Antimicrobial activity of long-chain (E)-3-alken-2-ones

(E)-3-Tridecen-2-one, a compound identified from the interdigital glands of black-tailed deer (Odocoileus hemionus columbianus), has been shown to inhibit the growth of bacteria and fungi. Homologues of (E)-3-tridecen-2-one were prepared and screened for antimicrobial activity. For the fungus, Trichophyton mentagrophytes, the minimum inhibitory concentration (MIC) of (E)-3-Tetradecen-2-one was 12.5 μg/mL, and for the bacteria, Propionibacterium acnes, the MIC of (E)-3-heptadecen-2-one was 3.13 μg/mL.     

Methylglyoxal upregulates Dictyostelium discoideum slug migration by triggering glutathione reductase and methylglyoxal reductase activity

Glutathione (GSH)-deprived Dictyostelium discoideum accumulates methylglyoxal (MG) and reactive oxygen species (ROS) during vegetative growth. However, the reciprocal effects of the production and regulation of these metabolites on differentiation and cell motility are unclear. Based on the inhibitory effects of γ-glutamylcysteine synthetase (gcsA) disruption and GSH reductase (gsr) overexpression on aggregation and culmination, respectively, we overexpressed GSH-related genes encoding superoxide dismutase (Sod2), catalase (CatA), and Gcs, in D. discoideum. Wild-type KAx3 and gcsA-overexpressing (gcsA^OE) slugs maintained GSH levels at levels of approximately 2.1-fold less than the reference GSH synthetase-overexpressing mutant; their GSH levels did not correlate with slug migration ability. Through prolonged KAx3 migration by treatment with MG and H₂O₂, we found that MG increased after the mound stage in this strain, with a 2.6-fold increase compared to early developmental stages; in contrast, ROS were maintained at high levels throughout development. While the migration-defective sod2- and catA-overexpressing mutant slugs (sod2^OE and catA^OE) decreased ROS levels by 50% and 53%, respectively, these slugs showed moderately decreased MG levels (36.2 ± 5.8 and 40.7 ± 1.6 nmol g⁻¹ cells wet weight, P < 0.05) compared to the parental strain (54.2 ± 3.5 nmol g⁻¹). Importantly, defects in the migration of gcsA^OE slugs decreased MG considerably (13.8 ± 4.2 nmol g⁻¹, P < 0.01) along with a slight decrease in ROS. In contrast to the increase observed in migrating sod2^OE and catA^OE slugs by treatment with MG and H₂O₂, the migration of gcsA^OE slugs appeared unaffected. This behavior was caused by MG-triggered Gsr and NADPH-linked aldolase reductase activity, suggesting that GSH biosynthesis in gcsA^OE slugs is specifically used for MG-scavenging activity. This is the first report showing that MG upregulates slug migration via MG-scavenging-mediated differentiation.     

Dermatomycosis in lower limbs of diabetic patients followed by podiatry consultation

BackgroundDiabetic patients are particularly susceptible to fungal infections due to modifications that occur in their immunological system. These modifications compromise natural defences, such as skin and nails, especially from lower limbs.AimsAssessing the presence of dermatomycosis in lower limbs of Portuguese diabetic patients followed on Podiatry consultation. Determination of possible predisposing factors and the most frequent fungal species associated with the cases are included in the study.MethodsA six-month prospective study was carried out in 163 diabetic patients with signs and symptoms of dermatomycosis followed by Podiatry at the Portuguese Diabetes Association in Lisbon. Samples from the skin and/or nails of the lower limbs were collected and demographic and clinical data of those patients were recorded.ResultsTrichophyton rubrum was the most frequently isolated dermatophyte (12.1%), followed by Trichophyton mentagrophytes (7.7%) and Trichophyton tonsurans (4.4%). Our study showed positive associations between type 2 diabetes and the presence of dermatomycosis in the studied population (p = 0.013); this association was also shown between the occurrence of dermatomycosis and the localization of the body lesion (p = 0.000). No other predisposing factor tested was positively associated with infection (p > 0.05).ConclusionsData on superficial fungal infections in diabetic patients are scarce in Portugal. This study provides information on the characterization of dermatomycosis in lower limbs of diabetic patients.     

