EvoCor: a platform for predicting functionally related genes using phylogenetic and expression profiles.

The wealth of publicly available gene expression and genomic data provides unique opportunities for computational inference to discover groups of genes that function to control specific cellular processes. Such genes are likely to have co-evolved and be expressed in the same tissues and cells. Unfortunately, the expertise and computational resources required to compare tens of genomes and gene expression data sets make this type of analysis difficult for the average end-user. Here, we describe the implementation of a web server that predicts genes involved in affecting specific cellular processes together with a gene of interest. We termed the server 'EvoCor', to denote that it detects functional relationships among genes through evolutionary analysis and gene expression correlation. This web server integrates profiles of sequence divergence derived by a Hidden Markov Model (HMM) and tissue-wide gene expression patterns to determine putative functional linkages between pairs of genes. This server is easy to use and freely available at http://pilot-hmm.vbi.vt.edu/.

Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load.

Mutations are the ultimate source of heritable variation for evolution. Understanding how mutation rates themselves evolve is thus essential for quantitatively understanding many evolutionary processes. According to theory, mutation rates should be minimized for well-adapted populations living in stable environments, whereas hypermutators may evolve if conditions change. However, the long-term fate of hypermutators is unknown. Using a phylogenomic approach, we found that an adapting Escherichia coli population that first evolved a mutT hypermutator phenotype was later invaded by two independent lineages with mutY mutations that reduced genome-wide mutation rates. Applying neutral theory to synonymous substitutions, we dated the emergence of these mutations and inferred that the mutT mutation increased the point-mutation rate by ∼150-fold, whereas the mutY mutations reduced the rate by ∼40-60%, with a corresponding decrease in the genetic load. Thus, the long-term fate of the hypermutators was governed by the selective advantage arising from a reduced mutation rate as the potential for further adaptation declined.

Ciclopirox olamine lotion 1%: bioequivalence to ciclopirox olamine cream 1% and clinical efficacy in tinea pedis.

Studies were conducted to assess the bioequivalence of a new antimycotic formulation, ciclopirox olamine lotion 1%, to an established compound, ciclopirox olamine cream 1%. Results of in vitro studies, using skin samples from human cadavers and domestic pigs, demonstrated that the two formulations equally penetrate all layers of the stratum corneum and inhibit the growth of Trichophyton mentagrophytes and Candida albicans. In vivo studies in guinea pigs and in human volunteers demonstrated the comparable therapeutic efficacy of the lotion and the cream in experimental trichophytosis. In addition, a multicenter, double-blind clinical trial was undertaken to compare ciclopirox olamine lotion 1% with the vehicle alone in the treatment of patients with tinea pedis. Patients with plantar, interdigital, or vesicular tinea pedis were enrolled in the studies. Patients were treated for 28 days. Clinical and mycological responses were determined during treatment and two weeks posttreatment. Ciclopirox olamine lotion 1% was found to be significantly more effective than its vehicle in the treatment of patients with common tinea pedis. Minor localized side effects (pruritus, burning sensation) were reported in 2% of 89 patients treated with ciclopirox olamine lotion 1%. The results demonstrate the bioequivalence of ciclopirox olamine lotion 1% and ciclopirox olamine cream 1% and confirm the clinical effectiveness and safety of the lotion in the treatment of tinea pedis, a generally recalcitrant fungal infection. It is concluded that ciclopirox olamine lotion 1% can be used as an alternative to ciclopirox olamine cream 1% for treatment of tinea pedis, tinea versicolor, tinea cruris, tinea corporis, and cutaneous candidiasis when the convenience and/or cosmetic elegance of a lotion is desired.

Evaluation of fungicidal action in vitro and in a skin model considering the influence of penetration kinetics of various standard antimycotics.

