Presence Of Neomycin Research Articles

The Ganglioside Monosialotetrahexosylganglioside Protects Auditory Hair Cells Against Neomycin-Induced Cytotoxicity Through Mitochondrial Antioxidation: An in vitro Study.

Neomycin is a common ototoxic aminoglycoside antibiotic that causes sensory hearing disorders worldwide, and monosialotetrahexosylganglioside (GM1) is reported to have antioxidant effects that protect various cells. However, little is known about the effect of GM1 on neomycin-induced hair cell (HC) ototoxic damage and related mechanism. In this study, cochlear HC-like HEI-OC-1 cells along with whole-organ explant cultures were used to establish an in vitro neomycin-induced HC damage model, and then the apoptosis rate, the balance of oxidative and antioxidant gene expression, reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) were measured. GM1 could maintain the balance of oxidative and antioxidant gene expression, inhibit the accumulation of ROS and proapoptotic gene expression, promoted antioxidant gene expression, and reduce apoptosis after neomycin exposure in HEI-OC-1 cells and cultured cochlear HCs. These results suggested that GM1 could reduce ROS aggregation, maintain mitochondrial function, and improve HC viability in the presence of neomycin, possibly through mitochondrial antioxidation. Hence, GM1 may have potential clinical value in protecting against aminoglycoside-induced HC injury.

Synthesis and characterization of a new magnetic adsorbent for removal of 4-nitrophenol: application of response surface methodology.

Magnetic modified graphene oxide was synthesized as a new modified magnetic nano-composite (MMNC) by a simple sonochemical-hydrothermal method. The sonochemical reaction was employed to exfoliate, functionalize and decorate neomycin on graphene oxide sheets. Nickel ferromagnetic particles were synthesized by hydrothermal co-precipitation method and decorated on neomycin-modified graphene oxide. The morphology and chemical structure of MMNC were characterized by scanning electron microscopy, energy dispersive spectroscopy and X-ray diffraction spectroscopy. The adsorption capability of MMNC for removal of phenolic compounds was assessed through adsorption of 4-nitrophenol (4-NP) from aqueous solution. The three-factor Box-Behnken design coupled with response surface method was applied to evaluate and optimize the important variables which affect the adsorption process. A significant quadratic model (p-value <0.05, R2 (adj) = 0.9593) was derived using analysis of variance. The maximum adsorption capacity of 125.4 mg 4-NP/g MMNC at pH 6 was obtained, which was comparable in some cases and higher than most adsorbents reported in the literature. The presence of neomycin on graphene oxide sheets improved the maximum adsorption capacity of the nano-sorbent up to 28% (from 98.7 to 125.4 mg 4-NP/g adsorbent). The adsorption isotherms fitted well with the Langmuir model (Langmuir constant b = 0.064 l/mg, R2 = 0.9989) and the kinetic study showed that the nitrophenol uptake process followed the pseudo-second-order rate expression (R2 ≥ 0.9960, pseudo-second-order constant K2 ≥ 1.7 × 10-3).

Alginate hydrogels incorporating neomycin or propolis as potential dressings for diabetic ulcers: Structure, swelling, and antimicrobial barrier properties

Alginate hydrogels have many attractive characteristics for potential use as wound dressing materials. However, they are not considered to possess any intrinsic activity against microbial infection, often present in neuropathic wounds. To overcome this, the effect of incorporating neomycin or propolis in alginate hydrogels was investigated, both by direct blending alone and also by further addition of loaded alginate microparticles prepared by the extrusion dripping method. The morphological, microstructural, thermal, mechanical, and swelling properties of each film were evaluated, as well as particle size distribution and antimicrobial penetration analysis. Microparticle size was considered suitable for drug delivery applications and incorporation in hydrogel films. The presence of neomycin and propolis, in both blended and microparticle form, interfered with film properties leading to hydrogels with different characteristics. All samples showed swelling degrees up to 100% and mechanical and thermal properties suitable for application as wound dressings. In addition, all samples acted as barriers to microbial penetration.

