Identification of antibiotics for use in selection of the chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans.

Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted treatments are necessary to manage the disease. Currently, no tools for genetic manipulation are available to identify and test specific drug targets in these fungi. To facilitate the development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin. We have identified effective concentrations of each for selection in both liquid culture and on solid media. These concentrations are within the range of concentrations used for selecting genetically modified cells from a variety of other eukaryotic species.

Antibiotic Treatment Reduces the Health Benefits of Soy Protein.

SCOPE: Soy protein is a high-quality protein and its consumption has been associated with a reduction of serum cholesterol and triglycerides and an improvement in insulin resistance. However, it is not known whether the effects of soy protein are mediated by the gut microbiota. Thus, the aim of this study is to assess whether using antibiotics to partially eradicate the gut microbiota can prevent the beneficial effects of soy protein in rats. METHODS AND RESULTS: Thus, rats are fed one of the following diets for 16 weeks: casein control, soy protein control, high-fat casein, and high-fat soy protein. The rats are then treated for 4 weeks with antibiotics. Body weight and composition, energy expenditure, glucose tolerance test, metabolic endotoxemia, and gut microbiota are measured before and after treatment with antibiotic. The results show that soy protein consumption decreases weight gain, body fat, metabolic endotoxemia, and increases energy expenditure and glucose tolerance. Antibiotic treatment suppresses all these metabolic effects. These changes are accompanied by modifying the diversity and taxonomy of the gut microbiota. CONCLUSION: In conclusion, the evidence suggests that the health benefits of soy protein are partly dependent of the gut microbiota.

Performance and intestinal microbiota of chickens receiving probiotic in the feed and submitted to antibiotic therapy.

The purpose of this study was to verify the ability of a probiotic in the feed to maintain the stability of the gut microbiota in chickens after antibiotic therapy and its association with growth performance. One thousand six hundred twenty 1-day-old Cobb male were housed in floor pens (36 pens, 45 birds/pen) and were fed corn-/soya bean meal-based diets supplemented with or without probiotic (Bacillus subtilis) during the entire rearing phase. From 21 to 24 days of age (three consecutive days), the chickens were submitted to antibiotic therapy via drinking water (bacitracin and neomycin) in order to mimic a field treatment and induce dysbiosis. Growth performance was monitored until 42 days of age. At 2, 4 and 6 days after antibiotic therapy, three chickens from each pen were euthanized and the contents of the small intestine and caeca were collected and pooled. The trial was conducted with four treatments and nine replicates in a 2 × 2 factorial arrangement for performance characteristics (with and without probiotic × with and without antibiotic therapy); for the intestinal microbiota, it was in a 2 × 2 × 3 factorial arrangement (with and without probiotic × with and without antibiotic therapy × 2, 4 and 6 days after the antibiotic therapy) with three replicates per treatment. Terminal restriction length polymorphism (T-RFLP) analysis showed that the structure of gut bacterial community was shaped by the intestinal segment and by the time after the antibiotic therapy. The number of 16S rDNAs copies in caecum contents decreased with time after the therapeutic treatment. The antibiotic therapy and dietary probiotic supplementation decreased richness and diversity indexes in the caecal contents. The improved performance observed in birds supplemented with probiotic may be related to changes promoted by the feed additive in the structure of the intestinal bacterial communities and phylogenetic groups. Antibiotic therapy modified the bacterial structure, but did not cause loss of broiler performance.

Chemical probing for examining the structure of modified RNAs and ligand binding to RNA.

Among different RNA modifications, the helix 69 (H69) region of the bacterial ribosomal RNA (rRNA) contains three pseudouridines (Ψs). H69 is functionally important due to its location in the heart of the ribosome. Several structural and functional studies have shown the importance of Ψ modifications in influencing the H69 conformation as well as maintaining key interactions in the ribosome during protein synthesis. Therefore, a need exists to understand the influence of modified nucleosides on conformational dynamics of the ribosome under solution conditions that mimic the cellular environment. In this review on chemical probing, we provide detailed protocols for the use of dimethyl sulfate (DMS) to examine H69 conformational states and the influence of Ψ modifications under varying solution conditions in the context of both ribosomal subunits and full ribosomes. The use of DMS footprinting to study the binding of aminoglycosides to the H69 region of bacterial rRNA as a potential antibiotic target will also be discussed. As highlighted in this work, DMS probing and footprinting are versatile techniques that can be used to gain important insight into RNA local structure and RNA-ligand interactions, respectively.

