Dose-response resistance to HIV-1/MuLV pseudotype virus ex vivo in a hairpin ribozyme transgenic mouse model

We have investigated the efficacy of a hairpin ribozyme targeting the 5′ leader sequence of HIV-1 RNA in a transgenic model system. Primary spleen cells derived from transgenic or control mice were infected with HIV-1/MuLV pseudotype virus. A significantly reduced susceptibility to infection in ribozyme-expressing transgenic spleen cells (P = 0.01) was shown. Variation of transgene-expression levels between littermates revealed a dose response between ribozyme expression and viral resistance, with an estimated cut off value below 0.2 copies of hairpin ribozyme per cell. These findings open up possibilities for studies on ribozyme efficacy and anti-HIV-1 gene therapy.

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1–1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)—this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

HIV type-1 infection of the cotton rat (Sigmodon fulviventer and S. hispidus)

Cotton rats (Sigmodon hispidus and S. fulviventer) are susceptible to many viruses that infect humans (e.g., poliovirus, respiratory syncytial virus, influenza virus, adenovirus, and parainfluenza virus) and have been influential in developing therapeutic clinical intervention strategies for many viral infections of man. This study set out to determine whether cotton rats are susceptible to infection with HIV type 1 (HIV-1). Results indicate that HIV-1 does infect the cotton rat and S. fulviventer is more susceptible than S. hispidus. The virus was passaged from animal to animal for a total of three serial passages; but HIV replicated poorly in vivo, was only detectable as proviral DNA, and never exceeded one provirus per 1.8 × 105 cotton rat peripheral blood mononuclear cells. Infection induced a distinct and characteristic anti-HIV antibody response that, in some animals, included neutralizing antibodies, recognized all of the major HIV-1 antigens and the antibodies lasted out to 52 wk post-infection. Neonate S. fulviventer were not more susceptible to infection than adults. In vitro culture studies produced indirect evidence of viral replication by detection of viral gag gene RNA in reverse transcriptase–PCR assays on viral culture supernatants. Collectively, these results indicate that HIV-1 can replicate in a nontransgenic rodent and that this system may have potential as an animal model for HIV-1 infection if viral replication rates can be improved in vivo.

Modulation of the chaperone-like activity of bovine α-crystallin

The effects of pantethine, glutathione, and selected chemical reagents on the anti-aggregation activity of α-crystallin was evaluated. Protein aggregation was monitored by light scattering of solutions of denatured βL-crystallin or alcohol dehydrogenase (ADH). The ratios of βL-crystallin/α-crystallin and ADH/α-crystallin were adjusted so that partial inhibition of protein aggregation at 60°C or 37°C, respectively, was observed and modulation of the chaperone action of α-crystallin could be evaluated easily with selected endogenous metabolites. Enhancement of the anti-aggregation activity in the βL-crystallin assay was strongest with pantethine, which appeared to interact with α-crystallin. Enhancement of the anti-aggregation activity in the ADH assay was strongest with glutathione which appeared to interact with ADH. The results indicated that the products of common metabolic pathways can modulate the chaperone-like effects of α-crystallin on protein aggregation.

Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein.

A recombinant rabies virus (RV) mutant deficient for the surface spike glycoprotein (G) gene was used to study the incorporation of envelope proteins from HIV-1 expressed from transfected plasmids. A hybrid HIV-1 protein in which the cytoplasmic domain was replaced with that of RV G was incorporated into the virus envelope and rescued the infectivity of the RV mutant. The RV(HIV-1) pseudotype viruses could infect only CD4+ cells, and their infectivity was neutralized specifically by anti-HIV-1 sera. In contrast to the chimeric protein, wild-type HIV-1 envelope protein or mutants with truncated cytoplasmic domains failed to produce pseudotyped particles. This indicates the presence of a specific signal in the RV G cytoplasmic domain, allowing correct incorporation of a spike protein into the envelope of rhabdovirus particles. The possibility of directing the cell tropism of RV by replacement of the RV G with proteins of defined receptor specificity should prove useful for future development of targetable gene delivery vectors.