Electrochemiluminecence nanogears aptasensor based on MIL-53(Fe)@CdS for multiplexed detection of kanamycin and neomycin

A dual gears electrochemiluminecence (ECL) aptasensing strategy for multiple selective determination of kanamycin and neocycin was designed on the basis of the combination of kanamycin and neocycin induced dual gears conversion, the loading platform of metal-organic frameworks (MOFs), surface plasmon resonance (SPR) and ECL resonance energy transfer (ERET) between CdS QDs and AuNPs (or PtNPs). In the absence of target, the dual gears were "off". Then the B1-AuNP (gear B) and aptamer 1-PtNPs acted as signal quenching elements to quench ECL intensity due to ERET process. Upon addition of kanamycin, the aptamer 1-PtNPs were removed from the gear gradually, the ECL was enhanced due to SPR process between AuNPs and CdS QDs. After the incubation of aptamer 2, the dual gears were "off" again and ECL intensity was decreased by ERET process between AuNPs and CdS QDs. In the presence of neomycin, dual gears were "on" again, the ECL signal was enhanced by SPR process between AuNPs and CdS QDs. Under optimal condition, the proposed aptasensor exhibited wide linear ranges of kanamycin (10−10–10−6 M) and neomycin (10−9–10−5 M), and relatively low detection limits to kanamycin (1.7 × 10–11 M) and neomycin (3.5 × 10−10 M). The developed aptasensor realized the multiple ECL detection of kanamycin and neomycin with single luminophore, and was successfully applied to the detection of kanamycin and neomycin in food samples.

The role of PLC-IP3 cascade on 4-aminopyridine (4-AP) contracture in electrically-driven rat atrial and diaphragmatic strips: new evidence by neomycin and heparin

Induction of cardiac contractures by 4-AP in Ca2+-free medium implied the involvement of SR and PLC-IP3 cascade. Thus, the role of PLC-IP3 cascade against contractile actions of 4-AP in electrically-driven rat atrial and diaphragmatic strips were studied both in the presence, and absence of Ca2+ using neomycin, a PLC inhibitor, and heparin, an IP3-R antagonist. 4-AP was applied cumulatively in logarithmically increasing concentrations in the range of 1-16µg/ml, and the preparations were treated with neomycin (400µM) or heparin (400µg/ml) for 3min prior to 4-AP injection. Post-rest potentiation in atrial strips was obtained by interruption of stimulation for 30min. 4-AP caused biphasic alteration in twitch amplitudes, as initially increased up to 16mM and then depressed due to contracture development, which were not affected significantly by neomycin and heparin. Both atrial and denervated diaphragmatic strips challenged to 4-AP in the presence and absence of Ca2+ developed dose dependent contractures which were significantly antagonized both by neomycin and heparin (p&lt;0.05). Post-rest first contractions in controls were found to be reduced by 2min exposure to 4mM 4-AP and augmented by 3min exposure to heparin alone. 4-AP responses in the presence of neomycin and heparin were significantly higher than with those only treated with 4-AP alone and lesser than controls. Because of the fact that 4-AP inducing contracture in Ca2+-free medium, Ca2+ causing contracture should be of SR in origin. Depending on these results, it was concluded that activation of PLC-IP3 cascade by 4-AP is involved in the mediation of contracture and contractile actions of this molecule.

Adaptive Cross-Resistance to Aminoglycoside Antibiotics in Pseudomonas aeruginosa Induced by Topical Dosage of Neomycin

