Lost in translation: endoplasmic reticulum stress and the decline of ß-cell health in diabetes mellitus.

Emerging data illustrate a pivotal role for activation of ß-cell endoplasmic reticulum (ER) stress pathways in diabetes pathophysiology. The purpose of this review is to appraise the evidence for ß-cell ER stress in human type 1 and 2 diabetes, review the molecular signalling pathways involved in the unfolded protein response and ER stress signalling, and to provide data from polyribosome profiling to illustrate the impact of ER stress on the mRNA translation response. Finally, we will discuss existing and novel therapeutic strategies that target ß-cell ER stress and discuss their use in rodent and human type 1 and 2 diabetes.

A novel protein that participates in nonself discrimination of malignant cells by homologous complement.

The human complement (C) system protects an individual against substances of nonself origin, including xenografts and microbial pathogens. Human cells express C-regulatory proteins, CD46 and CD55, thereby circumventing attack by C3, a major effector of C. Nevertheless, certain malignant cells, particularly those undergoing apoptotic stress, can activate homologous C, overcoming the regulatory actions of CD46 and/or CD55. The molecular mechanisms whereby malignant cells are tagged by homologous C3 remain largely unknown. We identified a novel gene product that converts human cells into targets for homologous complement. Only malignant cells and cell lines exposed to Fas or X-irradiation stimuli produced this protein, designated M161Ag, which was an unglycosylated 43-kDa protein. Analysis of cloned cDNAs indicated that this molecule was a secretory protein containing five amino acids encoded by TGA codons. Its functions were unique in that once secreted from the tumor cells, it bound back to the surface of these cells and activated homologous complement (C3) via the alternative pathway, allowing for C3 deposition on the membrane. This molecule may offer new insight into innate immunity; surveillance of tumor cells by complement is a common feature in the human immune system.

Tumorigenicity of Indian muntjac diploid cells by the proviral integration of sarcoma gene of a mouse retrovirus.

The transformed clonal isolates of Indian muntjac diploid cells by a mouse sarcoma virus, 43-2XV, were tested for tumorigenicity in athymic nude mice. In spite of the indistinguishable transformed morphology, the tumorigenicity exhibited four different patterns: (a) no tumor formation; (b) slowly growing regressive tumor formation; (c) rapidly growing regressive tumor formation; and (d) rapidly growing progressive tumor formation. This demonstrates that the same diploid host cells transformed by the same virus reveal variable patterns of tumorigenic expression and some transformed host cells lack the tumorigenicity entirely. The findings that there are at least two chromosomes and four recombinant sites assigned for the proviral integrations of the sarcoma gene into the Indian muntjac gene (M. Hatanaka, R. Klein, R. Kominami, T. Oikawa, H. Okabe, N. Tsuchida, E. C. Connors, and A. Carrano. Transformation of Indian muntjac diploid cells by the proviral integration of sarcoma gene of a mouse retrovirus. Manuscript in preparation.) lead us to propose a hypothesis that variable expressions of tumorigenicity under the neutral background of immune responses, may arise from variable integrations of the sarcoma gene into the host chromosome.

Mutants of nonproducer cell lines transformed by murine sarcoma virus. II. Relationship of tumorigenicity to presence of viral markers and rescuable sarcoma genome.

Tumorigenic and nontumorigenic mutants induced by a single 5'-bromodeoxyuridine (BrdU) treatment of a nonproducer (NP) tumorigenic cell line were isolated and characterized. Among the cloned derivatives were examples of virus-free and sarcoma virus-producing cell lines. Oncogenicity did not correlate with production of virus or ease of rescue of the sarcoma genome. All lines, including nononcogenic derivatives, retained the sarcoma genome. Phenotypic reversion of some cell mutants was observed after in vivo inoculation or long term in vitro cultivation. The M-50T cell line, obtained from a tumor induced by M-50 cells, had a sarcoma genome rescuable by direct superinfection; this was only achieved with parental M-50 cells by a cell fusion rescue technique. The M-43-2T cell, obtained from a single small static tumor induced by otherwise nononcogenic M-43-2 cells, shed sarcoma virus and became tumorigenic. M-58-4-48 became tumorigenic after passage 48 of the M-58-4 line, which was originally nontumorigenic. These observations of phenotypic reversion demonstrate that the presence of the sarcoma gene in cells is an essential but not sufficient condition of tumorigenesis.

Mutants of nonproducer cell lines transformed by murine sarcoma viruses. I. Induction, isolation, particle production, and tumorigenicity.

