Loss of Hrs in the Central Nervous System Causes Accumulation of Ubiquitinated Proteins and Neurodegeneration

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs^flox/flox;SynI-cre mice. Notably, the hrs^flox/flox;SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs^flox/flox;SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.     

Clinical usefulness of urinary diacetylpolyamines as novel tumor markers

N1,N12-Diacetylspermine(DiAcSpm) and N1,N8-diacetylspermidine(DiAcSpd) are excreted in the urine of healthy persons as minor components of urinary polyamine, with small individual variations in amount. They are promising tumor markers, since their excretion is frequently elevated in patients with various types of cancers. DiAcSpm is sensitive in cancer detection, while DiAcSpd is highly specific for cancer. Diacetylpolyamines were initially characterized and determined by HPLC fractionation, followed by enzymatic detection. More recently, antibodies highly specific for DiAcSpm and DiAcSpd were developed, and an ELISA system applicable to the determination of urinary DiAcSpm was established. Measurement of urinary DiAcSpm using this ELISA system revealed that DiAcSpm is a more sensitive tumor marker than CEA, CA19-9 and CA15-3 for colon and breast cancers. More importantly, DiAcSpm efficiently detects patients at early stages. On the other hand, CEA, CA19-9 and CA15-3 are quite insensitive for early stage cancers. The urinary DiAcSpm level tends to remain low even in tumor-bearing individuals when their cancerous lesions remain static, while it rises rapidly concomitant with recurrence. DiAcSpm may serve as a prognostic indicator and marker for recurrence of prostate and colon cancers. Diacetylpolyamines may turn out to be general tumor markers, since active proliferation of any cancer tissues would likely be accompanied by activation of polyamine metabolism.     

Modifying effects of 1'-acetoxychavicol acetate (ACA) and the novel synthetic retinoids Re-80, Am-580 and Am-55P in a two-stage carcinogenesis model in female rats

Effects of dietary administration of 1'-acetoxychavicol acetate (ACA) and the novel synthetic retinoids 4-[1-hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (Re-80); 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (Am-580); and 6-[(3,5-di-tert-butylphenyl) carbamoyl]nicotinic acid (Am-55P) were examined using a two-stage rat carcinogenesis model. A total of 190 female SD rats was treated sequentially with 1,2-dimethylhydrazine (DMH, s.c.); 7,12-dimethylbenz(a)anthracene (DMBA, i.g.); and 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN, in the drinking water) during the first three weeks (DDD-initiation), and an additional 60 rats received the vehicle alone (non-initiation). One week after the completion of the initiation period, they were divided into nine groups and administrated Re-80 (at dose levels of 1.0 or 0.4 ppm), Am-580 (20 or 4 ppm), Am-55P (20 ppm), ACA (100 ppm), all-trans-retinoic acid (10 or 2 ppm) or no supplement in the diet for 33 weeks, until survivors were euthanatized at week 37 weeks. After DDD-initiation, all-trans-retinoic acid at the high dose delayed the development of mammary tumors. The multiplicity of colon tumors in the group fed Am-55P and the incidences of nephroblastomas with ACA or Am-580 were decreased as compared with the control values, but the other chemicals had no modifying effects on tumor development in any organs. Thus, among ACA and the novel synthetic retinoids tested, only Am-55P showed a weak inhibitory effect on a neoplasm of general interest under the present experimental conditions.     

