Evaluation of the in vitro cytotoxicity of the antimicrobial peptide P34

The in vitro cytotoxicity of the antimicrobial peptide P34 was evaluated in different eukaryotic cells. The food‐grade bacteriocin nisin was also analysed for comparison. Vero cells were treated with different concentrations (0.02–2.5 μg·ml^?1) of antimicrobial peptide P34 and nisin. Cell viability and plasma membrane integrity were checked by MTT [3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide], NRU (Neutral Red dye uptake) and LDH (lactate dehydrogenase) assays. The EC₅₀ values of the peptide P34 in MTT and NRU assays were 0.60 and 1.25 μg·ml^?1 respectively, while values of nisin found were 0.50 and 1.04 μg·ml^?1. In the LDH assay, the EC₅₀ values were 0.65 and 0.62 μg·ml^?1 for P34 and nisin, respectively. The peptide P34 revealed similar haemolytic activity on human erythrocytes (5.8%) when compared with nisin (4.9%). The effects on viability, motility and acrosomal exocytosis of human sperm were also evaluated. Nisin and P34 showed similar effects on sperm parameters. The evaluation of cytotoxicity of antimicrobial peptides is a critical step to guarantee their safe use.     

Methylation of TMV RNA

Several plant and animal viral RNAs contain a tRNA like structure at their 3′ ends. In this communication we show that tobacco mosaic virus (TMV) RNA is an acceptable substrate for a specific tRNA methyltransferase. Using a crude preparation of $\text{E. coli}$ ribothymidine (rT) forming uracil methylase and (methyl ³H) S-adenosyl-L-methionine (SAM) as a methyl donor, 0.7 moles of methyl group is incorporated per mole of TMV RNA in 10 hours at 30°C. Upon T₂ RNAse digestion of the labeled RNA, all of the radioactivity was found to be in TMP. T₁ RNAse digestion of ³H methylated TMV RNA showed that all of the label was located in a tetranucleotide which co-migrated with authentic TpψpCpGp, an oligonucleotide characteristically found in normal cellular tRNA.The use of this specific methyl transferase reaction may provide a simple assay for the detection of tRNA like structures in large RNAs.     

Homology between the proteins encoded by tobacco mosaic virus and two tricornaviruses

Summary A comparison was made of the amino acid sequences of the proteins encoded by RNAs 1 and 2 of alfalfa mosaic virus (A1MV) and brome mosaic virus (BMV), and the 126K and 183K proteins encoded by tobacco mosaic virus (TMV). Three blocks of extensive homology of about 200 to 350 amino acids each were observed. Two of these blocks are located in the A1MV and BMV RNA 1 encoded proteins and the TMV encoded 126K protein; they are situated at the N-terminus and C-terminus, respectively. The third block is located in the A1MV and BMV RNA 2 encoded proteins and the C-terminal part of the TMV encoded 183K protein. These homologies are discussed with respect to the functional equivalence of these putative replicase proteins and a possible evolutionary connection between A1MV, BMV and TMV.     

Reduced levels of class 1 reversibly glycosylated polypeptide increase intercellular transport via plasmodesmata

Maize and Arabidopsis thaliana class 1 reversibly glycosylated polypeptides (^C1RGPs) are plasmodesmata-associated proteins. Previously, overexpression of Arabidopsis ^C1RGP AtRGP2 in Nicotiana tabacum was shown to reduce intercellular transport of photoassimilate, resulting in stunted, chlorotic plants, and inhibition of local cell-to-cell spread of tobacco mosaic virus (TMV). Here, we used virus induced gene silencing to examine the effects of reduced levels of ^C1RGPs in Nicotiana benthamiana. Silenced plants show wild-type growth and development. Intercellular transport in silenced plants was probed using fluorescently labeled TMV and its movement protein, P30. P30 shows increased cell-to-cell movement and TMV exhibited accelerated systemic spread compared with control plants. These results support the hypothesis that ^C1RGPs act to regulate intercellular transport via plasmodesmata.     

Analytical subcellular fractionation of rat pituitary homogenates with special reference to the subcellular localization and properties of alkaline phosphatases

Alkaline phosphatase activities of the virgin rat anterior pituitary were studied with a highly sensitive fluorometric assay. Tissue whole homogenates were fractionated on sucrose density gradients in a Beaufay automatic zonal rotor and the gradient fractions assayed for alkaline phosphatase, prolactin and various organelle marker enzymes. Alkaline phosphatase was distributed between two peaks on the gradient. The low-density (1.10–1.15 g·cm^?3) alkaline phosphatase component co-sedimented with the plasma membrane marker, 5′-nucleotidase, had an apparent K_m for 4-methylumbelliferyl phosphate of approx. 59 μM, and was inhibited by levamisole. The high-density (1.20–1.25 g·cm^?3) peak was resistant to levamisole-inhibition, had an apparent K_m of approx. 30 μM and its distribution was distinct from plasma membrane, Golgi, lysosome, endoplasmic reticulum, mitochondria and prolactin granule markers on the isopycnic gradients.     

Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

Antiviral properties and interaction of novel chalcone derivatives containing a purine and benzenesulfonamide moiety

A new concise and facile method was explored to synthesize a series of novel chalcone derivatives containing a purine and benzenesulfonamide moiety and their antiviral properties were evaluated against TMV and CMV. Biological assays indicated that several of the derivatives exhibited significant anti-TMV and anti-CMV activities in vivo. In particular, compound d2 displayed excellent inactivating activity against TMV, with the EC₅₀ value of 51.65?μg/mL, which was better than that of ribavirin (150.45?μg/mL). Molecular docking showed that there are four hydrogen bonds between compound d2 and TMV coat protein (TMV-CP). Compound d2 demonstrated strong binding capacity to TMV-CP with K_a?=?1.58?×?10⁵?L/mol and K_d?=?12.16?μM. These findings indicated that chalcone derivatives are worthy of further research and development as templates for new antiviral agents.     

Effects of thymol and diphenyliodonium chloride against Campylobacter spp. during pure and mixed culture in vitro

Aims: To determine if the purported deaminase inhibitors diphenyliodonium chloride (DIC) and thymol reduce the growth and survivability of Campylobacter. Methods and Results: Growth rates of Campylobacter jejuni and Camp. coli were reduced compared to unsupplemented controls during culture in Muellar–Hinton broth supplemented with 0·25 μmol DIC or thymol ml^?1 but not with 0·01 μmol monensin ml^?1 or 1% ethanol. Recovery of Camp. jejuni and Camp. coli was reduced >5 log₁₀ CFU from controls after 24 h pure culture in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Similarly, each test Campylobacter strain was reduced >3 log₁₀ CFU from controls after 24 h mixed culture with porcine faecal microbes in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Treatments with 0·25 μmol thymol ml^?1, 0·01 μmol monensin ml^?1 or 1% ethanol were less effective. Ammonia production during culture or incubation of cell lysates was reduced by 0·25 or 1·0 μmol DIC ml^?1 but only intermittently reduced, if at all, by the other treatments. Conclusions: Diphenyliodonium chloride and thymol reduced growth, survivability and ammonia production of Camp. jejuni and Camp. coli. Significance and Impact of the Study: Results suggest a potential physiological characteristic that may be exploited to develop interventions.     

Stimulation of caffeine metabolism in the rat by 3-methylcholanthrene

Groups of adult male rats treated with 3-methylcholanthrene, phenobarbital or vehicles alone, were administered caffeine either orally or intravenously. Serum caffeine concentrations were measured by radioimmunoassay. In vehicle and phenobarbital pretreated animals, caffeine elimination kinetics were non-linear. In control animals, the $\text{in}$$\text{vivo}$ apparent Km was 8 μg·ml^?1 (40 μM) and the apparent Vmax was 0.1 μg·ml^?1·min^?1 (0.5 μM·min^?1). Phenobarbital pretreatment did not change the apparent Km but slightly increased the apparent Vmax. 3-Methylcholanthrene pretreatment dramatically altered the elimination kinetics of caffeine, whether caffeine was given orally or intravenously. The elimination of caffeine from serum of 3-methylcholanthrene pretreated rats was first order with a $\text{t}\text{1}\text{2}$ of approximately 14 minutes.Our results are consistent with the proposed involvement of the cytochromes P-450 monooxygenase system in the elimination of caffeine. In addition, our results suggest that caffeine is a moderately poor substrate for the cytochromes P-450 present in control and phenobarbital-pretreated rats, but a particularly good substrate for the form(s) induced by 3-methylcholanthrene.     

Localization of N6-methyladenosine in the Rous sarcoma virus genome.

The 10,000-nucleotide RNA genome of the Prague strain, subgroup B (PR-B) of Rous sarcoma virus, was found to contain 11.6 ± 0.5 residues of m⁶Ap by quantitative analysis of ³²P-labeled virion RNA after complete RNAase digestion. Approximately ten of the m⁶Ap residues are located, without obvious clustering, in that region of the genome between 500 and 4000 nucleotides from the 3′ poly(A) end. The src gene, which is required for transformation, and part of the env gene, which codes for the major viral envelope glycoprotein, have previously been mapped in this region of the viral genome. A transformation-defective deletion mutant of PR-B Rous sarcoma virus, which lacks the src gene, has 7.0 ± 0.2 m⁶Ap residues per RNA subunit. This supports our mapping of a portion of the m⁶A residues in src and suggests that this methylation is specific to certain regions of the genome. The possible significance of this result for Rous sarcoma virus RNA processing and translation is discussed.     

