Microbial community and physicochemical dynamics during the production of ‘Chicha’, a traditional beverage of Indigenous people of Brazil

The microbial community of artisanal corn fermentation called Chicha were isolated, purified and then identified using protein profile by Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) and confirmed by partial ribosomal gene sequencing. Samples from Chicha beverage were chemically characterized by gas and liquid chromatography (HPLC and GC-MS). Aerobic mesophilic bacteria (AMB) (35.8% of total of isolated microorganisms), lactic acid bacteria (LAB) (21.6%) and yeast (42.6%) were identified. Species of the genera Klebsiella, Bacillus, Staphylococcus, Micrococcus, Enterobacter, and Weissella were identified. Rhodotorula mucilaginosa, Lodderomyces elongisporus, Candida metapsilosis, and C. bohicensis were the yeasts found. The LAB isolates detected were responsible for the high concentrations of lactic acid found during the fermentation process (1.2 g L^??1), which is directly related to the decrease in pH values (from 6.95 to 3.70). Maltose was the main carbohydrate detected during corn fermentation (7.02 g L^??1 with 36 h of fermentation). Ethanol was found in low concentrations (average 0.181 g L^??1), making it possible to characterize the beverage as non-alcoholic. Twelve volatile compounds were identified by gas chromatography; belonging to the groups acids, alcohols aldehydes, acetate and others. MALDI-TOF was successfully used for identification of microbiota. Weissella confusa and W. cibaria were detected in the final product (after 36 h of fermentation), W. confusa is often classified as probiotic and deserve further application studies.     

miR‐200/375 control epithelial plasticity‐associated alternative splicing by repressing the RNA‐binding protein Quaking

Members of the miR‐200 family are critical gatekeepers of the epithelial state, restraining expression of pro‐mesenchymal genes that drive epithelial–mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR‐200c and another epithelial‐enriched miRNA, miR‐375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA‐binding protein Quaking (QKI). During EMT, QKI‐5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI‐5 is both necessary and sufficient to direct EMT‐associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial‐derived cancer types. Importantly, several actin cytoskeleton‐associated genes are directly targeted by both QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR‐200/miR‐375/QKI axis that impacts cancer‐associated epithelial cell plasticity through widespread control of alternative splicing.     

Medroxyprogesterone acetate: receptor binding and correlated effects on steroidogenesis in rat granulosa cells

Medroxyprogesterone acetate (MPA), a widely used synthetic steroid, was studied to determine both its effects on steroid receptors and steroidogenesis in the well-characterized rat ovarian granulosa cell model. Initial receptor binding studies showed MPA was as potent as progesterone and 10-fold less potent than R-5020 (an active synthetic progestin) in binding to progesterone cytosolic receptors in rat ovarian granulosa cells. MPA was 20-fold less potent than testosterone, and 10-fold less potent than dexamethasone in binding to the androgen and glucocorticoid cytosolic receptors, respectively. The binding of MPA to progestrone, androgen and glucocorticoid receptors predicted direct effects of MPA on FSH-stimulated estrogen (E), progesterone (P), and 20 alpha-dihydroprogesterone (DHP) production by cultured rat ovarian granulosa cells. MPA at 10(-7) to 10(-6) M significantly augmented FSH-stimulated P and DHP production (a previously documented progestin, androgen and glucocorticoid effect). This augmentation was blocked by the concurrent addition to cell culture of 10-fold excess RU-486 (a potent anti-progestin and anti-glucocorticoid). At concentrations greater than 10(-6) M, MPA inhibited the production of P and DHP (a progestin effect), and the production of E (a progestin and glucocorticoid effect). MPA, structurally a progestin, has complex steroid hormone effects predicted by its interaction with progesterone, androgen and glucocorticoid receptors.     

A solid-state,portable instrument for measurement of chlorophyll luminescence induction in plants

A newly developed compact instrument is described for the measurement of chlorophyll luminescence induction in plants. The instrument operates with a pulsed light emitting diode (LED) as light source and a photodiode as luminescence detector. A special emitter-detector geometry provides for high irradiance of the sample and efficient collection of luminescence by the detector. With insertion of appropriate filters the same probe is also suited for measuring prompt chlorophyll fluorescence. The instrument shows considerable flexibility with respect to pulse frequency, relative lengths of light/dark intervals and luminescence sampling periods. Due to a selective amplifier system only that part of luminescence is processed which is induced by the individual excitation pulses. By this approach, the problem of slow phase accumulation, encountered with conventional phosphoroscopes, is eliminated. Some examples are given for system operation, demonstrating satisfactory performance in measurements with intact leaves and isolated chloroplasts.     

Monoclonal antibodies to the guanine-nucleotide binding proteins of adenylate cyclase.

Monoclonal antibodies (Mabs) to the stimulatory (Ns) and inhibitory (Ni) guanine nucleotide regulatory proteins associated with adenylate cyclase have been developed. Two Mabs (2A3 and 5G12), which are of the IgG2b subclass, recognize the beta-subunits (beta) of Ns, Ni and transducin. Iodinated beta can be immunoprecipitated by either Mab coupled to Affi-Gel 10 and this can be decreased by prior incubation of the Mabs with excess unlabelled beta. The Mabs stabilize the activated state of Ns while decreasing the rate of deactivation of activated Ns in the presence of beta.     

