Rikuo Machinami

Ganglioside composition of the human cranial nerves, with special reference to pathophysiology of Miller Fisher syndrome.

Total ganglioside fractions from the human cranial nerves purified on a Phenyl Sepharose column, were given mild alkaline treatment, after which their composition and amounts of lipid-bound sialic acid were determined by HPTLC-densitometry with resorcinol as the coloring reagent. The total amounts of lipid-bound sialic acid were 156.5 ng/mg of wet tissue in the Ist cranial nerve (olfactory tract) and 131.9 ng/mg in the IInd nerve, greater than the amounts in the other nerves (99.1-120.0 ng/mg). The Ist, IInd, and VIIIth nerves had GM4, but not LM1. It may reflect their histological feature of the central nervous system. The IIIrd, IVth, and VIth nerves, as well as the IInd, had significantly higher percentages of GQ1b (11.6-13.2%) than the other nerves (5.2-8.4%). The high proportion of GQ1b specific to these three cranial nerves involved in the ocular movement lends support to the role of serum anti-GQ1b antibody in the pathogenetic mechanisms of ophthalmoplegia in Miller Fisher syndrome and Guillain-Barré syndrome.

Overexpression of p53 as a possible prognostic factor in human thyroid carcinoma.

A total of 110 cases of thyroid carcinomas were examined immunohistochemically to evaluate the overexpression of mutant forms of p53 protein in the light of their relationship with their histological subtypes. A polyclonal antibody, CM-1, was applied to the routine formalin-fixed, paraffin-embedded tissues for this survey. Overall, immunohistochemically detected p53 expression confined to the nucleus was identified in 22.7% of the thyroid carcinomas. A significant difference in the positivity of p53 among histological subtypes was noted; the positivity was 11.1% of the cases in well-differentiated papillary carcinoma, 14.3% in well-differentiated follicular carcinoma, 40.9% in poorly differentiated carcinoma, and 63.6% in undifferentiated carcinoma. No immunohistochemical positivity was found in adjacent non-neoplastic tissues or in benign lesions, including follicular adenoma, adenomatous goiter, and chronic thyroiditis. These results suggest that overexpression of p53 is not a responsible factor for the oncogenesis itself, but rather that it plays a crucial role in aggressive subtypes of thyroid carcinomas. Additionally, the distinct entity of poorly differentiated carcinoma, previously categorized in the well-differentiated carcinoma under the name of papillary or follicular carcinoma, was statistically confirmed.

Increased expression of vascular endothelial growth factor in human hepatocellular carcinoma

BACKGROUND/AIMS Angiogenesis is critical for the development and progression of solid tumors. The purpose of this study was to evaluate the possible role of vascular endothelial growth factor (which is considered to be one of the most important factors involved in tumor-associated angiogenesis), in human hepatocellular carcinoma. METHODS Vascular endothelial growth factor gene and protein expression were analyzed by means of Northern hybridization and immunohistochemical methods in 5 hepatocellular carcinoma cell lines. Secretion of vascular endothelial growth factor was evaluated by immunoblotting of conditioned medium of these hepatocellular carcinoma cells. Further, we compared the level of vascular endothelial growth factor expression in hepatocellular carcinoma tissues along with that in surrounding tumor-free tissues obtained from 20 patients. We also analyzed mRNA expression of Flt-1, one of the vascular endothelial growth factor specific high-affinity receptors, in hepatocellular carcinoma cell lines. RESULTS Northern hybridization analysis and immunohistochemistry revealed that all cultured hepatocellular carcinoma cells exhibited a high level of vascular endothelial growth factor mRNA. In addition, vascular endothelial growth factor secretion by Hep G2, one of the hepatocellular carcinoma cell lines, was shown by Western blot. In vivo, we observed vascular endothelial growth factor expression in both hepatocellular carcinoma and non-hepatocellular carcinoma tissues. However, in 12 of 20 cases, vascular endothelial growth factor mRNA levels were significantly up-regulated in hepatocellular carcinoma tissues. In the majority of cases (10 out of 12 cases) with abundant tumor vascularity, vascular endothelial growth factor mRNA up-regulation in hepatocellular carcinoma tissues was observed. We failed to detect Flt-1 mRNA expression in hepatocellular carcinoma cells. CONCLUSIONS This study suggests that the possibility that hepatocellular carcinoma cells overexpress the vascular endothelial growth factor gene and protein. These findings support the hypothesis that vascular endothelial growth factor is one of the important factors involved in the angiogenesis of hepatocellular carcinoma, and may even be involved in the development and/or progression of hepatocellular carcinoma itself.