Background: Topical antimicrobial formulations containing neomycin are commonly used to prevent and treat burn infections. However, Pseudomonas aeruginosa shows rapid acquisition of adaptive resistance to neomycin. This study aimed to evaluate the survival of P. aeruginosa during exposure to neomycin at high concentrations comparable to those used in topical formulations, and to investigate the effect of adaptive resistance to neomycin on the susceptibility to other aminoglycosides. Methods: Strain IID1130 [neomycin minimal inhibitory concentration (MIC) = 4 µg/ml] was incubated on an agar medium containing neomycin at high concentrations (8-4,096 µg/ml), and growing colonies were macroscopically observed. Acquisition of adaptive resistance was examined for 5 P. aeruginosa strains. Cells were sequentially passaged on agar medium containing neomycin with step-wise increased concentrations (8-2,048 µg/ml). To assess reversion of antibiotic susceptibility, the resulting colonies were repeatedly subcultured on antibiotic-free agar plates. Results: Growing IID1130 colonies were macroscopically detected on a neomycin-containing (2,048 µg/ml) agar plate for 48 h. These cells showed increasing MIC for not only neomycin, but also gentamicin and amikacin; the MIC values were occasionally higher than the breakpoints. When the adapted cells were subcultured on antibiotic-free agar, several passages were required for reversion of susceptibility. Conclusions: Our findings suggest that P. aeruginosa can survive in the presence of neomycin with a concentration typically used in topical dosage forms, and that the acquired adaptive resistance is persistent and is accompanied by cross-resistance to other aminoglycosides.

Localization of angiotensin-II type 1(AT1) receptors on buffalo spermatozoa: AT1 receptor activation during capacitation triggers rise in cyclic AMP and calcium

The purpose of this study was to determine the role of Ang-II in buffalo spermatozoa; localize angiotensin type 1 (AT1) receptors on the sperm surface and understand the signaling mechanisms involved therein. Immunoblotting and immunocytochemistry using polyclonal Rabbit anti-AT1 (N-10) IgG were performed to confirm the presence of AT1 receptors. Intracellular levels of cyclic adenosine monophosphate (cAMP) were determined by non-radioactive enzyme immunoassay, while that of Calcium [Ca(2+)] were estimated by fluorimetry using Fura2AM dye. The results obtained showed that AT1 receptors were found on the post-acrosomal region, neck and tail regions. Immunoblotting revealed a single protein band with molecular weight of 40 kDa. Ang-II treated cells produced significantly higher level of cAMP compared to untreated cells (22.66 ± 2.4 vs. 10.8 ± 0.98 pmol/10(8) cells, p < 0.01). The mean levels of Ca(2+) were also higher in Ang-II treated cells compared to control (117.4 ± 6.1 vs. 61.15 ± 4.2 nmol/10(8) cells; p < 0.01). The stimulatory effect of Ang-II in both the cases was significantly inhibited in the presence of Losartan (AT1 antagonist; p < 0.05) indicating the involvement of AT1 receptors. Further, presence of neomycin (protein kinase C inhibitor) inhibited significantly the Ang-II mediated rise in Ca(2+) indicating the involvement of PKC pathway. These findings confirm the presence of AT1 receptors in buffalo spermatozoa and that Ang-II mediates its actions via the activation of these receptors. Ang-II stimulates the rise in intracellular levels of cAMP and Ca(2+) during capacitation.

Colorimetric and Fluorometric Detection of Neomycin Based on Conjugated Polydiacetylene Supramolecules

Utilizing the colorimetric and fluorogenic changes, a system based on polydiacetylenes (PDAs) is developed for the detection of neomycin. The PDA supramolecules polymerized from the mixed liposome composed of N-(3-hydroxyphenyl)pentacosa-10,12-diynamide (PCDA-AP) and pentacosa-10,12-diynoic acid (PCDA) at an optimized ratio of 1:9 display a unique colorimetric change (blue to red) and fluorescent enhancement in the presence of neomycin. The detection limit for neomycin is estimated to be 2.55 × 10(-7) M by the fluorogenic method. The optical changes induced by neomycin can be attributed to the disruption of the hydrogen bonding between phenol and carboxylic acid from PCDA-AP and PCDA.

β-Lactams and Florfenicol Antibiotics Remain Bioactive in Soils while Ciprofloxacin, Neomycin, and Tetracycline Are Neutralized

