Anticancer Activities of Antimicrobial BmKn2 Peptides Against Oral and Colon Cancer Cells

There is considerable current interest in developing antimicrobial and anticancer agents with a new mode of action. The antimicrobial peptides are regarded as a potential solution for treating cancer cells. The antimicrobial effect of 6 synthetic peptides against 7 bacterial species was evaluated. The result showed that IsCT, BmKn2 and BMAP-28 exhibited broad range of action against Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, methicillin resistant S. aureus DMST 20651, Staphylococcus epidermidis ATCC 12228, Acinetobacter baumanii ATCC 19066, Escherichia coli ATCC 25922 and Salmonella typhi DMST 562 at minimal inhibitory concentrations (MIC) of 2.97–24.28 μM. Neither AMP induced significant hemolysis, or showed cytotoxic on dental pulp stem cells and smooth muscle cells at their MICs. In addition, BmKn2 inhibited growth of human oral squamous carcinoma HSC4 cells and human colon cancer SW620 cells with IC₅₀ of 17.26 and 40 µM, respectively. Taken together, BmKn2 peptide from scorpion venom may offer a novel therapeutic strategy for development of cationic antimicrobial and anticancer peptides as potential new therapeutic agents.     

Influence of bovine lactoferrin on the growth of selected probiotic bacteria under aerobic conditions

Bovine lactoferrin (bLf) is a natural glycoprotein, and it shows broad-spectrum antimicrobial activity. However, reports on the influences of bLf on probiotic bacteria have been mixed. We examined the effects of apo-bLf (between 0.25 and 128 mg/mL) on both aerobic and anaerobic cultures of probiotics. We found that bLf had similar effects on the growth of probiotics under aerobic or anaerobic conditions, and that it actively and significantly (at concentrations of >0.25 mg/mL) retarded the growth rate of Bifidobacterium bifidum (ATCC 29521), B. longum (ATCC 15707), B. lactis (BCRC 17394), B. infantis (ATCC 15697), Lactobacillus reuteri (ATCC 23272), L. rhamnosus (ATCC 53103), and L. coryniformis (ATCC 25602) in a dose-dependent manner. Otherwise, minimal inhibitory concentrations (MICs) were 128 or >128 mg/mL against B. bifidum, B. longum, B. lactis, L. reuteri, and L. rhamnosus (ATCC 53103). With regard to MICs, bLf showed at least four-fold lower inhibitory effect on probiotics than on pathogens. Intriguingly, bLf (>0.25 mg/mL) significantly enhanced the growth of Rhamnosus (ATCC 7469) and L. acidophilus (BCRC 14065) by approximately 40–200 %, during their late periods of growth. Supernatants produced from aerobic but not anaerobic cultures of L. acidophilus reduced the growth of Escherichia coli by about 20 %. Thus, bLf displayed a dose-dependent inhibitory effect on the growth of most probiotic strains under either aerobic or anaerobic conditions. An antibacterial supernatant prepared from the aerobic cultures may have significant practical use.     

Description ofOceanospirillum kriegii sp. nov. andO. jannaschii sp. nov. and assignment of two species ofAlteromonas to this genus asO. commune comb. nov. andO. vagum comb. nov

Immunological studies of Fe-containing superoxide dismutase (FeSOD) and glutamine synthetase (GS) have established a close relationship betweenOceanospirillum linum (the type species of the genus),O. beijerinckii, Alteromonas communis, A. vaga, and two unnamed species of marine bacteria (groups H-1 and I-1). The four latter species have, consequently, been assigned to the genusOceanospirillum asO. commune comb. nov.,O. vagum comb. nov.,O. kriegii sp. nov. (group H-1; type strain 197, ATCC 27133), andO. jannaschii sp. nov. (group I-1; type strain 207, ATCC 27135). The phenotypic properties of these species are presented together with their distinguishing traits.     

Isolation and characterization of flagellar filaments from Bacillus cereus ATCC 14579

Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length.     

