Norifumi Ueda

Altered carbohydrate composition in colorectal adenomas and carcinomas: Histochemical characterization of N-acetylgalactosamine,l-fucose, and o-acetylated sialic acid

Changes of surface sugar residues in the large intestinal mucosa may be associated with malignant transformation and may be of importance in differentiating borderline lesions. To compare these changes in normal mucosa, adenomas, and carcinomas of the large intestine, we investigated modifications of carbohydrate composition, such as those of N-acetyl-galactosamine (GalNac),l-fucose, ando-acetylated sialic acid, by histochemical staining withDolichos biflorus agglutinin (DBA) andUlex europaeus agglutinin-1 (UEA-1) lectins, and with Cullings periodic acid-thionin Schiff/potassium hydroxide/periodic acid-Schiff (PAT/KOH/PAS), respectively. For stable staining, the sections stained with DBA and UEA-1 were pretreated with potassium hydroxide and neuraminidase. We conclude that the pattern of the two lectin stainings in carcinomas is quite different from that in normal mucosa and adenomas, and that it shows the carcinomatous features in some cases of adenoma with severe atypia (borderline lesions). In contrast, PAT/KOH/PAS staining demonstrates differences between normal mucosa and adenomas rather than differences between adenomas and carcinomas.

Induction of myasthenia by immunization against muscle-specific kinase

Muscle-specific kinase (MuSK) is critical for the synaptic clustering of nicotinic acetylcholine receptors (AChRs) and plays multiple roles in the organization and maintenance of neuromuscular junctions (NMJs). MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering at the postsynaptic membrane. Although autoantibodies against the ectodomain of MuSK have been found in a proportion of patients with generalized myasthenia gravis (MG), it is unclear whether MuSK autoantibodies are the causative agent of generalized MG. In the present study, rabbits immunized with MuSK ectodomain protein manifested MG-like muscle weakness with a reduction of AChR clustering at the NMJs. The autoantibodies activated MuSK and blocked AChR clustering induced by agrin or by mediators that do not activate MuSK. Thus MuSK autoantibodies rigorously inhibit AChR clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of MG.

NEDD8 protein is involved in ubiquitinated inclusion bodies.

Proteolysis by the ubiquitin–proteasome system is considered to play a pathological role in several degenerative diseases that involve ubiquitinated inclusion bodies. In recent years, several ubiquitin‐like proteins have been isolated, but it is uncertain whether their roles are associated with protein degradation through the ubiquitin–proteasome system. NEDD8 (neural precursor cell‐expressed and developmentally down‐regulated gene), which consists of 81 amino acid residues, possesses the highest sequence similarity to ubiquitin. Recent studies have indicated that NEDD8 is covalently ligated to cullin family proteins, which are components of certain ubiquitin E3 ligases, by a pathway analogous to that of ubiquitin. Thus, by focusing on the structural and functional association between NEDD8 and ubiquitin, it would be of interest to know whether the NEDD8 system is involved in pathological disorders of the ubiquitin–proteasome system. This study has examined the immunohistochemical distribution of NEDD8 protein by using a highly purified antibody in normal tissues and in tissues known to contain ubiquitinated inclusions. NEDD8 protein expression was widely observed in most types of tissues. Furthermore, accumulation of the NEDD8 protein was commonly observed in ubiquitinated inclusion bodies, including Lewy bodies in Parkinsons disease, Mallory bodies in alcoholic liver disease, and Rosenthal fibres in astrocytoma. Two of ten cases of neurofibrillary tangles and senile plaques from patients with Alzheimers disease showed intense staining for NEDD8 as well as for ubiquitin. These findings suggest the possibility that the NEDD8 system is involved in the metabolism of these inclusion bodies via the ubiquitin–proteasome system. Copyright