Synthesis and antimicrobial activity of polyhalo isophthalonitrile derivatives

A series of polyhalo isophthalonitrile derivatives (3 and 4) that incorporate a variety of substituents at the 2-, 4-, 5- and/or 6-positions of the isophthalonitrile moieties have been designed and synthesized. These derivatives were evaluated for their antimicrobial activity against Staphylococcus aureus, Bacillus cereus (Gram-positive bacteria), Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacteria); and Candida albicans (Fungi). Compounds 3 and 4 showed stronger inhibition of Gram-positive bacteria and fungi growth, and the antimicrobial ability of compound 3j (a 4-(benzylamino)-5-chloro-2,6-difluoro analog, MIC[SA] = 0.5 μg/mL; MIC[BC] = 0.4 μg/mL; MIC[CA] = 0.5 μg/mL) were close to nofloxacin and fluconazole and identified as the most potent antimicrobial agents in the series. The preliminary analysis of structure–activity relationships is also discussed.     

Isolation,structure and biological activity of phomafungin,a cyclic lipodepsipeptide from a widespread tropical Phoma sp.

We isolated a cyclic lipodepsipeptide, phomafungin, from a Phoma sp. The distinct antifungal activity of phomafungin in the crude extract was initially discovered by mechanistic profiling in the Candida albicans fitness test. The purified compound contains a 28 member ring consisting of eight amino acids and a β-hydroxy-γ-methyl-hexadecanoic acid, and displays a broad spectrum of antifungal activity against Candida spp., Aspergillus fumigatus and Trichophyton mentagrophytes with MIC of 2–8 μg/ml, and toxicity to mice at 25 mg/kg. The linear peptide derived from opening of the lactone ring was devoid of antifungal activity as well as toxicity. Phomafungin has been identified in a number of Phoma spp. collected from Africa and the Indian and Pacific Ocean islands.     

Effect of the contraction of medial rotators of the tibia on the electromyographic activity of vastus medialis and vastus lateralis

PurposeThis study attempted to assess if the resisted contraction of medial rotators of the tibia increases the ratio between the activity of vastus medialis (VM) and vastus lateralis (VL) during maximal isometric contractions (MIC) of the quadriceps femoral (QF) muscle at 90° of knee flexion.MethodsAbout 24 female subjects participated in this study, performing four series MIC of the QF. In the first series subjects performed only MIC of the QF muscle, whereas in the other three there was MIC of the QF with resisted contraction of medial rotators of the tibia, with the tibia positioned in medial, neutral and lateral rotation. During each contraction, VM and VL electromyographic signal (EMGs) and QF force were collected, being the EMGs root mean square (RMS) used to access the activity level of these muscles.ResultsThe use of the General Linear Model (GLM) test showed that for α = 0.05 there was a significant increase in the VM:VL ratio when the resisted contraction of medial rotators of the tibia was performed with the tibia in medial (p = <0.0001), neutral (p = <0.0001) and lateral rotation (p = 0.001). The same test showed that during MIC of the QF associated to resisted contraction of medial rotators of the tibia there were no significant differences in the VM:VL ratio between the three tibial rotation positions adopted (p = 0.866 [medial–neutral]; p = 0.106 [medial–lateral]; p = 0.068 [neutral–lateral]).ConclusionsThe resisted contraction of medial rotators of the tibia increases the VM:VL ratio during MIC of the QF and the tibial rotation position does not influence the VM:VL ratio during MIC associated to resisted contraction of medial rotators of the tibia.     

Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor

The continuing threat to biodiversity lends urgency to the need of identification of sustainable source of natural products. This is not so much trouble if there is a microbial source of the compound. Herein, violacein, a natural indolic pigment extracted from Chromobacterium violaceum, was evaluated for its antitumoral potential against the Ehrlich ascites tumor (EAT) in vivo and in vitro. Evaluation of violacein cytotoxicity using different endpoints indicated that EAT cells were twofold (IC₅₀ = 5.0 μM) more sensitive to the compound than normal human peripheral blood lymphocytes. In vitro studies indicated that violacein cytotoxicity to EAT cells is mediated by a rapid (8–12 h) production of reactive oxygen species (ROS) and a decrease in intracellular GSH levels, probably due to oxidative stress. Additionally, apoptosis was primarily induced, as demonstrated by an increase in Annexin-V positive cells, concurrently with increased levels of DNA fragmentation and increased caspase-2, caspase-9 and caspase-3 activities up to 4.5-, 6.0- and 5.5-fold, respectively, after 72 h of treatment. Moreover, doses of 0.1 and 1.0 μg kg^?1 violacein, administered intraperitoneally (i.p.) to EAT-bearing mice throughout the lifespan of the animals significantly inhibited tumor growth and increased survival of mice. In view of these results, a 35-day toxicity study was conducted in vivo. Complete hematology, biochemistry (ALT, AST and creatinine levels) and histopathological analysis of liver and kidney indicated that daily doses of violacein up to 1000 μg kg^?1 for 35 days are well tolerated and did not cause hematotoxicity nor renal or hepatotoxicity when administered i.p. to mice. Altogether, these results indicate that violacein causes oxidative stress and an imbalance in the antioxidant defense machinery of cells culminating in apoptotic cell death. Furthermore, this is the first report of its antitumor activity in vivo, which occurs in the absence of toxicity to major organs.     

Antimicrobial and cytotoxic activities of the crude extracts of Dietes bicolor leaves,flowers and rhizomes

The crude extracts of Dietes bicolor leaves, flowers and rhizomes were subjected to comparative antimicrobial screening against two Gram-positive, two Gram-negative bacteria and four fungal strains using the agar well diffusion method. The minimum inhibitory concentrations (MIC) of the tested extracts were also determined. Furthermore, the cytotoxic activity was evaluated. D. bicolor extracts generally demonstrated notable broad spectrum antimicrobial activities (MIC values ≤ 500 μg/mL) against all tested pathogens. D. bicolor leaf extract showed potent broad spectrum antimicrobial activity with MIC values ranging between 0.24 and 31.25 μg/mL against all tested pathogens. Moreover, the flowers extract exhibited promising antimicrobial activities, displaying MIC values ranging between 1.95 and 125 μg/mL against the tested bacteria and fungi. However, the rhizomes extract showed moderate antimicrobial activity with MIC values ranging between 31.25 and 500 μg/mL. Despite the potent antimicrobial activity of D. bicolor extracts, they were ineffective as cytotoxic agents against nine tested cancer cell lines, displaying 50% inhibitory concentration (IC₅₀) values above 100 μg/mL. The reported potent antimicrobial activity along with the lack of measurable cytotoxic activity indicated that the antimicrobial activity of D. bicolor crude extracts is mediated through a mechanism other than cytotoxicity. These results suggest that D. bicolor can act as a potential source for natural antibacterial and antifungal agents with a good safety profile at a preliminary level.     

Reactive oxygen species mediated modifications in Bacillus subtilis lipid membrane to improve protein productivities

The overall objective of this work was to investigate the modifications that occur in Bacillus subtilis lipid membrane during induced oxidative stress caused by reactive oxygen species (ROS), via an electrophysiological approach. Further, based on the results, we have developed and demonstrated a novel strategy to enhance specific enzyme production. Electrical parameters such as phase angle (θ), impedance (Z), capacitance (C) and breakdown voltage of reconstituted bilayer lipid membrane (BLM) composed of lipids extracted from non-stressed, mildly stressed (2.5 mM H₂O₂) and strongly stressed (2.5 mM H₂O₂ with 100 μM FeSO₄) B. subtilis were compared. Strongly stressed BLM showed lower values of θ (10°), Z (0.4 Mohm), and breakdown voltage (100 mV) in comparison with those observed for non-stressed BLM, i.e., 30°, 0.5 Mohm and 250 mV, respectively. The capacitance of strongly stressed BLM, however, was higher (2.28 nF) compared to that of the non-stressed BLM (0.4 nF). These results suggest that under strongly stressed conditions, the lipids were loosely packed that resulted in a more permeable BLM. The higher permeability seems to result, unexpectedly, from a higher unsaturated fatty acid (UFA) synthesis and membrane incorporation (UFA fraction increased by 227%), and expectedly, from increased lipid peroxidation (increased by nearly 200%) in the BLM. A strategy that is based on increased membrane permeability due to induced ROS, enhanced specific amylase and protease production under oxidative stress by 62% and 137%, respectively.     

