Comparative analysis and development of microsatellite markers on swine (Sus scrofa) chromosome 1qter.

Several quantitative trait loci (QTL) have been detected on SSC1qter (Sus scrofa chromosome 1qter), including QTL for the number of vertebrae, as reported in our previous study. To provide the tools for analysis of QTLs on SSC1qter, we constructed a comparative map of swine and human. In addition, we identified 26 swine STSs and mapped 16 of them on SSC1qter using the INRA - University of Minnesota porcine radiation hybrid (IMpRH) panel. We screened a BAC library using these swine STSs and developed 35 new polymorphic microsatellite markers from the BAC clones, of which 26 were informative in our reference family. We also mapped nine microsatellite markers we had isolated previously. Consequently a total of 44 new polymorphic microsatellite markers were located within a 60-cM region of SSC1qter, spanning from SW1092 to the telomere.

Assessment of the intestinal permeability following postoperative chemotherapy for human malignant disease.

PURPOSE: Administration of anticancer drugs may damage gastrointestinal epithelium, thereby increasing the permeability of the gastrointestinal mucosa. We estimated the usefulness of oral lactulose and mannitol (L/M) test for assessment the extent of mucosal damage following postoperative chemotherapy for human malignant disease. METHODS: The permeability index (PI): the urinary recovery ratio of excreted L to M was measured before and after chemotherapy in 31 patients with gastrointestinal cancers who underwent surgical resection. These findings were compared with data on 12 patients with breast cancer. The effect of chemotherapy was evaluated by the ratio of increase in PI, which was designed as post-chemotherapy value on day 7 divided by pre-chemotherapy value. RESULTS: The mean PIs before chemotherapy in patients who underwent gastrectomy or colectomy were significantly higher than the value in those treated with mastectomy (p < 0.05). In the gastrointestinal cancer patients, the mean PIs significantly increased after chemotherapy compared with the pre-chemotherapeutic value (p < 0.01), however no significant difference was seen in breast cancer patients. When the ratios of increase in PI were calculated among gastric cancer patients, the total gastrectomy group showed a significantly higher increase in PI compared with the partial gastrectomy group (p < 0.05). CONCLUSIONS: Since the oral L/M absorption test is useful for assessing the degree of mucosal damage and measurement of intestinal permeability, this analysis should be recommended to determine the optimum timing and the adequate dosage of the anticancer drug administration.

Differences in mutant p53 protein stability and functional activity in teniposide-sensitive and -resistant human leukemic CEM cells.

We examined p53 protein stability and DNA damage-induced p53-dependent responses in a human leukemic CEM cell line and two teniposide-resistant sublines, CEM/VM-1 and CEM/VM-1-5 ( approximately 40 and 400-fold resistant to teniposide, respectively). Although all cell lines contain the same p53 mutations at codons 175 (Arg-->His) and 248 (Arg-->Gln), the constitutive levels of p53 were progressively increased with the resistance of the cells to teniposide. By pulse-chase experiments, we found that the half-lives of mutant p53 protein were approximately 12, 17, and >30 h in CEM, CEM/VM-1, and CEM/VM-1-5 cells, respectively. The prolonged half-lives of p53 in these cells is consistent with the fact that the protein harbors the indicated mutations. Of note, however, is the fact that the increased p53 protein half-lives in the two drug-resistant cell lines corresponds to a proportional decrease in MDM2 protein levels but an increase in p53-MDM2 binding interactions. This suggests that MDM2-mediated p53 degradation may be altered in our leukemic cell lines. The DNA damage-induced p53 response is fully functional in the drug-sensitive CEM cells containing a mutant p53, but this pathway is attenuated in the drug-resistant cells. Specifically, while the mutant p53 was phosphorylated at serine-15 in response to ionizing radiation in all these cell lines, mutant p53 induction in response to teniposide or ionizing radiation and induction of the p53-target genes, p21 and GADD45 only occurred in the drug-sensitive CEM cells. As assessed by MTT cytotoxicity assay, CEM cells were also significantly more sensitive to ionizing radiation, compared to the drug-resistant cell lines, and this correlated with p53 induction. Collectively, these results suggest that changes in constitutive mutant p53 protein levels, p53-MDM2 binding interactions, and altered regulation of the DNA damage-inducible p53-dependent pathway may play a role in drug- and radiation-responsiveness in these cells.

Quantitative trait loci (QTL) analysis in a Meishan x Göttingen cross population.

In order to locate the genetic regions in the swine genome that are responsible for economically important traits, a resource population has been constructed by mating two female Meishan pigs with a male Göttingen miniature pig. In subsequent generations, 265 F2 offspring were produced from two F1 males and 19 F1 females. The F2 offspring were scored for eight traits including growth rate, teat number, vertebra number and backfat thickness, and genotyped for 318 genetic markers spanning the swine genome. Least-square analysis revealed quantitative trait loci (QTL) effects for vertebra number on chromosomes 1 and 2; for teat number on chromosomes 1 and 7; for birth weight on chromosome 1; for average daily gain between 4 and 13 weeks of age on chromosomes 9 and 10; for backfat thickness on chromosome 7; and for backskin thickness on chromosome 3.

A linkage map of 243 DNA markers in an intercross of Göttingen miniature and Meishan pigs.

A resource family of pigs has been constructed by using a boar of Göttingen miniature pig and two sows of Meishan pig as parents. In the construction of the family, two F1 males and 18 F1 females were intercrossed to generate 143 F2 offspring. The members of the family were genotyped using 243 genetic markers including 26 markers developed in our laboratory in order to generate a linkage map of markers for use in detecting quantitative trait loci (QTLs) in the family. The markers consisted of 237 microsatellites, five PRE-1 markers, and one RFLP marker. The linkage map was revealed to cover all 18 autosomes and the X chromosome; and the total length of the sex-averaged linkage map was calculated to be 2561.9 CM. Four out of the 26 markers developed in our laboratory exended the current linkage map at the termini of chromosomes 1p, 5p, 11p, and Xq. The linkage maps of all the chromosomes except for chromosome 1 were found to be longer in females than in males. Concerning chromosome 1, the length of the linkage map showed no difference between females and males, which was attributed to low recombination rates between markers localized in the centromeric region in females. The average ratio of female-to-male recombination was calculated to be 1.55.

Relation between intrinsic viscoelasticity and activation level of the human finger muscle during voluntary isometric contraction.

The purpose of the present study was to isolate the length perturbation-evoked force attributed to intrinsic muscle viscoelasticity, and to investigate the relation between muscle viscoelasticity and the level of muscle activation during isometric contraction in five healthy male subjects. A small length perturbation (stretching or contraction) was applied to the flexor pollicis longus muscle while the subject maintained constant isometric force; the time courses of the length perturbation was found to be almost identical in all the experiments. The force (Fv) induced by the muscle viscoelasticity was calculated using the equation Fv = F - Fc - Fp over an interval of 35 ms after the onset of perturbation, where F is the measured force, Fc is the tonic isometric force before the onset of perturbation and Fp is the force at rest obtained from the same length perturbation. The force response attributed to the stretch reflex is not included during this interval. These experiments were repeated at varying levels of isometric force. An almost linear relationship was obtained between the muscle viscoelasticity-induced force and the tonic isometric force during both the stretching and contraction of the muscle, i.e. the intrinsic muscle viscoelasticity varied almost linearly with the level of isometric contraction.

Clinical implications of serum anti-p53 antibodies for patients with gastric carcinoma.

BACKGROUND: Mutations of p53 can lead to the production of anti-p53 antibodies in sera of cancer patients. Before this study, the value of preoperative serum anti-p53 antibodies in determining the prognoses of patients with gastric carcinoma had yet to be determined. METHODS: The authors used a highly specific enzyme-linked immunosorbent assay (ELISA) kit (Pharma Cell, France) to determine the preoperative presence of serum anti-p53 antibodies in 120 patients with gastric carcinoma. The relation between the positivity of serum anti-p53 antibodies and p53 abnormal staining of gastric carcinoma tissues was examined. Clinicopathologic characteristics and prognoses of these patients were given attention. RESULTS: Anti-p53 antibodies were detected in 19.2%(23 of 120) of these patients with gastric carcinoma. Among those who were positive for anti-p53 antibodies, female patients were predominant, the depth of invasion was greater, and liver metastasis was present, as compared with those who were negative for anti-p53 antibodies. Regarding other factors, there were no differences between those who were positive or negative for anti-p53 antibodies. Gastric carcinoma tissues had a 60.9% (14 of 23) positivity rate of p53 staining with anti-p53 antibodies and a 33.0% (32 of 97) negativity rate, and this difference was statistically significant (P < 0.05). The survival time of patients with anti-p53 antibodies in their sera was shorter than that of subjects with sera negative for anti-p53 antibodies (P < 0.05). The presence of anti-p53 antibodies was not an independent prognostic factor in multivariate analysis. CONCLUSIONS: Serum assay of anti-p53 antibodies is a rapid and readily facilitated test for predicting tumor advancement, depth of invasion, and liver metastasis, and it will show a poorer prognosis for surgically treated patients with gastric carcinoma.