Ciclopiroxolamine (Loprox) was the first representative of hydroxpyridones to be developed as a topical antifungal. In the microtitration test and in a skin model using excised skin prices of slaughtered pigs, the fungistatic and fungicidal activity as well as the penetration kinetics of ciclopiroxolamine and ciclopirox were compared with those of the azole compounds bifonazole, clotrimazole, econazole, miconazole, oxiconazole, and tioconazole, and with other antimycotics such as naftifine, sulbentine, tolciclate, and tolnaftate. Clotrimazole had an in vitro inhibitory activity superior to that of the hydroxypyridone ciclopiroxolamine; the latter compound, however, exhibited faster penetration and higher inhibitory or fungicidal activity than the azoles and other antimycotics in the pig skin model. Studies of cream formulations with antimycotics at the bottom layer of the stratum corneum (close to the stratum granulosum) showed that ciclopiroxolamine cream had the most prominent inhibitory effect (93%) and the highest fungicidal activity (98%). The other antimycotics tested exhibited growth inhibition rates of 50% or less and lower fungicidal rates. Inoculated pig skin was treated with lacquer formulations to show that ciclopirox completely inhibited the growth of Trichophyton mentagrophytes, whereas the clotrimazole-treated samples allowed only 0.47% growth. The fungicidal activity of lacquer formulations was 99% for ciclopirox and around 90% for most of the azoles on the pig skin model. The necessity of models tackling the complex penetration kinetics in human skin was discussed.

In vitro mutagenicity of valepotriates.

Valepotriates are epoxide-bearing triesters of the monoterpene alcohol 4,7-dimethylcyclopenta-(c)-pyrane isolated from the roots of several Valerianacae species. They are regarded as the main tranquilizing constituents of these drugs. Although the valepotriates valtrate/isovaltrate (VAL) and dihydrovaltrate (DH-VAL) showed a strong alkylating activity against the nucleophilic agent 4-(p-nitrobenzyl)-pyridine (NBP), they were not clearly mutagenic for the strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium or for the strains WP2 and WP2 uvrA- of Escherichia coli in the absence of a metabolic activation system (S9-mix). However, the valepotriates were mutagenic for TA100, WP2 and WP2 uvrA- at concentrations up to about 1.0 mumole/plate when S9-mix was added to the test system. With more than 1 mumole/plate the valepotriates were toxic in the presence of a metabolic activation system for all strains tested. The mutagenicity of the valepotriates was inversely related to the protein content of the S9-mix used. The mutagenicity and toxicity of the valepotriates could be inhibited when the S9-mix was preincubated with the esterase inhibitor paraoxon (1 mM) for 5 min before the test compounds and bacteria were added. Therefore, bioactivation of the valepotriates by an enzymatic hydrolysis of their ester groups is considered. This could be proven by activating the valepotriates with purified esterase.

In vitro and in vivo investigations for the development of cytostatic methylhydrazones.

In in vitro short-term (3 h) assays, the beta-chloroethyl-methyl-hydrazones B 1 and B 2 inhibit the uptake of 3H-thymidine by EAC and L 1210 leukemia cells, B 2 being 5 to 10 times more effective than B 1. The growth inhibitory effect of both compounds was also confirmed in long-term (7 days) clonal assays using agar-containing glass capillaries, B 2 again being more effective than B 1. In contrast to these differences in vitro, in vivo both substances showed remission to the same degree in EAC- and complete resistance in L 1210-bearing mice. The diverging in vitro/in vivo sensitivities were thought to result from differences in the affinity of the methylhydrazones to the tumor cells: using short exposure periods (3 h) B 1 was more inhibitory than B 2 on both EAC and L 1210 colony growth; i.e., the more hydrophilic B 2 could more easily be washed off. To further test the idea of different cell membrane affinities, the methylhydrazones ZB 1 and P 1 with increasing lipophilic properties were synthesized. In vitro, after both pulse and continuous exposure ZB 1 and P 1 showed enforced inhibitory effects on colony growth. In vivo, ZB 1 and P 1 reduced the tumor weight of EAC mice, while only P 1 increased the survival time of L 1210 mice. The results suggest that from the combination of in vitro/in vivo assays mechanistic conclusions can be derived that are valuable for further development of these cystostatics.

