Coiled bodies (CBs) are nuclear organelles whose structures appear to be highly conserved in evolution.In rapidly cycling cells, they are typically located in the nucleoplasm but are often found in contact with the nucleolus. The CBs in human cells contain a unique protein, called p80-coilin. Studies on amphibian oocyte nuclei have revealed a protein within the "sphere" organelle that shares significant structural similarity to p80-coilin. Eukaryotic cells can be viewed as if they were a mixture of compartments, some of which can be defined physically, some functionally. In the cytoplasmic compartment, the roles of the various organelles and the interplay between them have been extensively studied. In contrast, the domains and their interconnections within the nucleus are poorly understood. However, it is now clear that the nucleus is a highly organized structure, permeated by a proteinaceous "matrix" and composed of many different subdomains. The so-called interchromatin space of somatic cells can be characterized at the ultrastructural level by several types of structures. These are perichromatin fibrils, interchromatin granule clusters, and nuclear bodies. Small nuclear ribonucleoproteins (snRNPs) are major components of each of these three subcompartments (see refs. 1 and 2 for reviews). One type of nuclear body, the coiled body (CB), contains relatively high concentrations of small RNPs as well as other proteins (see Table 1 for a list of components reported to be either enriched or not enriched in CBs). Despite (or perhaps because of) this rather lengthy roster, the list of putative CB functions is nearly as long (see refs. 3 and 4 for reviews). Early studies on amphibian oocyte nuclei also revealed morphologically distinct structures, including: nucleoli, lampbrush chromosomes, and "spheres." The subsequent finding that spheres (which are composed of B-and C-type "snurposomes") contain numerous snRNPs and snRNP proteins caused speculation that CBs and spheres were related organelles (reviewed in ref. 5). The final pieces to the puzzle linking CBs and spheres were revealed when SPH-1, an integral protein component ofThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.C snurposomes, was shown to have significant structural homology to a CB marker protein, p80-coilin (6, 7), and when Wu et al (8) showed that p80-coilin protein is targeted to spheres when the human coilin mRNA was injected into amphibian oocytes. Thus spheres and CBs are homologous organelles that are likely to have similar functions, but as yet no functions have been demonstrated (5).Of particular interest is the fact that some of the spheres within the amphibian oocyte are attached to the so-called "sphere-organizer" regions of the lampbrush chromosomes. These sphere-organizers are located at the histone gene clusters in two classes of amphibia, Anura and Urodela (9, 10). The fa...

Obesity is becoming the new pediatric epidemic. Non-alcoholic fatty liver disease (NAFLD) is frequently associated with obesity, and has become the most common cause of pediatric liver disease. The gut microbiome is the major metabolic organ and determines how calories are processed, serving as a caloric gate, and contributing towards the pathogenesis of NAFLD. The goal of this study is to examine gut microbial profiles in children with NAFLD using phylogenetic, metabolomic, metagenomic, and proteomic approaches. Fecal samples were obtained from obese children with or without NAFLD and healthy lean children. Stool specimens were subjected to 16S rRNA gene microarray, shotgun sequencing, mass spectroscopy for proteomics, and NMR spectroscopy for metabolite analysis. Children with NAFLD had more abundant gamma-proteobacteria and Prevotella and significantly higher levels of short chain fatty acids, and ethanol. This group also had increased genomic and protein abundance for energy production with a reduction in carbohydrate and amino acid metabolism and urea cycle and urea transport systems. The metaproteome and metagenome showed similar findings. The gut microbiome in pediatric NAFLD is distinct from lean healthy children with more alcohol production and pathways allocated to energy metabolism over carbohydrate and amino acid metabolism, which would contribute to development of disease.