Stepwise participation of p53 gene mutation during dedifferentiation of human thyroid carcinomas.

The spectrum of p53 gene mutations was investigated in thyroid carcinomas with respect to histopathological classification. In all histological subtypes of thyroid carcinoma that had previously revealed positivity in immunohistochemical staining for p53 protein, single-stranded conformation polymorphism analysis and direct sequencing were performed to detect point mutations between exons 5 and 8. In well differentiated papillary and follicular carcinomas, in which we had already known that 11.1 and 14.3% of the cases, respectively, revealed p53 overexpression as determined by immunohistochemistry, genetic aberrations were undetectable. In poorly differentiated carcinoma, in which 40.9% had revealed overexpression, two of six cases revealed point mutations at codon 244 in exon 7 and at codon 278 in exon 8. In undifferentiated carcinoma, in which 63.6% had revealed overexpression, four of six cases examined showed point mutations at codon 157 in exon 5, at codon 248 in exon 7, and at codon 273 and a two-base insertion between codons 266 and 267 in exon 8. These results strongly suggest the crucial role of p53 gene aberration and protein overexpression in a biologically aggressive subtype, possibly as a stepwise participation in the process of tumor dedifferentiation in human thyroid carcinomas.

Role of ras mutation in the progression of thyroid carcinoma of follicular epithelial origin.

The histological differentiation of thyroid carcinoma is known to correlate with prognosis. Ras oncogene mutations, which have been identified in various human cancers, have been suspected playing an important role in carcinogenesis and tumor progression. The purpose of this study was to clarify the mechanism of thyroid tumor progression, focusing on ras oncogenes. We examined ras mutations using nested polymerase chain reaction (PCR) and direct sequencing methods. The ras oncogene product was also examined immunohistochemically. Our results indicated that the incidence of ras mutations correlated with the histological differentiation of thyroid cancer. Three poorly differentiated carcinomas showed a higher rate of ras mutations than did 17 well-differentiated counterparts. Hot spots were not identified except for a relative accumulation of the N-ras gene at codon 61. There was a correlation between the immunoreactivity of the ras oncogene product and ras mutation, although the immunoreactivity of ras-p21 did not correlate with the histological differentiation. Mutation of the ras gene seemed to be one of the important events in the progression from well-differentiated carcinoma to poorly differentiated thyroid carcinoma.

A clinicopathological and immunohistochemical study of osteofibrous dysplasia, differentiated adamantinoma, and adamantinoma of long bones

A clinicopathological and immunohistochemical study of 12 cases of osteofibrous dysplasia (OFD), two cases of differentiated adamantinoma, and five cases of adamantinoma of long bones is presented. Although OFD and differentiated adamantinoma showed similar radiologic findings, differentiated adamantinoma was more likely to be a recurrent lesion than osteofibrous dysplasia and seemed to require a more extensive surgical procedure. Immunohistochemically, cytokeratin- and vimentin-positive cells were seen in both OFD and differentiated adamantinoma. The positive cells were scattered in the former, and were both scattered and nest-like in the latter. Both these lesions, however, were negative for epithelial membrane antigen. Excluding two cases of Ewing-like adamantinoma, the other three cases of adamantinoma were also positive for cytokeratin and vimentin. These results suggest that these three lesions share the same histogenetic origin. The two cases of Ewing-like adamantinoma differ from tibial adamantinoma in their radiological, histological and immunohistochemical aspects, and seem to constitute a distinct variant of adamantinoma with a different histogenesis.

Simultaneous expression of Cadherin-11 in signet-ring cell carcinoma and stromal cells of diffuse-type gastric cancer

We examined the expression of cadherin-11, a type II cadherin, in normal human tissues, cell lines and gastric cancer surgical specimens. Cadherin-11 was expressed widely in adult tissues, except the liver. It was expressed in fibroblast, mesothelial cell lines, and in only two signet ring cell carcinomas out of 16 various cancer cell lines. Cadherin-11 expression was detected in both signet ring cell carcinoma cells and surrounding fibroblasts of surgical specimens by in situ hybridization. These results suggest that cadherin-11 may play a role in the formation of diffuse-type gastric cancer through cancer-stromal interactions.

