Antimicrobial peptides of the vaginal innate immunity and their role in the fight against sexually transmitted diseases.

Some antimicrobial peptides (AMPs) are produced in the vaginal innate immune system and play an important role in protecting this organ against pathogenic agents. Moreover, sexually transmitted diseases have become a major problem in human societies and are rapidly spreading. The emergence of antibiotic-resistant microbes (superbugs) can pose a major threat to human societies and cause rapid spread of these diseases. Finding new antimicrobial compounds to fight superbugs is therefore essential. It has been shown that AMPs have good potential to become new antibiotics. The most important AMPs in the vaginal innate immune system are defensins, secretory leucocyte protease inhibitors, calprotectin, lysozyme, lactoferrin and elafin, which play an important role in host defence against sexually transmitted infections, modulation of immune responses and anticancer activities. Some AMPs, such as LL-37, magainin 2 and nisin, show both spermicidal and antimicrobial effects in the vagina. In this summary, we will discuss vaginal AMPs and continue to address some of the challenges of using peptides to control pathogens that are effective in sexually transmitted diseases.

Effect of mutations in putative hormone binding sites on V2 vasopressin receptor function.

The vasopressin V2 receptor belongs to the large family of the G-protein coupled receptors and is responsible for the antidiuretic effect of the neurohypophyseal hormone arginine vasopressin (AVP). Based on bioinformatic studies it seems that Ala300 and Asp297 of the V2 vasopressin receptor (V2R) are involved in receptor binding. Ala300Glu mutation resulted in lower energy while Asp297Tyr mutation resulted in higher energy in AVP-V2R docked complex rather than the wild type. Therefore we hypothesized that the Ala300Glu mutation results in stronger and Asp297Tyr mutation leads to weaker ligand-receptor binding. Site directed mutagenesis of Asp297Tyr and Ala300Glu was performed using nested polymerase chain reaction. After restriction enzyme digestion, the inserts were ligated into the pcDNA3 vector and Escherichia coli XL1-Blue competent cells were transformed using commercial kit and electroporation methods. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using ESCORT™ IV transfection reagent, the adenylyl cyclase activity assay was performed for functional studies. The cell surface expression of V2R was analyzed by indirect ELISA method. Based on the obtained results, the Ala300Glu mutation of V2R led to reduced levels of cAMP production without a marked effect on the receptor expression and the receptor binding. Effect of Asp297Tyr mutation on cell surface expression of V2R was the same as the wild type receptor. Pretreatment with 1 nM vasopressin showed an increased level of Asp297Tyr mutant receptor internalization as compared to the wild type receptor, while the effect of 100 nM vasopressin was similar in the mutant and wild type receptors. These data suggest that alterations in Asp297 but not Ala300 would affect the hormone receptor binding.

Antifungal activity of enynediesters and acetylenic compounds obtained by synthesis and in silico prediction pattern.

OBJECTIVE: In this paper, we describe the preparation and synthesis of several enynediesters, conjugated diynes and acetylenic compounds from starting materials comprising one hydroxyl and one acetylenyl group and their antifungal activity. MATERIALS AND METHODS: For synthesis of the compounds, a combined solution of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridin in CH2Cl2 was added to a solution of compound containing one hydroxyl group and propiolic acid at 0°C over a period of 1 hour. As the reaction occurs under very mild conditions this procedure offers easy access to 1,3-diynes in a very short reaction time. Some of the compounds are commercially available. The antifungal activity of these compounds against Candida albicans, Aspergillus niger and Saccharomyces cerevisiae was investigated. RESULTS: Among the compounds tested, some showed potent activity. CONCLUSION: The results of synthesis showed that the direct coupling of two acetylenes is suitable for the preparation of diynes. The in silico study used here was able to predict if the compound with triple bond can posses the antifungal activity with a reasonable prediction. The result of investigations at both in silico and in vitro levels confirmed that the position of the triple bond is important for antifungal activity.

Screening of the anticonvulsant activity of some plants from Fabaceae family in experimental seizure models in mice.

