Mutational screening of APP gene in patients with early-onset Alzheimer disease utilizing mismatched PCR-RFLP

To elucidate the frequency of mutations of the β/A4 amyloid protein precursor (APP) gene in early-onset Alzheimer disease, we designed a mismatched PCR-RFLP that can identify all kinds of missense mutations at codon 717 in addition to the seven kinds of known mutations at exon 17. When we screened mutations at exon 17 utilizing this method and the double missense mutations at exon 16 of the APP gene by PCR-RFLP, no cases revealed mutations of the APP gene among 13 familial and 54 sporadic cases, except one family (OS-1) that had previously been reported and used as a positive control of APP717(Val → Ile). Our results support the hypothesis that mutations in the APP gene are not major causes in early-onset Alzheimer disease.     

The structure of the avian fast skeletal muscle troponin T gene: seven novel tandem-arranged exons in the exon x region

To elucidate the mechanism that produces enormous molecular diversity in troponin T (TnT) of fast skeletal muscle, we determined the 5-half genomic sequence of the chicken fast muscle TnT gene. The sequence of ca. 16 kb included seven exons (exons 1, 2, 3, 4, w, 5, and 6), which have been reported previously and presumed by sequencing TnT cDNAs. Additionally we found six 15 nt and one 18 nt sequences in the region between exons 5 and 6 (i.e. the exon x region). They were encompassed by consensus splice donor and acceptor sites and preceded by putative branch sites, and designated herein as exons xa to xg. Our result shows that the sequence derived from exons x1, x2, and x3, the exons presumed previously by cDNA sequencing, is actually encoded by the seven exons xa to xg, establishing the precise gene structure in the exon x region. Based on our data, together with that on the 3-half genomic sequence of the quail fast muscle TnT gene, we conclude that the avian fast skeletal muscle TnT gene includes 27 exons, 16 of which are alternatively spliced.     

Regulation by Ca2+-calmodulin of the actin-bundling activity ofPhysarum 210-kDa protein

Summary From the plasmodia of a lower eukaryote,Physarum polycephalum, we have previously purified a 210-kDa protein that showed similar properties to those of smooth muscle caldesmon. Further characterization of the 210-kDa protein revealed that it bundled actin filaments. This bundling activity was inhibited by calmodulin in the presence of Ca²⁺. Unlike smooth muscle caldesmon, the 210-kDa protein bundled actin filaments whether or not a reducing agent, such as dithiothreitol, was present. The protein was shown to have two (or more) different actin-binding sites which were classified into salt-sensitive and salt-insensitive sites. Electron microscopy revealed that the 210-kDa protein was an elongated molecule (mean length, 97 ± 25 nm) which was bent in the middle. The Stokes radius and sedimentation coefficient of the 210-kDa protein were 130 Å and 2.9 S, respectively. An immunofluorescence study revealed that the 210-kDa protein colocalized with the bundles of actin filaments in thin-spread preparations ofPhysarum plasmodia, suggesting that the 210-kDa protein was regulating the appearance and disappearance of the actin bundles that are associated with the contraction-relaxation cycle of the plasmodia.     

Calcium pump expression in human bone and soft tissue tumours

Ninety-one cases of human bone and soft tissue tumours were studied for calcium pump expression by strepto-avidin-biotin immunohistochemical staining with a monoclonal antibody against sarcoplasmic reticulum calcium-ATPase (mAb6F5). Two out of 5 cases of embryonal rhabdomyosarcoma, 1 out of 5 cases of biphasic synovial sarcoma, 4 of 4 cases of chordoma and all of 3 chondrosarcoma cases were positive for mAb6F5. Although this novel monoclonal antibody can be used as a marker of myogenic tumours, the present positive result for endoplasmic reticulum calcium-ATPase (calcium pump) in other tumours including chordoma, chondrosarcoma and synovial sarcoma indicates a wider immunoreactivity. The findings further suggest that intracellular calcium may play an important role in cell proliferation and/or differentiation.     

HISTOPATHOLOGICAL STUDIES ON THROMBOTIC MICROANGIOPATHY WITH SPECIAL REFERENCES TO THREE CASES ASSOCIATED WITH ACUTE PROMYELOCYTIC LEUKAEMIA

Five autopsy cases of thrombotic microangiopathy, including 3 cases associated with acute promyelocytic leukaemia, were examined macroscopically, light-and electronmicroscopically.
The so-called hyaline thrombi In thrombotic microangiopathy were composed of fibrin and its degenerative products. Thrombocytes and other blood cells were not seen in the thrombi.
At the site of the formation of a thrombus, there was no conspicuous change in the walls of the capillaries and arterioles. It was considered, therefore, that the intravascular deposition of fibrin was the primary event in the development of thrombotic microangiopathy.
In regard to the distribution and morphologic findings, there was no basic difference between the microthrombi in cases associated with acute promyelocytic leukaemia and those without it.
The bone marrow and some other organs in cases of thrombotic microangiopathy associated with acute promyelocytic leukaemia macroscopically revealed a green colour. Many thrombi composed of leukaemic cells and fibrin were found in the pulmonary arteries of these cases. Furthermore, prominent erythrophagocytosis in the bone marrow and lymph nodes was a common finding in these cases.     

