Trihydrophobin 1 Phosphorylation by c-Src Regulates MAPK/ERK Signaling and Cell Migration

c-Src activates Ras-MAPK/ERK signaling pathway and regulates cell migration, while trihydrophobin 1 (TH1) inhibits MAPK/ERK activation and cell migration through interaction with A-Raf and PAK1 and inhibiting their kinase activities. Here we show that c-Src interacts with TH1 by GST-pull down assay, coimmunoprecipitation and confocal microscopy assay. The interaction leads to phosphorylation of TH1 at Tyr-6 in vivo and in vitro. Phosphorylation of TH1 decreases its association with A-Raf and PAK1. Further study reveals that Tyr-6 phosphorylation of TH1 reduces its inhibition on MAPK/ERK signaling, enhances c-Src mediated cell migration. Moreover, induced tyrosine phosphorylation of TH1 has been found by EGF and estrogen treatments. Taken together, our findings demonstrate a novel mechanism for the comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Serotonin-mediated protein carbonylation in the right heart

Pulmonary hypertension is a devastating disease, which leads to right heart failure. Serotonin (5-HT) plays important roles in the pathogenesis Of Pulmonary hypertension and pulmonary vascular remodeling. The role of 5-HT in right heart failure. however, is unknown. Since oxidative stress may mediate heart failure the, present study examined the effects of 5-HT on protein oxidation in the adult rat right heart ventricle, Treatment of perfused isolated hearts with 5-HT resulted in the promotion of protein carbonylation, specifically in the right ventricle, but not in the left. While no differences between right and left ventricular antioxidant enzymes and 5-HT receptors/transporter were detected, monoamine oxidase A (MAO-A) expression and activity were found to be lower in the right ventricle compared to the left. These results indicate that differences in neither the reactive oxygen species (ROS) scavenging ability, 5-HT membrane signaling capacity, nor MAO-dependent production of hydrogen peroxide are responsible for varied 5-HT-mediated protein carbonylation in right and left ventricles. Rather, lower MAO-A in the right heart might preserve cytosolic 5-HT which triggers other mechanisms for ROS production. Consistently, inhibition of MAO-A resulted in the promotion of protein carbonylation. We propose that low MAO-A, thus reduced degradation of 5-HT, increases the intracellular 5-HT activity in the right ventricle, leading to the promotion of protein carbonylation. (C) 2008 Elsevier Inc. All rights reserved

Free riding, public goods and cognitive dissonance: An experimental approach

Is the motivation to free-ride based mainly on naked cost-benefit considerations? Can psychological theories of cognitive dissonance help deepen our understanding of an individual\u27s free-riding behavior? In this paper, the authors modify a Voluntary Contributions Mechanism experiment in order to see which of 3 theories explaining free-riding behaviour best \u27organizes\u27 the experimental data. Sixty DLSU undergraduate form the subject pool. The researchers\u27 findings strongly suggest that people do not always take account into consideration the costs and benefits of their decisions. In fact, complaint behavior is observed after individuals have established norms or standards in contributions. Also, regression results that exposure to unexpected outcomes motivates an individuals\u27 free riding behavior

Microtubule-associated proteins (MAPs) in microtubule cytoskeletal dynamics and spermatogenesis

The microtubule (MT) cytoskeleton in Sertoli cells, a crucial cellular structure in the seminiferous epithelium of adult mammalian testes that supports spermatogenesis, was studied morphologically decades ago. However, its biology, in particular the involving regulatory biomolecules and the underlying mechanism(s) in modulating MT dynamics, are only beginning to be revealed in recent years. This lack of studies in delineating the biology of MT cytoskeletal dynamics undermines other studies in the field, in particular the plausible therapeutic treatment and management of male infertility and fertility since studies have shown that the MT cytoskeleton is one of the prime targets of toxicants. Interestingly, much of the information regarding the function of actin-, MT- and intermediate filament-based cytoskeletons come from studies using toxicant models including some genetic models. During the past several years, there have been some advances in studying the biology of MT cytoskeleton in the testis, and many of these studies were based on the use of pharmaceutical/toxicant models. In this review, we summarize the results of these findings, illustrating the importance of toxicant/pharmaceutical models in unravelling the biology of MT dynamics, in particular the role of microtubule-associated proteins (MAPs), a family of regulatory proteins that modulate MT dynamics but also actin- and intermediate filamentbased cytoskeletons. We also provide a timely hypothetical model which can serve as a guide to design functional experiments to study how the MT cytoskeleton is regulated during spermatogenesis through the use of toxicants and/or pharmaceutical agents

mTORC1/rpS6 and p-FAK-Y407 signaling regulate spermatogenesis: Insights from studies of the adjudin pharmaceutical/toxicant model

