the liver injury model. During the modeling, the Yi Gan Ling/Ge Hua Jie Cheng Decoction were administered intragastrically to the rats. So the histopathological changes were observed after eight weeks, meanwhile the serum γ- glutamyl endopeptidase (GGT), Glutathione (GSH) and aspartate aminotransferase mitochondrial isoenzyme (m-AST) were assayed by automatic biochemical analyzer. Results: under the light microscope, the groups of high and middle dosages of Ge Hua Jie Cheng Decoction , especially the high one, had apparent improvement of inflammatory infiltration in liver tissues. Compared with the normal group, the serum GGT and m-AST levels had elevated (P＜0.01), whereas the serum GSH level decreased (P＜0.01); compared with model group, the high and middle dosages of Ge Hua Jie Cheng Decoction groups had decreased serum GGT and m-AST (P＜0.01 or P＜0.05), as well as increased serum GSH level (P＜0.01). Conclusion: Ge Hua Jie Cheng Decoction has a protective effect for liver injuries induced by alcohol, and this effect is dose-dependent. The high dosage showed stronger protection effect, which might be related to the increased serum GSH and decreased serum GGT and m-AST.