Telomerase components as a diagnostic tool in human oral lesions

Telomerase activity is considered to be a diagnostic marker of malignancy since most malignant cells express this activity and most somatic cells do not. However, the detection of telomerase activity is rather complicated and is affected by many factors. Recently, human telomerase components were cloned and found to consist of 3 subunits. We assessed which component of telomerase best correlates with malignancy in order to study the possibilities for developing a new diagnostic marker. Telomerase activity was measured by a telomeric repeat amplification protocol (TRAP) assay, and the telomerase components hTR, hTRT-mRNA and TP1-mRNA were detected by the reverse transcriptase-polymerase chain reaction (RT-PCR). Twenty-five of 26 oral malignant lesions, 9 of 22 benign lesions and none of 19 normal control tissues exhibited distinct telomerase activity. hTR and TP1-mRNA expression levels were detected in all malignant lesions and normal control tissues and had no significant correlation with the telomerase activity results. In contrast, hTRT-mRNA expression was closely associated with telomerase activity. All lesions expressing hTRT were telomerase positive. In addition, some samples of dysplastic lesions, benign tumors, lichen planus and normal mucosa exhibiting poor telomerase activity revealed weak expression of hTRT. Expression levels of hTRT-mRNA positively correlated with clinical and pathological findings. Detection of hTRT-mRNA by RT-PCR appeared to be more sensitive for telomerase than measurement of telomerase activity by the TRAP assay. Detection of hTRT-mRNA may provide information useful in the diagnosis of oral malignancies.

Incidence of p53 and Ha‐ras gene mutations in chemically induced rat mammary carcinomas

To determine whether p53 alterations, which are frequent in human breast cancers, are also common in rat mammary tumors, we examined 40 tumors from 24 rats treated with 7,12‐dimethylbenz[a]anthracene (DMBA) and 34 tumors from 14 rats treated with N‐nitroso‐N‐methylurea (NMU) (an N‐nitroso compound). DMBA and NMU are known genotoxic mutagens. The entire coding regions of the p53 and Ha‐ras genes were examined for mutations by polymerase chain reaction single‐strand conformational polymorphism analysis and by direct sequencing. One of the 40 DMBA‐induced mammary tumors had a p53 mutation, a single‐base substitution (???AGC→???GGC) at codon 307, resulting in an amino‐acid change from Ser to Gly. No mutations were found in NMU‐induced tumors. The incidence of Ha‐ras gene mutation was 79% (27 of 34) at codon 12 in the NMU group and 23% (nine of 40) at codon 61 in the DMBA group. Thus, p53 mutation, in contrast to Ha‐ras mutation, did not seem to be a prerequisite for carcinogenesis in chemically induced rat mammary tumors.

MuSK antibodies in AChR Ab-seropositive MG vs AChR Ab-seronegative MG

Hoch et al.1 recently reported a novel specific target antigen, the muscle-specific receptor tyrosine kinase (MuSK), for the autoantibodies in acetylcholine receptor antibody (AChR-Ab)-seronegative myasthenia gravis (MG) patients. MuSK is expressed at the postsynaptic membrane of neuromuscular junctions and has multiple roles in the clustering of AChR by the agrin secreted from motoneuron terminus. Therefore, autoantibodies against MuSK may cause severe inhibition of AChR clustering, leading to reduced numbers, altered distribution of AChRs in mature muscle, or both. MuSK antibodies are found in ∼37.5 to 70% of patients with AChR-Ab-seronegative MG but not in those with AChR-Ab-seropositive MG, a clear differentiation of the two forms of disease.1–5⇓⇓⇓⇓ Furthermore, a lack of detectable MuSK antibodies was reported in MG patients with thymoma. To evaluate MuSK antibodies clinically in patients with MG, we developed a …

Expression and phosphorylation of TOPK during spermatogenesis

Among normal organs and tissues, the MAPKK‐like mitotic protein kinase TOPK is expressed exclusively in the testis. We analyzed the expression and phosphorylation of TOPK to address the functional role of this kinase during spermatogenesis. TOPK protein is expressed mainly in the cytosol of spermatocytes and spermatids, but not in spermatids and spermatogonia in situ. TOPK‐Thr‐9, a cdk1/cyclin B target residue, was specifically phosphorylated during mitotic and meiotic phases, while TOPK‐Thr‐198, a key amino acid for the ATP pocket, was constantly phosphorylated irrespective of the cell cycle. These data indicate that spermatogenic germ cells with vital proliferation activity express TOPK. As TOPK‐Thr‐9 was phosphorylated during both mitosis and meiosis, TOPK was indicted to play a role in cytokinesis and/or chromosomal segregation but not in DNA replication.

