Characteristics of the spin-trapping reaction of a free radical derived from AAPH: further development of the ORAC-ESR assay.

The characteristics of the spin-trapping reaction in the oxygen radical absorbance capacity (ORAC)-electron spin resonance (ESR) assay were examined, focusing on the kind of spin traps. 2,2-Azobis(2-amidinopropane) dihydrochloride (AAPH) was used as a free radical initiator. The spin adducts of the AAPH-derived free radical were assigned as those of the alkoxyl radical, RO· (R=H(2)N(HN)C-C(CH(3))(2)). Among the spin traps tested, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5,5-dimethyl-4-phenyl-1-pyrroline N-oxide (4PDMPO), 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were applicable to the ORAC-ESR assay. Optimal formation of spin-trapped radical adduct was observed with 1 mM AAPH, 10 mM spin trap, and 5 s UV irradiation. The calibration curve (the Stern-Volmer's plot) for each spin trap showed good linearity, and their slopes, k (SB)/k (ST), were estimated to be 87.7±2.3, 267±15, 228±9, and 213±16 for DMPO, 4PDMPO, CYPMPO, and DEPMPO, respectively. Though the k (SB)/k (ST) values for selected biosubstances varied with various spin traps, their ratios to Trolox (the relative ORAC values) were almost the same for all spin traps tested. The ORAC-ESR assay also had a very good reproducibility. The ORAC-ESR assay was conducted under stoichiometric experimental conditions. The present results demonstrate the superiority of the ORAC-ESR assay.

[Video-assisted thoracic surgery (VATS) for clinical stage I lung cancer in consideration of the diameters and characteristcs of each tumor and the technical limitations of VATS].

UNLABELLED: We planned an intervention study to investigate the late outcome of limited surgery for cStage IA lung cancer by several video-assisted thoracic surgery (VATS) procedures. METHODS: VATS partial resection was done for non-solid tumors less than 1.5 cm in maximum diameter with non-solid component on high resolution computed tomography (HRCT) [group A]. VATS segmentectomy with minor thoracotomy with ND1 + alpha lymph node dissection was done for tumors less than 2.0 cm in maximum diameter that was not included in the group A (group B). Tumors of less than 3.0 cm in diameter that did not fit into the other 2 groups were treated by VATS lobectomy with minor thoracotomy plus ND2 lymph node dissection (group C). RESULTS: A total of 159 patients were enrolled during the 5-year enrollment period (group A: 21 patients, group B: 43 patients, group C: 95 patients). The recurrence-free 5-year survival rate was 100% in the group A, 82.8% in the group B, and 78.4% in the group C, showing no significant differences between the groups. Twenty-eight % of patients was switched to surgical techniques involving more extensive resection in the group A and B. while 6% of the patients was switched to thoracotomy overall. The overall recurrence rate was 10.7% (n=17), while the locoregional and distant recurrence rate was 5.7% (n=9) and 5.0% (n=8), respectively. CONCLUSIONS: This controlled intervention study suggested that limited surgery by VATS approaches for cStage IA lung cancer are acceptable as cancer operation.

[Malignant pleural mesothelioma in which many asbestos bodies were counted in its specimen].

A 53-years-old woman was admitted to our hospital because of pleural effusion. She underwent pleural biopsy and diagnosed as mesothelioma. Right extrapleuralpneumonectomy was performed. We counted asbestos bodies in the resected lung. 443,571 asbestos bodies were counted in 1 gram of dry lung. We thought that she was heavily exposed to asbestos. Since high risk of incidence of mesothelioma is suggested among her fellow worker, special investigation is necessary for asbestos exposure.

[Paravertebral muscle metastasis from lung cancer; report of a case].

A 69-year-old man had undergone right S6 segmentectomy for lung cancer (poorly differentiated adenocarcinoma, pT2N0M0, stage IB). One years later, computed tomography (CT) showed abnormal shadow in the right paravertebral muscle of 7 x 5 cm in size. The patient underwent resection of the tumor. The tumor was white, solid and elastic hard mass. Histopathologically, the tumor was paravertebral muscle metastasis from lung cancer. The patient was discharged on 23rd postoperative day, but died of other disease after 1 and a half month postoperatively.

[Mycobacterium avium complex disease after conservative therapy for 3 years; report of a case].

