The influence of recombinant feline oviductin on different aspects of domestic cat (Felis catus) IVF and embryo quality

Oviductin is known to be a key player providing a convenient environment for the process of fertilization affecting this by direct interaction with oocytes and sperm. As in vitro embryo production in the context of assisted reproduction for endangered felids is still in the process of optimization, oviductin might be used to improve IVF results. Recombinant His-tagged feline oviductin was expressed by transformed Escherichia coli BL21DE3 cells. The protein was purified by immobilized metal ion affinity chromatography. The effect of the recombinant protein was characterized in three experiments: a hemizona assay for sperm binding analysis, the IVF outcome, and the relative mRNA expression levels in blastocysts after IVF. A significant higher number of bound sperm cells were found after incubation in oviductin. No significant effect on cleavage, morula, and blastocyst rates with or without oviductin incubation during IVF could be observed. However, the relative mRNA abundance of GJA1, a gene, whose expression level is known to be a marker of embryo quality, was significantly increased (P value less than 0.05) in blastocysts after oviductin treatment. In contrast to this, expression of OCT4, HSP70, DNMT1, DNMT3A, BAX, IGF1R, and GAPDH was not significantly affected. We assume that our recombinant oviductin in its current nonglycosylated form is able to enhance sperm binding. Despite of a missing significant effect on IVF outcome, embryo quality in terms of relative GJA1 expression is influenced positively. These promising results demonstrate the value of recombinant oviductin for the IVF in cats.     

Small molecule antagonists of CCR8 inhibit eosinophil and T cell migration

In this study, we demonstrate that in addition to T lymphocytes, human naïve eosinophils and the differentiated eosinophil-like cell line, AML14.3D10 express CCR8 and respond to CCL1 through CCR8 engagement. The responsiveness of cells was dependent on maturation stage, since CCL1 induced pronounced chemotaxis only in differentiated CCR8 positive AML14.3D10 cells. Despite the low CCR8 surface expression, human naïve eosinophils respond with a chemotaxis to high concentration CCL1. We further describe that Th2 clones in a maturation dependent fashion produce autocrine CCL1, which renders them unresponsive to further stimulation. An innovative method to enrich primary CCR8 reactive T cells was developed which demonstrates that primary peripheral CCR8 expressing T cells respond significantly to CCL1.We have developed novel small molecule CCR8 antagonists that are effective in inhibiting calcium mobilization and chemotaxis in differentiated AML cells as well as in human primary CCR8 positive T cells. Importantly, we demonstrate that the compounds can be divided into two subgroups: (i) compounds that are functional agonists for calcium mobilization and chemotaxis (ii) compounds that are pure antagonists. We demonstrate that agonism of these compounds does not correlate with their antagonistic potency. Taken together, we have identified a novel set of CCR8 compounds with antagonistic properties that inhibit CCL1 driven chemotaxis in both CCR8 expressing eosinophils as well as primary human T cells.     

Duration and variability of speech segments in fluent speech of children with and without stuttering

The purpose of this study was to compare the duration and variability of speech segments of children who stutter with those of children who do not stutter and to identify changes in duration and variability of speech segments due to the effect of utterance length. Eighteen children participated (ranging from 6.3 to 7.9 years of age). The experimental task required the children to repeat a single word in isolation and the same word embedded in a sentence. Durations of speech segments and Coefficients of variation (Cv) were defined to assess temporal parameters of speech. Significant differences were found in the variability of speech segments on the sentence level, but not in duration. The findings supported the assumption that linguistic factors pose direct demands on the speech motor system and that the extra duration of speech segments observed in the speech of stuttering adults may be a kind of compensation strategy.     

Distribution of major brain gangliosides in olfactory tract of frogs

Gangliosides are major cell-surface determinants in the central nervous system (CNS) of vertebrates, found both in neuronal and glial cell membranes. Together with cholesterol and glycosylphosphatidylinositol (GPI) - anchored proteins, gangliosides are involved in organization of plasma membrane microdomains. Based on biochemical studies, frog brain was previously described as having low quantities of gangliosides and their distribution pattern in specific brain regions was unknown. Using highly specific monoclonal antibodies generated against four major brain gangliosides (GM1, GD1a, GD1b and GT1b), we examined the distribution of these molecules in CNS of four different species of frogs (Rana esculenta, Rana temporaria, Bufo bufo and Bufo viridis). We also studied the distribution of myelin- associated glycoprotein (MAG), an inhibitor of axonal regeneration, which is a ligand for gangliosides GD1a and GT1b. Our results show that ganglioside GDla is expressed in neurons of olfactory bulb in all studied animals. In the brain of Rana sp., GD1a is expressed in the entire olfactory pathway, from olfactory bulbs to amygdala, while in Bufo sp. GD1a is restricted to the main olfactory bulb. Furthermore, we found that most of myelinated pathways in frogs express MAG, but do not express GD1a, which could be one of the reasons for better axon regeneration of neural pathways after CNS injury in amphibians in comparison to mammals.     

