Tetrahydrobiopterin from cyanide-degrading bacterium Bacillus pumilus strain SVD06 induces apoptosis in human lung adenocarcinoma cell (A549).

Tetrahydrobiopterin (BH4) is an essential biological cofactor and a derivative of pterin which is considered potent anticancer agents. In continuation of our previous study on the identification of BH4 from cyanide-degrading Bacillus pumilus, the present study focuses on evaluating the anticancer properties of BH4 on A549, a human lung adenocarcinoma. Anticancer activity analysis shows that BH4 inhibited A549 cell growth after 24 h of incubation with 0.02 mg/mL. In acridine orange/ethidium bromide staining, BH4-treated A549 cells showed apoptotic morphology. BH4 treatment caused cell cycle arrest at G0/G1 phase compared to control cells. BH4 augmented p53 expression in alveolar cancer cells by downregulating MDM2 levels. There was downregulation of casp-3 and upregulation of iNOS gene in BH4-treated A549 cells. Further, docking studies indicated that BH4 had significant interactions with the above proteins affirming the apoptosis mechanism. Thus, BH4 could be considered a potential anticancer drug.

In vitro and in vivo antimicrobial activity of self-assembled melittin nanoparticles: A comparative study with melittin peptide.

The aim of the present study was to analyse the efficacy of self-assembled melittin nanoparticles (MelNP) and compare with native melittin peptide (Mel). Self-assembly formation of the melittin was promoted by heating at 90 °C for 50 min followed by cooling at room temperature. SEM micrographs revealed the formation of nanovesicles. MIC of MelNP against E. coli, S. aureus and P. aeruginosa was found to be 4, 2, and 2 µM, respectively while it was 8, 8 and 4 µM for Mel peptide. Markedly, MelNP showed 12.6 % hemolysis at 8 µM whereas with Mel it was about 71.63 %. The lytic activity of MelNP was also higher in the presence of trypsin/serum than Mel. Both MelNP and Mel exhibited membranolytic activity with cellular disintegration. Further, toxicity analysis studied up to 72 h showed that MelNP was non-toxic to zebrafish embryos up to 6 µM; however, with Mel exposed embryos showed up 30 dead embryos. Bacterial load was markedly reduced in MelNP and Mel exposed infected embryos than compared to the infected one. Moreover, the peptides were also responsible for reducing the infection and prolonging the survivability in infected embryos. Thus, MelNP could be considered an efficient and safer therapeutic molecule that Mel and wherein further experiments are warranted to affirm the broad spectrum efficiency.

Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5.

Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of glutamate. It possesses several physiological functions such as neurotransmission, diuretic, and tranquilizer effects and also regulates cardiovascular functions such as blood pressure and heart rate in addition to playing a role in the reduction of pain and anxiety. The objective of this study was to evaluate the GABA producing ability and probiotic capability of certain lactic acid bacteria strains isolated from dairy products. Around sixty-four bacterial isolates were collected and screened for their ability to produce GABA from monosodium glutamate, among which nine isolates were able to produce GABA. The most efficient GABA producer was Enterococcus faecium BS5. Further, assessment of several important and desirable probiotic properties showed that Ent. faecium BS5 was resistant to acid stress, bile salt, and antibiotics. Ent. faecium BS5 may potentially be used for large-scale industrial production of GABA and also for functional fermented product development.

Dual antimicrobial and anticancer activity of a novel synthetic α-helical antimicrobial peptide.