[Diffuse panbronchiolitis with myeloperoxidase-specific antineutrophil cytoplasmic antibody-related vasculitis].

A 46-year-old woman was referred to our department in July 1996 with complaints of fever and myalgia in her calves. She had a 20-year history of purulent sputum; diffuse panbronchiolitis had been diagnosed in 1983. Physical examination revealed low-pithed rhonchi over the lung fieldis and hypesthesia of the right leg. She had a white blood cell count of 16,100/mm3, including 4% eosinophils, and a platelet count of 80.0 x 10(4)/mm3. The serum IgE level was 2,200 U/ml, and the cold hemagglutinin titer was high. Pulmonary-function tests showed mixed ventilatory dysfunction, and arterial blood gas analysis revealed a PaO2 of 55.8 Torr on room air. Pseudomonas aeruginosa was cultured from her sputum. A chest X-ray film and CT scan showed diffuse nodular shadows and bronchiectatic changes with mild hyperinflation. An infiltrative lesion in right S6 area could also be seen. Administration of broad-spectrum antibiotics did not alleviate her symptoms. The level of myeloperoxidase-specific antineutrophil cytoplasmic antibody (MPO-ANCA) in serum was 245 EU/ml, and 67Ga scintigraphy showed marked accumulation in the abdomen. Abdominal angiography demonstrated a bead-like appearance and irregularities in the peripheral branches of the hapatic artery, the splenic artery, the cystic artery, and the superior mesenteric artery. Because of the high MPO-ANCA level and the angiographic abnormalities, MPO-ANCA-related vasculitis was diagnosed. She was treated with 1 g of methylprednisolone daily for 3 days, followed by 60 mg of prednisolone and 50 mg of cyclophosphamide daily. Her condition improved dramatically, and the MPO-ANCA level became almost normal. During treatment, her blood pressure rose markedly with a normal serum creatinine level and normal urinalysis. Plasma renin activity was 13.3 ng/ml/hr. Renal angiography showed stenoses and irregularities in the peripheral branches of renal arteries bilaterally. These findings led to a diagnosis of renovascular hypertension due to vasculitis. Her blood pressure was controlled with an angiotensin-converting enzyme inhibitor and a calcium antagonist. Vasculitis associated with chronic supportive lung disease has occasionally been reported, which suggests a casual relation between chronic respiratory infection and ANCA-related vasculitis. Systemic vasculitis should be taken into account as a potential complication of chronic suppurative lung disease.

[Serum carcinoembryonic antigen doubling time in patients with recurrent gastrointestinal carcinoma and its relationship to tumor biology and life expectancy].

BACKGROUND: The present study was done to determine if carcinoembryonic antigen (CEA) concentration doubling time can predict the course of disease in patients with adenocarcinoma of the gastrointestinal tract and characterize tumor biology. METHODS: CEA doubling times were determined from semilogarithmic plots of CEA concentration time courses in 20 patients with recurrent gastric cancer and 17 with recurrent colorectal cancer. RESULTS: Gastric and colorectal carcinomas showed mean CEA doubling times of 229 days and 85 days, respectively. There were no significant differences with regard to patient age, tumor size, gross appearance and histological differentiation. However, women had shorter CEA doubling times than did men. Flow cytometric analysis showed that tumors with a higher proportion of cells in S-phase (> or = 15%) had significantly shorter CEA doubling times than those with a lower S-phase fraction (< 15%). There was a significant correlation between the CEA doubling time and the length of survival after the initial CEA concentration increase in patients with recurrent gastric and colorectal carcinomas. CONCLUSIONS: CEA doubling time predicts life expectancy in patients with adenocarcinoma of the gastrointestinal tract. Differences in survival time are closely associated with variations in the biological aggressiveness of individual tumors.