Antitubercular activity of quinolizidinyl/pyrrolizidinylalkyliminophenazines

Novel riminophenazine derivatives, characterized by the presence of the basic and cumbersome quinolizidinylalkyl and pyrrolizidinylethyl moieties, have been synthesized and tested (Rema test) against Mycobacterium tuberculosis H₃₇R_v and H₃₇R_a, and six clinical isolates of Mycobacterium avium and Mycobacterium tuberculosis. Most compounds exhibited potent activity against the tested strains, resulting more active than clofazimine, isoniazid and ethambutol.The best compounds (4, 5, 12 and 13) exhibited a MIC in the range 0.82–0.86 μM against all strains of Mycobacterium tuberculosis and, with the exception of 4 a MIC around 3.3 μM versus M. avium. The corresponding values for clofazimine (CFM) were 1.06 and 4.23 μM, respectively. Cytotoxicity was evaluated against three cell lines and compound 4 displayed a selectivity index (SI) versus the human cell line MT-4 comparable with that of CFM (SI = 5.23 vs 6.4). Toxicity against mammalian Vero 76 cell line was quite lower with SI = 79.     

New monomeric and dimeric uridinyl derivatives as inhibitors of chitin synthase

This study described the synthesis and in vitro evaluation of eight new derivatives of uridine as antifungal agents and inhibitors of chitin synthase. Dimeric uridinyl derivatives synthesized by us did not exhibit significant activity. One of the studied monomeric derivative, 5′-(N-succinyl)-5′-amino-5′-deoxyuridine methyl ester (compound 7) showed activities against several fungal strains (MIC range 0.06–1.00 mg/mL) and inhibited chitin synthase from Saccharomyces cerevisiae (IC₅₀ = 0.8 mM). Moreover compound 7 exhibited synergistic interaction with caspofungin against Candida albicans (FIC index = 0.28).     

Phenolic composition and medicinal usage of Psidium guajava Linn.: Antifungal activity or inhibition of virulence?

Psidium guajava is a Myrtaceae plant whose medicinal properties are recognized in several locations. The use of teas and tinctures prepared from their leaves has been used to combat infections caused by fungi of the genus Candida. In this study, aqueous extracts of leaves and hydroethanolic were tested to verify the antifungal potential and its chemical composition has been investigated. The microbiological assays were performed by broth microdilution to determine the minimum inhibitory concentration (MIC) and from these the minimum fungicidal concentration was performed (MFC) by subculturing on solid media. A cell viability curve was obtained for demonstration of inhibition of fungal growth of strains of Candida albicans and Candida tropicalis. Tests to check morphological changes by the action of the extracts were performed in microcultive cameras depleted environment at concentrations of MIC/2, MIC and MIC × 2. Extracts analyzed by high performance liquid chromatography demonstrated flavonoids and phenolic acids. The extracts showed fungistatic effect and no fungicide with MIC >8192 μg/mL, MFC above 8192 μg/mL. The IC₅₀ was calculated ranging from 1803.02 to 5623.41 μg/mL. It has been found that the extracts affect the morphological transition capability, preventing the formation of pseudohyphae and hyphae. Teas and tinctures, therefore, have the potential antifungal, by direct contact, causing inhibition of fungal multiplication and its virulence factor, the cell dimorphism, preventing tissue invasion. Further studies are needed to elucidate the biochemical pathways and genes assets involved in these processes.     

Synergysm of voriconazole or itraconazole with other antifungal agents against species of Fusarium

BackgroundInfections caused by Fusarium are difficult to treat because these fungi show in vitro and in vivo resistance to practically all the antifungal agents available, which explains the high mortality rates. An attempt to overcome fungal resistance is the combination of antifungal agents, especially those with different mechanisms of action.AimsEvaluate the in vitro interactions of combinations of voriconazole or itraconazole with other antifungal agents against 32 isolates of Fusarium spp.: Fusarium chlamydosporum, Fusarium oxysporum, Fusarium proliferatum and Fusarium solani.MethodsDrug interactions were assessed by a checkerboard microdilution method that also included the determination of the MIC of each drug alone according to CLSI (Clinical and Laboratory Standards Institute) document M38-A2, 2008.ResultsThe best combinations were voriconazole + terbinafine which showed synergism against 84% of Fusarium strains. Other synergistic combinations were voriconazole + itraconazole (50%), voriconazole + fluconazole (50%), voriconazole + miconazole (38%), voriconazole + flucytosine (22%) and voriconazole + ketoconazole (25%). The synergisms observed with itraconazole combinations were itraconazole + terbinafine (25%) and itraconazole + flucytosine (9.37%). The antagonisms observed were: voriconazole + fluconazole (3%) and itraconazole + flucytosine (12.5%).ConclusionsThe synergism showed by voriconazole + terbinafine was remarkable. To better elucidate the potential usefulness of our findings, new in vivo and in vitro studies deserve be performed.     