Antimicrobial peptides (AMPs) are increasingly sought-after and researched antimicrobial agents due to its desired pharmacological properties and the continuous diminishing efficacy of antibiotics. In addition to this line of research, the aim of the present study is to determine the antimicrobial and anticancer activity of a de novo designed α-helical peptide. Circular dichroism showed 100% helical nature of the peptide in 10 mM SDS. Notably, the peptide exerted significant antimicrobial activity against the reference and antibiotic-resistant clinical isolates belonging to Pseudomonas sp. at a MIC and MBC of 2 and 8 µM, respectively. The progressive disruption and disturbance of cell membrane in the overall topography was observed in the scanning electron microscopy (SEM) micrographs of Pseudomonas aeruginosa ATCC 27853 treated with the peptide as compared to untreated control. The results of time-kill kinetics showed complete lysis at 3x MIC after 50 min of incubation of the microbe with the peptide. Moreover, the peptide did not lyse human RBCs even at the highest concentration of the peptide (10 mM) and retained its activity upon treatment at 0.5 mg/ml trypsin. Cancer cell lines, viz. A549 and MCF-7 were also found to be sensitive to peptide activity showing 50% reduction in survivability at 4 and 2 µM, respectively; however, L929 cells were unaffected. Drastic membrane permeability and necrotic mode of lysis of peptide-treated-A549 cells were affirmed by propidium iodide and live/dead cell staining. The results showed that the designed peptide could be an efficient drug molecule for clinical studies subjected to successful experiments on animal models.

A Brief Review on the Non-protein Amino Acid, Gamma-amino Butyric Acid (GABA): Its Production and Role in Microbes.

Gamma-Aminobutyric acid (GABA) is a non-protein amino acid widely distributed in nature. It is produced through irreversible α-decarboxylation of glutamate by enzyme glutamate decarboxylase (GAD). GABA and GAD have been found in plants, animals, and microorganisms. GABA is distributed throughout the human body and it is involved in the regulation of cardiovascular conditions such as blood pressure and heart rate, and plays a role in the reduction of anxiety and pain. Although researchers had produced GABA by chemical method earlier it became less acceptable as it pollutes the environment. Researchers now use a more promising microbial method for the production of GABA. In the drug and food industry, demand for GABA is immense. So, large scale conversion of GABA by microbes has got much attention. So this review focuses on the isolation source, production, and functions of GABA in the microbial system. We also summarize the mechanism of action of GABA and its shunt pathway.

Biological characterization of omw1 and omw2: antimicrobial peptides derived from omwaprin.

Two cationic antimicrobial peptides (AMP) were designed based on the snake venom peptide, omwaprin, hypothesized to be shorter, cost effective and potent. Omw1 and omw2 demonstrated significant broad-spectrum antimicrobial activity against standard and clinical strains at a MIC ranging from 15.625 to 250 µg/ml for omw1 and from 31.3 to 500 µg/ml for omw2. Time-kill kinetics revealed that omw1 caused complete lysis of E. coli ATCC 25922 at 1× MIC and S. aureus ATCC 25923 at 2× MIC after 40 and 60 min of incubation, respectively. Membranolytic activity of the peptides was assessed by propidium iodide stain, where red fluorescence was observed in cells treated with the peptides compared to untreated cells. Notable morphological changes were observed in the microbes treated with peptides, as revealed by scanning electron micrographs. Omw1 and omw2 were also potent to inhibit the formation as well as dispersal of matured biofilms at 1/2× MIC against clinical strain, C. albicans. Further, minimal hemolytic activity demonstrated by both the peptides at microbicidal concentration against human erythrocytes proves that the designed peptides were less toxic and potent antimicrobial agents which could be considered for further studies with animal models to affirm its efficiency.

Real-time heterogeneous protein-protein interaction between αA-crystallin N-terminal mutants and αB-crystallin using quartz crystal microbalance (QCM).

The lens transparency depends on higher concentration of lens proteins and their interactions. α-Crystallin is one of the predominant lens proteins, responsible for proper structural and functional architecture of the lens microenvironment, and any alteration of which results in cataract formation. The R12C, R21L, R49C and R54C are the most significant and prevalent αA-crystallin congenital cataract-causing mutants worldwide. Protein-protein interaction, crucial for lens proper structure and function, was posited to be lost due to point mutation and the elucidation of which could shed light on the molecular basis of cataract. In this conjuncture, we report quartz crystal microbalance (QCM) as a warranted technique for real-time analysis of protein-protein interaction between the N-terminal mutants of αA-crystallin and αB-crystallin. The biophysical characteristics of the mutated proteins were determined by size-exclusion HPLC, far-UV circular dichroism and fluorescence studies. Far-UV circular dichroism spectral analysis displayed slight modifications in ß-sheet of R54C mutant. Altered intrinsic tryptophan fluorescence and decreased bis-ANS fluorescence were observed in all the N-terminal mutations revealing the tertiary structural changes and decreased exposure of surface hydrophobicity. An emphatic fall in the chaperone activity was observed in the N-terminal mutants, R12C, R21L and R54C. QCM analysis revealed the occurrence of strong heterogeneous interaction between αA-crystallin and αB-crystallin. Nevertheless, decreased interactions were observed with the N-terminal mutants. In summary, the present study concludes that the loss of interactions between αA-crystallin N-terminal mutants and αB-crystallin signifies quaternary structural alterations due to mutation in the arginine residues.