A 62-year old woman admitted to our hospital because of an abnormal shadow on chest X-ray. Chest X-ray and computed tomography (CT) scan revealed a 2 cm nodular shadow with cavity in the right upper lobe. Bronchofiberscopy was performed, and Mycobacterium avium complex was demonstrated bacteriologically. She had been followed-up for 3 years with chemotherapy, however slow but progressive enlargement of the lesion was noted on chest X-ray. Finally she reffered to our department for surgical treatment, and right upper lobectomy was performed. Ten months after operation, there is no sign of recurrence.

[Intrapulmonary and mediastinal, double bronchogenic cysts; report of a case].

A 59-year-old woman was pointed out abnormal shadows on chest film and has been followed up since 2001. In October 2006, she consulted our hospital for further examination. Computed tomography (CT) showed double cystic lesions located in the right lower lobe, and in the paraesophageal region. The patient underwent video-assisted thoracic surgery (VATS) right lower lobectomy and resection of the tumor in the mediastinum. The content of the tumor were greenish and whitish discharge. Histopathologically, both were bronchogenic cysts. The patient was discharged on 17th postoperative day and doing well for 6 months postoperatively.

[Granulocyte-colony stimulating factor producing tumor with high serum interleukin-6].

A 53-year-old man admitted to our hospital because of fever and chest abnormal shadow. Chest X-ray and computed tomography (CT) scan revealed large tumor on right upper lobe. Serum interleukin (IL)-6 and granulocyte-colony stimulating factor (G-CSF) were high. Right upper lobectomy and chest wall resection was performed. Histological diagnosis was large cell carcinoma. Immunohistological examination of lung tumor cells showed positive staining for G-CSF in only 1% of them. We diagnosed that tumor was G-CSF producing tumor and we thought that tumor produced IL-6.

Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants.

To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

Association of novel promoter single nucleotide polymorphisms in vasopressin V1a receptor gene with essential hypertension in nonobese Japanese.

We studied the association between four novel single nucleotide polymorphisms (SNPs) in the promoter region of V1aR gene and essential hypertension in 620 Japanese subjects (365 hypertensives and 255 healthy). A significant association was found between one of the genotypes and alleles at SNP -6951 and hypertension in a subsample of nonobese individuals. This association demonstrated an independent risk for nonobese hypertension.

A case of thyrotropin-producing pituitary adenoma, accompanied by an increase in anti-thyrotropin receptor antibody after tumor resection.

We describe a rare, but interesting, case of TSH-producing adenoma (TSHoma), accompanied by increases in both anti-TSH receptor antibody (TRAb) and thyroid-stimulating antibody (TSAb) after tumor resection. A 21-yr-old woman was referred to our department for further evaluation of pituitary tumor. In a nearby hospital, she had been diagnosed as having pituitary tumor. Her serum free T4, free T3, and TSH levels were all elevated concomitantly. On the basis of a diagnosis of pituitary adenoma with TSH production, transsphenoidal resection of the pituitary adenoma was performed. Two weeks after the operation, the blood concentrations of TSH were undetectable, whereas both TRAb and TSAb levels were elevated. TSAb levels gradually increased further from 2 weeks to 3 months after the operation, accompanied by an increase in TSH and free T4 levels. TSH is an important hormone in maintaining physiology and regulating immunomodulators in thyrocytes, as it can influence a variety of immune-regulating cytokine-like activities and inhibit expressions of Fas antigen, intracellular adhesion molecule-1, and class II trans-activator. Changes in TSH would modulate the immune circumstances in the thyroid, and then induce TRAb and TSAb. Autoimmune parameters with thyroid function should be observed carefully when managing patients with TSHoma.

[Mycobacterium avium complex (MAC) infection needed differential diagnosis of the recurrence after surgery for double lung cancer; report of a case].

A 62-year-old woman had undergone video-assisted thoracic surgery (VATS) -right upper lobectomy and right S8 segmentectomy for double lung cancers (papillary adenocarcinoma and bronchioloalveoler carcinoma, stage IA). Four years later, chest computed tomography (CT) showed abnormal shadow, 20 mm in size, along the staple-suture line. The 3 months later, new lesion, 15 mm in size, was observed in right lower lobe. CT-guided biopsy revealed no malignancy. The patient underwent partial resection of the right lower lobe and tumorectomy. The tumor was solid and cystic mass. Histopathologically, the tumor was granuloma infected by Mycobacterium avium. The patient was discharged on 17th postoperative day, and doing well without new lesion for 9 months postoperatively.

[Malignant solitary fibrous tumor of the pleura; report of a case].

