Interaction of 4-ethylphenol, pH, sucrose and ethanol on the growth and fermentation capacity of the industrial strain of Saccharomyces cerevisiae PE-2.

Volatile phenols such as 4-ethylphenol are produced from hydroxycinnamic acids by Dekkera bruxellensis, an important yeast contaminating alcoholic fermentations. 4-ethylphenol results from the decarboxylation and reduction of p-coumaric acid, a compound found in sugarcane musts. In wine, volatile phenols are responsible by sensorial alterations whereas in the context of bioethanol fermentation, little is known about their effects on the main yeast, Saccharomyces cerevisiae. Here we evaluated the interaction of 4-ethylphenol and pH, sucrose and ethanol on the growth and fermentation capacity of the industrial strain of S. cerevisiae PE-2. A central compound rotational design was utilized to evaluate the effect of 4-ethylphenol, pH, ethanol and sucrose concentration on the yeast maximum specific growth rate (µmax) in microplate experiments in YPS medium (Yeast extract-Peptone-Sucrose), at 30 °C. Following, single-cycle fermentations in YPS medium, pH 4.5, 17% sucrose, at 30 °C, with 4-ethylphenol in concentrations of 10 and 20 mg L-1 being added at the start or after 4 h of fermentation, were carried out. 4-ethylphenol affected µmax of S. cerevisiae in situations that resemble the conditions of industrial bioethanol production, especially the low pH of the fermentation medium and the high ethanol concentration because of the anaerobic sucrose uptake. The addition of 4-ethylphenol on fermentation resulted in significant effect on the cell yeast concentration, pH and alcohol production, with significant decrease from 86% to the range of 65-74% in the fermentative efficiency. The industrial yeast S. cerevisiae PE-2 growth and fermentative capacity were affected by the presence of 4-ethylphenol, a metabolite produced by D. bruxellensis, which may contribute to explain the impact of this yeast on bioethanol industrial production.

Production, structural characterization, and antiproliferative activity of exopolysaccharide produced by Scleroderma areolatum Ehrenb with different carbon source.

The effects of different three carbon sources, that is, glucose, fructose, and sucrose, on production, molecular properties and antiproliferative activity of exopolysaccharide (EPS), were evaluated in the submerged culture of Scleroderma areolatum Ehrenb. Among carbon sources examined, the addition of sucrose maximizes the mycelia production, while fructose could maximize the EPS yield. Although the predominant carbohydrate compositions identified were gluconic acid and mannose, the monosaccharide composition of EPSs was also different significantly. FT-IR spectral analysis revealed there was no significant difference among the prominent characteristic groups in three EPSs. The molecular weight of EPSs was also affected by carbon source, being generally lower compared with that with glucose. However, all EPSs molecule existed as nearly globular shape form in aqueous solution. The variation of carbon sources also affected antiproliferative activity examined in vitro using cell proliferation assay. Fructose was optimal carbon source giving higher antiproliferative activity probably due to the relatively high contents of xylose in the EPS with low molecular weight.

Different resistance patterns of reference and field strains of Brucella abortus.

The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 µg/mL and 80 µg/mL), thionin (2.5 µg/mL and 10 µg/mL), rifampicin (200 µg/mL) and safranin O (200 µg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO 2 . Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 µg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 µg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 ( B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75-0.80 (10 µg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.

Different resistance patterns of reference and field strains of Brucella abortus

The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 μg/mL and 80 μg/mL), thionin (2.5 μg/mL and 10 μg/mL), rifampicin (200 μg/mL) and safranin O (200 μg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO2. Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 μg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 μg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 (B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75–0.80 (10 μg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.

Different resistance patterns of reference and field strains of Brucella abortus

The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 μg/mL and 80 μg/mL), thionin (2.5 μg/mL and 10 μg/mL), rifampicin (200 μg/mL) and safranin O (200 μg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO2. Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 μg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 μg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 (B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75–0.80 (10 μg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.(AU)

Effects of progesterone on neurite growth inhibitors in the hippocampus following global cerebral ischemia.