Purification and characterization of antimicrobial substances from Bacillus licheniformis BFP011

Bacillus licheniformis BFP011 isolated from papaya (Thailand) could produce extracellular antimicrobial substances which were active against some important phytopathogens, pathogenics and spoilage microorganisms such as Colletotrichum capsici, Escherichia coli O157: H7 and Salmonella typhi ATCC 5784. The antimicrobial substances of this bacterium showed resistance to pronase enzyme and high temperature at 100 and 121°C for 15 min. They were purified by TLC on silica gel plates F254 using the different solvent mixtures. The best solvent mixture was revealed as n-butanol: ethanol: acetic acid: water (30: 60: 5: 30, v/v). The spots F4, F5 and F6 from TLC were able to inhibit growth of S. typhi ATCC 5784 assayed in vitro by the disc diffusion method. The characterization of the active fractions F4, F5 and F6 from TLC and reversedphase HPLC indicated that the antimicrobial substances of B. licheniformis BFP011 contain peptides and unsaturated fatty acids.     

Characterization and site-directed mutation of a novel aldo–keto reductase from Lodderomyces elongisporus NRRL YB-4239 with high production rate of ethyl (R)-4-chloro-3-hydroxybutanoate

A novel aldo–keto reductase (LEK) from Lodderomyces elongisporus NRRL YB-4239 (ATCC 11503) was discovered by genome database mining for carbonyl reduction. LEK was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity and the catalytic properties were studied. Among the substrates, ethyl 4-chloro-3-oxobutanoate was converted to ethyl (R)-4-chloro-3- hydroxybutanoate ((R)-CHBE), an important pharmaceutical intermediate, with an excellent enantiomeric excess (e.e.) (>99 %). The mutants W28A and S209G obtained by site-directed mutation were identified with much higher molar conversion yields and lower Km values. Further, the constructed coenzyme regeneration system with glucose as co-substrate resulted in a yield of 100 %, an enantioselectivity of >99 %, and the calculated production rate of 56.51 mmol/L/H. These results indicated the potential of LEK for the industrial production of (R)-CHBE and other valuable chiral alcohols.     

Microbial strain improvement for enhanced polygalacturonase production by Aspergillus sojae

Strain improvement is a powerful tool in commercial development of microbial fermentation processes. Strains of Aspergillus sojae which were previously identified as polygalacturonase producers were subjected to the cost-effective mutagenesis and selection method, the so-called random screening. Physical (ultraviolet irradiation at 254 nm) and chemical mutagens (N-methyl-N′-nitro-N-nitrosoguanidine) were used in the development and implementation of a classical mutation and selection strategy for the improved production of pectic acid-degrading enzymes. Three mutation cycles of both mutagenic treatments and also the combination of them were performed to generate mutants descending from A. sojae ATCC 20235 and mutants of A. sojae CBS 100928. Pectinolytic enzyme production of the mutants was compared to their wild types in submerged and solid-state fermentation. Comparing both strains, higher pectinase activity was obtained by A. sojae ATCC 20235 and mutants thereof. The highest polygalacturonase activity (1,087.2?±?151.9 U/g) in solid-state culture was obtained by mutant M3, which was 1.7 times increased in comparison to the wild strain, A. sojae ATCC 20235. Additional, further mutation of mutant M3 for two more cycles of treatment by UV irradiation generated mutant DH56 with the highest polygalacturonase activity (98.8?±?8.7 U/mL) in submerged culture. This corresponded to 2.4-fold enhanced polygalacturonase production in comparison to the wild strain. The results of this study indicated the development of a classical mutation and selection strategy as a promising tool to improve pectinolytic enzyme production by both fungal strains.     

Dry-heat inactivation ofBacillus subtilis spores by means of infra-red heating

An experimental equipment for dry-heat inactivation of bacterial spores in an open system using Infrared (IR) radiation for energy transfer was developed. The dry-heat-inactivation kinetics forBacillus subtilis ATCC 6633 spores were studied in the temperature range of 120–180C. The z value (z=23C) was constant in the temperature range investigated. The advantages offered by using IR radiation in sterilization systems are pointed out.     

