Dissection of the Ovulatory Process Using ex vivo Approaches.

Ovulation is a unique physiological phenomenon that is essential for sexual reproduction. It refers to the entire process of ovarian follicle responses to hormonal stimulation resulting in the release of mature fertilization-competent oocytes from the follicles and ovaries. Remarkably, ovulation in different species can be reproduced out-of-body with high fidelity. Moreover, most of the molecular mechanisms and signaling pathways engaged in this process have been delineated using in vitro ovulation models. Here, we provide an overview of the major molecular and cytological events of ovulation observed in frogs, primarily in the African clawed frog Xenopus laevis, using mainly ex vivo approaches, with the focus on meiotic oocyte maturation and follicle rupture. For the purpose of comparison and generalization, we also refer extensively to ovulation in other biological species, most notoriously, in mammals.

Localization-specific distributions of protein pI in human proteome are governed by local pH and membrane charge.

BACKGROUND: Whole-proteome distributions of protein isoelectric point (pI) values in different organisms are bi- or trimodal with some variations. It was suggested that the observed multimodality of the proteome-wide pI distributions is associated with subcellular localization-specific differences in the local pI distributions. However, the factors responsible for variation of the intracellular localization-specific pI profiles have not been investigated in detail. RESULTS: In this work, we explored proteome-wide pI distributions of 32,138 human proteins predicted to reside in 10 subcellular compartments, as well as the pI distributions of experimentally observed lysosomal and Golgi proteins. The distributions were found to differ significantly, although all of them adhered to the major recurrent bimodal pattern. Grossly, acid-biased and alkaline-biased patterns with various minor statistical features were observed at different subcellular locations. Bioinformatics analysis revealed the existence of strong statistically significant correlations between protein pI and subcellular localization. Most markedly, protein pI was found to correlate positively with nuclear and mitochondrial locations and negatively with cytoskeletal, cytoplasmic, lysosomal and peroxisomal environment. Further analysis demonstrated that subcellular compartment-specific pI distributions are greatly influenced by local pH and organelle membrane charge. Multiple nonlinear regression analysis identified a polynomial function of the two variables that best fitted the mean pI values of the localization-specific pI distributions. A high coefficient of determination calculated for this regression (R2 = 0.98) suggests that local pH and organelle membrane charge are the major factors responsible for variation of the intracellular localization-specific pI profiles. CONCLUSIONS: Our study demonstrates that strong correlations exist between protein pI and subcellular localization. The specific pI distributions at different subcellular locations are defined by local environment. Predominantly, it is the local pH and membrane charge that shape the organelle-specific protein pI patterns. These findings expand our understanding of spatial organization of the human proteome.

Autophosphorylation of MAP kinase disables the MAPK pathway in apoptotic Xenopus eggs.

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of various cellular processes, including cell survival and apoptosis. Here, we report that Xenopus p42 MAPK becomes phosphorylated in apoptotic eggs, however this modification does not activate the enzyme. Using phosphorylation residue-specific antibodies, we demonstrate that this modification occurs on the Tyr residue in the MAPK activation segment, pinpointing the autophosphorylation mechanism. Notably, MAPK phosphorylation in apoptotic Xenopus eggs coincides with prominent intracellular acidification accompanying apoptosis in these cells. Furthermore, autophosphorylation of recombinant Xenopus MAPK is stimulated and phosphorylation of a protein substrate is inhibited under low pH conditions. Thus, acidic intracellular conditions inactivate MAPK and effectively disable the MAPK-mediated survival pathway in the apoptotic eggs. Given that cell acidification is a rather common feature of apoptosis, we hypothesize that stimulation of MAPK autophosphorylation and shutdown of the MAPK pathway may represent universal traits of apoptotic cell death.

Postovulatory cell death: why eggs die via apoptosis in biological species with external fertilization.

Spawned unfertilized eggs have been found to die by apoptosis in several species with external fertilization. However, there is no necessity for the externally laid eggs to degrade via this process, as apoptosis evolved as a mechanism to reduce the damaging effects of individual cell death on the whole organism. The recent observation of egg degradation in the genital tracts of some oviparous species provides a clue as to the physiological relevance of egg apoptosis in these animals. We hypothesize that egg apoptosis accompanies ovulation in species with external fertilization as a normal process to eliminate mature eggs retained in the genital tract after ovulation. Furthermore, apoptosis universally develops in ovulated eggs after spontaneous activation in the absence of fertilization. This paper provides an overview of egg apoptosis in several oviparous biological species, including frog, fish, sea urchin, and starfish.

