Study of the relationship between applied transmembrane pressure and antimicrobial activity of lysozyme

During the processing of biomolecules by ultrafiltration, the lysozyme enzyme undergoes conformational changes, which can affect its antibacterial activity. Operational conditions are considered to be one of the main parameters responsible for such changes, especially when using the same membrane and molecule. The present study demonstrates that, the same cut-off membrane (commercial data) can result in different properties of the protein after filtration, due to their different pore network. The filtration of lysozyme, regardless of the membrane, produces a decrease in the membrane hydraulic permeability (between 10 and 30%) and an increase in its selectivity in terms of observed rejection rate (30%). For the filtrated lysozyme, it appears that the HPLC retention time increases depending on the membrane used. The antibacterial activity of the filtrated samples is lower than the native protein and decreases with the increase of the applied pressure reaching 55–60% loss for 12 bar which has not been reported in the literature before. The observed results by SEC-HPLC and bacteriological tests, suggest that the conformation of the filtrated molecules are indeed modified. These results highlight the relationship between protein conformation or activity and the imposed shear stress.

Study of the relationship between applied transmembrane pressure and antimicrobial activity of lysozyme

During the processing of biomolecules by ultrafiltration, the lysozyme enzyme suffers conformational changes, which can affect its antibacterial activity. Operational conditions are considered ones of the principal responsible for the modifications, especially when using the same membrane and molecule. The present study demonstrates that, the same cut-off membrane (commercial data) can produce different properties on the protein after filtration, due to their different pore network. The filtration of lysozyme, regardless of the membrane, produces a decrease in the membrane hydraulic permeability (between 10–30%) and an increase in its selectivity in terms of observed rejection rate (30%). For the filtrated lysozyme, it appears that the HPLC retention time increases depending on the membrane used. The antibacterial activity of the filtrated samples is lower than the native protein and decreases with the increase of the applied pressure reaching 55–60% loss for 12 bar which is not reported in the literature. The observed results by SEC-HPLC and bacteriological tests, suggest that the conformation of the filtrated molecules are indeed modified. These results highlight the relationship between protein conformation or activity and the imposed shear stress.

Marine Streptomyces sp. VITASP as a Source of New Bioactive Secondary Metabolites

Background:: The main aim of the study was to evaluate the bioactive properties of ethyl acetate crude extract of Streptomyces sp. VITASP with a view to assess their therapeutic potential. Methods: The morphological, physiological and the biochemical properties of the strain Streptomyces sp. VITASP were confirmed by conventional methods. The present study evaluated the antibacterial, antioxidant and cytotoxic activities. Results: The isolate was identified to be Streptomyces sp. (Genbank accession number: KR233807). The ethyl acetate extract of Streptomyces sp. VITASP showed maximum antibacterial activity of two Gram- positive and Gram- negative bacteria at 0.5mg/mL. The antioxidant potential of the crude extract exhibited strong reducing power activity at 0.5 mg/mL with 93±0.05% inhibition. The antiinflammatory and anti-diabetic properties were identified at 0.5 mg/mL concentration. The cytotoxic effect was found with an IC50 of 500μg/ mL on HeLa cell lines. The GC-MS analysis and the chromatogram patterns revealed major peak at 18.485 which corresponds to Pyrrolo[1,2-a]pyrazine-1,4-dione, Hexahydro-3-(2-methylpropyl. IR spectra showed the functional groups. HPLC Retention time of the peak was found to be 2.414 min. Conclusion: This work demonstrates that the extract should be considered as a useful source as an antioxidant and antimicrobial agent and gives further insight into the potential use of the compounds as drugs for various other bioactivities.