A variety of cell mutants were obtained by a single 5'-bromodeoxyuridine (BrdU) treatment of an nonproducer (NP) cell line transformed by the Kirsten strain of murine sarcoma virus (Ki-MSV). Isolation procedures of these cell See PDF for Structure mutants are described. The cell mutants obtained were classified by tumorigenic potential and shedding of Type C virus particles. The cell mutants were classified into four groups: (A) tumorigenic, without particles; (B) tumorigenic, with Type C particles; (C) nontumorigenic, without particles; and (D) nontumorigenic, with Type C particles. The tumorigenic cell lines showed variability in morphology with both flat and typical transformed appearing cell lines showing equal transplantability.

Mutants of nonproducer cell lines transformed by murine sarcoma virus. 3. Detection and characterization of RNA specific for helper and sarcoma viruses.

BALB/3T3 cells transformed by the Kirsten sarcoma virus (nonvirus producer BALB/3T3 cells) and mutant cell lines derived therefrom by treatment with bromodeoxyuridine (BrdU) were analyzed for expression of virus-specific RNA using single-stranded DNA transcripts of Rauscher leukemia virus (RLV), a virus activated in one of the cell lines (58-2T), and Ki-SV-specific DNA transcript; the latter transcript after removal of all sequences cross-reactive with RLV RNA. The Rauscher virus DNA detected multiple copies of viral RNA in virus-producing cells ( approximately 2.5 x 10(3)/cell) whether infected with RLV or activated to produce virus with BrdU. Nonproducer (NP) cells and normal BALB cells showed small numbers of RNA genomes (70-250/cell) and only partial saturation of the transcript. The intracellular RNA sedimented at 35S (main peak) with a variable minor peak at 20S with the exception of one mutant cell, M-43-2 (main peak at 26-27S). The 58-2T transcript reacted preferentially in NP cells and their derivatives with biphasic kinetics suggesting the possibility of sequences specific for the original transforming virus. The size of Ki-SV specific sequences were 30S in mutant cells whether or not complete virus was being produced and independent of in vivo transplantability.

Hyper IgM syndrome and complement Clq deficiency in an individual with systemic lupus erythematosus-like disease.

Many immunedeficiency syndromes are associated with autoimmune disorders. We here report on a girl with a systemic lupus erythematosus-like disease who suffered from both hyperimmunoglobulin M syndrome (HIGMS) and C1q deficiency. Despite severe central nervous system-lupus like disease, probably due to C1q deficiency, kidney function was relatively spared. IgM autoantibody might play a protective role against lupus-glomerulonephritis.

Catalytic Chemoselective O-Phosphorylation of Alcohols.

Phosphorylation of alcohols is a fundamentally important reaction in both life science and physical science. Product phosphate monoesters play key roles in living organisms, natural products, pharmaceuticals, and organic materials. Most of the chemical methods to date for synthesizing phosphate monoesters, however, require multistep sequences or are limited to specific types of substrates possibly due to harsh conditions. An alternative way to enable the simple production of phosphate monoesters from highly functionalized precursor alcohols is, thus, highly desired. We report herein a catalytic phosphorylation of alcohols with high functional group tolerance using tetrabutylammonium hydrogen sulfate (TBAHS) and phosphoenolpyruvic acid monopotassium salt (PEP-K) as the catalyst and phosphoryl donor, respectively. This method enables the direct introduction of a nonprotected phosphate group to the hydroxy group of a diverse menu of alcohol substrates, including functionalized small molecules, carbohydrates, and unprotected peptides. Nuclear magnetic resonance, mass spectrometric, and density functional theory analyses suggest that an unprecedented mixed anhydride species, generated from PEP-K and TBAHS, acts as an active phosphoryl donor in this reaction. This operationally simple and chemoselective catalytic phosphorylation allows for the efficient production of densely functionalized O-phosphorylated compounds, which are useful in diverse fields including biology and medicine.

Increased insulin demand promotes while pioglitazone prevents pancreatic beta cell apoptosis in Wfs1 knockout mice.