Antibacterial activity of Cladanthus arabicus and Bubonium imbricatum essential oils alone and in combination with conventional antibiotics against Enterobacteriaceae isolates.

Multidrug-resistant bacteria have become common all over the world, necessitating the development of new therapeutic strategies. Synergistic interactions between conventional antibiotics and natural bioactive may have therapeutic benefits in a clinical setting. There are plenty of medicinal plants that have proven efficacy against broad spectrum of micro-organisms. The aim of the work was to assess the antibacterial activity of Cladanthus arabicus, a Moroccan medicinal plant, and Bubonium imbricatum, a Moroccan endemic plant. The evaluation of the synergistic effect of extracted essential oils (EOs) together with some conventional antibiotics was also investigated. Checkerboard test was used to evaluate the interaction of EOs in combination with amoxicillin and neomycin. The results showed that EOs contain a potent activity against the tested Enterobacteriaceae isolates, with inhibition zones values in the range of 8·05 ± 0·1 and 13·1 ± 0·11 mm and MIC values between 200 µg ml-1 to 800 µg ml-1 for C. arabicus and from 400 µg ml-1 to 1600 µg ml-1 for B. imbricatum, respectively. Moreover, the current study allowed concluding that both EOs showed not only satisfactory antibacterial properties but also active effects combined with conventional antibiotics demonstrated by the Fractional Inhibitory Concentration Index (FICI). These findings are very interesting since there are no previous studies on synergistic interactions of these two plants with antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of antibiotic resistance is multifactorial, including the specific nature of the relationship of bacteria to antibiotics. This situation has forced scientists to search for new antimicrobial substances from various sources as novel antimicrobial chemotherapeutic agents. Recently, medicinal plants and their derivatives (essential oils, extracts) have become very important in therapeutics because they encounter minimal challenges of the emergence of resistance. In this direction, the antimicrobial activity of the endemic Bubonium imbricatum plant and medicinal Cladanthus arabicus plant essential oils against multidrug-resistant Enterobacteriaceae strains was demonstrated.

Aminoglycoside-based novel probes for bacterial diagnostic and therapeutic applications.

Specific detection of pathogens has long been recognized as a vital strategy in the control of infectious diseases. Two novel theranostic neomycin analogs exhibit efficient targeting, labelling and killing of broad spectrum bacteria while not damaging macrophage-like cells. Furthermore, lipidated probe 2 clearly showed antibacterial activity against methicillin-resistant S. aureus.

Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant.

The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-ß motif which consists of an α helix and a triple-stranded ß-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.

HEI-OC1 cells as a model for investigating drug cytotoxicity.

The House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few, and arguable the most used, mouse auditory cell line available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, it has been used to investigate, among other topics, apoptotic pathways, autophagy and senescence, mechanism of cell protection, inflammatory responses, cell differentiation, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. However, the use of different techniques with different goals resulted in apparent contradictions on the actual response of these cells to some specific treatments. We have now performed studies to characterize the actual response of HEI-OC1 cells to a battery of commonly used pharmacological drugs. We evaluated cell toxicity, apoptosis, viability, proliferation, senescence and autophagy in response to APAP (acetaminophen), cisplatin, dexamethasone, gentamicin, penicillin, neomycin, streptomycin, and tobramycin, at five different doses and two time-points (24 and 48 h), by flow cytometry techniques and caspase 3/7, MTT, Cytotoxicity, BrdU, Beclin1, LC3 and SA-ß-galactosidase assays. We also used HEK-293 and HeLa cells to compare some of the responses of these cells to those of HEI-OC1. Our results indicate that every cell line responds to the each drug in a different way, with HEI-OC1 cells showing a distinctive sensitivity to at least one of the mechanisms under study. Altogether, our results suggest that the HEI-OC1 might be a useful model to investigate biological responses associated with auditory cells, including auditory sensory cells, but a careful approach would be necessary at the time of evaluating drug effects.