Nuclear abnormalities in Pelger-Huet anomaly; progress in blood cell morphology

Gene abnormalities responsible for familial Pelger-Huet anomaly have been recently discovered. Abnormalities in sequence of Lamin B Receptor(LBR) gene results in a lack of LBR protein that is essential for chromatin-binding to nuclear membrane. In neutrophils lacking LBR protein shows abnormal bilobular or monolobular nuclear forms and hyper-condensed chromatin-aggregation. We re-analyzed distribution of such Pelger-Huet anomaly in other cell lineages; we found that not only neutrophils but erythroblasts, monocytes, lymphocytes, plasma cells, eosinophils and basophils are also carrying chromatin-hypercondensation. One third of megakaryocytes are also binucleated like neutrophils. We compared neutrophil morphology between familial Pelger-Huet anomaly and so called pseudo-Pelger-Huet anomaly observed in patients with myelodysplastic syndromes(MDS) and acute myeloid leukemia(AML). The neutrophils in MDS were much similar to those of the familial anomaly, but neutrophils of AML, such as t (8;21) M2-AML and t (15;17) M3-AML, showed more heterogeneous pattern in lobulation and chromatin-hypercondensation. Especially in M3, differentiation-induction by all-trans retinoic acid induced a marked neutrophilia with pseudo-Pelger-Huet anomaly without chromatin-hypercondensation. Lack of LBR protein in familial Pelger-Huet anomaly results in hypolobulation and chromatin-hypercondensation in neutrophils, but in other cells such as erythroblasts and lymphocytes only chromatin-hypercondensation can be observed. In contrast pseudo-Pelger-Huet anomaly are more heterogeneous in morphology compared to the familial anomaly. The lack of leukemic or MDS transformation in the familial anomaly is a sharp contrast to the neoplastic nature of the pseudo-Pelger-Huet anomaly. In conclusion, our morphological recognition of certain abnormality of cells shows an marked progression when genetic abnormality responsible for some of them are discovered, and often make us recognize a further heterogeneity in them. We, hematologists and technicians, must be well prepared to report our own observation of an un-explained morphological abnormality.     

Analysis of proliferating biliary epithelial cells in human liver disease using a monoclonal antibody against DNA polymeraseα

The proliferative activity and ultrastructural characteristics of proliferating biliary epithelial cells were analysed immunohistocytochemically in 39 biopsied liver specimens from patients with acute viral hepatitis, chronic hepatitis and liver cirrhosis using a monoclonal antibody against DNA polymerase (DNA-PA). In acute viral hepatitis with perivenular confluent necrosis, proliferation of typical bile ducts was found frequently in portal areas. In chronic aggressive hepatitis and cirrhosis, ductular proliferation of both typical and atypical forms was found in enlarged portal and periportal areas and in confluent necrotic areas. The number of proliferating biliary epithelial cells that stained positive for DNA-PA was small. There were very few positively stained cells in atypical bile ducts in confluent necrotic areas of cirrhosis. Atypical bile ducts seen in chronic aggressive hepatitis, cirrhosis and acute hepatitis with confluent necrosis were positively stained for both cytokeratins 8 and 19. In cirrhosis, the number of stained biliary epithelial cells in typical bile ducts was larger than the number of such cells in atypical bile ducts (P< 0.01). By electron microscopy, the cells positively stained for DNA-PA were mostly so-called clear cells with irregular nuclei containing coarse nucleoplasm, and a few small cells with scanty cytoplasm and few organelles.     

Oral Administration of Chlorella Vulgaris Augments Concomitant Antitumor Immunity

Chlorella vulgaris, an unicellular green algae, or its acetone-extract (Ac-Ex) were administered orally to Meth A tumor bearing BALB/c or (BALB/c DBA/2)F1 (CDF1) mice. When CDF1 mice were fed daily with 10% dried powder of Chlorella vulgaris (CVP) containing diet before and after Meth A tumor inoculation, the growth of rechallenged Meth A tumor was significantly suppressed in an antigen-specific manner. Augmentation of antitumor resistance was exhibited also by Winn assay using lymph node cells of tumor-bearing mice orally administered with CVP or Ac-Ex. Antigen-specific concomitant immunity in these mice were mediated by cytostatic T cells but not by cytotoxic T cells. Natural killer cells seemed not to contribute in antitumor resistance in this system.     

Detection of the putative E2 protein of hepatitis C virus in human liver

The question was asked whether a predicted envelope protein, considered to be processed from the polyprotein precursor encoded by the putative E2/NS1 region of the hepatitis C virus (HCV) genome, may be observed in HCV-infected humans. Two polyclonal antibodies against recombinant E2/NS1 proteins were prepared and their reactivity tested against liver extracts from HCV-infected patients by immunoblotting analysis. A band corresponding to a size of 44 kDa was detected in liver extracts from patients who were positive for the HCV-specific antibody anti-C100-3 but not in liver extracts from patients who did not have anti-C100-3 antibody. Additionally, no band was detected using preimmune sera or antisera which had been preabsorbed with recombinant E2/NS1 proteins. Deglycosylation studies demonstrated that the 44 kDa protein was a glycosylated form of a 38 kDa protein which corresponds to the predicted molecular weight of the putative E2/NS1 protein. These results suggest that the 44 kDa protein is a product of the E2/NS1 region. Frequent observation of the 44 kDa band in cases of chronic active hepatitis C suggests a correlation between the expression of this protein and the progression of hepatitis. © 1994 Wiley-Liss, Inc.     