Abstract. We have investigated the subcellular organization of the four human Y RNAs. These RNAs, which are transcribed by RNA polymerase III, are usually found complexed with the Ro autoantigen, a 60-kD protein. We designed 2'-OMe oligoribonucleotides that were complementary to accessible single-stranded regions of Y RNAs within Ro RNPs and used them in fluorescence in situ hybridization. Although all four Y RNAs were primarily cytoplasmic, oligonucleotides directed against three of the RNAs hybridized to discrete structures near the nucleolar rim. We have termed these structures "perinucleolar compartments" (PNCs). Double labeling experiments with appropriate antisera revealed that PNCs are distinct from coiled bodies and fibrillar centers. Co-hybridization with a genomic DNA clone spanning the human Y1 and Y3 genes showed that PNCs are not stably associated with the transcription site for these Y RNAs. Although 5S rDNA was often located near the nucleolar periphery, PNCs are not associated with 5S gene loci. Two additional pol III transcripts, the RNA components of RNase P and RNase MRP, did colocalize within PNCs. Most interestingly, the polypyrimidine tract-binding protein hnRNP I/PTB was also concentrated in this compartment. Possible roles for this novel nuclear subdomain in macromolecular assembly and/or nucleocytoplasmic shuttling of these five pol III transcripts, along with hnRNP I/PTB, are discussed.T HE nucleus, like the cytoplasm, is highly organized into discrete structural and functional domains. At the level of the light microscope, the most obvious structure in the interphase nucleus is the nucleolus, the site of rRNA transcription, processing and subunit assembly. With the use of antibodies and specific nucleic acid probes, it has become increasingly clear that many nucleoplasmic components, such as splicing factors and small ribonucleoprotein particles, are also nonrandomly distributed throughout the nucleus Spector, 1993).We have been studying a class of small ribonucleoprotein particles known as Ro RNPs. These RNPs were discovered because antibodies directed against these particles are present in many patients suffering from systemic lupus erythematosus and Sjogren's syndrome (Mattioli and Reichlin, 1974;Alspaugh and Tan, 1975;Lerner et al., 1981). Ro RNPs consist of several small cytoplasmic RNA molecules, each of which is complexed with a 60-kD protein (Wolin and Steitz, 1984 known as Y RNAs, are transcribed by RNA polymerase III and range in size from 69-112 nucleotides (Wolin and Steitz, 1983;O'Brien et al., 1993). In human cells, there are four distinct Y RNAs, called hY1, hY3, hY4, and hY5 (hY2 is a truncated version of hY1). Ro RNPs are present in mammalian cells at ~1% the level of ribosomes.Although Ro RNPs have been described in a variety of vertebrate species (Hendrick et al., 1981;Reddy et al., 1983;Mamula et al., 1989;O'Brien et al., 1993), as well as in the nematode (Van Horn et al., 1995), their function has remained obscure. However, the 60-kD Ro protein is also found comple...

SUMMARYThe gut barrier, composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, prevents the entrance of harmful microorganisms, antigens and toxins from the gut lumen into the blood. Small intestinal homeostasis is normally maintained by the rate of shedding of senescent enterocytes from the villus tip exactly matching the rate of generation of new cells in the crypt. However, in various localized and systemic inflammatory conditions, intestinal homeostasis can be disturbed as a result of increased IEC shedding. Such pathological IEC shedding can cause transient gaps to develop in the epithelial barrier and result in increased intestinal permeability. Although pathological IEC shedding has been implicated in the pathogenesis of conditions such as inflammatory bowel disease, our understanding of the underlying mechanisms remains limited. We have therefore developed a murine model to study this phenomenon, because IEC shedding in this species is morphologically analogous to humans. IEC shedding was induced by systemic lipopolysaccharide (LPS) administration in wild-type C57BL/6 mice, and in mice deficient in TNF-receptor 1 (Tnfr1−/−), Tnfr2 (Tnfr2−/−), nuclear factor kappa B1 (Nfκb1−/−) or Nfĸb2 (Nfĸb2−/−). Apoptosis and cell shedding was quantified using immunohistochemistry for active caspase-3, and gut-to-circulation permeability was assessed by measuring plasma fluorescence following fluorescein-isothiocyanate–dextran gavage. LPS, at doses ≥0.125 mg/kg body weight, induced rapid villus IEC apoptosis, with peak cell shedding occurring at 1.5 hours after treatment. This coincided with significant villus shortening, fluid exudation into the gut lumen and diarrhea. A significant increase in gut-to-circulation permeability was observed at 5 hours. TNFR1 was essential for LPS-induced IEC apoptosis and shedding, and the fate of the IECs was also dependent on NFκB, with signaling via NFκB1 favoring cell survival and via NFκB2 favoring apoptosis. This model will enable investigation of the importance and regulation of pathological IEC apoptosis and cell shedding in various diseases.