Enhancement of the antibiotic activity of aminoglycosides by extracts from Anadenanthera colubrine (Vell.) Brenan var. cebil against multi-drug resistant bacteria.

The aim of this work was to evaluate the antimicrobial activity of ethanol (EEAC) and hexane (HFAC) extracts from the stem bark of Anadenanthera colubrina (Vell.) Brenan var. cebil alone or in combination with aminoglycosides against multi-drug resistant (MDR) bacteria. Minimal inhibitory concentrations (MICs) of the extracts were determined by using microdilution assay. For the evaluation of extracts as modulators of antibiotic resistance, MICs of neomycin and amikacin were determined in presence or absence of each compound at sub-inhibitory concentrations. Both EEAC and HFAC did not show antimicrobial activity against MDR strains tested. However, the addition of EEAC and HFAC enhanced the activity of neomycin and amikacin against Staphylococcus aureus SA10 strain. When the natural products were replaced by chlorpromazine, the same effect was observed. Anadenanthera colubrine var. cebil may be a source of phytochemicals able to potentiate the aminoglycoside activity against MDR S. aureus by the inhibition of efflux pump.

Antimicrobial activity of different filling pastes for deciduous tooth treatment.

Guedes-Pinto paste is the filling material most employed in Brazil for endodontic treatment of deciduous teeth; however, the Rifocort® ointment has been removed. Thus, the aim of this study was to investigate the antimicrobial potential of filling pastes, by proposing three new pharmacological associations to replace Rifocort® ointment with drugs of already established antimicrobial power: Nebacetin® ointment, 2% Chlorhexidine Gluconate gel, and Maxitrol® ointment. A paste composed of Iodoform, Rifocort® ointment and Camphorated Paramonochlorophenol (CPC) was employed as the gold standard (G1). The other associations were: Iodoform, Nebacetin® ointment and CPC (G2); Iodoform, 2% Chlorhexidine Digluconate gel and CPC (G3); Iodoform, Maxitrol® ointment and CPC (G4). The associations were tested for Staphylococcus aureus (S. aureus), Streptococcus mutans (S. mutans), Streptococcus oralis (S. oralis), Enterococcus faecalis (E. faecalis), Escherichia coli (E. coli), and Bacillus subtilis (B. subtilis), using the methods of dilution on solid medium - orifice agar - and broth dilution. The results were tested using statistical analysis ANOVA and Kruskal-Wallis. They showed that all the pastes had a bacteriostatic effect on all the microorganisms, without any statistically significant difference, compared with G1. S. aureus was statistically significant (multiple comparison test of Tukey), insofar as G2 and G3 presented the worst and the best performance, respectively. All associations were bactericidal for E. coli, S. aureus, S. mutans and S. oralis. Only G3 and G4 were bactericidal for E. faecalis, whereas no product was bactericidal for B. subtilis. Thus, the tested pastes have antimicrobial potential and have proved acceptable for endodontic treatment of primary teeth.

Antimicrobial activity of different filling pastes for deciduous tooth treatment

Guedes-Pinto paste is the filling material most employed in Brazil for endodontic treatment of deciduous teeth; however, the Rifocort® ointment has been removed. Thus, the aim of this study was to investigate the antimicrobial potential of filling pastes, by proposing three new pharmacological associations to replace Rifocort® ointment with drugs of already established antimicrobial power: Nebacetin® ointment, 2% Chlorhexidine Gluconate gel, and Maxitrol® ointment. A paste composed of Iodoform, Rifocort® ointment and Camphorated Paramonochlorophenol (CPC) was employed as the gold standard (G1). The other associations were: Iodoform, Nebacetin® ointment and CPC (G2); Iodoform, 2% Chlorhexidine Digluconate gel and CPC (G3); Iodoform, Maxitrol® ointment and CPC (G4). The associations were tested for Staphylococcus aureus (S. aureus), Streptococcus mutans (S. mutans), Streptococcus oralis (S. oralis), Enterococcus faecalis (E. faecalis), Escherichia coli (E. coli), and Bacillus subtilis (B. subtilis), using the methods of dilution on solid medium – orifice agar – and broth dilution. The results were tested using statistical analysis ANOVA and Kruskal-Wallis. They showed that all the pastes had a bacteriostatic effect on all the microorganisms, without any statistically significant difference, compared with G1. S. aureus was statistically significant (multiple comparison test of Tukey), insofar as G2 and G3 presented the worst and the best performance, respectively. All associations were bactericidal for E. coli, S. aureus, S. mutans and S. oralis. Only G3 and G4 were bactericidal for E. faecalis, whereas no product was bactericidal for B. subtilis. Thus, the tested pastes have antimicrobial potential and have proved acceptable for endodontic treatment of primary teeth.