Proteome Characteristics of Non-Alcoholic Steatohepatitis Liver Tissue and Associated Hepatocellular Carcinomas.

To uncover mechanisms of nonalcoholic steatohepatitis (NASH) associated hepatocarcinogenesis, we compared the proteomes of human NASH-associated liver biopsies, resected hepatocellular carcinomas (HCCs) and HCCs of HCV⁺ patients with normal liver tissue of patients with gastrointestinal tumor metastasis, in formalin-fixed paraffin-embedded samples obtained after surgery in our hospital during the period from 2006 to 2011. In addition, proteome analysis of liver tumors in male STAM NASH-model mice was performed. Similar changes in the proteome spectrum such as overexpression of enzymes involved in lipid, cholesterol and bile acid biosynthesis and examples associated with suppression of fatty acid oxidation and catabolism, alcohol metabolism, mitochondrial function as well as low expression levels of cytokeratins 8 and 18 were observed in both human NASH biopsies and NASH HCCs, but not HCV⁺ HCCs. Alterations in downstream protein expression pointed to significant activation of transforming growth factor ß, SMAD family member 3, ß-catenin, Nrf2, SREBP-LXRα and nuclear receptor-interacting protein 1 (NRIP1), and inhibition of PPARs and p53 in human NASH biopsies and/or HCCs, suggesting their involvement in accumulation of lipids, development of fibrosis, oxidative stress, cell proliferation and suppression of apoptosis in NASH hepatocarcinogenesis. In STAM mice, PPARs inhibition was not obvious, while expression of cytokeratins 8 and 18 was elevated, indicative of essential differences between human and mouse NASH pathogenesis.

Calcium signaling and meiotic exit at fertilization in Xenopus egg.

Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation.

Effect of weak static magnetic fields on the development of cultured skeletal muscle cells.

We studied the effect produced on the development and functional activity of skeletal muscle cells from newborn Wistar rats in primary culture by weak static magnetic fields (WSMF; 60-400 µT) with a high capacity of penetrating the biological media. To reduce the impact of external magnetic fields, cells were cultured at 37 °C in a multilayered shielding chamber with the attenuation coefficient equal to 160. WSMF inside the chamber was created by a circular permanent magnet. We found that the application of WSMF with the magnetic field strength only a few times that of the geomagnetic field can accelerate the development of skeletal muscle cells, resulting in the formation of multinuclear hypertrophied myotubes. WSMF was shown to induce 1.5- to 3.5-fold rise in the concentration of intracellular calcium [Ca(2+)]i due to the release of Ca(2+) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyR), which increases in the maturation of myotubes. We also found that fully differentiated myotubes at late stages of development were less sensitive to WSMF, manifesting a gradual decrease in the frequency of contractions. However, myotubes at the stage when electromechanical coupling was forming dramatically reduced the frequency of contractions during the first minutes of their exposure to WSMF.

Activation of tubulin assembly into microtubules upon a series of repeated femtosecond laser impulses.

Tubulin, a globular protein, mostly distributed in nature in the dimeric alpha, beta form, can polymerize in vivo and in vitro into microtubules-longitudinal dynamic assemblies, involved in numerous cellular functions, including cell division and signaling. Tubulin polymerization starts upon binding Mg(2+) with the tubulin guanosine triphosphate (GTP) site. In the current study we show that a series of repeated femtosecond laser impulses activate the same site without adding Mg(2+). GTP site activation (without GTP no polymerization occurs) produces hydrated electrons (they are detected by the UV spectra), which are trapped in the shell of biological water, surrounding the tubulin. These electrons generate an additional, nonlinear by nature, polarization effect, responsible for the second harmonic generation at lambda=365 nm (the first harmonic is centered at lambda=730 nm) and manyfold increase in strength of the initial electric field. The results are supported by model calculations, based on the assumption of positive (negative) feedback, appearing on interaction of charge transfer exciton dipoles with the applied electromagnetic field.