Proteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface Proteins on Human Embryonic Stem Cells and Sperms

BACKGROUND: It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells. METHODS AND PRINCIPAL FINDINGS: Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells

Establishment of Rat Embryonic Stem Cells and Making of Chimera Rats

The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as hypertension, diabetes, neurological disorders, and cancer. The rats have long been used in extensive chemical carcinogenesis studies. Thus, the rat embryonic stem (rES) cell is an important resource for the study of disease models. Attempts to derive ES cells from various mammals, including the rat, have not succeeded. Here we have established two independent rES cells from Wister rat blastocysts that have undifferentiated characters such as Nanog and Oct3/4 genes expression and they have stage-specific embryonic antigen (SSEA) -1, -3, -4, and TRA-1-81 expression. The cells were successfully cultured in an undifferentiated state and can be possible over 18 passages with maintaining more than 40% of normal karyotype. Their pluripotent potential was confirmed by the differentiation into derivatives of the endoderm, mesoderm, and ectoderm. Most importantly, the rES cells are capable of producing chimera rats. Therefore, we established pluripotent rES cell lines that are widely used to produce genetically modified experimental rats for study of human diseases

Colocalization of connexin 36 and corticotropin-releasing hormone in the mouse brain

<p>Abstract</p> <p>Background</p> <p>Gap junction proteins, connexins, are expressed in most endocrine and exocrine glands in the body and are at least in some glands crucial for the hormonal secretion. To what extent connexins are expressed in neurons releasing hormones or neuropeptides from or within the central nervous system is, however, unknown. Previous studies provide indirect evidence for gap junction coupling between subsets of neuropeptide-containing neurons in the paraventricular nucleus (PVN) of the hypothalamus. Here we employ double labeling and retrograde tracing methods to investigate to what extent neuroendocrine and neuropeptide-containing neurons of the hypothalamus and brainstem express the neuronal gap junction protein connexin 36.</p> <p>Results</p> <p>Western blot analysis showed that connexin 36 is expressed in the PVN. In bacterial artificial chromosome transgenic mice, which specifically express the reporter gene Enhanced Green Fluorescent Protein (EGFP) under the control of the connexin 36 gene promoter, EGFP expression was detected in magnocellular (neuroendocrine) and in parvocellular neurons of the PVN. Although no EGFP/connexin36 expression was seen in neurons containing oxytocin or vasopressin, EGFP/connexin36 was found in subsets of PVN neurons containing corticotropin-releasing hormone (CRH), and in somatostatin neurons located along the third ventricle. Moreover, CRH neurons in brainstem areas, including the lateral parabrachial nucleus, also expressed EGFP/connexin 36.</p> <p>Conclusion</p> <p>Our data indicate that connexin 36 is expressed in subsets of neuroendocrine and CRH neurons in specific nuclei of the hypothalamus and brainstem.</p

Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation

Cardiac regeneration: different cells same goal

Cardiovascular diseases are the leading cause of mortality, morbidity, hospitalization and impaired quality of life. In most, if not all, pathologic cardiac ischemia ensues triggering a succession of events leading to massive death of cardiomyocytes, fibroblast and extracellular matrix accumulation, cardiomyocyte hypertrophy which culminates in heart failure and eventually death. Though current pharmacological treatment is able to delay the succession of events and as a consequence the development of heart failure, the only currently available and effective treatment of end-stage heart failure is heart transplantation. However, donor heart availability and immunorejection upon transplantation seriously limit the applicability. Cardiac regeneration could provide a solution, making real a dream of both scientist and clinician in the previous century and ending an ongoing challenge for this century. In this review, we present a basic overview of the various cell types that have been used in both the clinical and research setting with respect to myocardial differentiation

De Novo Missense Substitutions in the Gene Encoding CDK8, a Regulator of the Mediator Complex, Cause a Syndromic Developmental Disorder