Granulated lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology of 10-day-old pigs.

Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this experiment was to determine the effect of a purified granulated lysozyme, compared with antibiotics, on growth performance, small intestinal morphology, and Campylobacter shedding in 10-d-old pigs. Forty-eight pigs (n = 16 per treatment), with an initial BW of 4.0 ± 0.1 kg (P > 0.40), were weaned at 10 d of age, blocked by litter and sex, and assigned to pens (8 pigs/pen). Each block was randomly assigned to consume 1 of 3 liquid dietary treatments for 14 d: a control diet, the control diet + lysozyme (100 mg/kg of diet), or the control diet + antibiotics (neomycin and oxytetracycline, 16 mg/kg of diet). Pigs were weighed and blood was sampled on d 0, 7, and 14. Blood was analyzed for plasma urea N and IgA. After 14 d of treatment, pigs were killed and samples of the jejunum and ileum were collected and fixed to measure villus height and crypt depth. Rectal swabs were taken on d 0, 7, and 14 of treatment, and samples of ileal and cecal contents were taken at d 14 of treatment to determine the presence of Campylobacter. Pigs consuming lysozyme and antibiotics gained BW at a faster rate than did control pigs over the course of the study (402 ± 12 and 422 ± 14 g/d, respectively, vs. 364 ± 14 g/d; P < 0.02), resulting in heavier ending BW (9.9 ± 0.3, 9.9 ± 0.3, and 9.0 ± 0.2 kg for pigs in the lysozyme, antibiotic, and control groups, respectively; P < 0.03). Immunoglobulin A decreased and plasma urea N increased over the course of the study (P < 0.1), regardless of dietary treatment (P > 0.6). Crypt depth was increased in pigs fed lysozyme- and antibiotic-treated diets, compared with pigs fed the control diet, in both the jejunum (60.0 ± 2.8 and 62.2 ± 3.0 µm, respectively, vs. 50.7 ± 3.1 µm; P < 0.03) and ileum (76.0 ± 7.5 and 72.2 ± 5.0 µm, respectively, vs. 52.4 ± 3.5 µm; P < 0.02). Villus height did not differ in the jejunum (P > 0.2) but was increased in the ileum of pigs consuming the lysozyme- and antibiotic-treated diets, compared with pigs fed the control diet (312 ± 20 and 314 ± 10 µm, respectively, vs. 263 ± 15 µm; P < 0.4). Small intestinal total mucosa and mucosal protein concentrations, as well as disaccharidase-specific activities, were not altered by lysozyme or antibiotics (P > 0.05). Campylobacter was detected in 27% of control samples but in only 5% of samples from pigs fed antibiotics and 8% of samples from pigs fed lysozyme (P < 0.01). Thus, granulated lysozyme is a suitable alternative to antibiotics for 10-d-old pigs consuming manufactured liquid diets.