AIMS/HYPOTHESIS: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein called Wolfram syndrome 1 protein, homozygous mutations of which cause selective beta cell loss in humans. The function(s) of this protein and the mechanism by which the mutations of this gene cause beta cell death are still not fully understood. We hypothesised that increased insulin demand as a result of obesity/insulin resistance causes ER stress in pancreatic beta cells, thereby promoting beta cell death. METHODS: We studied the effect of breeding Wfs1 ( -/- ) mice on a C57BL/6J background with mild obesity and insulin resistance, by introducing the agouti lethal yellow mutation (A ( y ) /a). We also treated the mice with pioglitazone. RESULTS: Wfs1 ( -/- ) mice bred on a C57BL/6J background rarely develop overt diabetes by 24 weeks of age, showing only mild beta cell loss. However, Wfs1 ( -/- ) A ( y ) /a mice developed selective beta cell loss and severe insulin-deficient diabetes as early as 8 weeks. This beta cell loss was due to apoptosis. In Wfs1 ( +/+ ) A ( y ) /a islets, levels of ER chaperone immunoglobulin-binding protein (BiP)/78 kDa glucose-regulated protein (GRP78) and phosphorylation of eukaryotic translation initiation factor 2, subunit alpha (eIF2alpha) apparently increased. Levels of both were further increased in Wfs1 ( -/- ) A ( y ) /a murine islets. Electron micrography revealed markedly dilated ERs in Wfs1 (-/-) A ( y ) /a murine beta cells. Interestingly, pioglitazone treatment protected beta cells from apoptosis and almost completely prevented diabetes development. CONCLUSIONS/INTERPRETATION: Wfs1-deficient beta cells are susceptible to ER stress. Increased insulin demand prompts apoptosis in such cells in vivo. Pioglitazone, remarkably, suppresses this process and prevents diabetes. As common WFS1 gene variants have recently been shown to confer a risk of type 2 diabetes, our findings may be relevant to the gradual but progressive loss of beta cells in type 2 diabetes.

Induction of inflammatory arthropathy resembling rheumatoid arthritis in mice transgenic for HTLV-I.

Human T cell leukemia virus type-I (HTLV-I) is the etiologic agent of adult T cell leukemia and has also been suggested to be involved in other diseases such as chronic arthritis or myelopathy. To elucidate pathological roles of the virus in disease, transgenic mice were produced that carry the HTLV-I genome. At 2 to 3 months of age, many of the mice developed chronic arthritis resembling rheumatoid arthritis. Synovial and periarticular inflammation with articular erosion caused by invasion of granulation tissues were marked. These observations suggest a possibility that HTLV-I is one of the etiologic agents of chronic arthritis in humans.

Effect of Bacillus subtilis C-3102 on loose stools in healthy volunteers.

Ingestion of Bacillus subtilis C-3102 spores (C-3102) has relieved the symptoms of diarrhoea in piglets and changed the composition of gut microbiota in humans. Recently, it was suggested that the composition of the human gut microbiota affects stool consistency. In this study, a double-blind, randomised, placebo-controlled trial was conducted to assess the preventive effects of chronic diarrhoea in healthy volunteers with loose stools by ingestion of C-3102. The results showed that oral doses of C-3102 tablets significantly decreased the Bristol Stool Scale score and stool frequency, and also significantly improved abdominal sounds. With regard to gut microbiota, the relative abundance of Lachnospira, Actinomyces and SMB53 were significantly changed. This study shows that C-3102 could be effective for treating loose stools (Trial registration: UMIN000022583, http://tinyurl.com/ya4refqn ).

Overgrowth of human synovial cells driven by the human T cell leukemia virus type I tax gene.

One of the salient pathological features of rheumatoid arthritis is synovial cell proliferation with bone erosion. Despite extensive investigation, the factors essential for synovial cell proliferation remain to be identified. Recent studies suggest that human T cell leukemia virus type I (HTLV-I) may play an important role in synovial overgrowth observed in patients with one type of chronic inflammatory synovitis. In order to confirm and extend these observations, we have established synovial cell clones (SCCs) from three HTLV-I carriers who demonstrated synovial overgrowth but were otherwise asymptomatic. HTLV-I proviral DNA randomly integrated into the cellular genome was present in 20-30% of SCCs. The SCCs carrying HTLV-I proviral DNA and expressing the tax gene exhibited high levels of proliferative potential. HTLV-I was found to function as a transcriptional trans-activator in these SCCs. Moreover, transfection of the tax expression plasmid into SCCs resulted in the same phenotype of increased proliferation and cytokine expression as exhibited by HTLV-I provirus-carrying and tax-expressing SCCs. These data suggest that tax plays a critical role not only in leukemogenesis but also in synovial overgrowth in humans.

