Leishmania major: Cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family

In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (eg logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti-LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [³⁵S]methionine-labeled promastigote antigens after pulse chase experiments, using anti-LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted-secreted antigens (ESA). Moreover, immunoflurescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.     

Synthesis and Evaluation of Biphenyl Compounds as Kinesin Spindle Protein Inhibitors

Kinesin spindle protein (KSP), an ATP‐dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression. Antiproliferation (MTT (=3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide)) assays, combined with fluorescence‐assisted cell sorting (FACS) and Western blot studies analyzing cell‐cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far, compound 7 , exerts its pharmacologic effect specifically in the M phase and induces monoaster formation. These studies confirm that chemical scope remains for improving the potency and treatment efficacy of antimitotic KSP inhibition in this class of biphenyl compounds.     

Metal cation toxicity in the alga Gracilaria domingensis as evaluated by the daily growth rates in synthetic seawater

Macroalgae of the genus Gracilaria have considerable economic importance as raw material for agar production and belong to an important group of organisms that are tolerant of high concentrations of metal. The median inhibitory concentration (IC₅₀) values obtained by measuring the ratio of fresh mass variation (i.e., daily growth rates) of the red macroalga Gracilaria domingensis during a 48-h aquatic toxicity assay are reported here. The alga was exposed to 14 different metal cations as well as the molybdate anion in synthetic seawater. The actual concentrations of these ionic species (at IC₅₀ values) and the proportion of free ions (aqueous complexes) were determined by inductively coupled plasma atomic emission spectroscopy and the Environmental Protection Agency-recommended software, MINTEQA2, respectively. Based on the free IC₅₀ values (IC₅₀ ^F), the ions were ranked in terms of toxicity: Cd²⁺???Cu²⁺???Pb²⁺???Zn²⁺???Ni²⁺?>?Co²⁺?>?La³⁺???Mn²⁺?>?Ca²⁺?~?Li⁺???MoO₄ ^2????Sr²⁺?>?Mg²⁺???K⁺?>?Na⁺. As a member of the first trophic level in the marine food chain, G. domingensis is an appropriate target organism both for the development of toxicological assays and as a bioindicator of marine degradation.     

Structural characterization of the cell wall D-glucans isolated from the mycelium of Botryosphaeria rhodina MAMB-05

Three D-glucans were isolated from the mycelium of the fungus Botryosphaeria rhodina MAMB-05 by sequential extraction with hot-water and hot aqueous KOH (2% w/v) followed by ethanol precipitation. Following their purification by gel permeation chromatography on Sepharose CL-4B, the structural characteristics of the D-glucans were determined by FT-IR and 13C NMR spectroscopy and, after methylation, by GC-MS. The hot-water extract produced a fraction designated Q1A that was a beta-(1-->6)-D-glucan with the following structure: [Formula: see text] The alkaline extract, when subjected to repeated freeze-thawing, yielded two fractions: K1P (insoluble) that comprised a beta-(1-->3)-D-glucan with beta-D-glucose branches at C-6 with the structure: [Formula: see text] and K1SA (soluble) consisting of a backbone chain of alpha-(1-->4)-linked D-glucopyranosyl residues substituted at O-6 with alpha-D-glucopyranosyl residues: [Formula: see text]     

Interferon Regulatory Factor (IRF)-1 Is a Master Regulator of the Cross Talk between Macrophages and L929 Fibrosarcoma Cells for Nitric Oxide Dependent Tumoricidal Activity

Macrophage tumoricidal activity relies, mainly, on the release of Tumor Necrosis Factor alpha (TNFα) and/or on reactive oxygen or nitrogen intermediates. In the present work, we investigated the cytotoxic activity of resident peritoneal macrophages against L929 fibrosarcoma cell line in vitro and in vivo. Resident macrophages lysed L929 cells in a mechanism independent of TNFα and cell-to-cell contact. The cytotoxic activity was largely dependent on nitric oxide (NO) release since treatment with L-NAME (NOS inhibitor) inhibited L929 cells killing. Macrophages from mice with targeted deletion of inducible NO synthase (iNOS) together with L929 cells produced less NO and displayed lower, but still significant, tumoricidal activity. Notably, NO production and tumor lysis were abolished in co-cultures with macrophages deficient in Interferon Regulatory Factor, IRF-1. Importantly, the in vitro findings were reproduced in vivo as IRF-1 deficient animals inoculated i.p with L929 cells were extremely susceptible to tumor growth and their macrophages did not produce NO, while WT mice killed L929 tumor cells and their macrophages produced high levels of NO. Our results indicate that IRF-1 is a master regulator of bi-directional interaction between macrophages and tumor cells. Overall, IRF-1 was essential for NO production by co-cultures and macrophage tumoricidal activity in vitro as well as for the control of tumor growth in vivo.     