It is generally assumed that antibiotic residues in soils select for antibiotic-resistant bacteria. This assumption was tested by separately adding 10 different antibiotics (≥200 ppm) to three soil-water slurries (silt-loam, sand-loam, and sand; 20% soil [wt/vol]) and incubating mixtures for 24 h at room temperature. The antibiotic activity of the resultant supernatant was assessed by culturing a sensitive Escherichia coli strain in the filter-sterilized supernatant augmented with Luria-Bertani broth. We found striking differences in the abilities of supernatants to suppress growth of the indicator E. coli. Ampicillin, cephalothin, cefoxitin, ceftiofur, and florfenicol supernatants completely inhibited growth while bacterial growth was uninhibited in the presence of neomycin, tetracycline, and ciprofloxacin supernatants. High-performance liquid chromatography (HPLC) analysis demonstrated that cefoxitin and florfenicol were almost completely retained in the supernatants, whereas tetracycline and ciprofloxacin were mostly removed. Antibiotic dissipation in soil, presumably dominated by adsorption mechanisms, was sufficient to neutralize 200 ppm of tetracycline; this concentration is considerably higher than reported contamination levels. Soil pellets from the tetracycline slurries were resuspended in a minimal volume of medium to maximize the interaction between bacteria and soil particles, but sensitive bacteria were still unaffected by tetracycline (P = 0.6). Thus, residual antibiotics in soil do not necessarily exert a selective pressure, and the degree to which the pharmaceutical remains bioactive depends on the antibiotic. Efforts to control antibiotic contamination would be better directed toward compounds that retain biological activity in soils (e.g., cephalosporins and florfenicol) because these are the antibiotics that could exert a selective pressure in the environment.

Angiotensin-II induced nitric oxide production during buffalo sperm capacitation and acrosome reaction

The present study was designed to see the effects of Angiotensin-II (Ang-II) on buffalo sperm capacitation, acrosome reaction (AR), and its relation to nitric oxide (NO) production. The extent of capacitation or AR was determined by dual staining while the NO production was determined by spectrophotometry. The results thus obtained revealed that Ang-II induced capacitation in a concentration and time dependent manner and 200nM Ang-II was found to be optimal for capacitation as it was comparable to heparin treatment (50.7±2.45% vs. 51.66±2.33%). In capacitated cells the extent of AR induced by Ang-II was significantly higher than the untreated control (48.13±2.31% vs. 22.16±2.11%) and comparable to lysophosphatidyl Choline (LPC) treatment (51.56±1.94%). The NO production during Ang-II induced capacitation and AR was gradual and time dependent. These levels were significantly higher when compared to control (3.65±0.53nmoles/108cells vs. 9.12±0.30nmoles/108cells). All the actions of Ang-II were inhibited in the presence of Losartan but not PD123319, indicating the role of AT1 receptors in these actions. Further the NO production was also significantly inhibited in the presence of neomycin and trifluoperazine pointing towards the role of phosphoinositide pathway in this process. In conclusion, Ang-II has a concentration and time dependent effect on buffalo sperm capacitation and AR, mediated via the AT1 receptors. Its effect on NO production may be indirect involving the phosphoinositide pathway.

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

The Effects of Transformation and ZnT‐1 Silencing on Zinc Homeostasis in Cultured Cells

We have shown that the extracellular chelator DTPA (diethylenetriaminepentaaceticacid) promotes efflux of Zn65 from prelabeled rat primary hepatocytes, but increases retention of label in H4IIE hepatoma cells. To further understand this difference between primary cells and the corresponding cancer cell line, we investigated the effects of immortalizing primary cells on their zinc homeostasis. Freshly isolated rat hepatocytes were electroporated with the SV40 large T‐antigen‐coding‐plasmid, pSV3‐neo. Successful transformation was demonstrated by continued growth in the presence of neomycin. When these cells were pre‐labeled with Zn65, DTPA decreased efflux of Zn65, similarly to hepatoma cells and differently from primary hepatocytes. This homeostatic change may be required to account for the greater zinc requirements of rapidly dividing cells. To further understand the mechanism of DTPA inducedzinc retention we down‐regulated the expression of the plasma membrane located zinc efflux transporter, ZnT‐1, in rat hepatoma cells using vector‐based shRNA interference. Expression of ZnT‐1 protein was reduced to approximately 50%. Down‐regulation of ZnT‐1 resulted in greater retention of Zn65 in control cells. However, DTPA increased rather than decreased efflux of label from knockdown cells, suggesting that functional ZnT‐1 is required for the decreased efflux seen with DTPA in normal cells.Grant Funding Source: UConn Research Foundation