In this study, the effects of progesterone (P4) on the immunoreactivity to the neurite growth inhibitor Nogo-A, its receptor (Ng-R), and its effector Rho-A in the rat hippocampus, in association with parameters of spatial learning and memory following global cerebral ischemia, were assessed. Adult male rats were subjected to global cerebral ischemia (15 min), and treated with P4 or its vehicle at 15 min, 2, 6, 24, 48 and 72 h of reperfusion. Immunoreactivity to Nogo-A, Ng-R, and Rho-A was evaluated at 24 h, 72 h or 7 d, or at 14 d of reperfusion after rats were tested in the Morris Water Maze (MWM). Global cerebral ischemia induced an increase in Nogo-A, Ng-R, and Rho-A immunoreactivities in the cell bodies of CA1 pyramidal neurons at 24h after global cerebral ischemia, peaking at 72 h, and persisting 14 d later. In addition, at 72 h, a strong immunoreactivity was observed in the hippocampal layers where dendritic arborizations of CA1 pyramidal neurons are located. Treatment with P4 reduced Nogo-A, Ng-R, and Rho-A immunoreactivities in CA1, particularly at 72 h of reperfusion. These effects of P4 were consistent with the parameters of a more efficient spatial learning and memory in the MWM, as compared to vehicle-treated rats. Overall results suggest the reduction of neurite growth inhibitory molecules Nogo-A, Ng-R, and Rho-A, as a part of the restorative effects of progesterone possibly allowing the plastic phenomena to occur, able to support the functional preservation of the hippocampus following global cerebral ischemia.

Halotolerance, ligninase production and herbicide degradation ability of basidiomycetes strains

Fungi have been recently recognized as organisms able to grow in presence of high salt concentration with halophilic and halotolerance properties and their ligninolytic enzyme complex have an unspecific action enabling their use to degradation of a number of xenobiotic compounds. In this work, both the effect of salt and polyols on growth of the basidiomycetes strains, on their ability to produce ligninolytic enzyme and diuron degradation were evaluated. Results showed that the presence of NaCl in the culture medium affected fungal specimens in different ways. Seven out of ten tested strains had growth inhibited by salt while Dacryopinax elegans SXS323, Polyporus sp MCA128 and Datronia stereoides MCA167 fungi exhibited higher biomass production in medium containing 0.5 and 0.6 mol.L-1 of NaCl, suggesting to be halotolerant. Polyols such as glycerol and mannitol added into the culture media improved the biomass and ligninases production by D. elegans but the fungus did not reveal consumption of these polyols from media. This fungus degraded diuron in medium control, in presence of NaCl as well as polyols, produced MnP, LiP and laccase.

Halotolerance, ligninase production and herbicide degradation ability of basidiomycetes strains.

Fungi have been recently recognized as organisms able to grow in presence of high salt concentration with halophilic and halotolerance properties and their ligninolytic enzyme complex have an unspecific action enabling their use to degradation of a number of xenobiotic compounds. In this work, both the effect of salt and polyols on growth of the basidiomycetes strains, on their ability to produce ligninolytic enzyme and diuron degradation were evaluated. Results showed that the presence of NaCl in the culture medium affected fungal specimens in different ways. Seven out of ten tested strains had growth inhibited by salt while Dacryopinax elegans SXS323, Polyporus sp MCA128 and Datronia stereoides MCA167 fungi exhibited higher biomass production in medium containing 0.5 and 0.6 mol.L(-1) of NaCl, suggesting to be halotolerant. Polyols such as glycerol and mannitol added into the culture media improved the biomass and ligninases production by D. elegans but the fungus did not reveal consumption of these polyols from media. This fungus degraded diuron in medium control, in presence of NaCl as well as polyols, produced MnP, LiP and laccase.

Interferon-alpha conditioned sensitivity to an anti-epidermal growth factor receptor monoclonal antibody in a human lung cancer cell line with intermediate expression of the receptor.

