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'.      

Double antibody enzyme immunoassay for the quantitation of adenosine 3', 5' -cyclic monophosphate (cyclic AMP) and guanosine 3', 5'-cyclic monophosphate (cyclic GMP) in tissue and plasma

A sensitive double antibody enzyme immunoassay for the quantitation of cyclic AMP and cyclic GMP is presented. Specific antisera to each nucleotide were raised in rabbits by immunization with succinyl cyclic nucleotide-human serum albumin conjugates. For competitive reaction, antibodies were incubated with a mixture of succinyl cyclic nucleotide labelled with beta-D-galactosidase and unlabelled succinylated standard or sample cyclic nucleotides. The antibody-bound enzyme-hapten was separated from free hapten by anti-rabbit IgG immobilized to a polystyrene ball. Activity of the enzyme on the solid phase was fluorometrically determined. The assay system made it possible to ascertain values as low as 5 fmole of cyclic AMP or cyclic GMP. Cyclic nucleotides in plasma could be accurately determined by this method without requiring a deproteinizing reagent as the first step of assay.     

Proliferating cell nuclear antigen in malignant and pre-malignant lesions of epithelial origin in the oral cavity and the skin: an immunohistochemical study

Summary Proliferating cell nuclear antigen (PCNA) is a nuclear protein synthesized in the late G₁ and S phase of the cell cycle and immunohistochemical detection of the protein represents a useful marker for the proliferating fraction of cells in tissue specimens. A series of malignant and pre-malignant lesions of the oral cavity and skin were evaluated by the streptavidin biotin immunoperoxidase method for detection of this protein. Monoclonal anti-PCNA antibody (PC 10) labelled proliferating cells in all cases with varying intensity of nuclear staining. In squamous cell carcinoma (n=48), PCNA positivity correlated with the differentiation and atypia of the tumour cells; however, in poorly differentiated tumours, the relationship between PCNA expression and proliferation was lost. Basal cell carcinoma showed an increased growth fraction in tiny epithelial nests (mean 43.8, SD 6.0,n=20) than in neoplastic basal cells (mean 30.1, SD 6.9,n=8). The growth fractions were significantly higher in the pre-malignant lesions (leukoplakia, mean 22.3, SD 7.7,n=14; Bowen's disease, mean 45.2, SD 11.7,n=12; senile keratosis, mean 41.2, SD 7.0,n=12) than in the normal mucosa (mean 9.8, SD 4.9,n=10), suggesting that cellular growth fractions correlate with the degree of dysplasia in pre-malignant lesions.     

Atypical Glomus Tumor in the Mediastinum: A Case Report with Immunohistochemical and Ultrastructural Studies

A case is reported of atypical glomus tumor occurring in the posterior inferior mediastinum of a 26-year-old woman complaining of severe back pain. The tumor was composed of atypical small, round tumor cells with scattered mitotic figures. In addition to sheet-like, diffuse proliferation of the tumor cells, some areas of the tumor contained small “glo-moid” cells arranged in organoid and hemangiopericytoma-like patterns. Immunohistochemically, many tumor cells were positive for muscle-type actins and a few cells were focally positive for desmin. Ultrastructural studies revealed smooth muscle features of tumor cells, that is, pinocytotic vesicles, external laminas, dense plaques, and occasional thin filaments with dense bodies. The patient remained well for 5 years and 4 months after the operation without additional radiation and chemotherapy. The tumor was diagnosed as an atypical, or low-grade malignant, glomus tumor morphologically. It seems important to recognize the presence of this type of tumor in sites other than extremities and to differentiate it from other malignant small, round cell tumors.     

Ultraviolet B induces mast cell apoptosis: a hypothetical mechanism of ultraviolet B treatment for uraemic pruritus.

The pathogenesis of uraemic pruritus is unclear, although there is some evidence that an increased number of skin-infiltrating mast cells may play a role. Ultraviolet B reduces itchy sensation of uraemic patients by leading to depletion of cutaneous mast cells. This study presents data that both broad-band and narrow-band ultraviolet B irradiation are able to induce apoptosis in transformed mast cells (murine mastocytoma cell line P815) in a dose-dependent manner at a time point of 24 hours. The positive apoptotic rates were as follows: sham-exposed cells (controls) -- 13.3% +/- 0.6%; with broad-band ultraviolet B irradiation -24.5% +/- 1.1% with 10mJ/cm(2), 57.9% +/- 4.6% with 20mJ/cm(2) and 70.9% +/- 4.5% with 30mJ/cm(2); with narrow-band ultraviolet B irradiation -- 29.6% +/- 2.3% with 100mJ/cm(2), 57.3% +/- 4.1% with 200mJ/cm(2) and 81.5% +/- 1.9% with 300mJ/cm(2). The difference between the number of apoptotic cells in all groups of ultraviolet B-irradiated cells and sham-exposed cells was highly significant (P<0.001). Based on these findings, it is hypothesized that ultraviolet B induced mast cell apoptosis could be an important factor in phototherapy for the diseases dependent on increased number of cutaneous mast cells, including uraemic pruritus.     

