Comparison of sex ratio and cell number of IVM-IVF bovine blastocysts co-cultured with bovine oviduct epithelial cells or with Vero cells

The influence of 2 co-culture systems (BOEC and Vero cells) on the development rates, quality grades and sex ratios of IVM-IVF bovine embryos were studied. Zygotes obtained after IVF were co-cultured in each co-culture system for 7 and 8 d (Day 0 = day of insemination) in B2 medium. No effect of the co-culture system was observed on development rates measured on Days 7 and 8. However, Vero cell co-culture had a positive influence on embryo quality. Irrespective of their sex, embryos produced on Vero cells showed higher cells number than those co-cultured on BOEC (103.4 +/- 3.8 and 97 +/- 8.12 for BOEC vs 113.7 +/- 3.5 and 114 +/- 5.9 for Vero cells at Days 7 and 8, respectively; P < 0.05). The percentage of male embryos was increased in the two co-culture systems (60.7% males for BOEC; P < 0.05 vs 63% males for Vero cells; P < 0.01) on Day 7. In both co-culture systems the increase in the percentage of males was more obvious for embryos reaching the most advanced stage (expanded blastocysts). The results show that Vero cells improved the quality grade of bovine embryos produced in vitro, and thus are recommended for use as a safe co-culture system that does not contain pathogens.     

Fetal programming of neuropsychiatric disorders

Starting from the Developmental Origins of Health and Disease (DOHaD) hypotheses proposed by David Barker, namely fetal programming, in the past years, there is a growing evidence of the major role played by epigenetic factors during the intrauterine life and the perinatal period. Furthermore, it has been assessed that these factors can affect the health status in infancy and even in adulthood. In this review, we focus our attention on the fetal programming of the brain, analyzing the most recent literature concerning the epigenetic factors that can influence the development of neuropsychiatric disorders such as bipolar disorders, major depressive disorders, and schizophrenia. The perinatal epigenetic factors have been divided in two main groups: maternal factors and fetal factors. The maternal factors include diet, smoking, alcoholism, hypertension, malnutrition, trace elements, stress, diabetes, substance abuse, and exposure to environmental toxicants, while the fetal factors include hypoxia/asphyxia, placental insufficiency, prematurity, low birth weight, drugs administered to the mother or to the baby, and all factors causing intrauterine growth restriction. A better comprehension of the possible mechanisms underlying the pathogenesis of these diseases may help researchers and clinicians develop new diagnostic tools and treatments to offer these patients a tailored medical treatment strategy to improve their quality of life. Birth Defects Research (Part C) 108:207–223, 2016. © 2016 Wiley Periodicals, Inc.     

Differences in nitrate and ammonium uptake between Scots pine and European larch

In forest soils, ammonium is usually the predominant form of inorganic nitrogen. However, the capacity of trees to utilize both NO₃ ^- and NH₃ ⁺ may provide greater flexibility in responding to changes of nitrogen supply from the environment. Such capacity has been studied in seedlings of Scots pine (Pinus sylvestris L.) and European larch (Larix decidua Mill.) grown in the presence or absence of either nitrate or ammonium. Nitrate-induced plants showed a higher nitrate uptake rate than non-induced plants; this difference was almost negligible after 24 h of exposure to NO₃ ^-. Ammonium uptake in both species was consistently higher than that of nitrate, regardless of prior nitrogen provision. In both nutrient conditions, larch showed a more efficient transport system in comparison with Scots pine, with higher ammonium and nitrate uptake rates in both induced and non-induced plants. This was consistent also with the activity of nitrate reductase, measured in vivo in roots and leaves. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dasatinib, imatinib and staurosporine capture compounds - Complementary tools for the profiling of kinases by Capture Compound Mass Spectrometry (CCMS)

Capture Compound Mass Spectrometry (CCMS) is a platform technology for the functional isolation of subproteomes. Here we report the synthesis of two new kinase Capture Compounds (CCs) based on the tyrosine-kinase specific inhibitors dasatinib and imatinib and compare their interaction profiles to that of our previously reported staurosporine-CCs. CCs are tri-functional molecules: they comprise a sorting function (e.g. the small molecule or drug of interest) which interacts with target proteins, a photo-activatable reactivity function to covalently trap the interacting proteins, and a sorting function to isolate the CC-protein conjugates from complex biological samples for protein identification by liquid chromatography/mass spectrometry (LC-MS/MS). We present data of CCMS experiments from human HepG2 cells and compare the profiles of the kinases isolated with dasatinib, imatinib and staurosporine CC, respectively. Dasatinib and imatinib have a more selective kinase binding profile than staurosporine. Moreover, the new CCs allow isolation and identification of additional kinases, complementing the staurosporine CC. The family of kinase CCs will be a valuable tool for the proteomic profiling of this important protein class. Besides sets of expected kinases we identified additional specific interactors; these off-targets may be of relevance in the view of the pharmacological profile of dasatinib and imatinib.     