Anomalous Cadherin Expression in Osteosarcoma: Possible Relationships to Metastasis and Morphogenesis

Two isoforms of the human cadherin-11/OB-cadherin gene, the intact and the variant forms, had been isolated from an osteosarcoma cDNA library. The intact form has a typical cadherin structure, whereas the variant form, generated by alternative splicing, encodes a cytoplasmic domain that is completely different from that of the intact form and lacks a homophilic cell-cell adhesion ability. At the protein level, the secreted form generated from the intact cadherin-11 is present. We examined the expression of the intact and the variant forms of cadherin-11 in 23 primary and metastatic osteosarcomas from 22 patients by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, revealing that all 23 tumors in the patients expressed the variant form and three of them expressed it prominently. On the other hand, Western blot analyses of six tumors showed that the secreted form was strongly expressed, and furthermore, expression of N-cadherin was extremely low. Overexpression of the intact cadherin-11 cDNA in osteosarcoma cell lines demonstrated that the secreted form is derived from the intact form of cadherin-11 in osteosarcoma. Immunohistochemically, cadherin-11, N-cadherin, and beta-catenin were expressed at the cell surface of fetal osteoblasts, whereas in osteosarcoma cells, they were expressed only focally or weakly in the cytoplasm. Considering the function of cadherin in carcinomas, it is suggested that the anomalous expression of human cadherin-11 in osteosarcoma and the reduced expression of N-cadherin play a role in metastasis and the irregular morphology in the highly malignant mesenchymal tumor.

Advanced glycation end products in human optic nerve head

AIMS To localise advanced glycation end products (AGEs) in human optic nerve head. METHODS Optic nerve samples from 13 elderly individuals (seven diabetics and six non-diabetics) were obtained at necropsy. Pyrraline, an advanced glycation end product, was immunohistochemically localised in the optic nerve heads. RESULTS In the diabetic subjects, moderate to intense immunoreactivity for pyrraline was detected in sclera, pia mater, cribriform plates, connective tissues in the optic nerve, and around vessels in the optic nerve and pia mater. Immunoreactivity for pyrraline was also detected around retinal vessels. In the non-diabetic subjects, slight or no immunoreactivity for pyrraline was found in cribriform plates and around the optic nerve vessels. CONCLUSION Accumulation of AGEs in cribriform plates and around vessels in the optic nerve may contribute to the development of optic neuropathy in diabetic patients.

Expression and Function of the Splice Variant of the Human Cadherin‐11 Gene in Subordination to Intact Cadherin‐11

Cadherin‐11, a member of the type II classic cadherin subfamily, differs from type I family molecules such as P‐, E‐, and N‐cadherins. An isoform of the human cadherin‐11 gene, termed the variant form, encodes a truncated protein with a different cytoplasmic domain. The resulting protein does not possess any part of the cytoplasmic domain common to other cadherins. In the present study, analysis of the genomic organization of the cadherin‐11 gene revealed that an insertion of 179 bp in an intron generates an alternatively spliced form. The mRNA expression of the variant form of cadherin‐11 was examined in normal tissues by reverse transcription‐polymerase chain reaction and/or Northern blot analyses. The variant form was expressed in the heart, brain, placenta, lung, and bone, but not in the kidney, skeletal muscle, pancreas, and liver. Western blot analyses revealed that the variant form is expressed as an 85 kDa protein, and that an additional secreted form also exists as an 80 kDa protein originated from cleavage of the intact form. Gene transfer of the variant form into L cells demonstrated that it lacked the adhesion properties characteristic of the intact form of cadherin‐11 but enhanced the activity of Ca2+‐dependent adhesion of the intact form of cadherin‐11. The variant was expressed on the surface together with the intact form and stabilized the interaction between the intact form and β‐catenin. These findings suggest that expression of the variant form of human cadherin‐11 may regulate the intact cadherin‐11–mediated adhesion and alter the morphogenetic processes during mesenchymal cell differentiation including osteoblasts.