Integration of cytogenetic and genetic linkage maps of <Emphasis Type="Italic">Lotus japonicus</Emphasis>, a model plant for legumes

To construct a high-resolution pachytene chromosome map, we used the chromosome image analyzing system version 3 and fluorescence in situ hybridization. Two ribosomal RNA genes (45S rDNA and 5S rDNA), two major tandem repeat DNAs (LjTR1 and LjTR2), two major retroelements (LjRE1 and LjRE2), and 27 transformation-competent artificial chromosome clones were physically localized on Lotus japonicus (Miyakojima MG-20, 2n = 12) chromosomes. The distributions of heterochromatin and euchromatin along six chromosomes were compared based on the linkage map. Distortion between the recombination frequencies and physical chromosomal distance was recognized where the centromeric heterochromatic regions and constitutive heterochromatin are composed of the highest copy tandem repeat LjTR1 on the interstitial specific regions. Our study shows that the heterochromatin are composed of the specific repeated sequences, and the discrepancy between the recombination frequency and cytological information detected in L. japonicus chromosomes is due to the heterochromatin.     

Impact of histological subtype on survival of patients with surgically-treated stage IA2-IIB cervical cancer: Adenocarcinoma versus squamous cell carcinoma

Objectives

To evaluate the significance of adenocarcinoma (AC) compared with squamous cell carcinoma (SCC) with regard to the survival of surgically-treated early stage cervical cancer patients.

Methods

We retrospectively reviewed the medical records of 520 patients with FIGO stage IA2-IIB cervical cancer who were treated with radical hysterectomy with or without adjuvant radiotherapy between January 1998 and December 2008. The patients were classified according to (i) pathological risk factors (low-, intermediate-, or high-risk group) and (ii) adjuvant radiotherapy (concurrent chemoradiotherapy [CCRT group] or radiotherapy alone [RT group]). Survival outcomes were examined by Kaplan-Meier method and compared with Log-rank test. Multivariate analysis for disease-specific survival (DSS) was performed using Cox proportional hazards regression model to investigate the prognostic significance of histological subtype.

Results

AC histology was associated with significantly decreased DSS compared with SCC histology in the intermediate- and high-risk groups (hazard ratio: 3.06 and 2.88, respectively, both P < 0.05) while there was no survival difference in the low-risk group (P = 0.1). Among patients who received any types of adjuvant radiotherapy, DSS of AC histology patients were significantly poorer than SCC histology. Multivariate analysis demonstrated AC histology to be an independent predictor of decreased DSS in both CCRT and RT groups. Moreover, pelvic nodal metastasis significantly predicted the poor survival of patients with AC histology who received CCRT in multivariate analysis

Conclusions

Adenocarcinoma is an independent prognostic indicator of poor survival in early stage cervical cancer patients with intermediate- and high-risk factors, regardless of the type of adjuvant radiotherapy after radical hysterectomy.     

Vocal cord inflammatory myofibroblastic tumor with mucoid deposits harboring TIMP3–ALK fusion: A potential diagnostic pitfall

A 35‐year‐old Japanese man who had experienced hoarseness for 10 years presented with a vocal cord lesion. A gross examination revealed a left vocal cord polyp occupying two‐thirds of the vocal space. The endoscopically resected lesion contained scattered atypical fibroblastic, stellate, or ganglion‐like cells with mucoid stroma. Vacuolated cells were also seen. Lymphoplasmacytic infiltrate was largely undetectable. A vocal cord polyp was first suspected, but well‐differentiated liposarcoma and inflammatory myofibroblastic tumor (IMT) were included in the differential diagnoses. The tumor cells were positive for anaplastic lymphoma kinase (ALK), calponin, and vimentin, and negative for other smooth muscle markers by immunohistochemistry. Structures resembling myofibroblasts were not observed by electron microscopy, which confirmed abundant rough endoplasmic reticulum in the tumor cells and accumulated lipid droplets in some tumor cells. ALK gene rearrangement was detected by fluorescence in situ hybridization, and TIMP3–ALK fusion was confirmed by 5′ rapid ampli?cation of cDNA ends. We diagnosed the lesion as an IMT, and an ALK‐rearranged stellate cell tumor may be postulated. This is the first report of a fusion partner gene of ALK in a case of laryngeal IMT.     