A 68-year-old female was admitted to our hospital for further examination of abnormal shadow on chest X-ray. Needle biopsy could not establish pathological diagnosis. Three years later, chest computed tomography (CT) revealed the tumor was enlarged. We suspected it was a malignant tumor, and resected by video-assisted thoracoscopy. The tumor occurred from the right middle lobe, and intraoperative diagnosis was malignant tumor. We added middle lobectomy. Histological examination revealed that tumor was malignant solitary fibrous tumor.

[Lung metastasis of malignant peripheral nerve sheath tumor: report of a case].

A 37-year-old man with von Recklinghausen's disease admitted to our hospital because of chest abnormal shadow. He had underwent extended radical tumorectomy for malignant peripheral nerve sheath tumor (MPNST) in left lower limb 33 months before. Chest X-ray and computed tomography (CT) scan revealed solitary tumor on right S10. Tumor was resected under thoracoscopic surgery. Histological diagnosis was metastasis of MPNST. MPNST with lung metastasis showing very poor prognosis. The patient is doing well 2 years after pulmonary resection without recurrence. Careful follow up is important for MPNST.

Gene analysis of the calcium channel 1 subunit and clinical studies for two patients with hypokalemic periodic paralysis.

Hypokalemic periodic paralysis (HypoPP) is a skeletal muscle disorder in which episodic attacks of muscle weakness occur; they are associated with decreased serum potassium (K+) levels. Recent molecular approaches have clarified that the condition is caused by mutations in the skeletal muscle voltage-gated calcium channel 1 subunit (CACNA1S). We describe two unrelated patients with HypoPP, followed by their relevant clinical studies and gene analysis. Clinical studies included an oral glucose tolerance test (OGTT), food-loading and insulin tolerance tests (ITT). For Case 1, serum K+ levels were extremely decreased following insulin tolerance testing compared with levels for controls. These results support the hypothesis that no efflux of K+ ion occurs in patients because of low activity of adenosine triphosphate (ATP)-sensitive K+ channel (KATP) channels. Mutational analysis of the CACNA1S gene showed a duplicate insertion of 14 base pairs (bp) from 52 to 65 in intron 26, present in the heterozygous state in both patients. No other mutations were detected in the CACNA1S gene, the muscle sodium channel gene (SCN4A) or the voltage-gated K+ channel gene (KCN3) of either patient. Further analysis showed that this duplicate insertion of 14 bp in intron 26 of the CACNA1S gene was found in 23.7% of healthy subjects. K+ dynamics studies are useful for confirming this syndrome, while further gene analysis for various ion channels using amplification and direct sequencing are required to evaluate the molecular basis of the disorder in the individual patient.

A chimeric analog of human and salmon calcitonin eliminates antigenicity and reduces gastrointestinal disturbances.

The minimum region in salmon calcitonin (sCT) which induces antigenicity and gastrointestinal disturbances has been identified by examining the cross-reactivity of several sCT fragments and CT analogs with antisera from sCT-treated patients, and by examining inhibition of gastrointestinal motility of these sCT fragments and CT analogs in conscious dogs. Sixteen residues at the N-terminus of sCT comprised the minimum fragment capable of inducing both activities. Human CT (hCT) showed no antigenicity and a four-order weaker inhibition of gastrointestinal motility than sCT. Based on these data, we synthesized the human and salmon chimeric CT, ACT-15, in which the 16 N-terminal residues were those of hCT and the 16 C-terminal residues were those of sCT. ACT-15 had no cross-reactivity with the antisera and had almost the same weak gastrointestinal inhibition effect as hCT in dog and rat models. Nevertheless, it retained a hypocalcemic activity and an analgesic activity comparable to sCT. These results suggest that the amino acid residues in the N-terminal half of CT are responsible for the formation of antibodies and the induction of gastrointestinal disturbances, but may not influence calcium metabolism or analgesia. Clinical studies of ACT-15 will be needed to confirm this hypothesis.

Rapid and efficient cloning of cDNAs encoding Krüppel-like zinc finger proteins by degenerate PCR.

To isolate cDNAs encoding Krüppel-like zinc finger proteins consisting of several hundred members, most of which are yet to be identified, from a limited number of available cells, we developed a rapid and efficient zinc finger gene cloning method based on reverse transcription-polymerase chain reaction (RT-PCR) using tagged, degenerate oligonucleotide primers corresponding to the conserved H/C link followed by the reverse blue selection to identify clones containing properly amplified fragments. More than 5x103 blue colonies were obtained from only 1ng of total RNA. Eighty-eight out of 89 clones, which were randomly picked up from blue colonies and sequenced, encoded 60 different zinc fingers with the expected structure, and among them, only four have been previously described. Furthermore, it was possible to rapidly select clones that were differentially expressed in a tissue and stimulation-specific manner by a differential screening of the zinc-finger cDNA library using probes consisting of distinct sets of the zinc-finger PCR products. These results indicate that our PCR-based method is quite efficient and suitable for analyzing not only zinc finger genes but also other large gene families, especially when the available cells are very limited.