Surface Plasmon Resonance Analysis of Antibiotics Using Imprinted Boronic Acid-Functionalized Au Nanoparticle Composites

Au nanoparticles (NPs) are functionalized with thioaniline electropolymerizable groups and (mercaptophenyl)boronic acid. The antibiotic substrates neomycin (NE), kanamycin (KA), and streptomycin (ST) include vicinal diol functionalities and, thus, bind to the boronic acid ligands. The electropolymerization of the functionalized Au NPs in the presence of NE, KA, or ST onto Au surfaces yields bisaniline-cross-linked Au NP composites that, after removal of the ligated antibiotics, provide molecularly imprinted matrixes which reveal high sensitivities toward the sensing of the imprinted antibiotic analytes (detection limits for analyzing NE, KA, and ST correspond to 2.00 +/- 0.21 pM, 1.00 +/- 0.10 pM, and 200 +/- 30 fM, respectively). The antibiotics are sensed by surface plasmon resonance (SPR) spectroscopy, where the coupling between the localized plasmon of the NPs and the surface plasmon wave associated with the Au surface is implemented to amplify the SPR responses. The imprinted Au NP composites are, then, used to analyze the antibiotics in milk samples.

Calorimetric and spectroscopic studies of aminoglycoside binding to AT-rich DNA triple helices

Calorimetric and fluorescence techniques were used to characterize the binding of aminoglycosides-neomycin, paromomycin, and ribostamycin, with 5′-dA 12-x-dT 12-x-dT 12-3′ intramolecular DNA triplex (x = hexaethylene glycol) and poly(dA)·2poly(dT) triplex. Our results demonstrate the following features: (1) UV thermal analysis reveals that the T m for triplex decreases with increasing pH value in the presence of neomycin, while the T m for the duplex remains unchanged. (2) The binding affinity of neomycin decreases with increased pH, although there is an increase in observed binding enthalpy. (3) ITC studies conducted in two buffers (sodium cacodylate and MOPS) yield the number of protonated drug amino groups (Δn) as 0.29 and 0.40 for neomycin and paromomycin interaction with 5′-dA 12-x-dT 12-x-dT 12-3′, respectively. (4) The specific heat capacity change (Δ C p) determined by ITC studies is negative, with more negative values at lower salt concentrations. From 100 mM to 250 mM KCl, the Δ C p ranges from −402 to −60 cal/(mol K) for neomycin. At pH 5.5, a more positive Δ C p is observed, with a value of −98 cal/(mol K) at 100 mM KCl. Δ C p is not significantly affected by ionic strength. (5) Salt dependence studies reveal that there are at least three amino groups of neomycin participating in the electrostatic interactions with the triplex. (6) FID studies using thiazole orange were used to derive the AC 50 (aminoglycoside concentration needed to displace 50% of the dye from the triplex) values. Neomycin shows a seven fold higher affinity than paromomycin and eleven fold higher affinity than ribostamycin at pH 6.8. (7) Modeling studies, consistent with UV and ITC results, show the importance of an additional positive charge in triplex recognition by neomycin. The modeling and thermodynamic studies indicate that neomycin binding to the DNA triplex depends upon significant contributions from charge as well as shape complementarity of the drug to the DNA triplex Watson–Hoogsteen groove.

Molecular recognition of single-stranded RNA: Neomycin binding to poly(A)

Poly(A) is a relevant sequence in cell biology due to its importance in mRNA stability and translation initiation. Neomycin is an aminoglycoside antibiotic that is well known for its ability to target various nucleic acid structures. Here it is reported that neomycin is capable of binding tightly to a single-stranded oligonucleotide (A 30) with a K d in the micromolar range. CD melting experiments support complex formation and indicate a melting temperature of 47 °C. The poly(A) duplex, which melts at 44 °C (pH 5.5), was observed to melt at 61 °C in the presence of neomycin, suggesting a strong stabilization of the duplex by the neomycin.