Dual action of ionophore A23187 on intact chloroplasts

1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O₂ evolution by envelope-free chloroplasts and oxaloacetate-dependent O₂ evolution by intact chloroplasts. The half maximal concentration ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml^?1.2. Ionophore A23187 also induces inhibition of CO₂ and 3-phosphoglycerate-dependent O₂ evolution by intact chloroplasts in the presence of 3 mM MgCl₂. The half maximal concentrations ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for inhibition of O₂ evolution are 3 μM and 5 μM respectively · 100 μg^?1 chlorophyll · ml^?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg^?1 chlorophyll · ml^?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.     

Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

Properties and role of ferritin in the hemolymph of the chiton Clavarizona hirtosa

The major iron-binding protein found in the hemolymph of the chiton Clavarizona hirtosa has been purified for the first time and identified as ferritin. This ferritin, which is present at a concentration of approx. 400 μg·ml⁻¹, has a M_r of 28 000 and 25 500, exhibits microheterogeneity with isoelectric values in the range 5.3–6.0, binds 1500–2500 Fe atoms·mol⁻¹ and is immunologically distinct from horse spleen ferritin. The initial rate of iron accumulation by ferritin molecules was determined to be markedly higher than that exhibited by horse spleen ferritin. Taken together, these data suggest that ferritin found in the hemolymph serves as a key component of the high-capacity transport system necessary to deliver iron to the rapidly mineralizing tissue of the radula in these molluscs.     

Effect and control of glucose feeding on bacitracin production by fed-batch culture of Bacillus licheniformis

The effect of glucose feeding on bacitracin production was investigated by fed-batch culture of Bacillus licheniformis. In batch culture, bacitracin secretion was induced after the glucose initially contained in the medium was completely consumed. The concentration of bacitracin, however, increased to no more than 340 units·ml⁻¹ in the batch cultivations. Therefore, additional glucose was supplied after exhaustion of the initial glucose. The effect of glucose feeding on bacitracin biosynthesss was investigated in two ways, the pH-stat modal feeding method and the CO₂-dependent feeding method. A kinetic study of bacitracin production found that some glucose was necessary, even during the bacitracin production phase. Excessive feeding of glucose, however, caused a reduction in bacitracin biosynthetic activity. When 50 g·l⁻¹ of defatted soy bean meal (SBM) was used, the bacitracin concentration reached 670 units·ml⁻¹ with the pH-stat modal feeding method and 610 units·ml⁻¹ with the CO₂-dependent feeding method, respectively. The yield of bacitracin from consumed glucose was better for the pH-stat method. Using this control strategy, the highest concentration of bacitracin (940 units·ml⁻¹) was obtained with 150 g·l⁻¹ of SBM.     

Iodine from bacterial iodide oxidization by Roseovarius spp. inhibits the growth of other bacteria

Microbial activities in brine, seawater, or estuarine mud are involved in iodine cycle. To investigate the effects of the microbiologically induced iodine on other bacteria in the environment, a total of 13 bacteria that potentially participated in the iodide-oxidizing process were isolated from water or biofilm at a location containing 131 μg ml^?1 iodide. Three distinct strains were further identified as Roseovarius spp. based on 16 S rRNA gene sequences after being distinguished by restriction fragment length polymorphism analysis. Morphological characteristics of these three Roseovarius spp. varied considerably across and within strains. Iodine production increased with Roseovarius spp. growth when cultured in Marine Broth with 200 μg ml^?1 iodide (I^?). When 10⁶ CFU/ml Escherichia coli, Pseudomonas aeruginosa, and Bacillus pumilus were exposed to various concentrations of molecular iodine (I₂), the minimum inhibitory concentrations (MICs) were 0.5, 1.0, and 1.0 μg ml^?1, respectively. However, fivefold increases in the MICs for Roseovarius spp. were obtained. In co-cultured Roseovarius sp. IOB-7 and E. coli in Marine Broth containing iodide (I^?), the molecular iodine concentration was estimated to be 0.76 μg ml^?1 after 24 h and less than 50 % of E. coli was viable compared to that co-cultured without iodide. The growth inhibition of E. coli was also observed in co-cultures with the two other Roseovarius spp. strains when the molecular iodine concentration was assumed to be 0.52 μg ml^?1.     

