Distribution of lysosome-associated membrane proteins-1 and -2, and cathepsin D in eosinophilic granular bodies: possible relationship to cyst development in pilocytic astrocytomas.

Pilocytic astrocytomas are usually cystic; cyst formation within these tumours may result in increased intracranial pressure, due to the effect of their mass, and contribute to cerebral damage. Eosinophilic granular bodies (EGBs) are produced abundantly in pilocytic astrocytomas but their role in disease progression remains unknown. Immunohistochemistry studies showed EGBs to exhibit pronounced reactivity to antibodies against lysosome-associated membrane proteins (LAMP)-1 and LAMP-2, and the lysosomal enzyme cathepsin D. Both LAMP-1 and LAMP-2 showed peripheral rim and granular staining patterns. The EGBs were scattered widely across cysts and, where EGBs aggregated in clusters, were usually close to areas of fluid in the cysts. Most EGBs had nuclei either attached or close by, indicating that the EGBs may be derived from anucleated astrocytes. The results suggest that EGBs, together with other factors, may play a role in the development of cysts in pilocytic astrocytomas.

Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing.

The SNT309 gene was identified via a mutation that causes lethality of cells in combination with a prp19 mutation. We showed previously that Snt309p is a component of the Prp19p-associated complex and that Snt309p, like Prp19p, is associated with the spliceosome immediately after or concomitantly with dissociation of U4 from the spliceosome. We show here that extracts prepared from the SNT309-deleted strain (DeltaSNT309) were defective in splicing but could be complemented by addition of the purified Prp19p-associated complex. Isolation of the Prp19p-associated complex from DeltaSNT309 extracts indicated that the complex was destabilized in the absence of Snt309p and dissociated on affinity chromatography, suggesting a role of Snt309p in stabilization of the Prp19p-associated complex. Addition of the affinity-purified Prp19p-Snt309p binary complex to DeltaSNT309 extracts could reconstitute the Prp19p-associated complex. Genetic analysis further suggests that Snt309p plays a role in modulating interactions of Prp19p with other associated components to facilitate formation of the Prp19p-associated complex. A model for how Snt309p modulates such interactions is proposed.

Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing.

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.

Snt309p, a component of the Prp19p-associated complex that interacts with Prp19p and associates with the spliceosome simultaneously with or immediately after dissociation of U4 in the same manner as Prp19p.

The yeast protein Prp19p is essential for pre-mRNA splicing and is associated with the spliceosome concurrently with or just after dissociation of U4 small nuclear RNA. In splicing extracts, Prp19p is associated with several other proteins in a large protein complex of unknown function, but at least one of these proteins is also essential for splicing (W.-Y. Tarn, C.-H. Hsu, K.-T. Huang, H.-R. Chen, H.-Y. Kao, K.-R. Lee, and S.-C. Cheng, EMBO J. 13:2421-2431, 1994). To identify proteins in the Prp19p-associated complex, we have isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of prp19, using the ade2-ade3 sectoring system. A novel splicing factor, Snt309p, was identified through such a screen. Although the SNT309 gene was not essential for growth of Saccharomyces cerevisiae under normal conditions, yeast cells containing a null allele of the SNT309 gene were temperature sensitive and accumulated pre-mRNA at the nonpermissive temperature. Far-Western blot analysis revealed direct interaction between Prp19p and Snt309p. Snt309p was shown to be a component of the Prp19p-associated complex by Western blot analysis. Immunoprecipitation studies demonstrated that Snt309p was also a spliceosomal component and associated with the spliceosome in the same manner as Prp19p during spliceosome assembly. These results suggest that the functions of Prp19p and Snt309p in splicing may require coordinate action of these two proteins.

Adenocarcinoma combined with malignant peripheral nerve sheath tumor of the gallbladder.

Adenocarcinoma of the gallbladder combined with a malignant peripheral nerve sheath tumor (MPNST) in the gallbladder in an 81-year-old woman is reported. The resected gallbladder showed two distinct tumor components, the epithelioid type of MPNST and adenocarcinoma with areas of mucin production. Although the immediate postoperative course was uneventful, a pathologic fracture of her right upper femur developed 4 months after the cholecystectomy. The pathology was determined to be a feature of metastatic MPNST rather than of adenocarcinoma. A whole body bone scan revealed multiple metastases, including the left parietal skull, left ninth rib, seventh thoracic vertebra, and right upper third of the femur. Despite cholecystectomy and postoperative irradiation therapy, she died 6 months after diagnosis of the tumor. Without an autopsy the primary site of the MPNST was unknown. We found that the prognosis was very poor in patients with distal metastatic MPNST, especially in older patients.