Anthocyanins in the Bracts of Curcuma Species and Relationship of the Species Based on Anthocyanin Composition

Five anthocyanins, delphinidin 3- O-rutinoside, cyanidin 3- O-rutinoside, petunidin 3- O-rutinoside, malvidin 3- O-glucoside and malvidin 3- O-rutinoside, were identified. Three anthocyanins, delphinidin 3- O-glucoside, cyanidin 3- O-glucoside and pelargonidin 3- O-rutinoside, were putatively identified based on C18 HPLC retention time, absorption spectrum, including λmax, and comparisons with those of corresponding standard anthocyanins, as the compounds responsible for the pink to purple-red pigmentation of the bracts of Curcuma alismatifolia and five related species. Cluster analysis based on four major anthocyanins formed two clusters. One consisted of only one species, C. alismatifolia, and the other consisted of five. Each cluster further formed sub-clusters depending on either species or habitats.

HPLC Biogram Analysis

High-performance liquid chromatography (HPLC) biogram methodology is a powerful pharmaceutical screening hit confirmation strategy that couples analytical HPLC data with functional bioassay data. It is used primarily for screening hit chemical validation and triaging in support of early phase discovery programs and enables further investigation of the source of bioactivity in screening hits. The process combines semi-preparative separation technologies, automated compound handling and distribution, high-throughput biological screening, and informatics tools. The final output is an HPLC retention time versus bioactivity graphical overlay report. In this manner, biograms allow the analyst to determine which component in the sample is responsible for the biological activity, enabling decision making toward chemotype selection and prioritization from a pool of potential candidates. Another powerful aspect of the biogram assay lies in its utility in investigating biological activity in atypical samples, such as degraded samples or mixtures, for detection of minor active impurities or in addressing lot-to-lot activity discrepancies for a given test compound. Biograms are employed to track, isolate, and identify the source of biological activity in such samples, often yielding important information for program decisions.

HPLC Retention Time as a Diagnostic Tool for Hemoglobin Variants and Hemoglobinopathies: A Study of 60000 Samples in a Clinical Diagnostic Laboratory

Background: Previous evaluations of HPLC as a tool for detection of hemoglobin variants have done so within newborn-screening programs and/or by use of stored samples. We describe a 32-month prospective study in a clinical diagnostic laboratory in which we evaluated the imprecision of HPLC retention times and determined the retention times for hemoglobin variants seen in a multiethnic setting. Methods: We analyzed all samples on the Bio-Rad Variant II HPLC system. For normal hemoglobin fractions and hemoglobin variants, we recorded and analyzed their retention times, their proportion of the total hemoglobin (%), and the peak characteristics. We compared the imprecision of retention time with the imprecision of retention time normalized to the retention time of hemoglobin A0 (HbA0) and to the retention time of HbA2. Alkaline and acid hemoglobin electrophoresis, and in certain cases globin chain electrophoresis, isoelectric focusing, and DNA analysis, were performed to document the identities of the hemoglobin variants. Results: The mean (SD) imprecision (CV) of the retention time was 1.0 (0.7)% with no statistical difference compared with the imprecision for normalized retention times. Among 60293 samples tested, we encountered 34 unique hemoglobin variants and 2 tetramers. Eighteen variants and 2 tetramers could be identified solely by retention time and 3 variants by retention time and proportion of total hemoglobin. Four variants could be identified by retention time and peak characteristics and eight variants by retention time and electrophoretic mobility. One variant (HbNew York) was missed on HPLC. Retention time on HPLC was superior to electrophoresis for the differentiation and identification of six members of the HbJ family, four members of the HbD family, and three variants with electrophoretic mobilities identical or similar to that of HbC. Six variants with electrophoretic mobilities identical or similar to that of HbS could be differentiated and identified by retention time and proportion of total hemoglobin. HPLC detected two variants (HbTy Gard and HbTwin Peaks) missed on electrophoresis. Conclusions: The retention time on HPLC is reliable, reproducible, and in many cases superior to conventional hemoglobin electrophoresis for the detection and identification of hemoglobin variants. Confirmatory testing by electrophoresis can be eliminated in the majority of cases by use of retention time, proportion of total hemoglobin, and peak characteristics of HPLC.