Interaction of αA-crystallin F71L mutant with wild type αA- and αB-crystallins by mammalian two hybrid assay.

Incidence of age related cataract (ARC) increases by a variety of factors including metabolic and environmental factors. Nonetheless, genetic mutations are responsible for the altered structural stability of the proteins, especially; the F71L mutation in αA-crystallin has been shown to be responsible for the incidence of cataracts. However, structural characteristics and chaperone function of this mutant and its interaction with wild type (WT) crystallins may aid to decipher its role in cataractogenesis. The aim of the present study is to show the interaction of F71L mutant protein with the WT α-crystallins. The F71L mutant used in this study was created by site-directed mutagenesis, overexpressed and purified. Biophysical characteristics determined by size exclusion HPLC, DLS, CD spectrometry, tryptophan fluorescence and surface hydrophobicity did not show significant structural changes in the mutant protein compared to WT counterpart. Interestingly, the F71L mutant displayed a significant loss in homogenous interaction with WT αA-crystallin and F71L mutant as well as heterogeneous interaction with αB-crystallin as evaluated by mammalian two hybrid system. Our findings suggest that F71L loses the ability to form homo and hetero-oligomers seems to result in the loss in chaperone like activity (CLA) and refractive index resulting in the development of cataracts.

Transcriptional regulation of crystallin, redox, and apoptotic genes by C-Phycocyanin in the selenite-induced cataractogenic rat model.

PURPOSE: This study was designed to examine the constrictive potential of C-Phycocyanin (C-PC) in regulating changes imposed on gene expression in the selenite-induced cataract model. METHODS: Wistar rat pups were divided into three groups of eight each. On P10, Group I received an intraperitoneal injection of normal saline. Groups II and III received a subcutaneous injection of sodium selenite (19 µmol/kg bodyweight); Group III also received an intraperitoneal injection of C-PC (200 mg/kg bodyweight) on P9-14. Total RNA was isolated on P16, and the relative abundance of mRNA of the crystallin structural genes, redox components, and apoptotic cascade were ascertained with real-time PCR with reference to the internal control ß-actin. RESULTS: Real-time PCR analysis showed the crystallin genes (αA-, ßB1-, γD-) and redox cycle components (Cat, SOD-1, Gpx) were downregulated, the apoptotic components were upregulated, and antiapoptotic Bcl-2 was downregulated in Group II. Treatment with 200 mg/kg bodyweight C-PC (Group III) transcriptionally regulated the instability of the expression of these genes, thus ensuring C-PC is a prospective anticataractogenic agent that probably delays the onset and progression of cataractogenesis induced by sodium selenite. CONCLUSIONS: C-PC treatment possibly prevented cataractogenesis triggered by sodium selenite, by regulating the lens crystallin, redox genes, and apoptotic cascade mRNA expression and thus maintains lens transparency. C-PC may be developed as a potential antioxidant compound applied in the future to prevent and treat age-related cataract.

Characterization of Bacillus spp. from the gastrointestinal tract of Labeo rohita--towards to identify novel probiotics against fish pathogens.

