Potential of Pm11 antimicrobial peptide against bovine mastitis pathogens.

OBJECTIVE: To investigate an alternative treatment for bovine mastitis by using Pm11 antimicrobial peptide. SAMPLE: 5 bovine mastitis pathogens that were previously isolated from cows affected by either clinical or subclinical mastitis. PROCEDURES: The current study introduces Pm11 antimicrobial peptide as an alternative treatment for bovine mastitis. The antibacterial activity of Pm11 was tested against Escherichia coli strain SCM1249, Klebsiella spp strain SCM1282, Staphylococcus aureus strain CM967, Streptococcus agalactiae strain SCM1084, and Streptococcus uberis strain SCM1310 using minimum bactericidal concentrations (MBCs) and time-kill kinetics. The pathogens' morphological changes were demonstrated using a scanning electron microscope (SEM). The cytotoxicity of Pm11 was assessed using the minimum hemolytic concentration assay. RESULTS: MBCs ranged from 2.5 to 10 µM and IC50 ranged from 0.32 to 2.07 µM. Time-kill kinetics at MBC demonstrated that Pm11 reduced viable cell counts of S agalactiae strain SCM1084 and S uberis strain SCM1310 from 105 to 0 CFU/mL within 1 h. E coli strain SCM1249 and S aureus strain CM967 were reduced from 105 to 0 CFU/mL within 4 h. The average Pm11-induced hemolytic activity was < 10% for all Pm11 concentrations tested except at the maximum concentration tested (160 µM: 10.19 ± 2.29%). Based on SEM, Pm11 induced morphological and cellular changes in S aureus and E coli. CLINICAL RELEVANCE: Pm11 antimicrobial peptide demonstrated in vitro antibacterial activity against the common bovine mastitis pathogens E coli, S aureus, S agalactiae, and S uberis, except Klebsiella spp, and should be further investigated in vivo.

Aqueous Extracts of Lemon Basil Straw as Chemical Stimulator for Gray Oyster Mushroom Cultivation.

To reduce the burning of lemon basil straw (LBS)-the byproduct of basil seed production-we propose utilizing LBS as a replacement substrate for mushroom cultivation. LBS can stimulate both mycelial growth and percentage biological efficiency; however, the rigidity of this material limits particle size reduction. In this work, aqueous extractions were facilely performed without using either hazardous chemicals or complex procedures to valorize LBS as a stimulator for gray oyster mushroom cultivation. An aqueous extraction at solid-to-liquid of 50 g/L was employed. The macerated-LBS and decocted-LBS extracts were tested for mycelial growth in potato dextrose agar and sorghum grains. Following this, both aqueous extracts were applied as a wetting agent in cylindrical baglog cultivation to estimate mycelial growth, biological efficiency, and productivity. It was found that LBS extracts insignificantly enhanced the mycelia growth rate on all media, while the diluted LBS (1:1 v/v) extracts improved 1.5-fold of percentage biological efficiency. Gas chromatograph-mass spectrometer results indicated 9-octadecaenamide is a major component in LBS aqueous extract. Results demonstrated that the LBS extract is a good stimulator for the production of Pleurotus mushroom.

Investigation of major amino acid residues of anti-norfloxacin monoclonal antibodies responsible for binding with fluoroquinolones.

It is important to understand the amino acid residues that govern the properties of the binding between antibodies and ligands. We studied the binding of two anti-norfloxacins, anti-nor 132 and anti-nor 155, and the fluoroquinolones norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. Binding cross-reactivities tested by an indirect competitive enzyme-linked immunosorbent assay indicated that anti-nor 132 (22-100%) had a broader range of cross-reactivity than anti-nor 155 (62-100%). These cross-reactivities correlated with variations in the numbers of interacting amino acid residues and their positions. Molecular docking was employed to investigate the molecular interactions between the fluoroquinolones and the monoclonal antibodies. Homology models of the heavy chain and light chain variable regions of each mAb 3D structure were docked with the fluoroquinolones targeting the crucial part of the complementarity-determining regions. The fluoroquinolone binding site of anti-nor 155 was a region of the HCDR3 and LCDR3 loops in which hydrogen bonds were formed with TYR (H:35), ASN (H:101), LYS (H:106), ASN (L:92), and ASN (L:93). These regions were further away in anti-nor 132 and could not contact the fluoroquinolones. Another binding region consisting of HIS (L:38) and ASP (H:100) was found for norfloxacin, enrofloxacin, and ciprofloxacin, whereas only ASP (H:100) was found for ofloxacin.

Expression, purification and biological activity of monomeric insulin precursors from methylotrophic yeasts.

The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have been used for the production of recombinant monomeric insulin precursor (MIP). Recombinant plasmids with one, two and four cassettes of the MIP gene have been successfully constructed in the pPICZαA expression vector to study the effects of gene copy number on MIP production. The MIP protein can be detected by dot-blot analysis from the culture broth of P. pastoris KM71H 24 h after placement in MMH induction medium. The secretion levels of MIP protein in culture broth at 72 h after induction indicated that P. pastoris KM71H with one cassette of the MIP gene had highest MIP protein levels (4.19 ±â€¯0.96 mg L-1). The transcription levels of the MIP gene increased proportionately with copy number. However, the amount of secreted MIP protein showed no correlation. The MIP molecular mass was 5756.951 Da, as confirmed by typical MALDI-TOF mass spectrometry. The MIP protein in culture broth was purified by two steps purification including SP Sepharose Fast Flow chromatography followed by ultrafiltration (10 kDa MW cutoff). The percentage of MIP recovery after the two-step purification was 70%, with a single band in a native-PAGE. The biological activity of tryptic hydrolyzed MIP was determined via the expression of the glucose transporter 4 gene (GLUT4) in H9c2 (2-1) cell line by RT-qPCR, and the results demonstrated that the MIP protein can induce glucose uptake and upregulation of GLUT4 mRNA transcription at 3 h and that this activity was related to Humalog® insulin.

Production of an antioxidative peptide from hairy basil seed waste by a recombinant Escherichia coli.

OBJECTIVE: To circumvent the time-consuming and costly problems associated with natural product extraction, a potential antioxidative peptide selected from hairy basil waste after oil extraction was produced by recombinant DNA technology. RESULTS: Because the target peptide is short, the recombinant peptide containing seven repeats of the target sequence, QTFQYSRGWTN, and the DNA fragment coding this sequence was cloned into the pQE-30 Xa expression vector and transformed into Escherichia coli. After 6 h of recombinant peptide expression in E. coli, the target peptide was purified by Ni(2+) affinity chromatography and gel extraction. The expected 15 kDa recombinant target peptide construct was verified by modified dot blot analysis. Compared with the chemically synthesized peptide, the recombinant peptide revealed significantly higher antioxidant activities (p < 0.05), as determined by DPPH and ABTS radical scavenging assays, and in vitro DNA damage induced by hydroxyl radicals. CONCLUSION: This approach provides an alternative to produce an antioxidative peptide that provides a potential scaffold for the further development of antioxidative peptides for industrial applications.