Phenotypic conversion of T lymphoblastic lymphoma to acute biphenotypic leukemia composed of lymphoblasts and myeloblasts. Molecular genetic evidence of the same clonal origin.

Acute biphenotypic leukemia composed of lymphoblasts and myeloblasts developed in a patient with T lymphoblastic lymphoma (T-LBL) who had an anterior mediastinal mass. A novel myeloid cell line, termed TK-1, has been established from his peripheral blood after the leukemic conversion. The identical rearranged pattern of T cell receptor gamma-chain gene was observed among the DNAs derived from lymph node cells in the lymphoma phase, the myeloid cell line TK-1, and the subclones with different karyotypes (TK-1B and TK-1D), which showed that myeloid cells had been derived from the T-LBL of the same patient. This finding demonstrates that phenotypic conversion occurs in the clonally propagating tumor cells and suggests that some hematopoietic cells retain the capacity to adopt either lineage.

Diffusion Tensor Imaging MRI With Spin-Echo Sequence and Long-Duration Measurement for Evaluation of Renal Fibrosis in a Rat Fibrosis Model.

BACKGROUND: Renal fibrosis (RF) is a well-known marker for chronic kidney disease (CKD) progression, including chronic renal injury after renal transplantation. However, invasive biopsy is an available examination for evaluation of RF. Diffusion MRI was once recognized as a promising option for RF. However, it is now controversial for RF evaluation in a unilateral ureteral obstruction (UUO) model. METHODS: To seek an optimal imaging method applicable for RF in UUO model kidneys, we attempted a series of MRI methods, including proton density-weighted imaging, T1-weighted imaging, T2-weighted imaging, T2*-weighted imaging, diffusion-weighted imaging, and diffusion tensor imaging (DTI). RESULTS: We identified DTI MRI by spin-echo sequence plus a special kidney attachment as the best option for evaluation of renal UUO fibrosis, compared with normal kidney on the opposite side. To confirm these results, we applied this technique to a rat UUO therapeutic model with the anti-fibrotic reagent Fasudil. Fractional anisotropy values calculated from DTI MRI showed statistically significant linear correlation with the RF area measured by use of Sirius red or Masson trichrome staining of the positive area [cortex (r = 0.6397, P = .0283) and outer stripe of the outer medulla (r = 0.7810, P = .0039)]. CONCLUSIONS: By use of the DTI MRI with spin-echo sequence, it may be possible to accurately evaluate RF in CKD.

Anticancer drugs: effect on the cloning of Raji lymphoma cells in soft agar.

The effect of 11 anticancer drugs on the ability of Raji lymphoma cells to form colonies in soft agar was determined with the use of both a 1-hour and a continuous drug exposure. Three distinct patterns of drug sensitivities were observed: a) Dactinomycin, adriamycin, bleomycin, mitomycin C, vincristine, and cis-platinum II all produced a dose-dependent reduction in colony formation following a 1-hour exposure, which was further augmented by a continuous exposure to the drugs; b) the antimetabolites (methotrexate, beta-cytosine arabinoside, and 5-fluorouracil) and pentamethylmelamine had no suppressive effects on colony formation with a 1-hour exposure, but they produced marked cytotoxicity with continuous drug exposure; and c) L-phenylalanine mustard had the same degree of colony suppression with both a 1-hour and a continuous drug exposure. Preincubation of Raji cells with an enzyme mixture (DNase + pronase + collagenase) did not alter the degree of colony suppression observed with the anticancer drugs. These results indicate that continuous drug exposure should be compared to a 1-hour drug incubation to determine in vitro drug sensitivities of fresh human tumors in the soft agar clonogenic assay, because the 1-hour drug exposure may not identify certain drugs that are potentially clinically active.

Effects of hydroxyurea on murine type C virus-specific DNA synthesis in newly infected cells.

Cell transformation and replication of the Rauscher pseudotype of Moloney murine sarcoma virus in mouse embryo fibroblasts were inhibited by hydroxyurea within a critical time period of 30 to 90 min postinfection. In cells infected by Rauscher leukemia virus, treatment with 1mM hydroxyurea during the critical time period resulted in the accumulation of minus-strand DNA (molecular weight, 3 x 10(6)) in association with the parental viral genoma RNA. This 5 to 6 x 10(6) dalton RNA:DNA hybrid was found in the cytoplasm. Positive-strand DNA of genomic or smaller size was not detected in the presence of hydroxyurea, but virus-specific DNA was found in the nucleus 30 min after removal of drug.

