Single sperm analysis of the CAG repeats in the gene for Machado-Joseph disease (MJD1): evidence for non-Mendelian transmission of the MJD1 gene and for the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability

To investigate the mechanism of the meiotic instability of expanded CAG repeats in the gene for Machado-Joseph disease (MJD1), we analyzed the CAG repeat sizes of 1036 single sperm from six individuals with Machado- Joseph disease (MJD). The segregation ratio between single sperm with an expanded allele and those with a normal allele is significantly different (P <0.0001) from the expected 1:1 segregation ratio, which demonstrates segregation distortion of expanded alleles in male meiosis. In single sperm from individuals with the [expanded (CAG)n- CGG]/[normal (CAG)n-GGG] genotype, significantly greater instability of the CAG repeat was observed compared with single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype (F-test, P <0.001). These findings in single sperm confirm non-Mendelian transmission of the MJD1 gene and the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability of the CAG repeats in the MJD1 gene, which have been observed in clinical and genetic studies. Our results indicate similarities and dissimilarities between MJD and Huntington's disease or myotonic dystrophy in terms of the inter-allelic interaction, segregation distortions and size distribution of trinucleotide repeats in mutant alleles. Further study is required to determine whether there is a common mechanism underlying the instability of the triplet repeats in 'triplet repeat diseases'.      

Thymic sarcomatoid carcinoma with skeletal muscle differentiation: report of two cases, one with cytogenetic analysis

AIMS: Malignant thymic tumour histologically resembling a soft tissue sarcoma is extremely rare and defined as sarcomatoid carcinoma in the recent World Health Organization (WHO) classification. We report two such cases in which the tumour cells showed a prominent rhabdomyoblastic differentiation and analyse whether these tumours retain an epithelial nature at least in part. METHODS AND RESULTS: One tumour occurred in a 51-year-old man (Case 1) and the other in a 40-year-old woman (Case 2). Microscopically, both tumours consisted essentially of two types of tumour cells: spindle and large round cells, with no apparent epithelial components. Osteosarcomatous small foci were also found in Case 2. Immunohistochemically, desmin and muscle-specific actin were positive in the majority of both types of tumour cells, whereas myogenin was predominant in the spindle cells and myoglobin in the large round cells. Some of both types of cells expressed cytokeratin with co-expression of myoglobin in the large round cells, but with no myogenin in the spindle cells. Some cytokeratin-positive spindle cells were also negative for desmin. Ultrastructural examination of a recurrent tumour in Case 2 revealed some epithelial features among the spindle cells. Cytogenetic study of the same tumour showed a complex abnormality including der(16)t(1;16)(q12;q12.1), an identical pattern previously reported in a case of thymic squamous cell carcinoma. CONCLUSIONS: The findings support the definition in the WHO classification of sarcomatoid carcinoma that includes purely sarcomatous tumour as in the present cases. Occurrence of this type of tumour may indicate a relationship between thymic epithelial cells and myoid cells and/or a potential for divergent differentiation in thymic epithelial tumours.     

Potential role of phosphodiesterase 7 in human T cell function: comparative effects of two phosphodiesterase inhibitors

Even though the existence of phosphodiesterase (PDE) 7 in T cells has been proved, the lack of a selective PDE7 inhibitor has confounded an accurate assessment of PDE7 function in such cells. In order to elucidate the role of PDE7 in human T cell function, the effects of two PDE inhibitors on PDE7A activity, cytokine synthesis, proliferation and CD25 expression of human peripheral blood mononuclear cells (PBMC) were determined. Recombinant human PDE7A was obtained and subjected to cyclic AMP-hydrolysis assay. PBMC of Dermatophagoides farinae mite extract (Df)-sensitive donors were stimulated with the relevant antigen or an anti-CD3 monoclonal antibody (MoAb). PBMC produced IL-5 and proliferated in response to stimulation with Df, while stimulation with anti-CD3 MoAb induced CD25 expression and messenger RNA (mRNA) synthesis of IL-2, IL-4 and IL-5 in peripheral T cells. A PDE inhibitor, T-2585, which suppressed PDE4 isoenzyme with high potency (IC50 = 0.00013 microM) and PDE7A with low potency (IC50 = 1.7 microM) inhibited cytokine synthesis, proliferation and CD25 expression in the dose range at which the drug suppressed PDE7A activity. A potent selective inhibitor of PDE4 (IC50 = 0.00031 microM), RP 73401, which did not effectively suppress PDE7A (IC50 > 10 microM), inhibited the Df- and anti-CD3 MoAb-stimulated responses only weakly, even at 10 microM. PDE7 may play a critical role in the regulation of human T cell function, and thereby selective PDE7 inhibitors have the potential to be used to treat immunological and inflammatory disorders.     

Contribution to the morphology of mitochondria with prismatic cristae in astrocytes of the inferior olivary nucleus of the cat

The internal structure of mitochondria with prismatic cristae in astrocytes of the inferior olivary nucleus of the adult cat was examined. The interior of the mitochondria with prismatic cristae cut in cross-section can be divided into 2 areas: (1) a peripheral, rather structureless area, and (2) a central, highly organized area. The former is composed of the inner mitochondrial membrane and a small number of peripheral cristae protruding from the membrane and scattered dots. The latter is composed of numerous prismatic cristae arranged in almost hexagonal spacing and many dots which probably represent the transverse configuration of filaments oriented parallel to the cristae embedded in the matrix. For future comparative analysis, various quantitative observations on the fine structure of the central, highly organized area are described and discussed.     