Targeted therapy to the epidermal growth factor receptor (EGFR) seems to be related to its expression level on tumor cells. Interferon-alpha (IFN-alpha) induces growth inhibition but also may up-regulate the EGFR expression in some cancer cell lines. We aimed to determine whether the IFN-alpha combined with an EGFR-specific monoclonal antibody (nimotuzumab) may affect the growth of human tumor epithelial cell lines with different EGFR expression levels. H125, a lung adenosquamous carcinoma, and A431, a vulvar epidermoid carcinoma, cell lines express intermediate and high levels of EGFR, respectively, whereas MDA MB231, a breast adenocarcinoma cell line expresses undetectable levels of EGFR measured by flow cytometry/FACS. We found that IFN-alpha alone inhibited in a dose-dependent fashion the growth of all cell lines, but only up-regulated the EGFR expression in the lung carcinoma-derived cell line. Noteworthy, the combined treatment did not modify the complement-mediated cytotoxicity of the antibody although the antiproliferative activity of nimotuzumab in H125 cells in vitro increased when an IFN-alpha-conditioning treatment was used. In conclusion, this study may provide insights about the rational use of EGFR inhibitors into the immunopharmacological management of targeted therapies including the IFN-alpha for lung cancer.

Upregulation of EphA3 receptor after spinal cord injury.

Spinal cord injury (SCI) releases a cascade of events that leads to the onset of an inhibitory milieu for axonal regeneration. Some of these changes result from the presence of repulsive factors that may restrict axonal outgrowth after trauma. The Eph receptor tyrosine kinase (RTK) family has emerged as a key repellent cue known to be involved in neurite outgrowth, synapse formation, and axonal pathfinding during development. Given the nonpermissive environment for axonal regeneration after SCI, we questioned whether re-expression of one of these molecules occurs during regenerative failure. We examined the expression profile of EphA3 at the mRNA and protein levels after SCI, using the NYU contusion model. There is a differential distribution of this molecule in the adult spinal cord and EphA3 showed an increase in expression after several injury models like optic nerve and brain injury. Standardized semi-quantitative RT-PCR analysis demonstrated a time-dependent change in EphA3 mRNA levels, without alterations in beta-actin levels. The basal level of EphA3 mRNA in the adult spinal cord is low and its expression was induced 2 days after trauma (the earliest time point analyzed) and this upregulation persisted for 28 days post-injury (the latest time point examined). These results were corroborated at the protein level by immunohistochemical analysis and the cell phenotype identified by double labeling studies. In control animals, EphA3 immunoreactivity was observed in motor neurons of the ventral horn but not in lesioned animals. In addition, GFAP-positive cells were visualized in the ventral region of injured white matter. These results suggest that upregulation of EphA3 in reactive astrocytes may contribute to the repulsive environment for neurite outgrowth and may be involved in the pathophysiology generated after SCI.

Role of TLR in B cell development: signaling through TLR4 promotes B cell maturation and is inhibited by TLR2.

The role of TLR4 in mature B cell activation is well characterized. However, little is known about TLR4 role in B cell development. Here, we analyzed the effects of TLR4 and TLR2 agonists on B cell development using an in vitro model of B cell maturation. Highly purified B220(+)IgM(-) B cell precursors from normal C57BL/6 mouse were cultured for 72 h, and B cell maturation in the presence of the TLR agonists was evaluated by expression of IgM, IgD, CD23, and AA4. The addition of LPS or lipid A resulted in a marked increase in the percentage of CD23(+) B cells, while Pam3Cys had no effect alone, but inhibited the increase of CD23(+) B cell population induced by lipid A or LPS. The TLR4-induced expression of CD23 is not accompanied by full activation of the lymphocyte, as suggested by the absence of activation Ag CD69. Experiments with TLR2-knockout mice confirmed that the inhibitory effects of Pam3Cys depend on the expression of TLR2. We studied the effects of TLR-agonists on early steps of B cell differentiation by analyzing IL-7 responsiveness and phenotype of early B cell precursors: we found that both lipid A and Pam3Cys impaired IL-7-dependent proliferation; however, while lipid A up-regulates B220 surface marker, consistent with a more mature phenotype of the IgM(-) precursors, Pam3Cys keeps the precursors on a more immature stage. Taken together, our results suggest that TLR4 signaling favors B lymphocyte maturation, whereas TLR2 arrests/retards that process, ascribing new roles for TLRs in B cell physiology.

Growth inhibition of toxigenic fungi by a proteinaceous compound from Streptomyces sp. C/33-6.

A Streptomyces sp. strain named C/33-6, previously isolated from soil, presented a strong and specific antagonistic effect against toxigenic fungi. This action was attributed to a proteinaceous compound (molecular weight estimated to be 14 kDa) present in the supernatant of the culture of strain C/33-6, which was sensitive to proteinases (elastase, pronase E, proteinase K) and prolongated heat treatment (100 degrees C, for 20 min). This compound showed non-chitinolytic fungicidal activity.

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution.

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.

Assessment of seminiferous tubule function (anti-müllerian hormone).

Anti-müllerian hormone (AMH) is specifically produced by Sertoli cells in the male. The testes express a high level of AMH from early fetal life, driven by the transcription factors SOX9, SF1, WT1 and GATA4, until puberty, when AMH is downregulated by testosterone and meiosis. When androgen negative effect is absent, follicle-stimulating hormone increases the secretion of AMH. Serum AMH determination is useful in the evaluation of children with non-palpable gonads, with or without ambiguous genitalia. It signals the existence of functional testicular tissue and allows a distinction to be made between gonadal dysgenesis and dissociated tubular-interstitial dysfunction. Serum AMH is a useful marker in the follow-up of male patients with precocious puberty or hypogonadotrophic hypogonadism, as well as of patients with sex cord stromal tumours of the gonads. Finally, AMH determination on the seminal plasma of men with non-obstructive azoospermia may be used as a marker of the existence of testicular spermatozoa when intracytoplasmic sperm injection is considered.

Endometrial dating and determination of the window of implantation in healthy fertile women.

OBJECTIVE: To reassess endometrial morphological criteria of normality identifying the best morphological and molecular "implantation window" indicators in normal women. DESIGN: Prospective clinical study. SETTING: Assisted reproductive unit. PATIENT(S): Fourteen healthy volunteers. INTERVENTION(S): Blood sampling for LH, E(2), and progesterone (P4) determinations. Daily vaginal ultrasounds. Two endometrial biopsies per volunteer, 7 days apart, during luteal phase. MAIN OUTCOME MEASURE(S): Endometrial dating, pinopodes formation, immunohistochemical determination of integrins (alphavbeta3, alpha4beta1), leukemia inhibitory factor (LIF), interleukin-1 receptor type I (IL-1R tI), mouse ascites Golgi (MAG), the transmembrane mucin (MUC-1), and P4 receptor expression. RESULT(S): In 26 of 28 biopsies observers agreed; in two biopsies there was a discrepancy (difference of 72 hours). With use of LH peak, 24 of 26 samples were in phase, and 2 were 3 days behind. Pinopodes appeared on days 20-21 and persisted through day 28 in small groups or larger areas. beta3 Integrin was highly expressed in luminal and glandular epithelium from day 22 through 28; 48 hours thereafter pinopodes appeared. alpha4 Subunit exhibited luminal epithelium reaction positivity on days 22-23 and glands on days 18-23. LIF and IL-1R tI showed weak, erratic expression. MAG antibodies showed luminal epithelium expression up to day 22 and glands up to day 25. MUC-1 showed positivity during the whole luteal phase. P4 receptors were positive through day 20 and at the end of the luteal phase. CONCLUSION(S): The three most cited markers that frame the window of implantation do not correlate in our material. Pinopodes are present from day 20 on; beta3 and alpha4 integrin subunits indicate a window opening on days 22-23.

Embryology and endocrinology of genital development.

In the human male fetus, testes develop by the 7th week and begin to secrete two hormones: anti-müllerian hormone (AMH) induces the regression of müllerian ducts, the anlagen of the uterus, fallopian tubes and upper vagina, upon binding to a specific membrane receptor, whereas testosterone induces the differentiation of the wolffian ducts into the epididymes, vasa deferentia and seminal vesicles. In some target tissues, testosterone is converted to dihydrotestosterone, which is responsible for masculinization of the urogenital sinus and external genitalia. Both androgens act upon binding to the same nuclear receptor. In the absence of AMH and androgen action, or example in the female or in abnormal male differentiation, the internal and external genital primordia differentiate following the female pathway, even in the absence of ovaries. In males, an impaired function of the AMH-dependent pathway results in the persistent müllerian duct syndrome, a disorder characterized by the presence of uterus and fallopian tubes in otherwise normally virilized boys. Several mutations found in the AMH and AMH-receptor genes explain the pathophysiology of this syndrome.

Changes induced by non-enzymatic glycosylation of IGF-binding protein-3: effects on its binding properties and on its modulatory effect on IGF-I mitogenic action.

The aim of this study was to demonstrate the feasibility of in vitro non-enzymatic glycosylation of IGF-binding protein-3 (IGFBP-3) and whether this process affects its binding properties and its modulatory effect on IGF-I mitogenic activity. Swiss 3T3 fibroblasts were cultured and the IGFBP-3 released into the medium (CM) glycated with either labelled or unlabelled glucose. Parallel glycation studies were performed using standard human IGFBP-3. Both species of IGFBP-3 became effectively glycated in a dose-dependent manner. Glycated IGFBP-3 bound larger amounts of 125I-labelled IGF-I than its non-glycated form. According to Scatchard analysis this effect might be due to an increase in the number of binding sites of the IGFBP-3 molecule rather than to changes in its affinity constants, which remain unchanged. Preincubation of fibroblasts with CM containing IGFBP-3 for 16 h before the addition of IGF-I enhanced the stimulatory effect of the hormone on thymidine incorporation into cell DNA. This potentiation was blunted when in vitro glycated instead of non-glycated IGFBP-3 was employed. These results provide further evidence of the in vitro glycation of IGFBP-3 and demonstrate that this process affects both its binding properties and its enhancing effect on IGF-I mitogenic activity. These changes may explain, at least partially, the development of many alterations observed in poorly controlled diabetic patients.

[G1 spermatogonial chalone]. / Chalona espermatogonial G1.

Chalones are physiological inhibitors of cell proliferation that act either at the G1 or G2 phase of the cell cycle. They have been described for a variety of tissues, including the seminiferous epithelium. In vivo and in vitro characterization of rat G1 spermatogonial chalone demonstrate that it is a glycoprotein, heat-labile, molecular weight under 5,000 D, tissue specific but not species-specific, active at physiological pH, with a mechanism of cell action mediated by cyclic AMP. The origin of the substance are the differentiated cells of the spermatogenesis from primary spermatocytes up to round spermatids. The target cells are the type A (and perhaps only the A0) spermatogonia. The inhibitory effect, measured as a decrease in the uptake of H3-thymidine into testicular DNA, is not dependent on testicular steroids, Sertoli cell products (inhibin or the like) nor on the hypothalamic-hypophyseal-gonadal axis, since it occurs in vitro. In the mouse, the biological half-life (in vivo) of the G1 spermatogonial chalone is around 14 hs. Chronic administration for the entire length of mouse spermatogenesis does not alter spermatogenic kinetics nor does it result in azoospermia. The biological effect of the G1 spermatogonial chalone can be counteracted in vitro by means of an immune rabbit serum raised against a partially purified rat testicular extract (source of the chalone).

Inhibition of growth of Leishmania mexicana mexicana by Leishmania mexicana amazonensis during "in vitro" co-cultivation.

Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell cloning experiments with Leishmania mexicana mexicana and Leishmania mexicana amazonensis. Clones were identified using the technique of schizodeme analysis. The phenomenon observed is clearly relevant to studies of parasite isolation, leishmanial metabolism, cross-immunity and chemotherapy.