Human stomach alcohol and aldehyde dehydrogenases: comparison of expression pattern and activities in alimentary tract.

BACKGROUND & AIMS: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the major enzymes responsible for ethanol metabolism in humans. The human stomach has been documented to be involved in the metabolism of first-passed alcohol. The aim of this study was to determine ethanol-metabolizing activities in the stomach with regard to sex, age, enzyme pattern, and polymorphism. METHODS: A total of 209 surgical gastric mucosal specimens were investigated. The expression patterns of ADH and ALDH were identified by isoelectric focusing, and the activities were assayed spectrophotometrically. RESULTS: At 33 or 500 mmol/L ethanol, pH 7.5, the activities in the ADH3 1-1 phenotypic and mu-ADH-expressing mucosal specimens were significantly greater than that in the ADH3 1-2 phenotypic and mu-ADH absent mucosal specimens, respectively. The activities of the ALDH2-inactive phenotypes were significantly lower than that of the ALDH2-active phenotypes at 200 micromol/L acetaldehyde. The gastric ADH and ALDH activities were not significantly different between men and women with respect to age and genetic polymorphism. CONCLUSIONS: The stomach may contribute only a small portion of the alcohol metabolism observed in humans, and the liver may be the major site for first-pass metabolism. Differential expression patterns of ADH and ALDH in the alimentary tract suggest that different vulnerabilities to ethanol-induced mucosal injury may exist.

Imprint cytology of nasopharyngeal biopsies.

OBJECTIVE: To use an instant cytologic diagnostic method to examine touch imprints of nasopharyngeal (NP) biopsies. This method aimed to ensure the adequacy of biopsied specimens for histologic examination. This paper describes the morphologic findings of NP lesions examined by this method. STUDY DESIGN: Imprints were made from NP biopsies from patients suspicious for nasopharyngeal carcinoma (NPC). These imprints were air dried, stained with Diff-Quik and examined immediately. The adequacy of the specimens was assessed, and the findings of the imprints were interpreted as positive-, negative- or suspicious for NPC. Repeat biopsies and cytologic studies were done as indicated. In selected cases, immunocytochemical staining was done to identify cells on the imprints. Histologic examination of the biopsied specimens served as the control. RESULTS: With this method we could interpret the imprints within five minutes of their receipt and determine if repeat biopsy was needed. In benign lesions, the imprints often contained many cells, most mature and reactive lymphocytes. These cells and the numerous lymphoglandular bodies (fragments of lymphoid cytoplasm) intermingled with the ciliated and squamous epithelial cells. In cases of NPC, the appearance was discrete, or clusters of, carcinoma cells or naked nuclei. They were less cellular than those of benign lesions. The lymphocytes were markedly depleted. Cells of dubious lineage were identified by the additional use of immunocytochemical studies. CONCLUSION: Cytologic examination of imprints of NP biopsies helps to determine the adequacy of the specimen for histologic examination. It is a rapid, practical method with high diagnostic accuracy.

Expression of Epstein-Barr virus-encoded RNAs as a marker for metastatic undifferentiated nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr virus (EBV) is associated with undifferentiated nasopharyngeal carcinoma (NPC). EBV-encoded nonpolyadenlyated RNAs (EBERs) are often used as a marker to detect EBV-infected NPC cells. This study was conducted to document the expression and determine the significance of EBERs in NPC cells at various metastatic sites. METHODS: An in situ hybridization (ISH) technique was used to identify the presence of EBERs in paraffin embedded tissues of primary and metastatic sites obtained from 21 patients with NPC. Nineteen of these patients had undifferentiated lesions, and 2 had squamous cell carcinoma. One hundred and fifty specimens of normal tissues and tissues from patients with a variety of benign and malignant diseases other than NPC served as controls. In the NPC specimens, the expression of latent membrane protein (LMP) and a lytic protein, BZLF-1, were also examined by immunohistochemistry. RESULTS: Tissues from all patients with undifferentiated NPC and one patient with squamous cell carcinoma contained EBERs in the malignant cells; the other case of squamous cell carcinoma was negative. In metastatic NPCs, LMP was expressed in 18% (4 of 22) of tissues whereas BZLF-1 was not expressed in any tissues. EBERs were not detected in the 43 patients with normal tissues and benign lesions. In malignant diseases other than NPC, EBERs were detected in only 2 of 12 cases of non-Hodgkin's lymphoma, 1 of 2 cases of Hodgkin's lymphoma, and 1 of 6 cases of gastric cancer. CONCLUSIONS: By virtue of the direct correlation between latent EBV infection and NPC, the authors conclude that EBERs can be used as a sensitive marker to identify NPC cells at various metastatic sites by techniques of in situ hybridization, and that demonstration of EBERs in lesions of undifferentiated histology may be useful as a diagnostic adjunct for NPC presenting as metastatic cancer of unknown origin.