Myasthenia gravis experimentally induced with muscle-specific kinase.

Here we present the first evidence that muscle‐specific kinase (MuSK) antigen can cause myasthenia in animals. MuSK is expressed at the postsynaptic membranes of neuromuscular junctions (NMJ) and forms complexes with acetylcholine receptors (AChR) and rapsyn. MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering and subsequent formation of NMJ in embryos. Notably, autoantibodies against MuSK were found in a proportion of patients with generalized myasthenia gravis (MG) but without the characteristic AChR autoantibodies. However, MuSK autoantibodies had no known pathogenic potential, and animals immunized with purified MuSK proteins did not develop MG in former studies. In contrast, we have now injected rabbits with MuSK ectodomain protein in vivo and evoked a MG‐like muscle weakness with a reduction of AChR clustering at the NMJ. Our results showed that MuSK is required for maintenance of synapses and that interference with that function by MuSK antibodies causes myasthenic weakness. In vitro, AChR clustering in myotubes is induced by agrin and agrin‐independent inducers, which do not activate MuSK. Neither the receptor nor the activation mechanisms of AChR clustering induced by agrin‐independent inducers has been identified with certainty, but MuSK autoantibodies in myasthenic animals inhibited both agrin and agrin‐independent AChR clustering. MuSK plays multiple roles in pre‐patterning of the postsynaptic membrane before innervation and formation of NMJ in embryos. Some of these mechanisms may also participate in the maintenance of mature NMJ. This model system would provide new knowledge about the molecular pathogenesis of MG and MuSK functions in mature NMJ.

Modulation of S‐100 genes response to growth conditions in human epithelial tumor cells

Many new members of the S‐100 genes are known to be associated with cell differentiation, malignant transformation, and cell cycle. Of the S‐100 genes examined In the present study, calcyclin, calpactin I light chain and calvasculln were expressed In most human epithellal tumor cells, and their expression levels differed according to various growth conditions. Their transcribed levels differed depending on each cell line, but their expression patterns were similarly changed under growth‐modulatory conditions. Their messenger RNA levels increased parallel to the S phase population of cells, and decreased at G1/G2 phases. In contrast, this expression diminished in tumor cells under growth‐Inhibitory conditions, such as treatment with topolsomerase II inhibitor VP‐16 or phorbol 12‐myristate 13‐acetate.

Identification of anti-α-enolase autoantibody as a novel serum marker for endometriosis

Diagnosis of endometriosis needs invasive maneuvers. New serum marker that possesses both high sensitivity and high specificity has long been desired. To establish novel serum marker for endometriosis, serum autoantibodies (autoAbs) were investigated using proteomic approach. AutoAbs in sera of endometriotic patients and healthy controls were analyzed using a mesothelial cell line, 2‐DE and Western blotting. Proteins in reacted spots were identified using MALDI TOF‐MS with MASCOT analysis. ELISAs were established using recombinant proteins and autoAb‐titers were estimated in sera of endometriotic patients, disease and healthy controls. Several autoAbs were identified. Anti‐α‐enolase (Eno1)‐autoAb levels in endometriotic patients were significantly elevated compared with both healthy and disease controls. Sensitivity and specificity of serum anti‐Eno1‐autoAb was nearly comparable to serum CA125. When anti‐Eno1‐autoAb and CA125 assays were combined, diagnostic sensitivity and accuracy improved. Serum anti‐Eno1‐autoAb can be a new serum endometriotic marker and it is useful as a supplement assay for CA125. This study validates further clinical evaluation of this novel marker.