Cellular response induced by a galactose-specific adhesin of enteroaggregativeEscherichia coliin INT-407 cells

In the present study, the role of a fimbrial galactose-specific adhesin of the T7 strain of enteroaggregative Escherichia coli (EAEC-T7) in the signal transduction pathways in human small intestinal epithelial cells (INT-407) was explored. The adhesin was purified by anion exchange chromatography using a Mono Q HR5/5 column in the AKTA purifier system. The characteristic stacked brick pattern of aggregative adherence of EAEC-T7 to INT-407 cells was found to be inhibited in the presence of immunoglobulin G against the purified adhesin as well as d-galactose. The adhesin induced a significant increase in the intracellular calcium concentration [Ca(2+)](i) in INT-407 cells, which was reduced in the presence of dantrolene (inhibitor of intracellular calcium stores), verapamil, calciseptin (calcium channel blockers) as well as neomycin [inhibitor of phospholipase C (PLC)]. Further, an increased level of PLCgamma1 and inositol 1,4,5-tri phosphate as well as enhanced activity of protein kinase C (PKC) in the adhesin-stimulated cells were found to be downregulated in the presence of neomycin and U73122 (inhibitors of PLC) and H-7 (inhibitor of PKC), respectively. The adhesin could also induce interleukin-8 secretion from INT-407 cells, which was inhibited in the presence of dantrolene as well as staurosporin (inhibitor of PKC). Collectively, our results have suggested that the galactose-specific adhesin-induced signal transduction pathway might play a crucial role in the EAEC-induced pathogenesis.

The C-Terminal Hydrophobic Domain of Hepatitis C Virus RNA Polymerase NS5B Can Be Replaced with a Heterologous Domain of Poliovirus Protein 3A

Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.

Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.

Neomycin inhibits platelet functions and inositol phospholipid metabolism upon stimulation with thrombin, but not with ionomycin or 12-O-tetradecanoyl-phorbol 13-acetate

Gel-filtered human platelets that had been pre-labelled with [32P]Pi were stimulated with thrombin, ionomycin or the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The effect of the hexacationic aminoglycoside antibiotic, neomycin, on platelet physiological responses, such as aggregation and secretion, as well as changes in phosphoinositide metabolism was studied. Neomycin strongly inhibited thrombin-induced aggregation and secretion whereas the antibiotic had no effect on ionomycin- or TPA-induced platelet functions. The thrombin-induced enhancement of inositol phospholipid metabolism was strongly inhibited by the presence of neomycin whereas the TPA- or ionomycin-induced increase in inositol [32P]polyphospholipids remained unaffected. The inhibitory effect of some other aminoglycoside antibiotics was compared to that of neomycin and the data demonstrate that the inhibition of platelet secretion and phosphatidic acid production was dependent on the cationic charge of the antibiotic. It is suggested that neomycin inhibits signal transduction in platelets at a level prior to the inositol-phospholipid-specific phosphodiesterase.

Direct electrochemistry of blue copper proteins at boron-doped diamond electrodes

Boron-doped diamond (BDD) is a promising electrode material for use in the spectro-electrochemical study of redox proteins and, in this investigation, cyclic voltammetry was used to obtain quasi-reversible electrochemical responses from two blue copper proteins, parsley plastocyanin and azurin from Pseudomonas aeruginosa. No voltammetry was observed at the virgin electrodes, but signals were observed if the electrodes were anodised, or abraded with alumina, prior to use. Plastocyanin, which has a considerable overall negative charge and a surface acidic patch which is important in forming a productive electron transfer complex with its redox partners, gave a faradaic signal at pre-treated BDD only in the presence of neomycin, a positively charged polyamine. The voltammetry of azurin, which has a small overall charge and no surface acidic patch, was obtained identically in the presence and absence of neomycin. Investigations were also carried out into the voltammetry of two site-directed mutants of azurin, M64E azurin and M44K azurin, each of which introduce a charge into the protein's surface hydrophobic patch. The oxidizing and cleaning effects of the BDD electrode pre-treatments were studied electrochemically using two inorganic probe ions, Fe(CN) 6 3− and Ru(NH 3) 6 3+, and by X-ray photoelectron spectroscopy (XPS). All of the electrochemical results are discussed in relation to the electrostatic and hydrophobic contributions to the protein/diamond electrochemical interaction.