Studies of Pig Complement: Measurement of Pig CH50, ACH50, and Components.

BACKGROUND: On the basis of a comparison of the hemolytic complement titer in pigs with that in humans, the complement system of pigs was investigated. The response of innate immunity, such as the natural antibodies, against humans was also examined. METHODS: Hemolytic complement activity of pig serum was measured with the use of a microtitration technique. CH50 was determined according to the method of Mayer. ACH50 was assayed according to the methods of Platts-Milles and Ishizaka. Hemolytic activities of C1, C4, C2, C3, C5, C8, and C9 were estimated through the use of intermediate cells and reagents, as described previously. In addition, the pig natural anti-human antibody was studied with the use of human peripheral blood mononuclear cells (PBMCs). Human PBMCs were stained with 5% pig serum, followed by staining with fluorescein isothiocyanate-labeled goat anti-pig IgG and IgM. The resulting stained cells were quantified by use of a FACScalibur system. The alternative pathway of pig complement was also measured with the use of human erythrocytes and normal pooled pig serum with or without Mg(++)EGTA. RESULTS: Both the CH50 and ACH50 titers were lower than those of humans. Concerning the components, except for C3, each component, that is, C1, C4, C2, C5, C8, and C9, was also lower than that of humans, based on measured values for human complement components. Pig serum clearly contains natural antibodies, IgG and IgM, to human PBMCs. The alternative pathway of pig complement reacted with human erythrocytes. CONCLUSIONS: As a whole, pig innate immunity, the complement system and natural antibody, recognizes the surfaces of human cells.

Tax protein of human T-cell leukemia virus type I is required for maintenance of the transformed phenotype.

We have isolated and characterized revertants of a clonal cell line (40MRatcl-1) of human T-cell leukemia virus type I Tax-transformed Rat1 cells. The 40MRatcl-1 cells contain a single copy of tax gene, form large colonies in soft agar, elicit tumors rapidly in nude mice and revert to the normal phenotype at low frequency. From one of its subclones (B7) bearing pSV2gpt DNA as a marker gene, four morphologically reverse-transformed cell lines were isolated. They display contact inhibition at confluency, lose the ability to form colonies in soft agar, fail to form tumors in nude mice and restore the transformed phenotype similar to that of 40MRatcl-1 cells by transfection with the tax-expression plasmid. Southern blot analysis revealed that they have lost the tax gene. Our results indicate that transformation of Rat1 cells by Tax is not the consequence of secondary mutations of cellular genes and that tax functions are directly required for establishment and maintenance of the transformed phenotype.

Long cellular repeats flanking a defective HTLV-I provirus: implication for site-targeted integration.

Retroviruses generally integrate as proviruses which are flanked by long-terminal repeats (LTRs) on both 5' and 3' ends. Since these LTRs are required for the efficient integration mediated by the viral integrase, it is believed that defective proviruses with a single LTR are normally formed by deletion after integration. However, we found no deletion of cellular sequences around the integration site of such a defective HTLV-1. Rather, we identified 99 bp-long direct repeats adjacent to both ends of the defective provirus. The repeated cellular sequences contained a potential poly(A) signal followed by a retroviral primer-binding-site-like sequence. The presence of the direct repeats of cellular sequences can be explained by the integration of the defective virus through homologous recombination between cellular and viral read-through sequences.

Cis/trans-activation of the interleukin-9 receptor gene in an HTLV-I-transformed human lymphocytic cell.

The MT-2 cell-line, which had been established through in vitro cell to cell transmission of human T-cell leukemia virus type I (HTLV-I) among human primary lymphocytes, was shown to possess multiple copies of integrated proviruses, including defective proviral genomes. By analysing a genomic clone, we identified the integration site of a single HTLV-I long terminal repeat (LTR) in the interleukin-9 (IL-9) receptor (IL-9R) gene. The integrated HTLV-I-LTR was shown to be functional as a promoter and the integration site was located in an intron upstream of the first coding exon of the IL-9R gene. Upon analysis of total cellular RNA, specific expression of HTLV-I-LTR Il-9R chimeric mRNAs in MT-2 cells was demonstrated. Cloning and characterization of these cDNAs have identified HTLV-I-IL-9R chimeric splicing, using either intact or alternative splice sites within the IL-9R gene. The potential roles of multiple interactions between IL-9, IL-9R and HTLV-I in the monoclonal expansion and transformation of MT-2 cells are explored.