Inhibitors of HIV-1 replication that inhibit HIV integrase.

HIV-1 replication depends on the viral enzyme integrase that mediates integration of a DNA copy of the virus into the host cell genome. This enzyme represents a novel target to which antiviral agents might be directed. Three compounds, 3,5-dicaffeoylquinic acid, 1-methoxyoxalyl-3,5-dicaffeoylquinic acid, and L-chicoric acid, inhibit HIV-1 integrase in biochemical assays at concentrations ranging from 0.06-0.66 microgram/ml; furthermore, these compounds inhibit HIV-1 replication in tissue culture at 1-4 microgram/ml. The toxic concentrations of these compounds are fully 100-fold greater than their antiviral concentrations. These compounds represent a potentially important new class of antiviral agents that may contribute to our understanding of the molecular mechanisms of viral integration. Thus, the dicaffeoylquinic acids are promising leads to new anti-HIV therapeutics and offer a significant advance in the search for new HIV enzyme targets as they are both specific for HIV-1 integrase and active against HIV-1 in tissue culture.

Disparate actions of hydroxyurea in potentiation of purine and pyrimidine 2',3'-dideoxynucleoside activities against replication of human immunodeficiency virus.

We and other groups have recently reported the potentiation by ribonucleotide reductase inhibitors such as hydroxyurea of the anti-human immunodeficiency virus type 1 (HIV-1) activity of purine and pyrimidine 2',3'-dideoxynucleosides in both resting and phytohemagglutinin-stimulated peripheral blood mononuclear cells. Little agreement prevails, however, as to the mechanism of the synergistic effects described. We report here that in phytohemagglutinin-stimulated peripheral blood mononuclear cells, two mechanisms exist for the potentiation of the anti-HIV-1 activity by low-dose hydroxyurea of the purine-based dideoxynucleoside 2',3'-dideoxyinosine and the pyrimidine-based dideoxynucleosides 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine. For 2',3'-dideoxyinosine, the enhancement arises from a specific depletion of dATP by hydroxyurea, resulting in a favorable shift of the 2',3'-dideoxyadenosine 5'-triphosphate/dATP ratio. For the pyrimidine dideoxynucleosides 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, the more modest anti-HIV enhancement results from hydroxyurea-induced increases of pyrimidine kinase activities in the salvage pathway and, hence, increased 5'-phosphorylation of these drugs, while depletion of the corresponding deoxynucleoside 5'-triphosphates (dTTP and dCTP) plays no significant role.

Extração, determinação da composição fenólica e avaliação do potencial de desativação de espécies reativas de oxigênio e da atividade anti-inflamatória de resíduos de amendoim, pimenta-rosa e pimenta-do-reino

