Antimicrotubular effect of calvatic acid and of some related compounds

A structure-activity relationship has been established between calvatic acid and some related synthetic compounds, and their ability to inhibit GTP-induced microtubular protein polymerization in vitro. These compounds were effective in a dose- and a time-dependent manner. The most active drug was the p-chloro substituted compound, which showed its inhibitory activity without any preincubation period, which the others needed. Since if cysteine was present, polymerization was no longer affected, an involvement of titratable -SH groups of tubulin could be suggested. In contrast, taxol-induced polymerization was only slightly inhibited by these compounds, and colchicine-binding activity was not generally impaired.     

Relaxation of supercoiled DNA associated with induction of heat shock proteins in Escherichia coli

Bacillus subtilis 168 is known to possess two thymidylate synthase (TSase; EC 2.1.1.45) genes: thyA and thyB. thyB encodes a thermosensitive TSase (inactivated at 46° C) which, in wild-type cells, accounts for only 5–8% of the total cellular TSase activity. In order to investigate the thermal lability of TSaseB we have analyzed the thyB genes of B. subtilis 168 and of an unrelated strain B. subtilis ATCC6633, which is shown here to have a temperature-resistant TSaseB. This conclusion is supported by the frequency of appearance of spontaneous Thy^– mutants at 37° C and 46° C, and by the analysis of clones containing the thyB genes from the two strains. The nucleotide sequences of these two thyB genes were compared.     

Heat-stable and heat-labile thymidylate synthases B of Bacillus subtilis: comparison of the nucleotide and amino acid sequences

Heat treatment of wild-type Escherichia coli cells led to a transient relaxation of negatively supercoiled plasmid DNA and there was no recovery of DNA torsional strain in the DNA in gyrA mutant cells. After heat treatment, DnaK and GroEL proteins were synthesized continuously in the gyrA mutant cells, whereas they were synthesized only transiently in wild-type cells. Thus, change in superhelical density of the DNA correlated with the temperature-induced expression of heat shock proteins. Inhibitors of DNA gyrase (nalidixic acid, novobiocin), an organic solvent (ethanol) and a psychotropic drug (chlorpromazine) all stimulated relaxation of cellular DNA over the same concentration range that induces heat shock proteins. As DNA relaxation was induced by heat treatment or chemicals in an rpoH mutant, the process is not the result of induced synthesis of heat shock proteins.     

Simultaneous quantification of GMP, AMP, cyclic GMP and cyclic AMP by liquid chromatography coupled to tandem mass spectrometry

Phosphodiesterases are drug targets for treating various diseases. Inhibition of these can increase cAMP and cGMP levels, which can affect a variety of physiological responses. Here we report a new method for determining PDE activity by combining high-performance liquid chromatography and tandem mass spectrometry. Characteristic peaks of the substrates, cGMP or cAMP and products, GMP or AMP, were identified in positive-ion electrospray ionization using multiple reaction monitoring. The method can be applied to determine activity of PDE inhibitors. Our results showed that this new method was fast, sensitive and highly reproducible.     

AtGRP2, a cold-induced nucleo-cytoplasmic RNA-binding protein, has a role in flower and seed development

The glycine-rich protein AtGRP2 is one of the four members of the cold-shock domain (CSD) protein family in Arabidopsis. It is characterized by the presence of a nucleic acid-binding CSD domain, two glycine-rich domains and two CCHC zinc-fingers present in nucleic acid-binding proteins. In an attempt to further understand the role of CSD/GRP proteins in plants, we have proceeded to the functional characterization of the AtGRP2 gene. Here, we demonstrate that AtGRP2 is a nucleo-cytoplasmic protein involved in Arabidopsis development with a possible function in cold-response. Expression analysis revealed that the AtGRP2 gene is active in meristematic tissues, being modulated during flower development. Down-regulation of AtGRP2 gene, using gene-silencing techniques resulted in early flowering, altered stamen number and affected seed development. A possible role of AtGRP2 as an RNA chaperone is discussed.     