BACKGROUND AND PURPOSE OF THE STUDY: Fabaceae is the third largest family of flowering plants. Lack of essential oils in the plants of this family can be an advantage in search for safe and effective medicines. In this study the anticonvulsant effect of the leaves of Albizzia julibrissin, Acacia juliflora, Acacia nubica and aerial parts of Astragalus obtusifolius was evaluated in pentylenetetrazole (PTZ) and maximal electroshock (MES) seizure tests. METHODS: The hydroalcoholic extracts of the plants were obtained by percolation. Different doses of the extracts were injected to the mice intraperitoneally (i.p.) and occurrence of clonic seizures induced by PTZ (60 mg/kg, i.p.) or tonic seizures induced by MES (50 mA, 50Hz, 1sec) were monitored up to 30 min after administration. Acute toxicity of the extracts was also assessed. The safe and effective extract was then fractionated by dichloromethane and anticonvulsant activity of the fractions was determined. Finally, the constituents of the extract and the fractions were screened by thin layer chromatography. RESULTS: Among the extracts, only A. obtusifolius extract showed low toxicity and protective effect against clonic seizures with ED50 value of 3.97 g/kg. Fractionation of the extract led to increase in anticonvulsant activity and ED50 value of 2.86 g/kg was obtained for the aqueous fraction. Phytochemical screening revealed the presence of alkaloids, flavonoids, anthrones and saponins in the aqueous fraction. MAJOR CONCLUSION: The presence of anticonvulsant compounds in A. obtusifolius suggests further activity-guided fractionation and analytical studies to find out the potential of this plant as a source of anticonvulsant agent.

Screening of antimicrobial membrane-active metabolites of soil microfungi by using chromatic phospholipid/polydiacetylene vesicles.

OBJECTIVE: The focus of this study is screening of antimicrobial membrane-active metabolites of soil microfungi by using chromatic phospholipid/polydiacetylene vesicles. MATERIALS AND METHODS: In this work, soil samples were collected from desert, forest, farm, lake shore and mineral zones of Northern and Central parts of Iran. These parts were not studied for antimicrobial potential of the soil isolates producing metabolites with membrane activity in particular, from microfungi. In the primary screening that was performed to evaluate the antimicrobial activity, isolates were analyzed in terms of their general inhibition effects to indicator strains including Escherichia coli, Candida albicans, and Saccharomyces cerevisiae. In the secondary screening, isolates producing membrane-active metabolites were determined using a Rapid Chromatic Detection method. The chromatic technology is simple and this method provides a rapid and easy evaluation of interactions between antimicrobial membrane-active metabolites and lipid layers of vesicles as well. RESULTS: A total number of 59 species of fungi was isolated from the soil samples. It has been found that 20 isolates were effective against indicator strains. Based on color and fluorescence changes that are easily identified by the naked eye and fluorescent microscopy and can be recorded by UV-Vis spectrophotometery, one fungus showed antimicrobial membrane-activity effect against some of the indicator strains. This isolate was identified to the genus level that belonged to Aspergillus. CONCLUSION: As resistance is barely developed against membrane-active antibiotics, in this paper, we demonstrated the application of the chromatic vesicle model for screening of antimicrobial membrane-active metabolites as potential new antibiotics from soil microfungi.

Cytotoxic effect of drugs eluted from polymethylmethacrylate on stromal giant-cell tumour cells: an in vitro study.

Curettage and packing with polymethylmethacrylate cement is a routine treatment for giant-cell tumour (GCT) of bone. We performed an in vitro evaluation of the cytotoxic effect of a combination of cement and methotrexate, doxorubicin and cisplatin on primary cell cultures of stromal GCT cells obtained from five patients. Cement cylinders containing four different concentrations of each drug were prepared, and the effect of the eluted drugs was examined at three different time intervals. We found that the cytotoxic effect of eluted drugs depended on their concentration and the time interval, with even the lowest dose of each drug demonstrating an acceptable rate of cytotoxicity. Even in low doses, cytotoxic drugs mixed with polymethylmethacrylate cement could therefore be considered as effective local adjuvant treatment for GCTs.

Cheminformatics in anti-infective agents discovery.

