Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts.

Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (e.g., RPS19) or large (e.g., RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.

[FISH analysis in the differential diagnosis of flat urothelial lesions using tissue microarrays]. / FISH-untersuchungen an Multi-Tissue-Arrays zur Differenzierung flacher Läsionen des Urothels.

AIMS: FISH technology offers an additional tool in the diagnosis of precancerous and malignant lesions of the urinary tract from cytological specimens. Here, we examined the relevance of chromosomal imbalance in flat urothelial lesions using FISH on paraffin-embedded tissue. In addition, the status of Her2/neu and STK15, a key molecule in the development of aneuploidy, was evaluated. METHODS: Flat lesions (normal urothelium, hyperplasia, reactive atypia, dysplasia and Carcinoma in situlCis) of 73 patients were analyzed in respect of chromosome 3, 7, 17 polysomy, deletion of p16, status of HER2/neu and of STK15 by FISH (UroVysion, PathVysion, Vysis) and immunohistochemistry (HercepTest, DAKO) using tissue microarrays. The data were correlated with histology. RESULTS AND CONCLUSIONS: Aneusomy of at least one of the chromosomes usually correlates with the histology of carcinoma in situ or invasive tumor growth. Reactive atypias, rarely showing chromosomal imbalance, can be distinguished from Cis in the majority of investigated cases, but the FISH technique is not able to differentiate reactive atypia from mild dysplasia. About 30 % of the non-neoplastic lesions like urothelial hyperplasia and normal urothelium display polysomy of at least one chromosome in more than 20% of all cells, indicating an elevated risk towards a synchronous development of a higher dysplastic lesion (i.e. Cis). Polysomy of one of three investigated chromosomes (3, 7, 17) occurs randomly within all lesions. A deletion of the p16 locus is most frequently observed in aneuploid lesions. Altered Her2/neu expression patterns are frequently observed in malignant and dysplastic lesions but also in 25 % of the non-neoplastic lesions. An overexpression of Her2/ neu is found in 10-20% of invasive urothelial carcinomas and occasionally in Cis (5 %). However, the Her2/neu gene locus is not amplified in these samples. Gene amplification of STK15 was seen in tumors as well as in normal urothelium but it is still unclear whether it indicates an elevated risk towards the development of manifest urothelial tumors.

Disaggregation of polyribosomes in the spinal anterior horn cells in a patient with X-linked spinal and bulbar muscular atrophy.

The spinal anterior horn cells (AHCs) in a patient with X-linked spinal and bulbar muscular atrophy (SBMA) were examined by light and electron microscopy, giving special attention to alterations in the rough endoplasmic reticulum (ER). Seven age-matched subjects were used as controls. The patient with SBMA showed a severe decrease of AHCs, but the Nissl substance in the remaining AHCs appeared well preserved on light microscopy. Electron microscopy revealed a relatively well preserved parallel lamellar pattern of ER and marked disaggregation of the polyribosomes surrounding the ER in the remaining AHCs. These findings indicate that the Nissl substance was affected in spite of its light microscopic appearance in SBMA, and that the AHCs degenerate through disaggregation of the polyribosomes of the ER.

Characterization of Schwann cells from normal nerves and from neurofibromas in the bicolour damselfish.

Schwann cells are an important component of neurofibromas, one of the primary lesions encountered in neurofibromatosis type 1 in man. A central question in studies of neurofibromatosis type 1 has been whether the Schwann cells present in these tumours are intrinsically abnormal or exhibit abnormal phenotypes in response to stimuli from other cell types in these tumours. Damselfish neurofibromatosis is a naturally occurring disease in a species of marine fish, the bicolour damselfish, that is being developed as an animal model of neurofibromatosis type 1. Affected fish exhibit multiple neurofibromas and neurofibrosarcomas (malignant schwannomas). The present study compares the morphology, antigen expression and proliferative capacity in vitro of Schwann cells derived from peripheral nerves of normal, healthy fish with cells isolated from both spontaneously occurring and experimentally induced neurofibromas. Schwann cells from normal nerves expressed S100 antigens but not fibronectin or glial fibrillary acidic protein antigens and were similar in morphology and proliferative capacity to Schwann cells isolated from mammalian peripheral nerves. Tumour-derived cultures contained variable proportions (27-79%) of S100-positive cells that were identified as Schwann cells based on this feature. These tumour-derived Schwann cells exhibited a different morphology than normal Schwann cells, usually exhibited an increased reactivity to anti-S100 antibodies and were able to proliferate in vitro without added mitogens. Repeated subculturing of tumour-derived cultures led to the production of six cell lines all of which were composed exclusively of Schwann cells as indicated by S100 expression. These findings show that Schwann cells are an important component of tumours in Damselfish neurofibromatosis and that these cells are morphologically and physiologically altered in this disease. Observations of cell lines also suggest that tumour-derived Schwann cells are intrinsically abnormal and that this phenotype is not a result of stimuli from other cell types in the tumours.