Evidence for a free radical mechanism of styrene-glutathione conjugate formation catalyzed by prostaglandin H synthase and horseradish peroxidase

We have proposed, using styrene as a model, a new mechanism for the formation of glutathione conjugates that is independent of epoxide formation but dependent on the oxidation of glutathione to a thiyl radical by peroxidases such as prostaglandin H synthase or horseradish peroxidase. The thiyl radical reacts with styrene to yield a carbon-centered radical which subsequently reacts with molecular oxygen to give the styrene-glutathione conjugate. We have used electron spin resonance spin trapping techniques to detect the proposed free radical intermediates. A styrene carbon-centered radical was trapped using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and t-nitrosobutane. The position of the carbon-centered radical was confirmed to be at carbon 7 by the use of specific 2H-labeled styrenes. The addition of the spin trap DMPO inhibited both the utilization of molecular oxygen and the formation of styrene-glutathione conjugates. Under anaerobic conditions additional styrene-glutathione conjugates were formed, one of which was identified by fast atom bombardment mass spectrometry as S-(2-phenyl)ethylglutathione. The glutathione thiyl radical intermediate was observed by spin trapping with DMPO. These results support the proposed free radical-mediated formation of styrene-glutathione conjugates by peroxidase enzymes.     

Nitrobenzyl radical metabolites from microsomal reduction of nitrobenzyl chlorides

The o-, m-, and p-nitrobenzyl chlorides are reduced aerobically and anaerobically by NADPH and rat hepatic microsomes. Under aerobic conditions, these nitro anion radicals reduce oxygen to superoxide as demonstrated by oxygen consumption and spin trapping of superoxide with 5,5-dimethyl-1-pyrroline N-oxide. At low oxygen concentration, the p- and o-nitro anion radicals undergo intramolecular electron transfer and decompose to carbon-centered nitrobenzyl radicals, which can be spin-trapped with t-nitrosobutane. The p-nitrobenzyl (o-nitrobenzyl) radical adduct was characterized by a nitrogen hyperfine splitting of 16.5 G (17.1 G) and two equivalent beta-hydrogen hyperfine splittings of 10.6 G (14.4 G). The spin trap 5,5-dimethyl-1-pyrroline N-oxide also yields adducts characteristic of carbon-centered free radicals. This unimolecular decomposition is much faster than the disproportionation decay, which is characteristic of most nitro anion radicals, and the primary o- and p-nitrobenzyl chloride anion radicals never achieve detectable concentrations. The nitrobenzyl radical trapping is not inhibited by metyrapone or CO. In contrast, the m-nitrobenzyl anion radical does achieve a detectable steady-state concentration, which is increased 20% by either metyrapone or a CO atmosphere.     

Anti-idiotype-induced, lipopolysaccharide-specific antibody response to Pseudomonas aeruginosa

Antibody directed to the O-specific polysaccharide (Ps) side chain of Pseudomonas aeruginosa LPS provides immunotype-specific protection against infection by virtue of enhancing opsonophagocytosis. We have developed a syngeneic anti-idiotypic antibody (mAb2) directed to a functionally active monoclonal immunotype 1 Ps-antibody (mAb1). The mAb2 performed as a molecular mimic of Ps as evidenced by 1) blocking of mAb1/mAb2 interaction by Ps, 2) blocking of mAb1/Ps binding by mAb2, 3) cross-species binding of mAb2 to human Ps antibodies from individuals immunized with the same immunotype 1 Ps, and 4) induction of anti-LPS antibody by immunization with mAb2 in syngeneic mice. Our studies thus show that an anti-idiotypic antibody may functionally mimic the O-polysaccharide of P. aeruginosa LPS, and bind to cross-reactive Id present in human Ps antibodies. We have further shown that this anti-idiotypic antibody induces anti-LPS antibody when used as an Ag in syngeneic mice, suggesting that this approach may eventually be used to successfully immunize humans.     

Effect of graft-versus-host disease on anti-tumor immunity

BCL1, a spontaneous B cell leukemia of BALB/c origin, is rejected by C.B-20 (Ighb, H-40b) but not BALB/c (Igha, H-40a) mice. Adoptive transfer of C.B-20 anti-BCL1 effector cells specific for the minor histocompatibility Ag H-40a protects irradiated C.B-20 but not BALB/c recipients. Because C.B-20 donor cells could potentially generate graft-vs-host disease (GVHD) in BALB/c recipients, we investigated the possibility that GVHD prevents the anti-tumor effect. GVHD was induced in (C.B-20 X B10.D2)F1 [H-2d, H-40b X H-2d,H-40b] recipients after injection of B10.D2-primed C.B-20 donor cells. GVHD was indicated by the histologic appearance of tissue sections from C.B-20----F1 livers, target organs of GVHD, which showed a marked mononuclear cell infiltrate around the portal tracts and central veins. In addition, splenic lymphocytes from these mice had altered CD4/CD8 ratios and were unable to respond to the polyclonal activators Con A and LPS. The mitogen unresponsiveness was at least partially due to the presence of a suppressor cell, because proliferation of normal spleen cells to Con A and LPS was suppressed upon addition of C.B-20----F1 spleen cells. Further immune dysfunction was evident by the inability of T cells from mice with GVHD to generate a CTL response to H-2 alloantigens. Addition of C.B-20----F1 spleen cells to F1 responder cells at the induction of culture did not prevent generation of CTL, indicating that a suppressor cell was not responsible for the lack of CTL activity. In this setting of GVHD, F1 recipients were able to reject BCL1 upon adoptive transfer of C.B-20 anti-BCL1 effector cells. These data indicate that GVHD-induced immune dysfunction does not inhibit the activity of antileukemia T cells.