The existing chemical data such as those created by high throughput screening (HTS), structure-activity relationship (SAR) studies are converted into information as a result of storage and registration. Accessibility, manipulation, and data mining of such information make up the knowledge for drug development. Cheminformatics, exploiting the combination of chemical structural knowledge, biological screening, and data mining approaches is used to guide drug discovery and development and would assist by integrating complex series of rational selection of designed compounds with drug-like properties, building smarter focused libraries. This paper presents cheminformatics approaches and tools for designing and data mining of chemical databases and information. Many examples of success in lead identification and optimization in the area of anti-infective therapy have been discussed.

Application of radioisotopes in inflammation.

Since the discovery of artificially produced radioisotopes in the 1930's, an estimated 10-12 million nuclear medicine diagnostic and therapeutic procedures are currently performed each year only in the United States. Gamma emission imaging has been successfully applied to almost every organ of the body (brain, bone, heart, kidney, lung, neuroreceptors) as well as sites of inflammation, atherosclerosis, and thrombosis. FDG-PET has been used in some of the inflammatory diseases as well. On the other hand, both alpha- and beta-emitting isotopes have been evaluated for brachytherapy of rheumatoid diseases, each with different radiobiological effectiveness. The current status of radionuclides for imaging, therapy and research studies of inflammatory processes is reviewed here and a look into the future directions is described at the conclusion.

Modulatory effect of cAMP on fungal ergosterol level and inhibitory activity of azole drugs.

The functions and biosynthesis of sterols have been effective targets for fungal control in different areas, including pharmaceutical and agricultural applications. Fungi are among the organisms that synthesize sterols, principally ergosterol. In this paper, the effect of dibutyryl-cAMP (db-cAMP) on ergosterol level and the interaction of drugs that would change the concentration of cAMP with antifungal drugs have been investigated. Sterols were extracted from Candida albicans, and ergosterol was measured using the gas chromatography method. The interaction of different agents was measured by the broth dilution method. It was found that phosphodiesterase inhibitors reverse the inhibitory activity of azole antifungal drugs. Evaluating the ergosterol level of C. albicans incubated with db-cAMP revealed that it increased ergosterol level. Further experiments provided evidence attributing the observed interaction between azoles and phosphodiesterase inhibitors to the relationship between ergosterol and cAMP. The possible significance of this interaction includes potentiation of antifungal activity of drugs by manipulating the cAMP level.

Applications of artificial neural network in AIDS research and therapy.

In recent years considerable effort has been devoted to applying pattern recognition techniques to the complex task of data analysis in drug research. Artificial neural networks (ANN) methodology is a modeling method with great ability to adapt to a new situation, or control an unknown system, using data acquired in previous experiments. In this paper, a brief history of ANN and the basic concepts behind the computing, the mathematical and algorithmic formulation of each of the techniques, and their developmental background is presented. Based on the abilities of ANNs in pattern recognition and estimation of system outputs from the known inputs, the neural network can be considered as a tool for molecular data analysis and interpretation. Analysis by neural networks improves the classification accuracy, data quantification and reduces the number of analogues necessary for correct classification of biologically active compounds. Conformational analysis and quantifying the components in mixtures using NMR spectra, aqueous solubility prediction and structure-activity correlation are among the reported applications of ANN as a new modeling method. Ranging from drug design and discovery to structure and dosage form design, the potential pharmaceutical applications of the ANN methodology are significant. In the areas of clinical monitoring, utilization of molecular simulation and design of bioactive structures, ANN would make the study of the status of the health and disease possible and brings their predicted chemotherapeutic response closer to reality.

Evaluation of methylthio-, methylsulfinyl-, and methylsulfonyl-analogs of alkanes and alkanoic acids as cardiac inotropic and antifungal agents.