Emergence of rifampin-resistant Rhodococcus equi with several types of mutations in the rpoB gene among AIDS patients in northern Thailand

The antimicrobial susceptibilities of 30 Rhodococcus equi isolates obtained from 30 patients between 1993 and 2001 in northern Thailand were investigated. The MICs showed a tendency toward resistance to various antibiotics but sensitivity to imipenem, minocycline, vancomycin, and teicoplanin (MICs, /=64 micro g/ml) to rifampin. PCR amplification and DNA sequencing of the rpoB gene and molecular typing by pulsed-field gel electrophoresis (PFGE) were performed for eight R. equi isolates from eight AIDS patients with pneumonia or lung abscess caused by R. equi between 1998 and 2001, including one low- and three high-level rifampin-resistant isolates. As a result, two high-level rifampin-resistant strains with PFGE pattern A had a Ser531Trp (Escherichia coli numbering) mutation, and one high-level rifampin-resistant strain with PFGE pattern B had a His526Tyr mutation, whereas one low-level rifampin-resistant strain with PFGE pattern C had a Ser509Pro mutation. Four rifampin-susceptible strains with PFGE patterns D and E showed an absence of mutation in the rpoB region. Our results indicate the presence of several types of rifampin-resistant R. equi strains among AIDS patients in northern Thailand.     

Evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver dysfunction, and its prevalence has markedly increased; however, the mechanisms involved in the pathogenesis of NAFLD have not been thoroughly investigated in humans. In this study, we evaluated the expression of fatty acid metabolism-related genes in NAFLD. Real-time RT-PCR was performed using liver biopsy samples from 12 NAFLD patients. The target genes studied were: acetyl-CoA carboxylase (ACC) 1, ACC2, and fatty acid synthase (FAS) for the evaluation of de novo fatty acid synthesis; carnitine palmitoyltransferase 1a (CPT1a), long-chain acyl-CoA dehydrogenase (LCAD), and long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase alpha (HADHalpha) for beta-oxidation in the mitochondria; peroxisome proliferator-activated receptor- (PPAR-) alpha and cytochrome P450 2E1 (CYP2E1) for oxidation in peroxisomes and microsomes (endoplasmic reticulum) respectively; and diacylglycerol O-acyltransferase 1 (DGAT1), PPAR-gamma, and hormone sensitive lipase (HSL) for triglyceride synthesis and catalysis. In NAFLD, expression of ACC1 and ACC2, but not FAS was increased, indicating that de novo fatty acid synthesis is enhanced in NAFLD. In contrast, expression of CTP1a, a rate-limiting enzyme, was remarkably decreased, indicating that beta-oxidation in the mitochondria was decreased, although the expression of LCAD and HADHalpha was increased. Expression of PPAR-alpha was increased, whereas that of CYP2E1 was reduced. The expression of DGAT1, PPAR-gamma, and HSL was enhanced. These data suggest that in NAFLD, increased de novo synthesis and decreased beta-oxidation in the mitochondria lead to accumulation of fatty acids in hepatocytes, although the extent of oxidation in peroxisomes and microsomes remains unclear.     

Immunocytochemical localization of secretogranin III in the endocrine pancreas of male rats

Secretogranin III (SgIII), a member of the granin protein family, is expressed specifically in neuronal and endocrine cells. To examine the precise localization of SgIII in the endocrine pancreas, pancreatic tissues of rats were analyzed immunocytochemically with a polyclonal anti-serum raised against rat SgIII. By light microscopy of semithin sections, the immunoreactivity for SgIII was readily detected in pancreatic A- and B-cells, faintly so in D-cells, and not at all in the exocrine pancreas. By immunoelectron microscopy, immunogold particles indicative of SgIII were observed in the peripheral regions of secretory granules, and universally in the pancreatic endocrine cells. Morphometrical analyses indicated that SgIII is most preferentially localized in the periphery of the secretory granule among granins. These findings suggest that SgIII is closely associated with the secretory granule membrane, serving to anchor the aggregates of other soluble constituents to the membrane.     

Cytokeratin expression in normal human thymus at different ages

Subsets of thymic epithellal cells were examined Immuno-histochemically to determine whether or not their pheno-types change during thymic growth and at early involution in terms of cytokeratin (CK) expression. Five monoclonal antibodies specific for CK4, CK8, CK13, CK18 and CK19 were used and applied for 16 neonatal, three Infantile and one adult thymus speeimen, which had been obtained at autopsy, that were normal macroscopically and microscopicaily. CK4, CK8, CK13, CK18 and CK19 were expressed simultaneously in the cortex, medulla and subcapsular area with the exception of CK4, which showed expression on the adult thymus. Light and electron microscopy showed that CK8 and CK19 expression was overlapped. Thus, It was thought that CK8 and CK19 formed complexes in the cytoplasm of thymic epithelial cells. The Immunoreactivity to CK4, CK13 and CK18 were attenuated or disappeared In the subcapsular area during the early involution stage. Interestingly, two patterns of CK18 expression were observed in the neonatal and Infantile thymus tissues, which Indicated that the thymic microenvironment was changeable even under normal conditions.