A comparison of genes involved in sphingan biosynthesis brought up to date

Microbial polysaccharides have a wide range of functional properties and show high relevance in industrial applications. The possibility to create tailor-made polysaccharides by genetic engineering will further enhance the product portfolio and may open new fields of application. Here, we have examined in detail the recently sequenced genome of the welan-producing strain Sphingomonas sp. ATCC 31555 to identify the complete welan cluster and further genes involved in EPS production. The corresponding genes were compared on the nucleotide and amino acid sequence level to the EPS clusters of the described gellan-producing Sphingomonas elodea ATCC 31461, diutan-producing Sphingomonas sp. ATCC 53159, and the S-88-producing Sphingomonas sp. ATCC 31554 strains. We also compared the previously mentioned strains to each other and included the genes upstream of the main cluster in gellan and welan cluster. The cluster organization of Sphingomonas strain S-7 was also compared based on previous hybridization experiments, without nucleotide sequences. We have found that the occurrence of genes in all biosynthesis clusters is connected to the structures of the various produced sphingans. Along these lines, homologous genes responsible for the assembly of the identical repeating unit generally show high sequence identity, whereas genes for putative side chain attachment urf31, urf31.4, and urf34 vary more in distinct areas. Moreover, gene clusters for biosynthesis of diutan, welan, gellan, and S-88 as well as S-7 are similar in general organization but differ in location and arrangement of some genes. Finally, we summarized genetic and mutational engineering approaches toward modified sphingan variants as described in literature.     

Nonspecificity of some commonly used inhibitors of DNA synthesis in cultures ofStreptococcus faecalis

The specificity of three commonly used inhibitors of DNA synthesis were tested in the batch culture ofStreptococcus faecalis ATCC 8043 in rich broth medium. It was shown that nalidixic acid, mitomycin C and 6-(4-hydroxyphenylazo)uracil inhibit the cell mass as much as they decrease net DNA synthesis. Hence the drugs tested are highly unspecific inhibitors of DNA synthesis inS. faecalis; i.e. they all interfere with other processes as well as with DNA synthesis.     

Role ofβ-oxidation in inhibitingLactobacillus leichmanii by fatty acids

The effect of saturated fatty acids from 6∶0 to 16∶0 and oleic acid onLactobacillus leichmanii ATCC 4797 growing in non-skim-milk media was determined. The inhibition by lauric acid was higher than that obtained with any other fatty acid. A mutant (MC12) resistant to the fatty acid inhibition with high β-oxidation activity was also studied. A positive correlation between the ability ofL. leichmanii ATCC 4797 and its derivative MC12 to degrade fatty acids and their resistance to the fatty acid inhibition is shown in this report.     

A Laboratory Case Study of Efficient Polyhydoxyalkonates Production by Bacillus cereus,a Contaminant in Saccharophagus degradans ATCC 43961 in Minimal Sea Salt Media

A contaminating bacterium growing along with the stock culture of Saccharophagus degradans ATCC 43961 (Sde 2-40) on marine agar plate was isolated and investigated for its ability to produce polyhydoxyalkonates (PHA). Preliminary screening by Sudan black B and Nile blue A staining indicated positive characteristic of the isolate to produce PHA. The isolate was able to grow and produce PHA in minimal sea salt medium broth. PHA quantification studies with gas chromatographic analyses of the dry cells derived from culture broths revealed accumulation of PHA in bacterial cells. PHA production started after 20 h and increased with cell growth and attained maximum values of 61 % of dry cell weight at 70 h of cultivation. After 70 h, a slight decrease in the level of PHA content was observed. The nature/type of PHA was found to be poly(3-hydroxybutyraye) by Fourier transform-infrared spectroscopy. Microbiological and 16S rRNA gene sequencing analyses suggested that the PHA producing bacterial isolate belongs to Bacillus genera and shows 100 % nucleotide sequence similarity with Bacillus cereus species in GenBank. This study is a first report for ability of Bacillus species to grow in marine sea salt media and produce PHA. The media used for the polymer production was novel in the context of the genus Bacillus and the production of PHA was three-fold higher than Sde 2-40 using same growth medium. This study shows that the contaminant bacteria once properly investigated can be used for advantageous characteristic of metabolites production in place of original cultures.     

Killer behavior within the Candida parapsilosis complex

A group of 29 isolates of Candida parapsilosis sensu stricto, 29 of Candida orthopsilosis, and 4 of Candida metapsilosis were assayed for the presence of killer activity using Saccharomyces cerevisiae ATCC 26609 as a sensitive strain. All C. metapsilosis isolates showed killer activity at 25 °C while strains of C. parapsilosis sensu stricto or C. orthopsilosis did not exhibit this activity. Sensitivity to killer toxins was evaluated using a set of previously reported killer strains of clinical origin. Only 11 isolates of the C. parapsilosis complex were inhibited by at least one killer isolate without resulting in any clear pattern, except for C. parapsilosis sensu stricto ATCC 22019, which was inhibited by every killer strain with the exception of C. parapsilosis and Candida utilis. The lack of sensitivity to killer activity among isolates of the genus Candida suggests that their toxins belong to the same killer type. Differentiation of species within the C. parapsilosis complex using the killer system may be feasible if a more taxonomically diverse panel of killer strains is employed.     

Identification of a Napin-Like Peptide from Eugenia malaccensis L. Seeds with Inhibitory Activity Toward Staphylococcus aureus and Salmonella Enteritidis

This study aimed to purify and characterize peptides from the seeds of Eugenia malaccensis, L. (jambo) with inhibitory activity against the foodborne pathogens Staphylococcus aureus and Salmonella Enteritidis. Crude extract (CE), precipitate fraction 30–60 % and molecules between 3.5 and 10 kDa obtained from precipitate fraction 30–60 % (Em2) showed inhibitory activity against the tested bacterial strains. The highest antibacterial activity was observed for Em2 against S. aureus. The major peak eluted at approximately 30 % in an acetonitrile gradient in reverse-phase chromatography of Em2 (Em2-F1 to Em2-F19), and it showed the highest antibacterial activity, which was twofold higher against S. aureus than against S. Enteritidis. MALDI-ToF spectra of Em2-F18 revealed a molecular mass of 1,231.1 Da and the amino acid sequence showed high identity to the napin family. These findings report for the first time a napin-like peptide from E. malaccensis L. seeds with potential to be applied as a new anti-Staphylococcus molecule.     

Effects of catechins on Agrobacterium-mediated genetic transformation of Camellia sinensis

In this study, recalcitrance of tea plant ( Camellia sinensis) to Agrobacterium-mediated genetic transformation was investigated with an emphasis on specialized compounds in tea. Chemical constituents in tea leaves and calli were extracted into liquid Luria–Bertani (LB) medium to determine their biological activities on Agrobacterium growth, virulence, and plant transformation efficiency. Compared to the control Agrobacterium grown in LB medium, tea leaf extract containing 6.5 mg mL^?1 catechins resulted in an 84.6 % reduction of Agrobacterium growth, a 73–36 % suppression of expression for the six virulence (vir) genes, browning of infected tobacco explant wounds, and an absence of transient or stable transformation events. Tea callus extract, containing 0.22 mg mL^?1 catechins, did not significantly affect Agrobacterium growth or tobacco transgenic hairy root generation, whereas it enhanced the expression of some vir genes. Treatment with authentic catechin mixtures (other than caffeine) dissolved in LB resulted in suppression of Agrobacterium growth, vir gene expression, and tobacco transformation efficiency. Our data suggest that catechins are the key active constituents in tea leaves. Transient transformation efficiencies of tea leaves were much lower than those of tobacco leaves as indicated by the GUS (β-glucuronidase) assay, probably a result of inhibition by the catechins present in tea leaves. Lower transformation efficiencies of tea calli suggested that additional plant factor(s) might also exert inhibitory effects on tea plant transformation. Agrobacterium rhizogenes ATCC 15834 induced transgenic roots from the tea explants with 15–20 % efficiency. Our data suggested catechins inhibition of tea gene transformation could be overcome by using optimized strains of Agrobacterium.     

Naphthalene oxygenase fromCorynebacterium renale: Characterisation and mechanism of oxygenation

The formation ofcis-l,2,-dihydroxy-l,2,-dihydronaphthalene from naphthalene by naphthalene oxygenase, purified fromCorynebacterium renale ATCC 15075, was demonstrated to involve oxidation of a mol NADH and consumption of one mol oxygen. The enzyme contains one g-atom Fe²⁺ and one FAD. Catalase inhibited product formation and H2O2 could substitute for NADH in the reaction. Superoxide dismutase inhibited enzyme activity when either NADH or H₂O₂ was present; the generation of superoxide anion on addition of NADH to the enzyme, in the absence of naphthalene, was detected by the nitro blue tetrazolium reduction method. Hydroxyl radical scavengers, ethanol, mannitol and sodium benzoate, inhibited product formation when either NADH or H₂O₂ was present. Electron spin resonance studies, under aerobic conditions, indicated that iron of the enzyme underwent valence changes during the course of the reaction