Correlates of preserved CD4(+) T cell homeostasis during natural, nonpathogenic simian immunodeficiency virus infection of sooty mangabeys: implications for AIDS pathogenesis

In contrast to HIV-infected humans, naturally SIV-infected sooty mangabeys (SMs) very rarely progress to AIDS. Although the mechanisms underlying this disease resistance are unknown, a consistent feature of natural SIV infection is the absence of the generalized immune activation associated with HIV infection. To define the correlates of preserved CD4(+) T cell counts in SMs, we conducted a cross-sectional immunological study of 110 naturally SIV-infected SMs. The nonpathogenic nature of the infection was confirmed by an average CD4(+) T cell count of 1,076 +/- 589/mm(3) despite chronic infection with a highly replicating virus. No correlation was found between CD4(+) T cell counts and either age (used as a surrogate marker for length of infection) or viremia. The strongest correlates of preserved CD4(+) T cell counts were a low percentage of circulating effector T cells (CD28(-)CD95(+) and/or IL-7R/CD127(-)) and a high percentage of CD4(+)CD25(+) T cells. These findings support the hypothesis that the level of immune activation is a key determinant of CD4(+) T cell counts in SIV-infected SMs. Interestingly, we identified 14 animals with CD4(+) T cell counts of <500/mm(3), of which two show severe and persistent CD4(+) T cell depletion (<50/mm(3)). Thus, significant CD4(+) T cell depletion does occasionally follow SIV infection of SMs even in the context of generally low levels of immune activation, lending support to the hypothesis of multifactorial control of CD4(+) T cell homeostasis in this model of infection. The absence of AIDS in these "CD4(low)" naturally SIV-infected SMs defines a protective role of the reduced immune activation even in the context of a significant CD4(+) T cell depletion.     

The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA

The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.     

The Vinculin-ΔIn20/21 Mouse: Characteristics of a Constitutive,Actin-Binding Deficient Splice Variant of Vinculin

Background

The cytoskeletal adaptor protein vinculin plays a fundamental role in cell contact regulation and affects central aspects of cell motility, which are essential to both embryonal development and tissue homeostasis. Functional regulation of this evolutionarily conserved and ubiquitously expressed protein is dominated by a high-affinity, autoinhibitory head-to-tail interaction that spatially restricts ligand interactions to cell adhesion sites and, furthermore, limits the residency time of vinculin at these sites. To date, no mutants of the vinculin protein have been characterized in animal models.

Methodology/Principal Findings

Here, we investigate vinculin-ΔEx20, a splice variant of the protein lacking the 68 amino acids encoded by exon 20 of the vinculin gene VCL. Vinculin-ΔEx20 was found to be expressed alongside with wild type protein in a knock-in mouse model with a deletion of introns 20 and 21 (VCL-ΔIn20/21 allele) and shows defective head-to-tail interaction. Homozygous VCL-ΔIn20/21 embryos die around embryonal day E12.5 showing cranial neural tube defects and exencephaly. In mouse embryonic fibroblasts and upon ectopic expression, vinculin-ΔEx20 reveals characteristics of constitutive head binding activity. Interestingly, the impact of vinculin-ΔEx20 on cell contact induction and stabilization, a hallmark of the vinculin head domain, is only moderate, thus allowing invasion and motility of cells in three-dimensional collagen matrices. Lacking both F-actin interaction sites of the tail, the vinculin-ΔEx20 variant unveils vinculin''s dynamic binding to cell adhesions independent of a cytoskeletal association, and thus differs from head-to-tail binding deficient mutants such as vinculin-T12, in which activated F-actin binding locks the protein variant to cell contact sites.

Conclusions/Significance

Vinculin-ΔEx20 is an active variant supporting adhesion site stabilization without an enhanced mechanical coupling. Its presence in a transgenic animal reveals the potential of splice variants in the vinculin gene to alter vinculin function in vivo. Correct control of vinculin is necessary for embryonic development.     

Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits.     

Identification of mammalian protein quality control factors by high-throughput cellular imaging

Protein Quality Control (PQC) pathways are essential to maintain the equilibrium between protein folding and the clearance of misfolded proteins. In order to discover novel human PQC factors, we developed a high-content, high-throughput cell-based assay to assess PQC activity. The assay is based on a fluorescently tagged, temperature sensitive PQC substrate and measures its degradation relative to a temperature insensitive internal control. In a targeted screen of 1591 siRNA genes involved in the Ubiquitin-Proteasome System (UPS) we identified 25 of the 33 genes encoding for 26S proteasome subunits and discovered several novel PQC factors. An unbiased genome-wide siRNA screen revealed the protein translation machinery, and in particular the EIF3 translation initiation complex, as a novel key modulator of misfolded protein stability. These results represent a comprehensive unbiased survey of human PQC components and establish an experimental tool for the discovery of genes that are required for the degradation of misfolded proteins under conditions of proteotoxic stress.