The aim of the present study is to screen and characterize endogenous microbiota Bacillus spp. from the gastrointestinal (GI) tract of Labeo rohita in order to evaluate their probiotic attributes. A total of 74 isolates from the GI of L. rohita were evaluated for their antimicrobial properties by agar well-diffusion method against fish pathogens. Based on the better antibacterial features, three isolates (KADR1, KADR3, and KADR4) were selected for further delineation. The three selected isolates exhibited higher tolerance to bile salt, moderate tolerance to low pH, high surface hydrophobicity to solvents, and capable to autoaggregate. All three isolates demonstrated notable proteolytic, catalase activity and susceptibility to various antibiotics. Partial 16S rRNA sequencing revealed that the isolates exhibited 99 % sequence homology with Bacillus subtilis, Bacillus aerophilus, and Bacillus firmus of the database substantiating morphological and physiological characterization. Survivability in low pH and bile salt ensures their adaptability in the fish intestinal microenvironment. The ability to autoaggregate reveals colonization potential in the GI of the fish. Absence of hemolytic activity, antibiotic susceptibility to certain antibiotics, presence of protease and catalase activity, and non-pathogenic caliber of the above-mentioned isolates could be feasible characteristics when considering them as probiotics in the aquaculture industry.

Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.

PURPOSE: To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays. METHODS: Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A (60)Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay. RESULTS: αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed ß-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant. CONCLUSIONS: Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.

Antimicrobial and antibiofilm activity of designed and synthesized antimicrobial peptide, KABT-AMP.

Lysine-rich peptide, designated as KABT-AMP, was designed and synthesized to supersede the irrational use of chemical antibiotics as standard therapy. KABT-AMP is a 22-amino acid helical cationic peptide (+10) and amphipathic in nature. The antimicrobial kinetics of the peptide was ascertained in the representative strains of gram-positive, gram-negative, and fungal strains, viz., Staphylococcus aureus MTCC 2940, Escherichia coli MTCC 2939, and Candida albicans MTCC 227, respectively. KABT-AMP was synthesized by solid-phase synthesis and purified using reverse-phase high-performance liquid chromatography which resulted in >95 % purity, and matrix-assisted laser desorption/ionization time of flight revealed the mass of the peptide to be 2.8 kDa. KABT-AMP showed significant broad-spectrum antimicrobial activity against the bacterial and fungal strains analyzed in the present study with survivability of 30.8, 30.6, and 31.7 % in E. coli, S. aureus, and C. albicans, respectively, at 6 h. KABT-AMP also demonstrated antibiofilm activity against the tested biofilm forming clinical isolate, Candida tropicalis. The putative membranolytic activity of the peptide was substantiated by electron microscopic analysis. Results reveal that KABT-AMP will exhibit noteworthy antimicrobial activity against multidrug-resistant bacteria and fungus at micromolar concentrations with minimal cytotoxicity and thus could be conceived for biomedical application.

C-phycocyanin modulates selenite-induced cataractogenesis in rats.

The present investigation is aimed to evaluate the anticataractogenic potential of C-phycocyanin (C-PC), extracted and purified from Spirulina platensis. Enucleated rat lenses were maintained in vitro in Dulbecco's modified Eagle medium (DMEM). Group I contained DMEM, Group II and Group III contained 100 µM of sodium selenite, Group III was subdivided into three viz IIIa, IIIb, IIIc supplemented with 100, 150, 200 µg of C-PC respectively. In the in vivo study, on tenth day post partum: Group I rat pups received an intraperitoneal injection of saline, Group II, IIIa, IIIb, and IIIc rat pups received a subcutaneous injection of sodium selenite (19 µmol/kg bodyweight) Group IIIa, IIIb, IIIc also received an intraperitoneal injection of 100, 150, 200 mg/kg body weight of C-PC, respectively, from postpartum days 9-14. On termination of the experiment, the lenses from both in vitro and in vivo studies were subjected to morphological examination and subsequently processed to estimate the activities of antioxidant enzymes namely superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, levels of reduced glutathione and lipid peroxidation products. Sodium selenite-exposed, C-PC-treated rat lenses (Group IIIc), showed significant restoration of antioxidant enzyme activity (p < 0.05) when compared to their counterpart Group II. Group IIIc conserved the levels of GSH and lipid peroxidation products at near to normal levels as compared with Group II. Results conclude the possible role of C-PC in modulating the antioxidant enzyme status, thereby retarding sodium selenite-induced cataract incidence both in vitro and in vivo.