O setor agroindustrial tem se expandido muito nos últimos anos, levando o país a um aumento na geração de resíduos agroindustriais, sendo que a maior parte deles ainda é descartada no meio ambiente, sem tratamento adequado, ou utilizada na alimentação animal, destinos que a priori, não geram ganhos econômicos para a agroindústria além de representarem gargalos logísticos e ambientais na sua disposição. Nesse sentido, o presente trabalho teve como objetivos otimizar o processo de extração de compostos bioativos, avaliar in vitro as atividades antioxidante, por meio da desativação de espécies reativas de oxigênio, e anti-inflamatória, bem como determinar a composição fenólica dos resíduos agroindustriais a saber: película de amendoim (Arachis hypogaea) (cultivares IAC886 e IAC505), pimenta-rosa (Schinus terebinthifolius Raddi) e pimenta-do-reino (Piper Nigrum L). O processo de otimização da extração de compostos antioxidantes foi realizado utilizando dois processos de extração, extração convencional e subcrítica, em delineamento composto central rotacional, utilizando como variáveis a temperatura e tempo e a pressão e temperatura, respectivamente, com os solventes etanol 80%, água e propilenoglicol 80%. Durante o processo de otimização a atividade antioxidante foi avaliada pelo método de sequestro do radical ABTS. Os melhores resultados foram obtidos para a extração convencional com os solventes etanol 80%, água e propilenoglicol 80%. A película de amendoim (IAC505) apresentou as maiores atividades antioxidantes (1.396,67, 580,44 e 859,89 μmol.g-1 em equivalentes de trolox, para os solventes etanol 80%, água e propilenoglicol 80%, respectivamente). A partir dos resultados obtidos para os solventes testados, utilizando a extração convencional, foram feitas outras análises de atividade antioxidante considerando o tempo e temperatura ideal de extração. Foram realizadas análises de avaliação da capacidade de redução de Folin-Ciocalteau e potencial de desativação dos radicais livres sintéticos (DPPH e ABTS) e espécies reativas de oxigênio (radicais peroxila, superóxido e ácido hipocloroso). O solvente de extração que apresentou melhores resultados em todos os ensaios foi o etanol 80%, sendo, portanto o solvente utilizado nas análises subsequentes. A partir da definição do melhor sistema extrator foram realizadas análises da composição fenólica, por meio das técnicas de cromatografia líquida de alta eficiência em modo analítico (HPLC-RP), cromatografia gasosa acoplada com espectrometria de massas (GC-MS), e avaliação in vitro da atividade anti-inflamatória. Foram identificados nos resíduos estudados procianidinas B1 e B2, ácido p-cumárico, miricetina, ácido ferúlico, ácido siríngico, ácido sinápico, epicatequina e catequina. A pimenta-do-reino diminui significativamente os níveis de TNF-α e nitritos, reduzindo assim o processo inflamatório gerado. Os resultados obtidos neste trabalho demonstram que estes resíduos agroindústrias possuem grande potencial biológico, podendo assim ser melhores aproveitados tanto pela indústria de alimentos quanto pela indústria farmacêutica.

An in vitro study of the anti-microbial efficacy of a 1% silver sulphadiazine and 0.2% chlorhexidine digluconate cream Silvazine (TM), 1% silver sulphadiazine cream Flamazine (TM) and a silver coated dressing Acticoat (TM)

Burn sepsis is a leading cause of mortality and morbidity in patients with major burns. The use of topical anti-microbial agents has helped improve the survival in these patients. There are a number of anti-microbials available, one of which, Silvazine(TM) (1% silver sulphadiazine (SSD) and 0.2% chlorhexidine digluconate), is used only in Australasia. No study, in vitro or clinical, had compared Silvazine(TM) with the new dressing Acticoat(TM). This study compared the anti-microbial activity of Silvazine(TM), Acticoa(TM) and 1% silver sulphadiazine (Flamazine(TM)) against eight common burn wound pathogens. Methods: Each organism was prepared as a suspension. A 10 mul inoculum of the chosen bacterial isolate (representing approximately between 104 and 105 total bacteria) was added to each of four vials, followed by samples of each dressing and a control. The broths were then incubated and 10 mul loops removed at specified intervals and transferred onto Horse Blood Agar. These plates were then incubated for 18 hours and a colony count was performed. Results: The data demonstrates that the combination of 1% SSD and 0.2% chlorhexidine digluconate (Silvazine(TM)) results in the most effective killing of all bacteria. SSD and Acticoat(TM) had similar efficacies against a number of isolates, but Acticoat(TM) seemed only bacteriostatic against E. faecalis and methicillin-resistant Staphylococcus aureus. Viable quantities of Enterobacter cloacae and Proteus mirabilis rei named at 24 h. Conclusion: The combination of 1% SSD and 0.2% chlorhexidine digluconate (Silvazine(TM)) is a more effective anti-microbial against a number of burn wound pathogens in this in vitro study. A clinical study of its in vivo anti-microbial efficacy is required. (C) 2003 Elsevier Ltd and ISBI. All rights reserved.