Elevation of antibodies to cytomegalovirus and other herpes viruses in pulmonary fibrosis.

The aim of this study was to determine whether latent viral infection is associated with idiopathic pulmonary fibrosis (IPF), an interstitial lung disease whose aetiology remains to be elucidated. Cytomegalovirus (CMV) immunoglobulin G (IgG) and complement fixation (CF), Epstein-Barr (EB) viral capsid antigen (VCA) IgG, herpes simplex virus (HSV) IgG, adenovirus CF, and parainfluenza 3 virus haemagglutinin inhibition (HI) titres were measured in the serum from patients with pulmonary diseases. The study included five subject groups: 35 normal controls (aged (mean +/- SD) 38 +/- 17 yrs); 43 IPF (63 +/- 10 yrs), seven collagen vascular disease-related interstitial pneumonitis (CVD-IP) (62 +/- 12 yrs); 22 sarcoidosis (36 +/- 14 yrs); and 17 emphysema (66 +/- 11 yrs). Levels of CMV IgG in IPF (87.6 +/- 51.7) and CVD-IP (101.2 +/- 69.9) were significantly elevated compared to those in the control (30.9 +/- 24.1), sarcoidosis (34.4 +/- 38.3) and emphysema groups (40.3 +/- 24.6), whereas CMV immunoglobulin M (IgM) was generally below the limit of detection. Similarly, CMV CF titres in IPF and CVD-IP were elevated compared to those in other diseases. EB VCA IgG titres in IPF, CVD-IP and emphysema and HSV IgG in IPF were also elevated. In contrast, adenovirus CF and parainfluenza 3 HI titres demonstrated no significant difference among all of the groups investigated. Increases in cytomegalovirus immunoglobulin G and complement fixation titres with negative cytomegalovirus immunoglobulin M suggest that latent cytomegalovirus infection may be more prominent in idiopathic pulmonary fibrosis or collagen vascular disease-related interstitial pneumonitis. Together with the elevation of Epstein-Barr virus viral capsid antigen and herpes simplex virus immunoglobulin G in idiopathic pulmonary fibrosis and/or collagen vascular disease-related interstitial pneumonitis, it is rational to assume that these viruses may be implicated in the development of pulmonary fibrosis. Further study is necessary to investigate the relationship between latent viral infection and pulmonary fibrosis.

Analysis of ornithine decarboxylase messenger ribonucleic acid expression in colorectal carcinoma.

PURPOSE: Ornithine decarboxylase (ODC) is a rate-limiting enzyme for polyamine synthesis. An elevated protein level of ODC was observed in the tumors. There has been, however, little information reported so far on the expression of ODC messenger ribonucleic acid (mRNA) in clinical colorectal carcinomas. In vitro studies disclosed that the transcriptions of the ODC gene is regulated by the c-myc gene. METHODS: The expression of ODC and c-myc mRNA in biopsy specimens obtained from both tumor tissue and the corresponding normal tissue was examined by the reverse transcriptase polymerase chain reaction method in 40 cases of colorectal carcinoma. RESULTS: The expression of ODC mRNA was observed in both tumor tissue and normal tissue. The tumor to normal ratio of ODC mRNA was higher in cases with deeply invasive tumors than in cases with shallow tumors, and it was also higher in Dukes B or C cases than in Dukes A cases. There was a significant correlation between the tumor to normal ratio of c-myc mRNA and that of ODC mRNA in each case. CONCLUSIONS: These findings suggested that 1) the study of the expression of ODC mRNA may be useful for preoperatively predicting more advanced disease of colon carcinoma, and 2) there was a significant correlation between expression of ODC and c-myc mRNA in the clinical samples, which was similar to the findings of a previous in vitro study.