A group of alkane and alkanoic acid compounds of general formula MeS(O)m(CH2)nR [m = 0-2; n = 1, 5, 13; R = Me, CO2H(Na)] were synthesized for evaluation as cardiac inotropic and antifungal agents. Inotropic activity was determined as the ability of the test compound to modulate in vitro guinea pig atrium contractility. The oxidation state of the S-atom was an important determinant of inotropic modulation since the thio (m = 0) analogs exhibited a positive inotropic effect. In contrast, the sulfinyl (m = 1) and sulfonyl (m = 2) analogs exhibited a negative inotropic effect. A pentyl spacer (n = 5) provided the largest positive or negative inotropic effect. The relative positive, and negative, inotropic potency orders with respect to the R-substituent were Me > or = CO2H, and CO2Na > or = Me, respectively. The most potent positive inotrope MeS(CH2)5Me (EC50 = 4.49 x 10(-6) M) could serve as a useful lead-compound for the design of a new class of positive inotropic agents. In a broad spectrum antifungal screen, the minimal inhibitory concentration (MIC) range for the five most active compounds was MeSO2(CH2)5Me (0.46-1.83 mM), MeS(CH2)13Me (0.31-1.23 mM), MeSO(CH2)13Me (< 0.009-1.87 mM), MeSO2(CH2)13Me (0.27-1.09 mM), and MeS(CH2)13CO2H (0.27-1.09 mM), relative to the reference drug Ampotericin B (< 0.0002-0.002 mM). The most active antifungal agent MeSO(CH2)13Me was selective against C. guillermondi, C. neoformans, S. cerevisiae, and A. fumigatus (strain TIMM 1776).

Synthesis and antifungal activity of coumarins and angular furanocoumarins.

Angelicin, a naturally occurring furanocoumarin, that showed antifungal activity, was considered as a lead structure for a group of synthetic coumarins. Antifungal activities of the synthesized coumarins and angelicin derivatives were reported against Candida albicans, Cryptococcus neoformans, Saccharomyces cerevisiae and Aspergillus niger. Human cell line cytotoxicity of several coumarins was evaluated against KB cells. Angelicin and several potent antifungals showed to be non-toxic in this assay.

In vitro dissociation of antifungal efficacy and toxicity for amphotericin B-loaded poly(ethylene oxide)-block-poly(beta benzyl L aspartate) micelles.

Amphotericin B (AmB) is a membrane-active drug used frequently for the treatment of systemic fungal diseases. Limitations for the use of AmB include poor water solubility and potential for serious systemic toxicities. Recently, it has been demonstrated that the aggregation state of AmB is a determinant factor for toxicity. To increase its therapeutic index, AmB has been solubilized in micelles based on poly(ethylene oxide)-block-poly(beta-benzyl-l-aspartate) (PEO-block-PBLA), using a dialysis method of drug loading. The aggregation state of AmB has been investigated by electronic absorption spectroscopy. AmB loaded in PEO-block-PBLA micelles is non-hemolytic for concentrations up to 15 microgram/ml. AmB as Fungizone(R) initiates hemolysis at 1.0 microgram/ml. The onset of hemolysis correlates with the respective critical aggregation concentrations (CACs) of AmB. The antifungal activity of the AmB-loaded PEO-block-PBLA micelles is four to eight times higher than Fungizone(R) in terms of minimal inhibitory concentrations (MICs). PEO-block-PBLA has no antifungal activity for concentrations up to 200 microgram/ml. The basis for the increase in antifungal activity of AmB-loaded PEO-block-PBLA micelles is unclear, but may be related to a stabilizing effect of the polymeric micelles against auto-oxidation of the AmB heptaene moiety or alternatively, an enhancement in membrane perturbation of fungal cells.

Synthesis and antifungal activities of myristic acid analogs.

Myristic acid analogs that are putative inhibitors of N-myristoyl-transferase were tested in vitro for activity against yeasts (Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans) and filamentous fungi (Aspergillus niger). Several (+/-)-2-halotetradecanoic acids including (+/-)-2-bromotetradecanoic acid (14c) exhibited potent activity against C. albicans (MIC = 39 microM), C. neoformans (MIC = 20 microM), S. cerevisiae (MIC = 10 microM), and A. niger (MIC < 42 microM) in RPMI 1640 media. Improved synthetic methods have been developed for the synthesis of 12-fluorododecanoic acid (12a) and 12-chlorododecanoic acid (12c). Three novel fatty acids, 12-chloro-4-oxadodecanoic acid (8a), 12-phenoxydodecanoic acid (12i), and 11-(4-iodophenoxy)-undecanoic acid (13d) were also synthesized and tested.