Chemical synthesis and biosynthesis of the cyclotide family of circular proteins

Cyclotides are a recently discovered class of proteins that have a characteristic head-to-tail cyclized backbone stabilized by a knotted arrangement of three disulfide bonds. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. Cyclotides have a range of bio-activities, including uterotonic, anti-HIV, anti-bacterial and cytotoxic activity but their insecticidal properties suggest that their natural physiological role is in plant defense. They are genetically encoded as linear precursors and subsequently processed to produce mature cyclic peptides but the mechanism by which this occurs remains unknown. Currently most cyclotides are obtained via direct extraction from plants in the Rubiaceae and Violaceae families. To facilitate the screening of cyclotides for structure-activity studies and to exploit them in drug design or agricultural applications a convenient route for the synthesis of cyclotides is vital. In this review the current chemical, recombinant and biosynthetic routes to the production of cyclotides are discussed.

Automated synthesis and evaluation of potential new anti-microbial agents

A series of N1-benzylideneheteroarylcarboxamidrazones was prepared in an automated fashion, and tested against Mycobacterium fortuitum in a rapid screen for antimycobacterial activity. Many of the compounds from this series were also tested against Mycobacterium tuberculosis, and the usefulness as M.fortuitum as a rapid, initial screen for anti-tubercular activity evaluated. Various deletions were made to the N1-benzylideneheteroarylcarboxamidrazone structure in order to establish the minimum structural requirements for activity. The N1-benzylideneheteroarylcarbox-amidrazones were then subjected to molecular modelling studies and their activities against M.fortuitum and M.tuberculosis were analysed using quantitative structure-analysis relationship (QSAR) techniques in the computational package TSAR (Oxford Molecular Ltd.). A set of equations predictive of antimycobacterial activity was hereby obtained. The series of N1-benzylidenehetero-arylcarboxamidrazones was also tested against a multidrug-resistant strain of Staphylococcus aureus (MRSA), followed by a panel of Gram-positive and Gram-negative bacteria, if activity was observed for MRSA. A set of antimycobacterial N1-benzylideneheteroarylcarboxamidrazones was hereby discovered, the best of which had MICs against m. fortuitum in the range 4-8μgml-1 and displayed 94% inhibition of M.tuberculosis at a concentration of 6.25μgml-1. The antimycobacterial activity of these compounds appeared to be specific, since the same compounds were shown to be inactive against other classes of organisms. Compounds which were found to be sufficiently active in any screen were also tested for their toxicity against human mononuclear leucocytes. Polyethylene glycol (PEG) was used as a soluble polymeric support for the synthesis of some fatty acid derivatives, containing an isoxazoline group, which may inhibit mycolic acid synthesis in mycobacteria. Both the PEG-bound products and the cleaved, isolated products themselves were tested against M.fortuitum and some low levels of antimycobacterial activity were observed, which may serve as lead compounds for further studies.

Effect of medications with anti-cholinergic properties on cognitive function, delirium, physical function and mortality:a systematic review

Objectives: to determine the effect of drugs with anti-cholinergic properties on relevant health outcomes.Design: electronic published and unpublished literature/trial registries were systematically reviewed. Studies evaluating medications with anti-cholinergic activity on cognitive function, delirium, physical function or mortality were eligible.Results: forty-six studies including 60,944 participants were included. Seventy-seven percent of included studies evaluating cognitive function (n = 33) reported a significant decline in cognitive ability with increasing anti-cholinergic load (P < 0.05). Four of five included studies reported no association with delirium and increasing anti-cholinergic drug load (P > 0.05). Five of the eight included studies reported a decline in physical function in users of anti-cholinergics (P < 0.05). Three of nine studies evaluating mortality reported that the use of drugs with anti-cholinergic properties was associated with a trend towards increased mortality, but this was not statistically significant. The methodological quality of the evidence-base ranged from poor to very good.Conclusion: medicines with anti-cholinergic properties have a significant adverse effect on cognitive and physical function, but limited evidence exists for delirium or mortality outcomes. © The Author 2014. Published by Oxford University Press on behalf of the British Geriatrics Society. All rights reserved.