Release of granule proteins from human eosinophils stimulated with mast-cell mediators

Background It has been suggested that mast cells and eosinophils are major effector cells in the pathogenesis of allergic diseases. However, the interaction of these cells has not been thoroughly elucidated. We examined eosinophil cationic protein (ECP) release and cytosolic free calcium concentration ([Ca²⁺]) in human eosinophils induced by the major mast-cell mediators including cytokines. Methods Eosinophils from healthy donors were stimulated with the major mast-cell mediators for 20 min after preincubation with cytochalasin B for 10 min. ECP in supernatants was measured by radioimmunoassay. Moreover, t o examine changes of [Ca²⁺]i in eosinophils, Fura-2-loaded eosinophils were monitored for fluorescence changes after stimulus addition. Results Of the tested mediators (prostaglandin [PG]D₂, leukotriene (LT)B₄, platelet-activating factor (PAF), histamine, LTQ, and eosinophil chemotactic factor of anaphylaxis [ECF-A]), LTB₄ and PAF induced ECP release from eosinophils. Any cytokines produced by human mast cells, i.e., interleukin (IL)-4, IL-5, IL-8, tumor necrosis factor (TNF), or granulocyte-macrophage colony-stimulating factor (GM-CSF), did not induce ECP release in our system. ECP release triggered with LTB₄ and PAF occurred at concentrations of 10^?8-10^?6 M concentration-dependently. LTB4 and PAF also elicited a rise in [Ca²⁺]_i in eosinophils. Neither PGDj, histamine, nor LTC₄ induced ECP release, although they increased cytosolic calcium in eosinophils. Conclusions Of mast-cell mediators, LTB₄ and PAF induced eosinophil degranulation. The contribution of LTB4 and PAF from mast cells to eosinophil degranulation may be important in the pathogenesis of allergic inflammatory diseases.     

A nested PCR-RFLP method for high-resolution typing of HLA-A alleles

We developed a nested polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) method for high-resolution typing of HLA-A alleles. HLA-A alleles can be identified by this method without the need for other information such as serological type. The first PCR was performed using outer primers, ASP5 and ASP3, specific for the HLA-A gene, and a 991-bp DNA fragment extending from exon 1 through exon 3 was amplified. In the second PCRs, exon 2 and exon 3 of the HLA-A gene were amplified separately from the diluted first PCR product using nested primers. Computer analysis of cleavage patterns for 78 HLA-A alleles showed that 31 RFLP patterns could be obtained by digestion of the exon 2 PCR product using eight restriction endonucleases and 42 RFLP patterns by digestion of the exon 3 PCR product using 11 restriction endonucleases, and all alleles could be discriminated based on combinations of these RFLP patterns except for nine allele groups or pairs: A*0201/ 0207/ 0215N/0220/0222, A*0205/0208/0214, A*0206/ 0221, A*0212/ 0213, A*2402/2405, A*2406/2413, A*2601 / 2605, A*2603/2606 and A*7401/7402. Thus, 65 PCR-RFLP patterns were predicted from the results of analysis of digestion patterns of 78 HLA-A alleles. Among 2145 possible homozygous and heterozygous combinations of the 65 distinguishable PCR-RFLP patterns, 82 combinations were predicted to have the same PCR-RFLP patterns. In PCR-RFLP analysis, although the nested primers were not specific for the HLA-A gene, clear RFLP banding patterns were obtained because specificity was guaranteed by the use of the outer primers, ASP5 and ASP3 in the first PCR. A*0201 and A*0207 occur relatively frequently in the Asian populations among indistinguishable allele groups or pairs using the present PCR-RFLP method. We also developed a PCR sequence-specific primers (PCR-SSP) method for distinguishing between A*0201/0220/0222 and A*0207/0215N. We could identify 39 alleles (groups) upon HLA-A typing of 50 Japanese individuals, 40 cell lines of the Fourth Asia-Oceania Histocompatibility Workshop, and 80 cell lines of the UCLA International Cell Exchange Program using the present PCR-RFLP and PCR-SSP methods.     