Analysis of MT1-MMP and MMP2 expression in human gastric cancers.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a presumed activator of MMP2, which is one of the major proteinases in tumor cell invasion. In this study, we determined the clinico-pathologic significance of MT1-MMP expression in 68 human gastric carcinomas. The tumor-normal ratio (T/N ratio) of MTI-MMP expression was determined by reverse transcription-polymerase chain reaction analysis. To visualize the localization of MT1-MMP, an immunohistochemical study was performed. In addition, a gelatin zymography was done to examine the activation ratio of MMP2, and a correlation between MT1-MMP expression and activation of MMP2 was studied. The expression of MT1-MMP mRNA was higher in tumor tissue than in corresponding normal tissue in most cases. The mean value of the T/N ratio was 4.8. Twenty cases with T/N > or = 4.8 showed significantly deeper invasion and higher frequency of lymph node metastasis than 48 cases with T/N < 4.8. MT1-MMP expression was an independent factor influencing both tumor invasion of the gastric wall and lymph node metastasis. Although MT1-MMP expression was not an independent prognostic factor, the patients with T/N > or = 4.8 showed a significantly worse prognosis than those with T/N < 4.8. An immunohistochemical study demonstrated that MT1-MMP expression was mainly recognized in the tumor cells. There was a significant correlation between MT1-MMP expression and activation of MMP2. Our findings suggest that: 1) the expression of MT1-MMP may influence prognosis via tumor invasion of the gastric wall and lymph node metastasis, and 2) MT1-MMP activation of MMP2 may be clinically relevant in gastric carcinoma tumors.

Diffuse alveolar hemorrhage associated with proteinase 3-specific anti-neutrophil cytoplasmic antibodies.

A 31-year-old man was referred to our hospital for the management of progressive diffuse alveolar hemorrhage associated with renal dysfunction. Leukocytoclastic vasculitis was shown by skin biopsy and crescentic glomerulonephritis was also detected, in addition to positivity for proteinase 3-specific anti-neutrophil cytoplasmic antibodies (PR3-ANCA). The patient was diagnosed as a rare case of PR3-ANCA-positive pulmonary-renal vasculitic syndrome without granulomatous lesions. There was a good response to combination therapy with steroids and cyclophosphamide.

[Successful treatment of chronic eosinophilic pneumonia with suplatast tosilate].

The patient was a 59-year-old woman who complained of coughing, sputum production, and dyspnea. Abnormal moving shadows were found by chest radiography. There was prolonged eosinophilia in blood. Eosinophilia was also found in bronchoalveolar lavage fluid, and examination of a specimen obtained by transbronchial lung biopsy revealed eosinophil infiltration in alveolar septal walls, which led to a diagnosis of chronic eosinophilic pneumonia. Because of an attack of bronchial asthma and a high level of IL-5 in serum on admission, the patient was given suplatast tosilate. Symptoms were relieved, eosinophil counts in blood and in bronchoalveolar lavage fluid decreased, and the radiographic abnormality disappeared. The patient was not treated with steroids, and there has been no reccurrence to date, one year after discharge.

[Use of a bronchoscope for thoracoscopic observation and diagnosis of pleural plaques].

A 62-year-old man was admitted to our hospital for evaluation of bilateral tumor-like shadows along the ribs. He had been a diamond-grinder for twenty years. Thoracoscopic examination with a flexible bronchoscope revealed many well-circumscribed tumors with shiny, smooth, convex surfaces on the parietal pleura. The biopsy specimen had hyaline collagen with no cellular components. Based on these findings the tumors were diagnosed as pleural plaques. Cases of pleural plaques are usually followed up with chest radiography only, but the clinical course may be complicated by malignant mesothelioma and other malignant disease. To differentiate these conditions we performed biopsy during intrathoracic observation. The flexible bronchoscope can be useful in such procedures because it is relatively easy to operate and the procedure is relatively non invasive.

Protein kinase inhibitor attenuates an increase in endothelial monolayer permeability induced by tumour necrosis factor-alpha.

We questioned the mechanism of the increase in pulmonary endothelial permeability induced by tumour necrosis factor-alpha (TNF-alpha), a cytokine implicated in the pathogenesis of adult respiratory distress syndrome. As a measure of permeability, we determined the albumin transferred across cultured pulmonary endothelial monolayers prepared on a porous filter. The agents evaluated included protein kinase inhibitors H-7 and H-8, a calmodulin antagonist W-7, and protein kinase C (PKC) activators, phorbol myristate acetate (PMA) and SC-9. H-7, more potent in inhibiting PKC than H-8, failed to attenuate the increase in permeability induced by TNF-alpha. Neither PMA nor SC-9 increased permeability. However, H-8, which is a potent inhibitor of cyclic nucleotide-dependent protein kinases, prevented the increase in permeability induced by TNF-alpha. These results suggest that protein kinase other than PKC are involved in the signal transduction in endothelial permeability increase induced by TNF-alpha. Calmodulin pathway may not be implicated in the increase in permeability induced by TNF-alpha.