Spindle cell lipoma in a 14-month-old girl.

This case report concerns a girl with spindle cell lipoma of the neck. Spindle cell lipoma is a variant of lipomas and was first described by Enzinger and Harvey in 1975. It occurs chiefly in males between 40 and 70 years of age. It is a benign lesion that can be cured by excision, and local recurrence is rare. Spindle cell lipoma is composed of adipocytes and non-fat-storing immature mesenchymal cells. The condition is uncommon in adults and had not been reported to occur in children.

Diagnosis of disseminated candidiasis by fine needle aspiration of lymph node and by splenic imprint in a patient with acute promyelocytic leukemia.

Cytologic studies were done on fine needle aspirates of the lymph node and imprints of splenic biopsies from a patient with acute promyelocytic leukemia who was febrile while being treated with chemotherapy. Examination of the lymph node aspirates revealed pus and numerous pseudohyphae which were later identified as Candida tropicalis. When multiple nodular lesions were detected in the spleen by abdominal sonography and CT scan, needle biopsy of the spleen was done. Cytologic examination of touch imprints of the biopsy disclosed intracellular fungal blastospores. The patient was treated with and responded well to amphotericin B and 5-fluorocytosine. As a result of our experience with this patient we emphasize the importance of close incorporation of clinical information and diagnostic cytology. With such a cooperation, cytologic studies become a most useful method for diagnosis.

PRP19: a novel spliceosomal component.

We have isolated the gene of a splicing factor, PRP19, by complementation of the temperature-sensitive growth defect of the prp19 mutant of Saccharomyces cerevisiae. The gene encodes a protein of 502 amino acid residues of molecular weight 56,500, with no homology to sequences in the data base. Unlike other PRP proteins or mammalian splicing factors, the sequence of PRP19 has no discernible motif. Immunoprecipitation studies showed that PRP19 is associated with the spliceosome during the splicing reaction. Although the exact function of PRP19 remains unknown, PRP19 appears to be distinct from the other PRP proteins or other spliceosomal components.

High-level production of human alpha- and beta-globins in insect cells.

High-level production of human alpha- and beta-globins in cultured Spodoptera frugiperda (Sf-9) cells infected with recombinant baculoviruses is described. The expressed globins are produced to 70-140 mg protein/liter of cell culture or 5-10% of the total cellular protein. Two recombinant baculoviruses for alpha-globin, H alpha and H beta alpha, differ in their construction in that the 5'-untranslated region of the beta-globin gene is inserted 5' to the alpha-globin mRNA coding region in H beta alpha. This insertion results in a 40% increase in yield of alpha-globin over that of H alpha. Consistent with previous observations of the processing of recombinant proteins in Sf-9 cells, both alpha- and beta-globins expressed in Sf-9 cells are correctly processed to remove the initiating methionine from the amino termini of the globins. Sequencing of the expressed globins in Sf-9 cells confirms their identity with globins purified from human normal adult hemoglobin.

Extracellular matrix alteration in chronic hepatitis.