Administration of interleukin -5 or -10 activates peritoneal B-1 cells and induces autoimmune hemolytic anemia in anti-erythrocyte autoantibody-transgenic mice

Activation mechanisms of B-1 (Ly-1 B) cells have been suggested to be different from those of conventional B cells. To assess the role of various interleukins (IL) in the activation of B-1 cells, we injected IL-4, IL-5 or IL-10 into nonanemic anti-red blood cells (RBC) autoantibody-transgenic mice, in which conventional B cells are clonally deleted but peritoneal B-1 cells persist without secreting Ig. Intraperitoneal or intramuscular injection of IL-5 or IL-10, but not IL-4, increased the number of antibody-producing peritoneal B-1 cells by four- to five-fold, resulting in increased anti-RBC serum autoantibody and induction of hemolytic anemia. These results suggest that IL-5 or IL-10 may play an important role in the terminal differentiation of B-1 cells into antibody-producing cells in vivo.     

Genome-wide array-based comparative genomic hybridization of natural killer cell lymphoma/leukemia: different genomic alteration patterns of aggressive NK-cell leukemia and extranodal Nk/T-cell lymphoma, nasal type

Natural killer (NK) cell lymphomas/leukemias are highly aggressive lymphoid malignancies, but little is known about their genomic alterations, and thus there is an urgent need for identification and analysis of NK cell lymphomas/leukemias. Recently, we developed our own array-based comparative genomic hybridization (array CGH) with an average resolution of 1.3 Mb. We performed an array CGH analysis for 27 NK-cell lymphoma/leukemia cases that were classified into two disease groups based on the World Health Organization Classification (10 aggressive NK-cell leukemia cases and 17 extranodal NK/T-cell [NK/T] lymphomas, nasal type). We identified the differences in the genomic alteration patterns of the two groups. The recurrent regions characteristic of the aggressive NK-cell leukemia group compared with those of the extranodal NK/T lymphoma, nasal-type group, were gain of 1q and loss of 7p15.1-p22.3 and 17p13.1. In particular, gain of 1q23.1-24.2 (P = 0.041) and 1q31.3-q44 (P = 0.003-0.047), and loss of 7p15.1-p22.3 (P = 0.012-0.041) and 17p13.1 (P = 0.012) occurred significantly more frequently in the former than in the latter group. Recurrent regions characteristic of the extranodal NK/T lymphoma, nasal-type group, compared with those of the other group were gain of 2q, and loss of 6q16.1-q27, 11q22.3-q23.3, 5p14.1-p14.3, 5q34-q35.3, 1p36.23-p36.33, 2p16.1-p16.3, 4q12, and 4q31.3-q32.1. Our results can be expected to provide further insights into the genetic basis of lymphomagenesis and the clinicopathologic features of NK-cell lymphomas/leukemias.     

Urine diacetylspermine as a novel tumor marker for pancreatobiliary carcinomas

A novel urine tumor marker, diacetylspermine, was compared with two conventional serum tumor markers, carcinoembryonic antigen (CEA) (highly specific for pancreatic cancer) and carbohydrate antigen (CA) 19-9 (highly sensitive for pancreatic cancer), in 125 patients with bilio-pancreatic tumors. When the diagnoses of benign or malignant conditions were examined, the sensitivity of urine diacetylspermine (75%) was shown to be higher than that of CEA (44%; P = 0.044) and CA19-9 (75%). The specificity of urine diacetylspermine (81%) was lower than that of CEA (92%) and as high as that of CA19-9 (80%). These results suggest that urine discetylspermine is a highly sensitive and specific novel marker for bilio-pancreatic carcinoma.