A simple and rapid method for HLA-DP genotyping by digestion of PCR-amplified DNA with allele-specific restriction endonucleases

We previously reported a simple and rapid method for HLA-DQA genotyping by digestion of polymerase chain reaction-amplified DQA genes with allele-specific restriction endonucleases. Here we report the application of this method to DP genotyping. The second exon of the HLA-DPB genes was selectively amplified from genomic DNAs of 72 HLA-D homozygous B-cell lines by the polymerase chain reaction method. Amplified DNAs were digested with ApaI, SacI, BstUI, FokI, and RsaI, which can recognize allelic sequence variations in the polymorphic segments of the DPB second exon and then subjected to electrophoresis in polyacrylamide gels. Sixteen different polymorphic patterns of the restriction fragments were found, and twelve were identical to patterns predicted from the known DNA sequences correlating with each HLA-DPw specificity defined by cellular typing. The other four patterns were distinct from those of the known DPw specificities, suggesting the presence of novel DP alleles. This polymerase chain reaction-restriction fragment length polymorphism method provides a simple and rapid technique for accurate definition of HLA-DP types at the nucleotide level, replacing the technically demanding method of primed lymphocyte typing.     

The effect of AH 21-132 on airway hyperresponsiveness induced by ozone exposure

We examined the effect of AH 21-132, which has been reported to relax airway smooth muscle and inhibit platelet activating factor (PAF)-induced airway hyperreactivity, on ozone-induced airway hyperresponsiveness (AHR) with airway inflammation in dogs. Airway responsiveness (AR) to methacholine was measured by modified Astograph (7 Hz oscillation method) before and after ozone exposure, and the numbers of neutrophils in the peripheral blood and total cell counts, differential cell counts and TXB2 in BALF were measured before and after ozone exposure. Ozone exposure was carried out for 2 hr at an ozone level of 3.46 +/- 0.10 ppm (mean +/- SE). There was a significant increase in AR to methacholine after ozone exposure (p less than 0.01), and the numbers of neutrophils in the peripheral blood and the total cell and neutrophil counts in BALF increased significantly (p less than 0.05). Pretreatment with AH 21-132 at an oral dose of 20 mg/kg significantly prevented the ozone-induced AHR to methacholine (p less than 0.01), and also inhibited the increase of neutrophil counts in the peripheral blood, and the total cell counts and the neutrophil counts in BALF after ozone exposure. There was no significant change in the levels of TXB2 in BALF before and after ozone exposure. In dogs not exposed to ozone, AR to methacholine and respiratory resistance to methacholine significantly decreased after administration of AH 21-132 at an oral dose of 20 mg/kg (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

HLA antigens are candidate markers for prediction of lymph node metastasis in gastric cancer

We investigated the association between human leucocyte antigen (HLA) antigens and lymph node metastasis in 724 gastric cancer patients. Among patients who had poorly differentiated adenocarcinoma with or without HLA-DR4 antigen, lymph node metastasis was detected in 80.8 and 54.9%, respectively (relative risk (RR)=3.5, P = 0.0005, corrected P = 0.0285). It was more common in patients with a family history of cancer death (RR = 7.7). Among signet ring cell carcinoma patients with or without HLA-1152 antigen, lymph node metastasis was detected in 57.7 and 19.7%, respectively (RR =5.6, P=0.0001, corrected P=0.0086). It was more common in patients who were smokers (RR = 8.3). Our findings suggest that HLA-DR4 and HLA-1152 antigens are associated with lymph node metastasis in gastric cancer.     

Effect of low-frequency electric stimulation on in vivo release of cholecystokinin-like immunoreactivity in medial thalamus of conscious rat

Release of cholecystokinin-like immunoreactivity (CCK-LI) in the medial thalamus of conscious rats was measured by brain dialysis and enzyme immunoassay. Analgesia caused by low-frequency electric stimulation of the tibial muscle, the tsusanli acupuncture point, was judged by change of pain threshold due to the stimulation. Medical thalamic CCK-LI released was increased by peripheral electric stimulations of both the acupuncture point and the non-acupuncture point. Results suggest that CCK acts as a neurotransmitter in the medial thalamus, a part of the analgesia inhibitory system.     

Exponential hyperbolic sine function fitting of heart rate response to constant load exercise

We attempted to fit heart rate (HR) changes induced by constant exercise loads of different intensities to an exponential hyperbolic sine curve by the least-squares method, and we compared the results with the fitting of the changes to exponential curves. Seven healthy male volunteers performed three different intensities of constant-load exercise on a bicycle ergometer. The exponential hyperbolic sine function adequately fitted the HR responses induced by all three different intensities of loads: low (30 W: correlation coefficient, r = 0.68 +/- 0.13, mean +/- SD), moderate (75 W: r = 0.93 +/- 0.07) and high (125 W: r = 0.97 +/- 0.02). The first-order exponential curve fitted only the moderate load response. Although the second-order exponential equation fitted the HR response for both the moderate and high loads, the equation did not fit the low-load response (r = 0.43 +/- 0.26). In low-load exercise, the sum of the power of the residuals for the exponential hyperbolic sine curve fitting was significantly smaller than that for the first- or second-order exponential curve fitting. In conclusion, the exponential hyperbolic sine function is useful for quantitative analyses of the HR response to exercise loads of various intensities.