In rodents and humans, the major cellular events at spermatogenesis include self-renewal of spermatogonial stem cells and undifferentiated spermatogonia via mitosis, commitment of spermatogonia to differentiation and transformation to spermatocytes, meiosis, spermiogenesis, and the release of spermatozoa at spermiation. While details of the morphological changes during these cellular events have been delineated, knowledge gap exists between the morphological changes in the seminiferous epithelium and the underlying molecular mechanism(s) that regulate these cellular events. Even though many of the regulatory proteins and biomolecules that modulate spermatogenesis are known based on studies using genetic models, the underlying regulatory mechanism(s), in particular signaling pathways/proteins, remain unexplored since much of the information regarding the signaling regulation is unknown. Studies in the past decade, however, have unequivocally demonstrated that the testis is using several signaling proteins and/or pathways to regulate multiple cellular events to modulate spermatogenesis. These include mTORC1/rpS6/Akt1/2 and p-FAK-Y407. While selective inhibitors and/or agonists and antagonists are available to examine some of these signaling proteins, their use have limitations due to their specificities and also potential systemic cytotoxicity. On the other hand, the use of genetic models has had profound implications for our understanding of the molecular regulation of spermatogenesis, and these knockout (null) models have also revealed the factors that are critical for spermatogenesis. Nonetheless, additional studies using in vitro and in vivo models are necessary to unravel the signaling pathways involved in regulating seminiferous epithelial cycle. Emerging data from studies, such as the use of the adjudin pharmaceutical/toxicant model, have illustrated that this non-hormonal male contraceptive drug is utilizing specific signaling pathways/proteins to induce specific defects in spermatogenesis, yielding mechanistic insights on the regulation of spermatogenesis. We sought to review these recent data in this article, highlighting an interesting approach that can be considered for future studies

Microtubule cytoskeleton and spermatogenesis—Lesson from studies of toxicant models

Studies have shown that mammalian testes, in particular the Sertoli cells, are highly susceptible to exposure of environmental toxicants, such as cadmium, PFOS (perfluorooctanesulfonate), phthalates, 2,5-hexanedione and bisphenol A. However, important studies conducted by reproductive toxicologists and/or biologists in the past have been treated as toxicology reports. Yet many of these studies provided important mechanistic insights on the toxicant-induced testis injury and reproductive dysfunction, relevant to the biology of the testis and spermatogenesis. Furthermore, recent studies have shown that findings obtained from toxicant models are exceedingly helpful tools to unravel the biology of testis function in particular spermatogenesis, including specific cellular events associated with spermatid transport to support spermiogenesis and spermiation. In this review, we critically evaluate some recent data, focusing primarily on the molecular structure and role of microtubules in cellular function, illustrating the importance of toxicant models to unravel the biology of microtubule cytoskeleton in supporting spermatogenesis, well beyond toxicity studies. These findings have opened up some potential areas of research which should be carefully addressed in the years to come

The non-hormonal male contraceptive adjudin exerts its effects via MAPs and signaling proteins mTORC1/rpS6 and FAK-Y407

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a nonhormonal male contraceptive, since it effectively induces reversible male infertility without perturbing the serum concentrations of follicle stimulating hormone (FSH), testosterone, and inhibin B based on studies in rats and rabbits. Adjudin was shown to exert its effects preferentially by perturbing the testis-specific actin-rich adherens junction (AJ) at the Sertoli–spermatid interface known as apical ectoplasmic specialization (apical ES), thereby effectively inducing spermatid exfoliation. Adjudin did not perturb germ cell development nor germ cell function. Also, it had no effects on Sertoli cell–cell AJ called basal ectoplasmic specialization (basal ES), which, together with tight junction constitute the blood-testis barrier (BTB), unless an acute dose of adjudin was used. Adjudin also did not perturb the population of spermatogonial stem cells nor Sertoli cells in the testis. However, the downstream signaling protein(s) utilized by adjudin to induce transient male infertility remains unexplored. Herein, using adult rats treated with adjudin and monitored changes in the phenotypes across the seminiferous epithelium between 6 and 96 h in parallel with the steady-state protein levels of an array of signaling and cytoskeletal regulatory proteins, recently shown to be involved in apical ES, basal ES and BTB function. It was shown that adjudin exerts its contraceptive effects through changes in microtubule associated proteins (MAPs) and signaling proteins mTORC1/rpS6 and p-FAK-Y407. These findings are important to not only study adjudin-mediated male infertility but also the biology of spermatogenesis