Polyribosome concentration in human skeletal muscle after starvation and parenteral or enteral refeeding.

Posttraumatic and septic states cause a loss of body proteins resulting in a negative nitrogen balance. The major part of the excreted nitrogen is derived from the proteins of skeletal muscle. The loss in proteins is due to a decrease in protein synthesis rather than an increase in protein degradation. Nutritional support may increase protein synthesis, and determination of its activity in skeletal muscle will give information on the utilization of nutrients in catabolic patients. The effect of nutritional support on healthy subjects was studied to achieve a background for future clinical studies. Male volunteers between 20 and 40 years old were refed parenterally or enterally after three days of starvation. Muscle biopsies (50 mg) were analyzed for the size distribution of ribosomes in a sucrose density gradient, and the ribosome concentration was determined per mg of DNA. Changes in the percentage content of polyribosomes preceded those of the total ribosome concentration. The total polyribosome concentration per gram wet weight of skeletal muscle decreased significantly during starvation. After one and two days of refeeding, a significant increase was observed, but the original level of the nonstarved subjects was not reached. The total ribosome concentration increased upon refeeding, but was not significantly different from that of the starved condition. The nitrogen balance was negative during starvation but attained equilibrium after two days of refeeding. Nutrition administered by the parenteral or enteral route were equally effective in restoring protein synthesis.

Dysregulation of protein synthesis in injured liver. A comparative study on microsomal and cytosole enzyme activities, microsomal lipoperoxidation and polysomal pattern in D-galactosamine and carbon tetrachloride-injured livers.

Despite the presence of a marked decrease in liver protein content 48 h after a single injection of D-galactosamine, increased activities of glucose-6-phosphate dehydrogenase, low-Km hexokinase and pyruvate kinase type M2 were observed in the injured liver. Microsomal aniline hydroxylase activity and cytochrome P-450 content in liver decreased significantly in 48 h of galactosamine treatment but not in the first 2 h in contrast with carbon tetrachloride (CCL4) intoxication. The extents of those changes were not so great as in CCl4-treated rats. The disaggreation of polyribosomes in liver was observed in 24 h of galactosamine treatment. However, the formation of microsomal lipoperoxidation did not increase in the entire course of acute liver injury by the amino sugar. These results taken together with our previous observations indicate that the dysregulation of protein synthesis is an essential biochemical event of hepatocyte injury induced by treatment of rats with galactosamine as well as CCl4.

Age dependence of viral expression. Electron microscopic and immunoperoxidase studies of Measles virus replication in mice.

The hamster neurotropic strain of measles virus inoculated intracerebrally into BALB/c mice causes a uniformly fatal encephalitis with production of infectious virus in 1- to 2-day-old suckling mice but a 31 per cent mortality with production of measles virus antigen without production of infectious virus in 4-week-weanling mice (8). These two groups of animals were studied using routine electron microscopy and the immunoperoxidase technique in order to determine the nature of the measles virus antigen produced in each situation. Suckling animals showed numerous cytoplasmic viral inclusions in neurons and glia. This nucleocapsid material stained specifically with rabbit antiserum to measles virus antigen overlayed with peroxidase-conjugated goat antirabbit gamma-globulin. Histologic and electron microscopic study of weanling mice revealed rare necrotic cells and occasional loose clusters of neurons with depolymerized ribosomes. Immunoperoxidase staining showed diffuse cytoplasmic staining of similar groups of neurons and dendritic processes without evidence of nucleocapsid material. These differences in virus replication appear to be a function of the maturation of the host cell rather than the measles virus. Hamster neurotropic virus infection of the weanling mouse is an example of a potentially fatal virus infection in which viral replication is defective at a stage prior to assembly of nucleocapsid material; thus, no direct morphologic evidence that the cause of the clinical disease is a viral infection is present.