Influence of valtrate/isovaltrate on the hematopoiesis and metabolic liver activity in mice in vivo.

Recently, cytotoxic effects of valepotriates with an epoxide moiety have been described on mouse bone marrow early progenitor cells IN VITRO. Consequently, the possible IN VIVO toxicity of valtrate on hematopoietic precursor cells was investigated. Mice were treated i.p. with 45 and 65 mg/kg or p.o. with 45 and 1350 mg/kg of the drug. Three days after treatment, colony formation of progenitor cells (CFC-S, GM-CFC, E-CFC) was not significantly different for control and experimental groups. Furthermore, the effect of valtrate on the ability of the liver to metabolize [ (14)C]methacetin was investigated by measuring the (14)CO (2) exhalation (breath test). There was a distinct reduction of the initial exhalation of (14)CO (2) following i.p. injection of 50 mg/kg of valtrate, but no effect was found after 50 or 1500 mg/kg of p.o. These results suggest that toxicity of valtrate in vivo is restricted because the distribution of the drug via circulation is obviously small.

Considerations on the carcinogenicity of the mushroom poison gyromitrin and its metabolites.

Monomethylhydrazine (MMH) and unsymmetrical dimethylhydrazine (UDMH), but not N-methyl-N-formylhydrazine (MFH), a mushroom poison, can be oxidized by the weak oxidant 2,6-dichlorophenolindophenol. Hydrogen peroxide is formed by this oxidation, and has been found to cause decay of DNA in aqueous solution as measured by the decreased viscosity of a DNA solution in the presence of the hydrazines. Furthermore, MMH and UDMH but not MFH inhibit the incorporation of [3H]thymidine and [3H]uridine into DNA and RNA of Ehrlich ascites carcinoma (EAC) cells. These experiments show that MFH is an inactive compound in contrast to MMH and UDMH. Since the hydrolysis rate of MFH, to produce MMH, is low, it is concluded that MFH must be activated in vivo into toxic metabolites which are ultimately responsible for the known high carcinogenicity of MFH.

On interactions of cytostatic benzylidine hydrazines with SH-groups.

The cytostatic and mutagenic compound N'-methyl-N'-cyano-(p-chloro)-benzaldehyde hydrazone (CyB4) has been found to be a strong SH-blocking agent since it reacts with the thiol groups of glutathione and of the cell membrane of Ehrlich ascites carcinoma cells (EAC). Furthermore, it decreases the intracellular, non-proteinogenic SH(NPSH)-level of tumor cells. CyB4 is not able to alkylate 4-(p-nitrobenzyl)-pyridine (NBP) by a nucleophilic substitution reaction, but it could be shown that the reactivity of CyB4 with thiol groups is due to a Michael-addition-type reaction of SH-groups with the cyano-group of CyB4. On the other hand, cytostatic beta-chloroethyl hydrazones showed a negligible reactivity against glutathione and led even to an increase of thiol groups, detectable by the 5,5'-dithiobis-2-nitrobenzoic acid (DTNB)-method, at the cell membrane of EAC when incubated in the presence of beta-chloroethyl hydrazones N-benzylidene-N'-methyl-N'-(2-chloroethyl) hydrazine (B1) and N-(4-dimethylaminobenzylidene)-N'-methyl-N'-(2-chloroethyl) hydrazine (B2). Therefore, it is concluded that the cytostatic efficiency of CyB4 is due to its SH-blocking while that of the beta-chloroethyl hydrazones is due to a rearrangement of the tumor cell membrane, as indicated by the increased level of reactive SH-groups.

[The chemistry of antimicrobially active 1-hydroxy-2-pyridones (author's transl)]. / Zur Chemie von antimikrobiell wirksamen 1-Hydroxy-2-pyridonen.