Role of Repeat I in the fast inactivation kinetics of the Ca(V)2.3 channel

The molecular basis for inactivation in Ca(V)2.3 (alpha 1E) channels was studied after expression of alpha 1E/alpha 1C (Ca(V)2.3/Ca(V)1.2) chimeras in Xenopus oocytes. In the presence of 10 mM Ba(2+), the CEEE chimera (Repeat I+part of the I-II linker from Ca(V)1.2) displayed inactivation properties similar to Ca(V)1.2 despite being more than 90% homologous to Ca(V)2.3. The transmembrane segments of Repeat I did not appear to be crucial as inactivation of EC(IS1-6)EEE was not significantly different than Ca(V)2.3. In contrast, EC(AID)EEE, with the beta-subunit binding domain from Ca(V)1.2, tended to behave like Ca(V)1.2 in terms of inactivation kinetics and voltage dependence. A detailed kinetic analysis revealed nonetheless that CEEE and EC(AID)EEE retained the fast inactivation time constant (tau(fast) approximately equal to 20-30 ms) that is a distinctive feature of Ca(V)2.3. Altogether, these data suggest that the region surrounding the AID binding site plays a pivotal albeit not exclusive role in determining the inactivation properties of Ca(V)2.3.     

Buddleja davidii and Buddleja yunnanensis: Exploring features associated with commonness and rarity in Buddleja

Buddleja davidii is a widespread shrub in Asia while B. yunnanensis is a narrowly endemic species limited to Yunnan Province, China. To explore whether floral volatiles, morphological characters of flower and seed and breeding system are correlated with their distributions, we measured length and width of corolla, trichome density at corolla throat, level of stigma/anthers relationship, seed size and weight. The results indicated that these characteristics were significantly different between the two species (P < 0.01). Bagging experiments revealed that B. davidii is a self-incompatible plant while B. yunnanensis is self-compatible. Thick trichome density at the corolla throat may reduce out-crossing in B. yunnanensis. Autogamy plays an important role in fruit production of this species while B. davidii requires pollinators for fruiting. Scents were collected using dynamic headspace adsorption method and identified with coupled gas chromatography and mass spectrometry. In total, 27 floral scent compounds were identified. The volatile composition in the two species was very different. We attempted to determine if these features, associated with commonness and with rarity found in these two taxa, could also help to explain the distribution pattern of other species of the genus Buddleja.     

Identification of a novel protonation pattern for carboxylic acids upon Q(B) photoreduction in Rhodobacter sphaeroides reaction center mutants at Asp-L213 and Glu-L212 sites

In the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides, light energy is rapidly converted to chemical energy through coupled electron-proton transfer to a buried quinone molecule Q(B). Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations that are observable using light-induced Fourier transform infrared (FTIR) difference spectroscopy. Upon formation, Q(B)(-) induces protonation of Glu-L212, located within 5 A of Q(B), resulting in a IR signal at 1728 cm(-1). However, no other IR signal is observed within the classic absorption range of protonated carboxylic acids (1770-1700 cm(-1)). In particular, no signal for Asp-L213 is found despite its juxtaposition to Q(B) and importance for proton uptake on the second electron-transfer step. In an attempt to uncover the reason behind this lack of signal, the microscopic electrostatic environment in the vicinity of Q(B) was modified by interchanging Asp and Glu at the L213 and L212 positions. The Q(B)(-)/Q(B) FTIR spectrum of the Asp-L212/Glu-L213 swap mutant in the 1770-1700 cm(-1) range shows several distinct new signals, which are sensitive to (1)H/(2)H isotopic exchange, indicating that the reduction of Q(B) results in the change of the protonation state of several carboxylic acids. The new bands at 1752 and 1747 cm(-1) were assigned to an increase of protonation in response to Q(B) reduction of Glu-L213 and Asp-L212, respectively, based on the effect of replacing them with their amine analogues. Since other carboxylic acid signals were observed, it is concluded that the swap mutations at L212 and L213 affect a cluster of carboxylic acids larger than the L212/L213 acid pair. Implications for the native reaction center are discussed.     

Detection of resistance genes and susceptibility patterns in Bacteroides and Parabacteroides strains

Susceptibility to five antimicrobials was determined for Bacteroides spp. (n = 52) and Parabacteroides distasonis (n = 8). All isolates were susceptible to metronidazole. The resistance rates to ampicillin, cefoxitin, tetracycline and clindamycin were 98%, 9.6%, 65.3% and 19.2% of the Bacteroides strains, respectively. The genes cepA, cfiA, cfxA, tetQ, ermF and nim were found in 69.2%, 17.3% 9.6%, 50%, 7.7% and 3.8% for these strains respectively. All P. distasonis strains were resistant to ampicilin. Cefoxitin, tetracycline and clindamycin resistance rates were 75%, 87.5% and 50%, respectively. The ermF and nim genes were absent and 37.5%, 12.5%, 12.5% and 87.5% of this strains possessed cepA, cfiA, cfxA and tetQ genes, respectively. Ten cfiA gene positive strains of Bacteroides and Parabacteroides were submitted to E-test with imipenem and amoxicillin–clavulanate. The resistance rate to imipenem was 4.1% and 8.3% to amoxicillin–clavulanate. This feature is for the first time described in Brazil.