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI₂) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 μg ml^?1). Both 15-HPAA (1–20 μg ml^?1 min^?1) and 13-hydroperoxy linoleic acid (13-HPLA, 20 μg ml^?1 min^?1) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI₂ (5 μg ml^?1 min^?1) or 6-oxo-prostaglandin F_1α (6-oxo-PGF_1α, 5 μg ml^?1 min^?1). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI₂ (10 ng - 10 μg ml^?1 min^?1) but was inhibited by PGE₂ (5 and 10 μg ml^?1 min^?1). Antiserum raised to 5,6-dihydro prostacyclin (PGI₁) in rabbits, which also binds PGI₂, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

Renierol from marine sponge Haliclona.SP.: A natural inhibitor of xanthine oxidase with hypouricemic effects

The purpose of this study was to evaluate the inhibitory effect of renierol, extracted from marine sponge Halicdona.SP., on xanthine oxidase (XO) and its hypouricemic effect in vivo. Renierol and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid and superoxide radical from xanthine. Renierol inhibited XO in a concentration-dependent and competitive manner. IC₅₀ value was 1.85 μg·ml^{? 1} through the measuring of uric acid and was 1.36 μg.ml^{? 1} through the measuring of superoxide radical. Renierol was found to have an in vivo hypouricemic activity against potassium oxonate-induced hyperuricaemia in mice. After oral administration of renierol at doses of 10, 20 and 30 mg.kg^{? 1}, there was a significant decrease in the serum urate level (4.08 ± 0.09 mg.dl^{? 1}, P < 0.01), (3.47 ± 0.11 mg.dl^{? 1}, P < 0.01) and (3.12 ± 0.08 mg.dl^{? 1}, P < 0.01), when compared to the hyperuricaemic control (6.74 ± 0.23 mg.dl^{? 1}). Renierol was a potent XO inhibitor with hypouricemic activity in mice.     

Expression of the acidic-subunit of amarantin, carrying the antihypertensive biopeptides VY, in cell suspension cultures of Nicotiana tabacum NT1

A modified version of amarantin, main seed storage protein of Amaranthus hypochondriacus, carrying four antihypertensive biopeptides Val-Tyr into the acidic-subunit of the protein, was expressed in cell suspension cultures of Nicotiana tabacum L. NT1. Cell growth and viability kinetics were assessed to determine optimal conditions for genetic transformation via Agrobacterium tumefaciens. Selection of putative transgenic calli was conducted using 25 μg ml^?1 hygromycin. Presence of the transgene was confirmed using histological glucuronidase assay and PCR analysis. Accumulation and expression of the recombinant protein was detected using Western blot analysis. Protein hydrolysate of transgenic calli showed high levels of inhibition of the angiotensin converting enzyme, with an IC₅₀ value of 3.5 μg ml^?1. This was 10-fold lower than that of protein extracts of wild-type cells, with an IC₅₀ of 29.0 μg ml^?1.     

Structure of the RNA in tobacco mosaic virus

A model of the RNA of tobacco mosaic virus has been built using computer model-building techniques. The model has good stereochemistry, and fits the electron density map of the virus obtained by fiber diffraction methods considerably better than did earlier models. The three sugar rings in the asymmetric unit all have the A (3′-endo) conformation, One of the bases is in the syn conformation, a conformation observed only rarely in nucleic acid structures.     

In vitro anti‐enterovirus 71 activity of gallic acid from Woodfordia fruticosa flowers

Aims: The anti‐enterovirus 71 (EV71) activity of six Nepalese plants’ extracts and gallic acid (GA) isolated from Woodfordia fruticosa Kurz (family; Lythaceae) flowers were evaluated in Vero cells. Methods and Results: The anti‐EV71 activity of tested compounds was evaluated by a cytopathic effect reduction method. Our results demonstrated that flowers’ extracts of W. fruticosa exerted strong anti‐EV71 activity, with a 50% inhibitory concentration (IC₅₀) of 1·2 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived therapeutic index (TI) was more than 83·33. Rivabirin showed no antiviral activity against EV71. Furthermore, GA isolated from W. fruticosa flowers exhibited a higher anti‐EV71 activity than the extract of W. fruticosa flowers, with an IC₅₀ of 0·76 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived TI was 99·57. Conclusions: This study demonstrated that flower extracts of W. fruticosa possessed anti‐EV71 activity and GA isolated from these flowers showed stronger anti‐EV71 activity than that the extracts. Significance and Impact of the Study: Our results suggest that the GA from W. fruticosa flowers may be used as a potential antiviral agent.