Photosynthesis is improved by exogenous calcium in heat-stressed tobacco plants

Effects of exogenous calcium chloride (CaCl₂) (20 mM) on photosynthetic gas exchange, photosystem II photochemistry, and the activities of antioxidant enzymes in tobacco plants under high temperature stress (43 °C for 2 h) were investigated. Heat stress resulted in a decrease in net photosynthetic rate (P_n), stomatal conductance as well as the apparent quantum yield (AQY) and carboxylation efficiency (CE) of photosynthesis. Heat stress also caused a decrease of the maximal photochemical efficiency of primary photochemistry (F_v/F_m). On the other hand, CaCl₂ application improved P_n, AQY, and CE as well as F_v/F_m under high temperature stress. Heat stress reduced the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), whereas the activities of these enzymes either decreased less or increased in plants pretreated with CaCl₂; glutathione reductase (GR) activity increased under high temperature, and it increased more in plants pretreated with CaCl₂. There was an obvious accumulation of H₂O₂ and O₂⁻ under high temperature, but CaCl₂ application decreased the contents of H₂O₂ and O₂⁻ under heat stress conditions. Heat stress induced the level of heat shock protein 70 (HSP70), while CaCl₂ pretreatment enhanced it. These results suggested that photosynthesis was improved by CaCl₂ application in heat-stressed plants and such an improvement was associated with an improvement in stomatal conductance and the thermostability of oxygen-evolving complex (OEC), which might be due to less accumulation of reactive oxygen species.     

Neuropeptide Y modulates functions of inflammatory cells in the rat: distinct role for Y1, Y2 and Y5 receptors

Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay.     

Visualization in simulation tools: requirements and a tool specification to support the teaching of dynamic biological processes

Simulation tools are playing an increasingly important role behind advances in the field of systems biology. However, the current generation of biological science students has either little or no experience with such tools. As such, this educational glitch is limiting both the potential use of such tools as well as the potential for tighter cooperation between the designers and users. Although some simulation tool producers encourage their use in teaching, little attempt has hitherto been made to analyze and discuss their suitability as an educational tool for noncomputing science students. In general, today's simulation tools assume that the user has a stronger mathematical and computing background than that which is found in most biological science curricula, thus making the introduction of such tools a considerable pedagogical challenge. This paper provides an evaluation of the pedagogical attributes of existing simulation tools for cell signal transduction based on Cognitive Load theory. Further, design recommendations for an improved educational simulation tool are provided. The study is based on simulation tools for cell signal transduction. However, the discussions are relevant to a broader biological simulation tool set.     

Identification of an interaction between the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor with protein kinase C-related kinase (PRK) 1: implications for prostate cancer

In humans, thromboxane (TX) A(2) signals through the TPα and TPβ isoforms of the TXA(2) receptor or TP. Here, the RhoA effector protein kinase C-related kinase (PRK) 1 was identified as an interactant of both TPα and ΤPβ involving common and unique sequences within their respective C-terminal (C)-tail domains and the kinase domain of PRK1 (PRK1(640-942)). Although the interaction with PRK1 is constitutive, agonist activation of TPα/TPβ did not regulate the complex per se but enhanced PRK1 activation leading to phosphorylation of its general substrate histone H1 in vitro. Altered PRK1 and TP expression and signaling are increasingly implicated in certain neoplasms, particularly in androgen-associated prostate carcinomas. Agonist activation of TPα/TPβ led to phosphorylation of histone H3 at Thr(11) (H3 Thr(11)), a previously recognized specific marker of androgen-induced chromatin remodeling, in the prostate LNCaP and PC-3 cell lines but not in primary vascular smooth muscle or endothelial cells. Moreover, this effect was augmented by dihydrotestosterone in androgen-responsive LNCaP but not in nonresponsive PC-3 cells. Furthermore, PRK1 was confirmed to constitutively interact with TPα/TPβ in both LNCaP and PC-3 cells, and targeted disruption of PRK1 impaired TPα/TPβ-mediated H3 Thr(11) phosphorylation in, and cell migration of, both prostate cell types. Collectively, considering the role of TXA(2) as a potent mediator of RhoA signaling, the identification of PRK1 as a bona fide interactant of TPα/TPβ, and leading to H3 Thr(11) phosphorylation to regulate cell migration, has broad functional significance such as within the vasculature and in neoplasms in which both PRK1 and the TPs are increasingly implicated.     

Lipid body formation during maturation of human mast cells

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.