The unsaturated delta-keto esters (3) obtained by condensation of acid chlorides with esters of di- or trialkyl-acrylic acids can be cyclized with hydroxylamine to yield 1-hydroxy-2-pyridones (4). However, in many cases a two-steps synthesis may be of advantage in preparative respect, the ketoesters being cyclized to 2-pyrones (7), which then are reacted with hydroxylamine in the presence of certain bases to give 4. The hydroxy-pyridones show pronounced antifungal activity in vitro as well as in experimental guinea pig dermatophytosis.

[Microbiological laboratory studies with ciclopiroxolamine (author's transl)]. / Mikrobiologische Laboruntersuchungen mit Ciclopiroxolamin.

A survey is given on the microbiological laboratory results obtained up to date with 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone, 2-aminoethanol salt (ciclopiroxolamine, Cic, Hoe 296, Batrafen). The activity spectrum of the drug on fungi, bacteria and other organisms is described. Various properties by which Cic differs from commercial antimycotics are mentioned. They concern the great uniformity of MICs in pathogenic fungi, their consistency under various test conditions, the importance of the nutrient medium for the level of in vitro activity, and the steepness of dose-response curves. In addition, previously described properties of Cic are summarized, including findings on its mode of action in fungi, its effect in model mycoses, and its high efficacy in hornified skin types. The relevance of laboratory findings for therapy is discussed.

[Penetration and antifungal activity of ciclopiroxolamine in hornified tissue (author's transl)]. / Zur Penetration und antimyzetischen Wirksamkeit von Ciclopiroxolamin in verhorntem Körpergewebe.

A brief survey is given on microbiological methods commonly used in investigations on the penetration of effective amounts of antimycotics into the epidermis. By means of more recent and widely improved techniques, 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone, 2-aminoethanol salt (ciclopiroxolamine, Batrafen) when applied in a sufficient amount--was shown to penetrate (enter) to an exceptional extent both average and very thick horny layers of skin as well as compact horny material.

[Open clinical trial in dermal mycoses of a 1% ciclopiroxolamine solution in polyethylene glycol 400 carried out in FR Germany/Study with shortened therapy (author's transl)]. / Offene Prüfung einer 1% igen Ciclopiroxolamin-Lösung auf Polyethylenglykol 400-Basis bei Dermatomykosen in der Bundesrepublik Deutschland. Studie mit verkürzter Therapiedauer.

In an open multicentre study, a 1% solution of 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone, 2-aminoethanol salt (ciclopiroxolamine, Cic. Hoe 296, Batrafen) in polyethylene glycol 400 was examined for its topical activity in 344 cases of dermal mycoses. 65.4% of them were caused by dermatophytes and 19.2% by yeast-like fungi. In most cases, the treatment period was two weeks, with one or two daily applications. The examiners found global therapeutic effectiveness in 97.5% of the mycoses, the rate of very good and good results being 84.0%, and a very good tolerance in 99.0%. Individual side effects were predominantly subjective and transient.

[Non-European open clinical studies on the efficacy and tolerance of ciclopiroxolamine in dermatomycoses (author's transl)]. / Offene, aussereuropäische Studien zur Wirksamkeit und Verträglichkeit von Ciclopiroxolamin bei Dermatomykosen. Zusammemfassendes Referat.

A survey is given of twenty published open clinical studies carried out outside Europe. They concern the topical action of 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone, 2-aminoethanol salt (ciclopiroxolamine, Cic, Hoe 296, Batrafen) in 991 cases of skin disorders, mostly mycoses. 70.7% of the disorders were dermatophytoses, 18.9% candidoses. 6.1% pityriasis versicolor, and 4.2% other skin infections. Four trials were done with 1% Cic cream (o/w-type-emulsion) in 157 dermal disorders and 16 trials with 1% Cic solution (vehicle: polyethylene glycol 400) in 834 cases. Several selected studies are reported in detail, the other are parts of a summarizing survey. After a treatment period which, as a rule, was limited to 3 weeks, the global success rate was 96% (healing and distinct improvement). Any side effects were only seldom recorded.