The Mediator is an evolutionarily conserved, multi-subunit complex that regulates multiple steps of transcription. Mediator activity is regulated by the reversible association of a four-subunit module comprising CDK8 or CDK19 kinases, together with cyclin C, MED12 or MED12L, and MED13 or MED13L. Mutations in MED12, MED13, and MED13L were previously identified in syndromic developmental disorders with overlapping phenotypes. Here, we report CDK8 mutations (located at 13q12.13) that cause a phenotypically related disorder. Using whole-exome or whole-genome sequencing, and by international collaboration, we identified eight different heterozygous missense CDK8 substitutions, including 10 shown to have arisen de novo, in 12 unrelated subjects; a recurrent mutation, c.185C>T (p.Ser62Leu), was present in five individuals. All predicted substitutions localize to the ATP-binding pocket of the kinase domain. Affected individuals have overlapping phenotypes characterized by hypotonia, mild to moderate intellectual disability, behavioral disorders, and variable facial dysmorphism. Congenital heart disease occurred in six subjects; additional features present in multiple individuals included agenesis of the corpus callosum, ano-rectal malformations, seizures, and hearing or visual impairments. To evaluate the functional impact of the mutations, we measured phosphorylation at STAT1-Ser727, a known CDK8 substrate, in a CDK8 and CDK19 CRISPR double-knockout cell line transfected with wild-type (WT) or mutant CDK8 constructs. These experiments demonstrated a reduction in STAT1 phosphorylation by all mutants, in most cases to a similar extent as in a kinase-dead control. We conclude that missense mutations in CDK8 cause a developmental disorder that has phenotypic similarity to syndromes associated with mutations in other subunits of the Mediator kinase module, indicating probable overlap in pathogenic mechanisms

Identification of key residues involved in adrenomedullin binding to the AM 1 receptor

BACKGROUND AND PURPOSE:Adrenomedullin (AM) is a peptide hormone whose receptors are members of the class B GPCR family. They comprise a heteromer between the GPCR, the calcitonin receptor-like receptor and one of the receptor activity-modifying proteins 1-3. AM plays a significant role in angiogenesis and its antagonist fragment AM22-52 can inhibit blood vessel and tumour growth. The mechanism by which AM interacts with its receptors is unknown.EXPERIMENTAL APPROACH:We determined the AM22-52 binding epitope for the AM1 receptor extracellular domain using biophysical techniques, heteronuclear magnetic resonance spectroscopy and alanine scanning.KEY RESULTS:Chemical shift perturbation experiments located the main binding epitope for AM22-52 at the AM1 receptor to the C-terminal 8 amino acids. Isothermal titration calorimetry of AM22-52 alanine-substituted peptides indicated that Y52, G51 and I47 are essential for AM1 receptor binding and that K46 and P49 and R44 have a smaller role to play. Characterization of these peptides at the full-length AM receptors was assessed in Cos7 cells by cAMP assay. This confirmed the essential role of Y52, G51 and I47 in binding to the AM1 receptor, with their substitution resulting in ≥100-fold reduction in antagonist potency compared with AM22-52 . R44A, K46A, S48A and P49A AM22-52 decreased antagonist potency by approximately 10-fold.CONCLUSIONS AND IMPLICATIONS:This study localizes the main binding epitope of AM22-52 to its C-terminal amino acids and distinguishes essential residues involved in this binding. This will inform the development of improved AM receptor antagonists

Mechanosensitive channels transduce osmosensitivity in supraoptic neurons.

Vasopressin is a peptide hormone synthesized by neurons of the supraoptic and paraventricular nuclei, which project axon terminals to the neurohypophysis. Consistent with its antidiuretic properties, vasopressin release rises as a function of plasma osmolality, a response that results from accelerated action potential discharge. Previous studies have shown that increases in fluid osmolality depolarize supraoptic neurons in the absence of synaptic transmission, suggesting that these cells behave as intrinsic osmoreceptors. The mechanism by which changes in osmolality are transduced into an electrical signal is unknown, however. Here we report that changes in cell volume accompany physiological variations in fluid osmolality and that these modulate the activity of mechanosensitive cation channels in a way that is consistent with the macroscopic regulation of membrane voltage and action potential discharge. These findings define a function for stretch-inactivated channels in mammalian central neurons