Enhanced ethanol and chitosan production from wheat straw by Mucor indicus with minimal nutrient consumption

Recently, Mucor indicus was introduced as a promising ethanol producing microorganism for fermentation of lignocellulosic hydrolysates, showing a number of advantages over Saccharomyces cerevisiae. However, high nutrient requirement is the main drawback of the fungus in efficient ethanol production from lignocelluloses. In this study, application of fungal extract as a potential nutrient source replacing all required nutrients in fermentation of wheat straw by M. indicus was investigated. Wheat straw was pretreated with N-methylmorpholine-N-oxide (NMMO) at 120 °C for 1–5 h prior to enzymatic hydrolysis. Hydrolysis yield was improved at least by 6-fold for 3 h pretreated straw compared with that of untreated one. A fungal extract was produced by autolysis of M. indicus biomass, an unavoidable byproduct of fermentation. Maximum free amino nitrogen (2.04 g/L), phosphorus (1.50 g/L), and total nitrogen (4.47 g/L) as well as potassium, magnesium, and calcium in the fungal extract were obtained by autolysis of the biomass at 50 °C and pH 5.0. The fungal extract as a nutrient-rich supplement substituted yeast extract and all other required minerals in fermentation and enhanced the ethanol yield up to 92.1% of the theoretical yield. Besides, appreciate amounts of chitosan were produced as another valuable product of the autolysis.     

Search for new pharmacophores for antimalarial activity. Part II: Synthesis and antimalarial activity of new 6-ureido-4-anilinoquinazolines

Synthesis of new 6-ureido-4-anilinoquinazolines have been accomplished and their in vitro antimalarial activity against chloroquine-sensitive P. falciparum have been examined. Out of 64 compounds evaluated, the IC₅₀ of 16 compounds which have displayed MIC of 0.25 μg/mL were also recorded. One of the compounds (24g) had IC₅₀ value of 2.27 ng/mL which was equipotent to the standard drug chloroquine used in the bioassay. The in vivo evaluation of a few compounds among the series led to discovery of one analog (30g) displaying 40% curative activity (28 days) against mdr P. yoeilli nigeriensis at an oral dose of 100 mg/kg × 4days.     

Diversity of fungal endophytes in shrubby medicinal plants of Malnad region,Western Ghats,Southern India

A total of 6125 fungal endophytes were isolated from 9000 leaf segments of 15 medicinal shrubs growing in Malnad region of Western Ghats, Southern India, during winter, monsoon, and summer seasons. These fungal isolates belonged to Ascomycota (8.6 %), Coelomycetes (26.0 %), Hyphomycetes (28.0 %), Mucoromycotina (0.3 %) and sterile forms (4.9 %). Alternaria, Chaetomium, Fusarium, Colletotrichum, Cladosporium, Penicillium, Phyllosticta and Xylaria were the most frequently isolated. Significantly more isolates were obtained during the winter season than monsoon and summer seasons.     

Deletion and site-directed mutagenesis of laccase from Shigella dysenteriae results in enhanced enzymatic activity and thermostability