Lindane may modulate the female reproductive development through the interaction with ER-beta: an in vivo-in vitro approach

Lindane (gamma-HCH) is a persistent environmental pollutant that may act as endocrine disrupter, affecting the nervous, immune and reproductive system, possibly through endocrine-mediated mechanisms. Since both estrogen receptors (ER-alpha and -beta) have shown to be target for endocrine disruption, we investigated the role of gamma-HCH on the development of female reproductive system. For an in vivo evaluation of gamma-HCH effects during prenatal period, pregnant CD1 mice were treated p.o. on gestational days 9-16 with 15 mg/kg bw/day of gamma-HCH and vehicle. The in vivo findings in treated F1 pups - in the absence of signs of systemic toxicity - included increase in the absolute and relative and absolute uterus weight revealed on post-natal day 22, earlier vaginal patency and reduced diameters of primary oocytes at fully sexual maturity. No effects on steroid hormone metabolism (aromatase, testosterone catabolism) were observed. Thus, gamma-HCH elicited subtle effects on female reproductive development likely mediated by ER-beta-mediated pathway(s), without a concurrent impairment of steroid hormone metabolism. Furthermore, to verify whether the endocrine interference of gamma-HCH is attributable to stimulation of ER-beta-mediated pathway(s), its effect has been evaluated in vitro on a cell line, LNCaP, expressing only functional ER-beta. In vitro treatments revealed a concentration-related effect on LNCaP cell viability and proliferation. Significantly, the contemporary addition of a pure anti-estrogen, the ER antagonist ICI 182,780, completely reversed gamma-HCH effects indicating an ER-beta-mediated action. Our findings indicate that gamma-HCH may act as endocrine disruptor during the female reproductive system development and ER-beta as a potential target for this compound and other endocrine disrupting chemicals as well.     

Antimycobacterial Activity of a New Peptide Polydim-I Isolated from Neotropical Social Wasp Polybia dimorpha

Mycobacterium abscessus subsp. massiliense, a rapidly growing mycobacteria (RGM) that is becoming increasingly important among human infectious diseases, is virulent and pathogenic and presents intrinsic resistance to several antimicrobial drugs that might hamper their elimination. Therefore, the identification of new drugs to improve the current treatment or lower the risk of inducing resistance is urgently needed. Wasp venom primarily comprises peptides that are responsible for most of the biological activities in this poison. Here, a novel peptide Polydim-I, from Polybia dimorpha Neotropical wasp, was explored as an antimycobacterial agent. Polydim-I provoked cell wall disruption and exhibited non-cytotoxicity towards mammalian cells. Polydim-I treatment of macrophages infected with different M. abscessus subsp. massiliense strains reduced 40 to 50% of the bacterial load. Additionally, the Polydim-I treatment of highly susceptible mice intravenously infected with M. abscessus subsp. massiliense induced 0.8 to 1 log reduction of the bacterial load in the lungs, spleen, and liver. In conclusion, this is the first study to show the therapeutic potential of a peptide derived from wasp venom in treating mycobacteria infections. Polydim-I acts on the M. abscessus subsp. massiliense cell wall and reduce 40–90% of the bacterial load both in vitro and in vivo. The presented results encourage further studies on the use of Polydim-I as one of the components for M. abscessus subsp. massiliense treatment.     

Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatoza by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization. © Wiley-Liss, Inc.     

Intraspecies Variability Affects Heterotypic Biofilms of Porphyromonas gingivalis and Prevotella intermedia: Evidences of Strain-Dependence Biofilm Modulation by Physical Contact and by Released Soluble Factors

It is well known that strain and virulence diversity exist within the population structure of Porphyromonas gingivalis. In the present study we investigate intra- and inter-species variability in biofilm formation of Porphyromonas gingivalis and partners Prevotella intermedia and Prevotella nigrescens. All strains tested showed similar hydrophobicity, except for P. gingivalis W83 which has roughly half of the hydrophobicity of P. gingivalis ATCC33277. An intraspecies variability in coaggregation of P. gingivalis with P. intermedia was also found. The association P. gingivalis W83/P. intermedia 17 produced the thickest biofilm and strain 17 was prevalent. In a two-compartment system P. gingivalis W83 stimulates an increase in biomass of strain 17 and the latter did not stimulate the growth of P. gingivalis W83. In addition, P. gingivalis W83 also stimulates the growth of P. intermedia ATCC25611 although strain W83 was prevalent in the association with P. intermedia ATCC25611. P. gingivalis ATCC33277 was prevalent in both associations with P. intermedia and both strains of P. intermedia stimulate the growth of P. gingivalis ATCC33277. FISH images also showed variability in biofilm structure. Thus, the outcome of the association P. gingivalis/P. intermedia seems to be strain-dependent, and both soluble factors and physical contact are relevant. The association P. gingivalis-P. nigrescens ATCC33563 produced larger biomass than each monotypic biofilm, and P. gingivalis was favored in consortia, while no differences were found in the two-compartment system. Therefore, in consortia P. gingivalis-P. nigrescens physical contact seems to favor P. gingivalis growth. The intraspecies variability found in our study suggests strain-dependence in ability of microorganisms to recognize molecules in other bacteria which may further elucidate the dysbiosis event during periodontitis development giving additional explanation for periodontal bacteria, such as P. gingivalis and P. intermedia, among others, to persist and establish chronic infections in the host.