Freeze-fracture studies of muscle plasma membranes in myopathic patients with hypo-and hyperthyroidism

To examine the influence of thyroid hormone on the skeletal muscle plasma membrane, we analyzed the changes in ultrastructural architecture and membrane area complexed with digitonin of muscle plasma membrane in myopathic patients with hypo-and hyperthyroidism by the conventional freeze-fracture (F-F) technique and F-F cytochemistry using the sterol-specific ligand digitonin. The densities of flask-shaped invaginations, which are mainly thought to correspond to caveolae, intramembranous particles, and orthogonal arrays, and the changes of digitoninreacted membrane areas in the muscle plasma membranes in three patients with hypothyroid myopathy and one patient with both myasthenia gravis and hyperthyroidism were compared with those in age-matched controls. In the conventional F-F study, the muscle plasma membrane of hypothyroid patients showed increased invagination density, whereas that of the hyperthyroid patient was normal ultrastructurally. In the F-F cytochemistry study, however, the ratio of digitonin-reacted membrane areas versus fractured membrane areas was not different between hypothyroid patients and controls, whereas that of the hyperthyroid patient was lowered in comparison with that of control. These results suggest that thyroid hormone may alter the biochemical properties and ultrastructural architecture of muscle plasma membrane.This study was presented at the 29th annual meeting of the Clinical Electron Microscopy Society of Japan, Sapporo, October 2–3, 1997     

Genetic heterogeneity of uncharacterized childhood autoimmune diseases with lymphoproliferation

Autoimmune diseases in children are rare and can be difficult to diagnose.  Single causative genes have been identified for some pediatric autoimmune diseases. Such orphan diseases may not be diagnosed properly due to the variability of patients' phenotypes. Guidelines for the diagnostic process need to be developed. Fifteen patients with uncharacterized childhood autoimmune diseases with lymphoproliferation that had negative testing for autoimmune lymphoproliferative syndrome were subjected to whole‐exome sequencing to identify genes associated with these conditions. Five causative genes, CTLA4, STAT3, TNFAIP3, IKZF1, and PSTPIP1, were identified. These genes should be considered as candidates for uncharacterized childhood autoimmune diseases with lymphoproliferation.     

Prognostic value of positron emission tomography in cryptogenic West syndrome

The relationship between positron emission tomography (PET) findings and developmental or seizure outcome was examined in 17 infants (11 males, six females; mean age at onset of spasms 7 months, range 3 to 26 months) with newly diagnosed cryptogenic West syndrome. The predictive value of PET in these infants was assessed. PET was performed in the infants at the onset of spasms and 3 months after initial therapy using 18F-labelled 2-deoxy-2-fluoro-D-glucose. A third PET was performed at 18 months of age if the second scan was abnormal. All infants were followed up until at least 3 years of age. Cortical hypometabolism was detected in 11 infants on the first PET and in five infants on the second. Rate of developmental delay at the last follow-up was significantly higher in infants with hypometabolism on the second PET than in those without PET abnormalities (p<0.05). Rate of seizure occurrence after initial treatment was higher in infants with cortical hypometabolism on the second PET, but the difference was not statistically significant. Results suggest that when PET after the initial treatment shows no abnormalities, even though the first PET shows hypometabolism, infants with cryptogenic West syndrome may have a favourable developmental or seizure outcome. PET may be a useful tool in evaluating the prognosis in infants with cryptogenic West syndrome.