THE IN VIVO PROTEIN SYNTHETIC ACTIVITIES OF FREE VERSUS MEMBRANE-BOUND RIBONUCLEOPROTEIN IN A PLASMA-CELL TUMOR OF THE MOUSE

Cytoplasmic extracts of the transplantable RPC-20 plasma-cell tumor were fractionated by sucrose density gradient centrifugation. Four major fractions were distinguished: (a) microsomes and mitochondria; (b) membrane-free polyribosomes; (c) free monomeric ribosomes; and (d) soluble fraction. The fractions were analyzed for RNA and lipid phosphorus, and their particulate components were characterized by electron microscopy. Particular attention was paid to the problem of membrane contamination of the free polyribosome fraction. It was shown that this contamination was small in relation with the total content of ribosomes in the fraction, and that it consisted primarily of smooth-surfaced membranes which were not physically associated with the polyribosomes themselves. In vivo incorporation studies were carried out by injecting tumor-bearing animals intravenously with leucine-C14, removing the tumors at various times thereafter, and determining the distribution of protein radioactivity among the gradient-separated cytoplasmic fractions. The free polyribosome and the microsome-mitochondria fractions constituted active centers for protein synthesis. It was shown that nascent protein of the free polyribosome fractions was not associated significantly with the contaminating membranes. The kinetics of labeling during incorporation times up to 11 min suggested that protein synthesized on the free polyribosomes was rapidly transferred in vivo to the soluble fraction of the cell, while protein synthesized by the microsomes and mitochondria remained localized within these elements. It was estimated that the free polyribosome fraction and the microsome-mitochondria fraction accounted for approximately equal proportions of the total cytoplasmic protein synthesis in vivo

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Organs to Cells and Cells to Organoids: The Evolution of in vitro Central Nervous System Modelling

With 100 billion neurons and 100 trillion synapses, the human brain is not just the most complex organ in the human body, but has also been described as “the most complex thing in the universe.” The limited availability of human living brain tissue for the study of neurogenesis, neural processes and neurological disorders has resulted in more than a century-long strive from researchers worldwide to model the central nervous system (CNS) and dissect both its striking physiology and enigmatic pathophysiology. The invaluable knowledge gained with the use of animal models and post mortem human tissue remains limited to cross-species similarities and structural features, respectively. The advent of human induced pluripotent stem cell (hiPSC) and 3-D organoid technologies has revolutionised the approach to the study of human brain and CNS in vitro, presenting great potential for disease modelling and translational adoption in drug screening and regenerative medicine, also contributing beneficially to clinical research. We have surveyed more than 100 years of research in CNS modelling and provide in this review an historical excursus of its evolution, from early neural tissue explants and organotypic cultures, to 2-D patient-derived cell monolayers, to the latest development of 3-D cerebral organoids. We have generated a comprehensive summary of CNS modelling techniques and approaches, protocol refinements throughout the course of decades and developments in the study of specific neuropathologies. Current limitations and caveats such as clonal variation, developmental stage, validation of pluripotency and chromosomal stability, functional assessment, reproducibility, accuracy and scalability of these models are also discussed

Intracisternal type a particles in murine pancreatic b cells. Immunocytochemical demonstration of increased antigen (p73) in genetically diabetic mice.

Intracisternal Type A particles (IAPs) are retroviruslike structures identified by a core protein antigen (p73) and found in mouse embryos, in many mouse tumor cells, and in pancreatic B cells of some strains of genetically diabetic mice. Using both peroxidase-antiperoxidase and protein A-gold immunocytochemical techniques to localize p73, the authors have observed differences in intracellular antigen distribution between MOPC-104E, a mouse tumor cell line rich in IAP, and B cells from genetically diabetic (db/db) mice of the CBA/LtJ and C57BL/KsJ strain. In MOPC-104E cells studied by electron microscopy, localization of protein A-gold complex label was almost exclusively limited to IAP and their sites of assembly on the rough endoplasmic reticulum. In contrast, p73 appeared widely distributed throughout the cytoplasm of B cells from hyperglycemic db/db mice but not normal littermate controls. In addition to distribution over budding IAP, label was also found dispersed through other cytoplasmic organelles involved in secretion, including Golgi complexes and secretory granules. Patch labeling of B cell surfaces was sometimes observed. An ultrastructural survey of islets isolated from normal mice of 7 inbred genetic backgrounds on which the "diabetes" (db) gene has been studied showed that constitutive ability to produce IAP was associated with strain susceptibility to severe diabetes (eg, C57BL/KsJ, DBA/2J, CBA/LtJ, and C3HeB/FeJ). Strains whose B cells failed to show constitutive expression in situ or glucose-inducible expression in cell culture were resistant to the diabetogenic action of db genes. The possibility is discussed that p73 may represent a "neoantigen" which sensitizes the diabetic mouse to reject, by autoimmune mechanisms, the B cells expressing it

Intracisternal Type A particles in murine pancreatic B cells. Immunocytochemical demonstration of increased antigen (p73) in genetically diabetic mice.

Intracisternal Type A particles (IAPs) are retroviruslike structures identified by a core protein antigen (p73) and found in mouse embryos, in many mouse tumor cells, and in pancreatic B cells of some strains of genetically diabetic mice. Using both peroxidase-antiperoxidase and protein A-gold immunocytochemical techniques to localize p73, the authors have observed differences in intracellular antigen distribution between MOPC-104E, a mouse tumor cell line rich in IAP, and B cells from genetically diabetic (db/db) mice of the CBA/LtJ and C57BL/KsJ strain. In MOPC-104E cells studied by electron microscopy, localization of protein A-gold complex label was almost exclusively limited to IAP and their sites of assembly on the rough endoplasmic reticulum. In contrast, p73 appeared widely distributed throughout the cytoplasm of B cells from hyperglycemic db/db mice but not normal littermate controls. In addition to distribution over budding IAP, label was also found dispersed through other cytoplasmic organelles involved in secretion, including Golgi complexes and secretory granules. Patch labeling of B cell surfaces was sometimes observed. An ultrastructural survey of islets isolated from normal mice of 7 inbred genetic backgrounds on which the diabetes: (db) gene has been studied showed that constitutive ability to produce IAP was associated with strain susceptibility to severe diabetes (eg, C57BL/KsJ, DBA/2J, CBA/LtJ, and C3HeB/FeJ). Strains whose B cells failed to show constitutive expression in situ or glucose-inducible expression in cell culture were resistant to the diabetogenic action of db genes. The possibility is discussed that p73 may represent a neoantigen: which sensitizes the diabetic mouse to reject, by autoimmune mechanisms, the B cells expressing it