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.     

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     

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.     

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.     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

The amino-terminal sequence ofStreptomyces griseus carboxypeptidase

The amino-terminal sequence of carboxypeptidase fromStreptomyces griseus was determined using a new protocol for automatic Edman degradation that reduced background noise. The sequence of the first 48 residues is: Asp-Phe-Pro-Pro-Ala-Asp-Ser-Arg-Tyr-His-Asn-Tyr-Ala-Glu-Met-Asn-Ala-Ala-Ile-Asp-Ala-Arg-Ile-Ala-Ala-Asn-Pro-Ser-Ile-Met-Ser-Lys-Arg-Val-Ile-Gly-Lys-Thr-Tyr-Gln-Gly-(Arg)-Asp-Val-Ile-Ala-Val-Lys, which is homologous to that of other zinc-containing carboxypeptidase from vertebrate and invertebrate sources.     

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.     

The investigation of the effect of pollination on ribosomal RNA,transfer RNA and DNA contents in styles ofNicotiana alata.

Using the phenol extraction method and MAK column chromatography the contents of nucleic acids in styles ofNicotiana alata were estimated before and up to 48 h after compatible pollination. As a result of pollination, high-molecular-weight rRNA and DNA registered a significant increase in their content approximating 30% and 16% respectively. The change in sRNA (4-S tRNA and 5-S rRNA) level was very slight and non-significant. In pollen grains there is an unusually high amount of rRNA with respect to the content of other nucleic acids and the rRNA/tRNA ratio approximates 14 : 1. On the other hand, in non-pollinated styles the content of rRNA is only about 6 times higher than that of tRNA. The changes in nucleic acid level found in styles after pollination are at least in a major part the result of the addition of the nucleic acids present in the amount of pollen used for pollination. The high rRNA level relatively to tRNA being generally associated with rapidly growing cells, its significance in pollen may be related to the rapid growth of pollen tubes.     

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.     

On the existence of two GABA pools associated with newly synthesized GABA and with newly taken up GABA in nerve terminals

[¹⁴C]Glutamic acid and [³H]GABA were injected into the lateral ventricle of mouse and then [¹⁴C]GABA and [³H]GABA in synaptosomes isolated from the animals were analysed. The [¹⁴C]GABA was interpreted to be newly synthesized GABA from [¹⁴C]glutamic acid while the [³H]GABA to be newly taken up GABA. We have obtained the following results: (1) when the animals were pretreated with aminooxyacetic acid and thus the GABA content in synaptosomes increased to about 2 times of the control level, only the [³H]GABA was enhanced to 3 times of the control level without any change of [¹⁴C]GABA, (2) the release of [¹⁴C]GABA from synaptosomes by high K⁺ depolarization was 1.5 times greater than that of [³H]GABA, (3) the releases of both [¹⁴C]GABA and [³H]GABA were increased in the presence of cold GABA,l-2,4-diaminobutyric acid or γ-amino-β-hydroxybutyric acid, but only slightly increased in the presence of β-alanine. These results would suggest that newly synthesized GABA and newly taken up GABA localized individually in different pools, which might localize either in different nerve terminals or separately in the same nerve terminal.     

Expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the Spinal Tuberculous Focus and Its Impact on the Disease

To investigate the expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the spinal tuberculous focus and its relationship with the lesions type, severity, and bone destruction. The pathological samples of patients with spinal tuberculosis (TB) were divided into hyperplasia group and necrosis group according to their intra-operative and post-operative pathological findings. Normal bone tissues were taken as the control group. Pathology and expression of TNF-α, IFN-γ, TGF-β, and IL-4 in different tissues were compared among these three groups using immunohistochemical staining, quantitative image analysis, and measurement of bone tissue. 286 granulomas observed in the 14 samples in the hyperplasia group, which included 84 necrotizing and 202 non-necrotizing granulomas. As for the 20 samples in the necrosis group, there were 356 necrotizing and 186 non-necrotizing granulomas among all the 542 granulomas. The proportion of necrotizing granulomas in the necrosis group was significantly higher than that of the hyperplasia group. By inter-group comparison, expression of TNF-α, IFN-γ of granulomas in the hyperplasia group was significantly higher than that of the necrosis group, while the expression of TGF-β, IL-4 of granulomas in the necrosis group was significantly higher than that of the hyperplasia group. Also, expression of IFN-γ of non-necrotizing granulomas was significantly higher than that of necrotizing granulomas in the hyperplasia group, and expression of TGF-β in necrotizing granulomas was significantly higher than that of non-necrotizing granulomas in the necrosis group. The lesions were mainly bone resorption in the hyperplasia group, whereas mostly necrotic bones accompanied by local fibrosis in the necrosis group. Expression levels of TNF-α, IFN-γ in the hyperplasia group have a positive correlation to bone loss, whereas expression levels of TGF-β, IL-4 in the necrosis group have a positive correlation to the bone formation. The high expressions of TNF-α, IFN-γ in the spinal tuberculous focus were associated with protective immune cells. TGF-β and IL-4 were related to allergic lesions, fibrosis and osteogenesis. Expression imbalance of TNF-α, IFN-γ, TGF-β, and IL-4 might aggravate the allergy of TB.     

cDNA library construction from meristematic tissue of finger millet panicle

A protocol to obtain full-length cDNA using a SuperScript® Full-Length cDNA Library Construction Kit II (Invitrogen, United States) was developed, and a high quality cDNA library of meristem tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was constructed. The titer of the constructed cDNA library was 3.01 × 10⁵ CFU/mL, the average length of the insert was approximately 1070 base pairs, and the average efficiency of insertion of cDNA fragments was 99.5%. The sequencing of randomly selected clones created cDNA library was carried out. The cDNA sequences of clones were identified by BLAST search. The cDNA library analysis and selective sequencing indicate good functionality and full size of cDNA inserts of the clones. The constructed cDNA library from meristematic tissue of finger millet panicle is a good and reliable source for isolation and identification of key genes of metabolism and development of meristem as well for creation of new genetic markers for genetic research and molecular selection.