We used immunofluorescence and immunohistochemical PAP methods on 22 paraffin-embedded liver tissue specimens for observation and analysis of the distribution of extracellular matrix (ECM) elements in chronic hepatitis and cirrhosis. Our study revealed that in CLH there was only mild increases of types III, V collagen and fibronectin in spotty necrosis areas. In CPH, types III, V collagen and fibronectin revealed mild to moderate increase in portal area and lobular sinusoid. In CAH, moderate to marked increases of types III, V collagen and fibronectin and mild increase of type IV collagen in portal area, sinusoid lining, piecemeal necrosis and fibroseptum were found. Types I, IV collagen in fibroseptum were also noted. Some periportal hepatocytes showed abundant intracellular fibronectin. In cirrhosis, cases expressed similar finding to CAH except much more type IV collagen deposition. In addition, the basement membrane components including type IV collagen and laminin were found in the "capillarization" of periportal sinusoids in fibrotic liver tissue. In areas of piecemeal necrosis, the hepatocytes, single or assembled in "rosettes", were underlined by linear deposits of laminin and type IV collagen. Our study revealed that, histologically, the ECMs distribution of CAH is similar to that of cirrhosis but could be clearly distinguished from CPH and CLH. The prominent changes of ECMs, especially the basement membrane components, in case of CAH and cirrhosis are consistent with the fact that ECM may play a central role in liver function impairment and fibrogenesis.

Inflammatory pseudotumor of the liver with occlusive endophlebitis: report of a case.

Inflammatory pseudotumor (IPT) of the liver is an extremely rare benign tumor. Only 12 cases have so far been reported in the English literature. The etiology and pathogenesis of IPT remain obscure. We herein present an additional case of IPT of the liver with occlusive endophlebitis in a 36-year-old female. Immunohistochemical studies demonstrated the polyclonal nature of the plasma cells in the tumor, including IgA, IgM, IgG, kappa and lambda. The inflammatory pseudotumor should be kept in mind in the differential diagnosis of hepatic space-occupying malignant lesions.

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.

Monoclonal antibodies to mitotic cells.

Certain proteins or activities are present in mitotic cells but not in interphase cells. These proteins may be synthesized or activated, or both, just prior to mitosis and are responsible for the breakdown of the nuclear envelope and the condensation of chromosomes. To learn more about the nature of these proteins, we raised monoclonal antibodies to mitotic cells. Spleen cells from mice immunized with a 0.15 M NaCl extract of synchronized mitotic HeLa cells were fused with SP2/0-Ag14 mouse myeloma cells, and hybrids were selected in medium containing hypoxanthine, methotrexate, thymidine, and glycine. Two different hybridoma clones secreting antibodies reactive with mitotic and meiotic cells from every species tested were isolated. Chromosomes as well as cytoplasm in mitotic cells reacted with the antibodies, as detected by indirect immunofluorescence. The proteins from mitotic cells were separated by electrophoresis in NaDodSO4/polyacrylamide slab gels, transferred to nitrocellulose sheets, and stained immunochemically. The two antibodies, designated MPM-1 and MPM-2, recognize a family of polypeptides with apparent molecular masses of 0.40 to greater than 200 kilodaltons (kDa). Both antibodies reacted strongly with three polypeptide bands of 182 kDa, 118 kDa, and 70 kDa. Only mitotic cells exhibited the protein bands that were recognized by the antibodies. All these bands were found to be phosphoproteins as shown by 32P labeling and autoradiography and their removal by alkaline phosphatase treatment.

Effects on mouse blastocysts of in vitro exposure to methylnitrosourea and 3-methylcholanthrene.

Mouse blastocysts were exposed in vitro to various concentrations of N-methyl-N-nitrosourea and 3-methylcholanthrene. The incorporation of [3H]thymidine, [3H]uridine, and [3H]leucine into the exposed blastocysts was determined either immediately following exposure or after 18 hr of culture. The concentrations of N-methyl-N-nitrosourea or 3-methylcholanthrene used had no effect on blastocyst viability in either situation. There was a concentration-dependent decrease in the incorporation of the precursors into blastocysts exposed to N-methyl-N-nitrosourea which appears to be more pronounced after 18 hr in culture. This effect was not demonstrated for 3-methylcholanthrene, which requires metabolic activation for reactivity. Blastocysts exposed to N-methyl-N-nitrosourea were injected into the uterine horns of surrogate mothers; a significant increase in the resorption rate of these blastocysts was seen when compared with controls. Similar experiments were performed to determine birth rate. A dose-dependent decrease in birth rate was observed which correlated well with the effects on incorporation of thymidine, uridine, and leucine but not with the effect of implantation rate.