Cavernous structures composed of hepatocytes in experimentally induced hepatocellular carcinoma

Cavernous structures composed of hepatocytes were observed for the first time in trabecular-patterned hepatocellular carcinomas by light, scanning and transmission electron microscopy. The carcinomas were induced by a single dose of diethylnitrosamine combined with subsequent administrations of 2-acetylaminofluorene and partial hepatectomy. The cavernous structures had numerous cavities walled by flat hepatocytes. In the cavities, dendritic hepatocytes, connected with each other by their long and slender processes, formed a network and were attached to the fenestrated sinusoidal endothelium via intercellular spaces between the flat hepatocytes. Dendritic hepatocytes had smooth surfaces, prominent nucleoli, decreased glycogen, and well organized cisternae. Dilated bile canaliculi were sometimes seen among flat and dendritic cells. Dendritic cells showed more morphological dearrangement than flat cells. Blood cells were observed in the cavities. Kupffer cells were not seen in the sinusoids among the cavernous structures. The cavernous structures in the hepatocellular carcinomas resembled the hepatocyte arrangement in fetal liver in which intercellular spaces of hepatocytes are extremely widened.     

Disseminated Atypical Mycobacteriosis

Two cases of disseminated infection caused by Mycobacterium intracellulare were reported and discussed. In the first case, the patient was a fifty-seven-year-old male who complained of general fatigue, weight loss, and fever. Biopsy of the right inguinal lymph nodes and the liver revealed infiltration by histiocytes engulfing many acid-fast bacilli. At autopsy an egg-sized abscess was found is the region of the right Iliac lymph nodes. Histological examination showed histiocytic infiltration in the abscess wall, neighboring lymph nodes, liver, and spleen. In the second case, the patient was a four-year-old boy, who had persistent fever and splenomegaly. Splenectomy was performed and histological examination of the spleen revealed multiple nodular infiltration by swollen histiocytes with many acid-fast bacilli in their cytoplasm. The bone marrow aspirates and liver tissue obtained in the necropsy also showed many histiocytes containing many acid-fast bacilli. The authors emphasized the importance of paying special attention to atypical mycobacteriosis in feverish patients having lesions with a proliferation of histiocytes.     

Rapid tumor regression and induction of tumor-regressing activity in serum by various immune-modulating agents

A rapid decrease in the number of tumor cells from S180 tumors was caused by several antitumor polysaccharides including the beta (1-3)glucans lentinan and TAK-N and a mannoglucan MGA, but not by those lacking antitumor activity. MGA was demonstrated to induce potent tumor-regressing activity in the serum of tumor-bearing mice similar to that reported previously to be induced after an injection of CM-TAK, a carboxymethylated beta (1-3)glucan. It is probable that the induction of rapid regression of established tumors is a phenomenon common to antitumor polysaccharides and some microbiological products and that the tumor-regressing factor in the serum underlies a common mechanism.     

A combination analysis of p53 and p21 in gastric carcinoma as a strong indicator for prognosis

We studied p53 and p21 expression simultaneously in gastric carcinoma tissues to investigate the clinical significance of p53-p21 pathway in this disease. One hundred sixty-four primary gastric carcinoma specimens were immunohistochemically stained for p53 and p21 protein, and clinicopathological features of the cases were examined. P53 was stained negatively, while p21 was stained positively in each normal stomach epithelium. P53, and p21 positive staining was observed in 82 (50%) and 61 (37.2%) tumors, respectively. Unexpectingly, no correlation was found between p53 and p21 staining status. Tumors demonstrating preserved p53-p21 pathway [p53(-)/p21(+)], observed in 20.1% of the tumors, displayed less aggressive characteristics, and no recurrent disease after curative resection. While tumors demonstrating disrupted p53-p21 pathway [p53(+)/p21(-)], observed in 32.9% of the tumors, displayed significantly more aggressive characteristics, poorer survival and higher recurrence rate than the tumors demonstrating other staining patterns. P53-p21 pathway was widely altered in gastric carcinomas. The combined evaluation of p53 and p21 expression in gastric carcinoma tissues is suggested to have clinical importance by indicating not only the malignant potential of each tumor, but also the prognosis of this disease.     

Radiation-induced reactive oxygen species formation prior to oxidative DNA damage in human peripheral T cells

Previously, we demonstrated that human peripheral T lymphocytes revealed early apoptotic changes (annexin V-positive) and late apoptotic changes (propidium iodide-positive), at 13 and 24 h, respectively, after irradiation of 5 Gy. Changes in mitochondrial membrane potential were observed at 10 h after irradiation of 5 Gy. Subsequently, mitochondrial cytochrome c-release was confirmed. In order to elucidate the mechanism which acts prior to the mitochondrial membrane potential changes, we examined in the previous study the radiation dose and the timing of oxidative DNA damage induced in human peripheral T lymphocytes following 10 MV X-ray irradiation. As a result, the production of 8-oxoguanine, i.e., the product of oxidative DNA damage, was clearly identified starting at 10, 6, and 3 h, after 2, 5, and 20 Gy of irradiation, respectively. Therefore, we examined in the present study reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation.