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The clinical manifestations of type 1 glycogen storage disease (GSD-1) in patients deficient in the glucose-6-phosphatase (G6Pase) system (e.g. growth retardation, hepatomegaly, hyperlipidemia, and renal dysfunction) are shared by Hnf1alpha(-/-) mice deficient of a transcriptional activator, hepatocyte nuclear factor 1alpha (HNF1alpha). However, the molecular mechanism is unknown. The G6Pase system, essential for the maintenance of glucose homeostasis, is comprised of glucose 6-phosphate transporter (G6PT) and G6Pase. G6PT translocates G6P from the cytoplasm to the lumen of the endoplasmic reticulum where it is metabolized by G6Pase to glucose and phosphate. Deficiencies in G6Pase and G6PT cause GSD-1a and GSD-1b, respectively. Hnf1alpha(-/-) mice also develop noninsulin-dependent diabetes mellitus caused by defective insulin secretion. In this study, we sought to determine whether there is a molecular link between HNF1alpha deficiency and function of the G6Pase system. Transactivation studies revealed that HNF1alpha is required for transcription of the G6PT gene. Hepatic G6PT mRNA levels and microsomal G6P transport activity are also markedly reduced in Hnf1alpha(-/-) mice as compared with Hnf1alpha(+/+) and Hnf1alpha(+/-) littermates. On the other hand, hepatic G6Pase mRNA expression and activity are up-regulated in Hnf1alpha(-/-) mice, consistent with observations that G6Pase expression is increased in diabetic animals. Taken together, the results strongly suggest that metabolic abnormalities in HNF1alpha-null mice are caused in part by G6PT deficiency and by perturbations of the G6Pase system.

Deficiencies in glucose 6-phosphate (G6P) transporter (G6PT), a 10-helical endoplasmic reticulum transmembrane protein of 429 amino acids, cause glycogen storage disease type 1b. To date, only three missense mutations in G6PT have been shown to abolish microsomal G6P transport activity. Here, we report the results of structure-function studies on human G6PT and demonstrate that 15 missense mutations and a codon deletion (delta F93) mutation abolish microsomal G6P uptake activity and that two splicing mutations cause exon skipping. While most missense mutants support the synthesis of G6PT protein similar to that of the wild-type transporter, immunoblot analysis shows that G20D, delta F93, and I278N mutations, located in helix 1, 2, and 6, respectively, destabilize the G6PT. Further, we demonstrate that G6PT mutants lacking an intact helix 10 are misfolded and undergo degradation within cells. Moreover, amino acids 415-417 in the cytoplasmic tail of the carboxyl-domain, extending from helix 10, also play a critical role in the correct folding of the transporter. However, the last 12 amino acids of the cytoplasmic tail play no essential role(s) in functional integrity of the G6PT. Our results, for the first time, elucidate the structural requirements for the stability and transport activity of the G6PT protein.

Porcine genomic DNA encoding a 55 kDa subunit of interleukin-2 receptor (IL-2R), which is termed alpha chain (IL-2Ralpha), was cloned by repeated plaque hybridization using IL-2Ralpha cDNA as a probe. Two different lambda phage clones, one of which encoded exon 1 and the 5'-upstream flanking region of IL-2Ralpha gene and another encoded the sequence from exon 2 to exon 8, were isolated. By analysis of the 5'-upstream region of the gene, putative binding motifs for transcription factors such as GATA family proteins, Ikaros, NF-kappaB, NF-IL2Ralpha and SRF, were found as described in human, murine and bovine genes. Two additional motifs for STAT4 binding were also found in this region. Moreover, using the FISH technique, we assigned the porcine IL-2Ralpha locus to the distal end of the long arm of chromosome 10 (10q6-qter) where the vimentin gene had been assigned nearby.

Infantile hemangioendothelioma of the thymus is a rare disease. We describe a patient who developed a large anterior mediastinal mass, severe thrombocytopenia and massive pleural effusion at 1 month of age. Glucocorticosteroid and irradiation therapy had no effect on either the tumor size or clinical symptoms and the tumor was resected subtotally. Three months after the subtotal resection, the remaining tumor had almost disappeared and the symptoms had resolved. The patient has now been well for 1 year after surgery without evidence of recurrence. The tumor tissue was characterized by prominent vascular endothelial proliferation intermixed with a normal thymic structure, producing a picture consistent with that of an infantile hemangioendothelioma in the thymus. Immunohistochemically, the tumor cells showed positive staining for vimentin, factor VIII and CD34. The DNA stemline and proliferative activity were examined by flow cytometry, which revealed a diploid stemline with a low growth fraction. DNA content and cell cycle analyses of the tumor tissue may be useful for predicting the biological behavior of the tumor.

Glycogen-storage disease type 1 (GSD-1), also known as "von Gierke disease," is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase) activity. There are four distinct subgroups of this autosomal recessive disorder: 1a, 1b, 1c, and 1d. All share the same clinical manifestations, which are caused by abnormalities in the metabolism of glucose-6-phosphate (G6P). However, only GSD-1b patients suffer infectious complications, which are due to both the heritable neutropenia and the functional deficiencies of neutrophils and monocytes. Whereas G6Pase deficiency in GSD-1a patients arises from mutations in the G6Pase gene, this gene is normal in GSD-1b patients, indicating a separate locus for the disorder in the 1b subgroup. We now report the linkage of the GSD-1b locus to genetic markers spanning a 3-cM region on chromosome 11q23. Eventual molecular characterization of this disease will provide new insights into the genetic bases of G6P metabolism and neutrophil-monocyte dysfunction.

Background: A mass screening system in Japan, which involves measuring urinary catecholamine metabolites, has resulted in an increasing number of cases of neuroblastoma, most of which have favorable biologic properties and some of which are associated with tumor regression or involution. At the time this study was begun, the characteristics and biologic nature of the neuroblastomas had not been fully defined, because a cell line had not yet been established with tumor tissue taken from a neuroblastoma detected in the mass screening., Methods: The authors established a cell line by tissue culture for over 50 generations from a neuroblastoma found during mass screening, which was characterized by favorable histology, with a triploid DNA stemline and without N-myc gene amplification. The morphologic and biologic characteristics of the new cell line were investigated in vitro., Results: The cell line, designated MASS-NB-SCH-1, has neuronal properties, such as neurite-like processes and neurofilaments, as well as the expression of vimentin and fibronectin in studies of the cell morphology and immunohistochemistry. Karyotype analysis detected the presence of 42-46 chromosomes, with a deletion of the short arm of 1 of the 3 copies of chromosome 1. DNA ploidy was near-diploid in association with 20-fold amplification of N-myc genes., Conclusions: The cell line has a nature distinct from the original tumor tissue. It may be characterized by phenotypic change caused by clonal selection or evolution of aggressive, proliferative properties in vitro. This cell line will be a useful model to investigate the properties of the neuroblastoma in relation to the N-myc amplification mechanism.

Epstein-Barr virus (EBV) infections are common in Japanese children, with infectious by EBV type 1. The relationships between EBV infection and lymphadenopathies in Hodgkin's disease (HD), non-Hodgkin's lymphomas (NHL), reactive follicular hyperplasia (RFH), and infectious mononucleosis (IM) in 37 Japanese children were evaluated. Formalin-fixed, paraffin-embedded lymph node specimens that were obtained at surgical resection or biopsy were evaluated for the presence of EBV DNA and the latent membrane protein-1 (LMP-1) using polymerase chain reaction (PCR) and immunohistochemical staining. The PCR detected EBV DNA in nine of 13 (69.2%) patients with RFH, including a case of IM, all three (100%) patients with HD, and one of 21 (4.8%) patients with NHL. All EBV-positive samples contained EBV type 1. Reed-Sternberg's cells in HD were immunohistochemically positive for LMP-1, whereas all cases of RFH and NHL were negative for LMP-1. Results suggest that EBV infection may be related to HD. Although no proof exists that EBV infection contributes to the transformation of cells, thus causing RFH or NHL, the present authors suggest that the EBV-positive cases in Japanese children demonstrate a relationship between the clinical and histopathological features of the lymphadenopathy and EBV-type 1 infection.

We investigated the prevalence of appendectomy in Japan to determine the tendency of familial aggregation on appendicitis using a large scale study. A total of 517 Japanese women (aged 20 to 45 years) served as the subjects and were questioned regarding their family history of appendectomy due to appendicitis. We compared the prevalence of appendectomy in children (subjects+their siblings) of three groups of families: (i) both parents affected; (ii) one parent affected; (iii) no parents affected. The differences between groups were statistically significant using contingency table and Chi-squared test. Our results suggest that about 40% of children with both parents affected, and about 20% of children with one parent affected may develop the disorder during childhood. This study may provide useful information in the nature of the development of appendicitis.