Sequence and chemistry requirements for a novel aptameric oligonucleotide inhibitor of EGF receptor tyrosine kinase activity

We have previously identified a phosphorothioate oligonucleotide (PS-ODN) that inhibited epidermal growth factor receptor tyrosine kinase (TK) activity both in cell fractions and in intact A431 cells. Since ODN-based TK inhibitors may have anti-cancer applications and may also help understand the non-antisense mediated effects of PS-ODNs, we have further studied the sequence and chemistry requirements of the parent PS-ODN (sequence: 5′-GGA GGG TCG CAT CGC-3′) as a sequence-dependent TK inhibitor. Sequence deletion and substitution studies revealed that the 5′-terminal GGA GGG hexamer sequence in the parent compound was essential for anti-TK activity in A431 cells. Site-specific substitution of any G with a T in this 5′-terminal motif within the parent compound caused a significant loss in anti-TK activity. The fully PS-modified hexameric motif alone exhibited equipotent activity as the parent 15-mer whereas phosphodiester (PO) or 2′-O-methyl-modified versions of this motif had significantly reduced anti-TK activity. Further, T substitutions within the two 5′-terminal G residues of the hexameric PS-ODN to produce a sequence, TTA GGG, representing the telomeric repeats in human chromosomes, also did not exhibit a significant anti-TK activity. Multiple repeats of the active hexameric motif in PS-ODNs resulted in more potent inhibitors of TK activity than the parent ODN. These results suggested that PS-ODNs, but not PO or 2′-O-methyl modified ODNs, containing the GGA GGG motif can exert potent anti-TK activity which may be desirable in some anti-tumor applications. Additionally, the presence of this previously unidentified motif in antisense PS-ODN constructs may contribute to their biological effects in vitro and in vivo and should be accounted for in the design of the PS-modified antisense ODNs. © 2002 Published by Elsevier Science Inc.

Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate

Background: We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors. Methods: We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)). Results: We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA. Conclusions: In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required. © 2014 Cancer Research UK. All rights reserved.

Antipyretic, analgesic and anti-oxidative activities of Aquilaria crassna leaves extract in rodents

In Thailand, the leaves of Aquilaria crassna have been used traditionally for the treatments of various disorders, but without any scientific analysis. In this study, the antipyretic, analgesic, anti-inflammatory and anti-oxidative properties of A. crassna leaves extract were investigated at a wide dose range in rodents. Experimental animals were treated orally with an aqueous extract of Aquilaria crassna leaves (ACE). They were tested for antipyretic (Baker′s yeast-induced fever in rats), analgesic (hot plate test in mice) and anti-inflammatory (carrageenan-induced paw edema in rats) activities. An anti-oxidative effect of ACE was evaluated by using the DPPH anti-oxidant assay. The results showed that, after 5 hours of yeast injection, 400 and 800 mg/kg ACE significantly reduced the rectal temperature of rats. Mice were found significantly less sensitive to heat at an oral dose of 800 mg/kg ACE, after 60 and 90 min. No anti-inflammatory activity of ACE at an 800 mg/kg dose could be observed in the rat paw assay. An anti-oxidative activity of ACE was observed with an IC 50 value of 47.18 g/ ml. No behavioral or movement change could be observed in mice after oral administration of ACE (800 or 8,000 mg/kg) for seven consecutive days. Interestingly, from the second day of treatment, animals had a significant lower body weight at the 8,000 mg/kg dose of ACE compared to the control. No toxicity was identified and the results of this study state clearly that Aquilaria crassna leaves extracts possess antipyretic, analgesic and anti-oxidative properties without anti-inflammatory activity.