Wound healing efficacy of Jatyadi Taila: in vivo evaluation in rat using excision wound model.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Indian medicinal treatise there are several Ayurvedic formulations mentioned which have been claimed as potential wound healing agents like Madhu Ghrita and Jatyadi Taila. Jatyadi Taila (JT) is a medicated oil formulation (Taila) popularly used in the treatment of various topical wounds. AIM OF THE STUDY: Though JT has its composition recorded in ancient Ayurvedic texts, there have been minimal attempts to standardize its use in the management of wound. The current work evaluates the wound healing efficacy of JT and also provides evidence of the dermal absorption kinetics of Karanjin from JT. MATERIALS AND METHODS: JT was subjected to preliminary phytochemical evaluation. Therapeutically active marker components ß-sitosterol, lupeol and karanjin were detected and separated using HPTLC. As a part of safety evaluation, skin irritation potential of JT was evaluated on rabbit skin. Excision wound model in rats were used to evaluate the wound healing efficacy of JT. Histopathological and biochemical evaluations of excised skin tissues at wound sites were carried out. The HPTLC method developed was also validated to evaluate the pharmacokinetics of Karanjin from JT after topical application on pinna of rabbit. RESULTS: Preliminary phytochemical evaluation of JT revealed presence of flavonoids, essential oils, tannins, glycosides, steroids and alkaloids while resins were found to be absent. HPTLC confirmed the presence of karanjin, lupeol and ß-sitosterol in JT. JT was found to be non-irritant when applied to the skin of rabbits. Topical application of JT on excision wounds caused significantly faster reduction in wound area as compared to the application of modern topical formulation (Neosporin(®)) and untreated control wounds. Animals treated with JT showed significant increase in protein, hydroxyproline and hexosamine content in the granulation tissue when compared with the untreated controls. Wound healing potential of JT was found to be dose dependant. HPTLC method was successfully used to evaluate the pharmacokinetics of Karanjin after topical application of JT on rabbit pinna. CONCLUSIONS: Current work demonstrates a modern approach towards standardization of the use of traditional topical formulation JT. The results justify the traditional claim of JT for its use in the management of wounds.

Termite usage associated with antibiotic therapy: enhancement of aminoglycoside antibiotic activity by natural products of Nasutitermes corniger (Motschulsky 1855).

BACKGROUND: Several species from Insecta are used as remedies. Among these species, the termite Nasutitermes corniger is commonly used in traditional medicine in Northeast Brazil. The present work tests the modifying antibiotic activity of Nasutitermes corniger, a termite used in folk medicine in Northeastern region of Brazil. METHODS: Chlorpromazine and decocts of N. corniger were collected from two different plant species used in the traditional medicine were tested for their antimicrobial activity against strains of Escherichia coli resistant to aminoglycosides. The growth of two bacterial strains of E. coli was tested using decocts and chlorpromazine alone or associeted with aminogycosides. RESULTS: The MIC and MBC values were > or =1024 microg/ml for both strains of E. coli assayed. A significant synergism was observed between both decocts and chlorpromazine when assyed with neomycin. This synergism with neomycin indicates the involvement of an efflux system in the resistance to this aminoglycoside. CONCLUSION: Therefore it is suggested that natural products from N. corniger could be used as a source of zoo-derived natural products with modifying antibiotic activity to aminoglycosides, being a new weapon against the bacterial resistance to antibiotics.

Proanthocyanidin from blueberry leaves suppresses expression of subgenomic hepatitis C virus RNA.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanol-HCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.

Establishment of a cell-based assay to screen regulators of the hypoxia-inducible factor-1-dependent vascular endothelial growth factor promoter.

In this study, we established a drug screening system based on transcriptional regulation of vascular endothelial growth factor (VEGF) under hypoxia-inducible factor-1alpha control. We cloned the neomycin-resistance gene into the plasmid GL (pGL)3-promoter vector to generate the pGL3-promoter-neo vector. The 3 copies of the 47-bp fragment that contained the hypoxia response element of VEGF were synthesized and inserted in front of the minimal promoter of the pGL3-promoter-neo vector to generate p3HRE-luc-neo. The recombinant reporter gene vectors were transfected into EAhy926 cells, and stable cell lines were obtained. The positive cell line was selected for its ability to express luciferase in response to hypoxia. We demonstrated that CoCl(2) significantly enhances luciferase activity in a concentration-dependent fashion. We then optimized the cell density and incubation time under hypoxia which were used to screen. The assay exhibited a low background and was an ideal model for high-throughput screening for human VEGF regulators.

The block of intracellular calcium release affects the pollen tube development of Picea wilsonii by changing the deposition of cell wall components.