A putative laccase gene was cloned from Shigella dysenteriae W202 and expressed in Escherichia coli as a soluble fusion protein with high yield. The purified product (Wlac) was characterized as the CueO-like laccase from E. coli, a monomer of molecular mass 55 kDa, with a maximum activity of 24.4 U/mg (K_m = 0.086) and a pH optimum of 2.5, in a standard assay using ABTS (2,2′-azino-di(3-ethyl-benzthiazoline-6-sulfonate) as the substrate. Activity was stable at 0–25 °C but inhibited above 40 °C. Purified Wlac was completely inhibited by 200 mM EDTA and partially by 32 mM SDS, 50 mM NaN₃ and 60 mM thioglycolic acid. Activity was stimulated by Cu²⁺; other metal ions had only slight or negative effects. Two mutated variants, WlacS and WlacD, were obtained by substituting Glu 106 with Phe 106, and adding a deletion of an α-helix domain (from Leu 351 to Gly 378). WlacS had a 2.2-fold (52.9 U/mg) and WlacD a 3.5-fold (85.1 U/mg) higher enzyme activity than the wild-type laccase and WlacD showed greater thermostability at higher temperatures. Sce VMA intein-associated fusion proteins maintained ～80% of total enzyme activity. Thus, deletion and site-directed mutagenesis of laccases are capable of promoting both enzymatic activity and thermostability.     

A new fungal peroxidase with alkaline-tolerant,chloride-enhancing activity and dye decolorization capacity

A new fungal peroxidase (Pspd) from Perenniporia subacida was purified by ammonium sulfate precipitation, DEAE-cellulose DE52 anionic exchange and Sepharose GL-6B chromatography, resulting in a high specific activity of 9.138 U mg⁻¹, 3.622-fold higher than that of crude enzyme at the same level. Polyacrylamide gel electrophoresis and UV–vis adsorption spectrum analysis showed that the purified enzyme is a heme-containing monomer with a molecular mass of 43.0 kDa. Optimal peroxidase activity was obtained at pH 5.5 and 30 °C when using 100.0 mM n-propanol as substrate, and under these conditions, the catalytic efficiency (k_cat/K_m) is 1.57 s⁻¹ μM⁻¹. Pspd was inhibited by l-cysteine, dithiothreitol, EDTA and sodium azide, but stimulated by Mn²⁺, Na⁺, Mg²⁺ and K⁺. The enzyme is stable over a broad pH range of 7.0–8.5 after incubation for 72 h, which indicated that the enzyme is lasting alkaline-tolerant. It was worth noting that the chloride at relatively low concentrations can enhance the peroxidase activity, with concomitant increase in substrate affinity. Additionally, Pspd performed high decolorization capability toward structurally various dyes and the capability was independent of the oxidizing mediators, with 75.31% of Neutral Red (50.0 mg L⁻¹) being decolorized by 1.5 U mL⁻¹ pure enzyme after incubation for 72 h. These properties demonstrated that Pspd has potentials for textile dyes decolorization applications.     

Structure–activity relationships of 2-aminothiazoles effective against Mycobacterium tuberculosis

A series of 2-aminothiazoles was synthesized based on a HTS scaffold from a whole-cell screen against Mycobacterium tuberculosis (Mtb). The SAR shows the central thiazole moiety and the 2-pyridyl moiety at C-4 of the thiazole are intolerant to modification. However, the N-2 position of the aminothiazole exhibits high flexibility and we successfully improved the antitubercular activity of the initial hit by more than 128-fold through introduction of substituted benzoyl groups at this position. N-(3-Chlorobenzoyl)-4-(2-pyridinyl)-1,3-thiazol-2-amine (55) emerged as one of the most promising analogues with a MIC of 0.024 μM or 0.008 μg/mL in 7H9 media and therapeutic index of nearly ～300. However, 55 is rapidly metabolized by human liver microsomes (t_1/2 = 28 min) with metabolism occurring at the invariant aminothiazole moiety and Mtb develops spontaneous low-level resistance with a frequency of ～10^?5.     

Selective activity against Mycobacterium tuberculosis of new quinoxaline 1,4-di-N-oxides

New series of 3-phenylquinoxaline 1,4-di-N-oxide with selective activity against Mycobacterium tuberculosis have been prepared and evaluated. Thirty-four of the seventy tested compounds showed an MIC value less than 0.2 μg/mL, a value on the order of the MIC of rifampicin. Furthermore, 45% of the evaluated derivatives showed a good in vitro activity/toxicity ratio. The most active and selective compounds carry a fluorine atom in the quinoxaline 7-position or in the phenyl substituent para-position. In conclusion, the potency, low cytotoxicity and selectivity of these compounds make them valid lead compounds for synthesizing new analogues, particularly compound 7-methyl-3-(4’-fluoro)phenylquinoxaline-2-carbonitrile 1,4-di-N-oxide (MIC <0.2 μg/mL and SI >500).