Tenascin is an extracellular matrix glycoprotein produced in response to epithelial-mesenchymal interactions that initiate fetal organogenesis, and it is also found in the stroma of benign and malignant neoplasms. Thirty-five human cell lines representing a variety of cancers were examined by immunoprecipitation and polyacrylamide gel electrophoresis of radiolabeled tenascin proteins from conditioned media. Two forms of tenascin with relative molecular masses of 190,000 and 250,000 were identified. Eight cell lines produced both forms. With the exception of myeloid and lymphoid leukemias and Burkitt's lymphoma, all of the mesodermal and neuroectodermal tumor lines were found to synthesize tenascin. Unexpectedly, tenascin was secreted by several mammary and colonic adenocarcinomas as well as by a line derived from normal mammary epithelial cells, and in some cases increased production was induced by transforming growth factor beta in serum-free medium. Cells producing fibronectin but not tenascin attached and spread on plastic culture dishes, while those producing tenascin alone remained suspended in the medium or were rarely attached. Tenascin also inhibited fibronectin-mediated adhesion of MCF7 breast carcinoma cells in vitro. The results suggest that tenascins synthesized and secreted by some cancer cells, especially those of epithelial origin, may have specific roles in determining tumor cell adhesion and ultimately the ability to form invasive outgrowths.

Heat shock proteins 60 (hsp60) and 10 (hsp10) are essential for the formation and restoration of many supramolecular structures. For reconstitution of these structures, we isolated stable hsps of 61kDa and 12kDa, which are similar to hsp60 and hsp10, respectively, from the supernatant fraction of thermophilic bacterium PS3 by ATP-Agarose chromatography. Using synthetic DNA of the deduced sequence, the 1.6kbp double stranded DNA encoding both proteins was obtained by the polymerase chain reaction (PCR). The complete sequence of the resulting reading frames showed high homology to those of the genes encoding GroEL (hsp60) and GroES (hsp10) of E. coli, and hsp60s and hsp10s of several other species. The genes for the 12K and 61K were present in the same operon. 61K was also partially similar to the F1 alpha subunit of thermophilic ATP synthase, which is highly reconstitutable to form the alpha beta complex.

We investigated oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between lysine-82 beta 1 and lysine-82 beta 2 by a fumaryl group, which is prepared by reaction of the CO form with bis(3,5-dibromosalicyl) fumarate. The cross-linked hemoglobin preparation isolated by the previous purification method, namely, gel filtration in the presence of 1 M MgCl2 followed by ion-exchange chromatography, was found to be contaminated with about 20% of an electrophoretically silent impurity that shows remarkably high affinity for oxygen. This impurity was separated from the desired cross-linked hemoglobin by a newly developed purification method, which utilizes a difference between the authentic hemoglobin and the impurity in reactivity of the sulfhydryl groups of cysteine-93 beta toward N-ethylmaleimide under a deoxygenated condition. After this purification procedure, the oxygen equilibrium properties of purified cross-linked hemoglobin in the absence of organic phosphate became very similar to those of unmodified hemoglobin with respect to oxygen affinity, cooperativity, and the alkaline Bohr effect. The functional similarity between the cross-linked hemoglobin and unmodified hemoglobin allows us to utilize this cross-linking for preparing asymmetric hybrid hemoglobin tetramers, which are particularly useful as intermediately liganded models. Previous studies on this type of cross-linked hemoglobin should be subject to reexamination due to the considerable amount of the impurity.

We have developed a biochemical method for purifying human tenascin from cultured fibroblasts or the culture medium. The method is a series of biochemical procedures including gel filtration, gelatin gel affinity chromatography and ion-exchange high performance liquid chromatography. The final preparation was identified as tenascin from its immunological cross-reactivity to antibody against chicken tenascin, strong hemagglutination activity which has been reported to be one of the biological functions of chicken tenascin, and from the electron microscopic study demonstrating a six-armed structure. Gel chromatography showed that intact human tenascin has an apparent molecular weight of over one million. Analysis of the purified tenascin with SDS-PAGE under reducing conditions demonstrated that tenascin consists of two kinds of subunits (250K and 190K). We established rat x mouse heterohybridoma cell lines which produce tenascin-specific antibodies. One monoclonal antibody (RCB1) was selected for immunohistochemical study and partially characterized. RCB1 bound native tenascin but not reduced and alkylated tenascin. Immunohistochemistry of normal and neoplastic tissues demonstrated that RCB1 bound the connective tissues surrounding the cancer nests and various normal tissues including interstitium of renal distal tubule, periosteum, endosteum, smooth muscles of digestive tract and media of arteries and arterioles.