Two potent drugs, neomycin and TMB-8, which can block intracellular calcium release, were used to investigate their influence on pollen tube growth and cell wall deposition in Picea wilsonii. Apart from inhibiting pollen germination and pollen tube growth, the two drugs largely influenced tube morphology. The drugs not only obviously disturbed the generation and maintenance of the tip-localized Ca(2+) gradient but also led to a heavy accumulation of callose at the tip region of P. wilsonii pollen tubes. Fourier transform infrared (FTIR) spectroscopy analysis showed that the deposition of cell wall components, such as carboxylic acid, pectins, and other polysaccharides, in pollen tubes was changed by the two drugs. The results obtained from immunolabeling with different pectin and arabinogalactan protein antibodies agreed well with the FTIR results and further demonstrated that the generation and maintenance of the gradient of cross-linked pectins, as well as the proportional distribution of arabinogalactan proteins in tube cell walls, are essential for pollen tube growth. These results strongly suggest that intracellular calcium release mediates the processes of pollen germination and pollen tube growth in P. wilsonii and its inhibition can lead to abnormal growth by disturbing the deposition of cell wall components in pollen tube tips.

Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators.

The transient receptor potential channel of the ankyrin-binding repeat subfamily, TRPA1, is a Ca(2+)-permeable non-selective cation channel that depolarizes the plasma membrane and causes Ca(2+) influx. A typical feature of TRPA1 is its rapid desensitization following activation by agonists such as mustard oil (MO), cinnamaldehyde, and a high intracellular Ca(2+) concentration. In whole-cell recordings on Chinese hamster ovary (CHO) cells expressing TRPA1, desensitization was delayed when phosphatidylinositol 4,5-biphosphate (PIP(2)) was supplemented via the patch pipette, whereas the PIP(2) scavenger neomycin accelerated desensitization. Preincubation with the PI-4 kinase inhibitor wortmannin reduced both constitutive TRPA1 channels activity and the response to MO. Run down was also accelerated by high intracellular Mg(2+) concentrations, whereas chelating intracellular Mg(2+) with 10 mM ethylenedinitrilotetraacetic acid (EDTA) increased the basal channel activity. In inside-out patches, we observed a rapid run down of TRPA1 activity, which could be prevented by application of diC8-PIP(2) or 2 mM Mg-ATP but not Na(2)-ATP to the cytosolic side of the excised patches. In isolated trigeminal ganglion neurons, preincubation with wortmannin resulted in inhibition of endogenous TRPA1 activation by MO. Taken together, our data indicate that PIP(2) modulates TRPA1, albeit to a lesser extent than other known PIP(2)-dependent TRP channels, and that tools modifying the plasma membrane PIP(2) content often have direct effects on this channel.

NeoR6 inhibits HIV-1-CXCR4 interaction without affecting CXCL12 chemotaxis activity.

Aminoglycoside-arginine conjugates (AACs) are multi-target HIV-1 inhibitors. The most potent AAC is neomycin hexa-arginine conjugate, NeoR6. We here demonstrate that NeoR6 interacts with CXCR4 without affecting CXCL12-CXCR4 ordinary chemotaxis activity or loss of CXCR4 cell surface expression. Importantly, NeoR6 alone does not affect cell migration, indicating that NeoR6 interacts with CXCR4 at a distinct site that is important for HIV-1 entry and mAb 12G5 binding, but not to CXCL12 binding or signaling sites. This is further supported by our modeling studies, showing that NeoR6 and CXCL12 bind to two distinct sites on CXCR4, in contrast with other CXCR4 inhibitors, e.g. T140 and AMD3100. This complementary utilization of chemical, biology, and computation analysis provides a powerful approach for designing anti-HIV-1 drugs without interfering with the natural function of CXCL12/CXCR4 binding.

Effect of dried oregano leaves versus neomycin in treating newborn calves with colibacillosis.

Treatment with neomycin (as a positive control) and dried oregano leaves on mortality, number of days scouring and severity of scours due to Escherichia coli were examined in 30 Holstein calves. Calves were assigned to one of the treatments following clinical signs of diarrhoea (i.e. faecal score >2), and treated either with an oral solution of neomycin sulphate, to provide 10 mg neomycin sulphate per kg calf body weight per 24 h, or dried oregano leaves, to provide 10 mg oregano essential oil per kg calf body weight per 24 h. The number of scouring days, severity of scouring and mortality rates were similar between the treatments. This study indicates that dried oregano leaves administered as an oral solution to calves with diarrhoea may be as effective in the treatment of colibacillosis as neomycin.