Endogenous 5-HT tonically inhibits spontaneous firing activity of dorsal hippocampus CA1 pyramidal neurons through stimulation of 5-HT(1A) receptors in quiet awake rats: in vivo electrophysiological evidence.

The present study was performed to examine an overall effect of endogenous serotonin (5-HT) on the spontaneous firing activity of the dorsal hippocampus CA1 pyramidal neurons in quiet awake rats. A selective 5-HT(1A) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohe xanecarboxamide (WAY-100635: 0.03-0.2 mg/kg, s.c.) significantly increased the firing activity. A depletion of 5-HT with parachlorophenylalanine (PCPA: 500 mg/kg/day x 3 days) completely abolished this increasing effect of WAY-100635. The baseline spike frequency of the PCPA-treated rats (3.90 +/- 0.39 Hz) was significantly higher than that of the vehicle-treated rats (2.09 +/- 0.19 Hz). A 5-HT(2A) antagonist ritanserin (1 mg/kg, i.p.) and a 5-HT(3/4) antagonist 2-methoxy-4-amino-5-chloro benzoic acid 2-(diethylamino) ethyl ester (SDZ-205557: 3 mg/kg, s.c.) did not modify the firing activity and the increasing effect of WAY-100635. These results suggest that, in quiet awake rats, endogenous 5-HT would tonically inhibit the spontaneous firing activity of the CA1 pyramidal neurons mainly through stimulating 5-HT(1A) receptors.

A rapid and sensitive method for HLA-DRB1 typing by acridinium-ester-labeled DNA probes.

A rapid and accurate detection of HLA class II antigen is essential for transplantation and for the understanding of disease susceptibility. Recent molecular genetic studies have revealed that the number of class II loci and the number of alleles at these loci are greater than had been previously detected. It is, therefore, of great importance to detect these extensive polymorphisms. A great deal of effort has been made on identification of individual HLA class II specificities at the DNA level, called "DNA typing." What seems to be lacking, however, is handling simplicity. Here we accomplished a simple method for HLA-DRB1 typing based on hybridization of acridinium-ester (AE)-labeled DNA probes to amplified DNA. This method is called hybridization protection assay (HPA). By using 13 AE-labeled probes, 20 homozygous B-cell lines and leukocytes from 80 healthy individuals were typed by HPA. The results were completely consistent with those obtained by polymerase chain reaction--restriction fragment length polymorphism. This method is suitable for mass screening because of its procedural simplicity and swiftness.

Effect of type-1 plasminogen activator inhibitor on human leukocyte elastase.

When human leukocyte elastase (HLE) activity (1.0 microgram/ml) was analysed in the presence of PAI-1 (0.15.0 micrograms/ml), HLE activity, measured with the low molecular weight paranitroanilide substrate L-pyroglutamyl-prolyl-L-valine-p-nitroanilide was increased time and dose dependently (a plateau of stimulation was reached after 30 minutes) with a simultaneous decrease in PAI-1 inhibitory activity. This effect was neither influenced by the presence of vitronectin nor heparin. When PAI-1 was converted into its latent form by incubation for 48 hours at 37 degrees C or incubated with an excess of recombinant t-PA to convert free PAI-1 into t-PA-PAI-1 complexes, the stimulatory effect of both the latent and the complexed form of PAI-1 was significantly greater than that of the active form. Analysing HLE PAI-1 interaction on a molecular level using SDS-PAGE, no SDS stable complex formation between HLE and PAI-1 could be observed but lower molecular weight cleavage products of PAI-1 were generated. The stimulatory effect of PAI-1 on HLE activity was not restricted to the low molecular weight pNA-substrate but was also revealed using a natural substrate assay (bovine neck ligament elastin solubilization). Therefore interaction of HLE and PAI-1 seems not to be restricted just to decrease PAI-1 activity but would simultaneously also increase HLE activity, thereby supporting enzymatic activity necessary for migration of leukocytes, dissolution of blood clots and tissue remodelling.

Immunocytochemical localization of cathepsins B and G in odontoclasts of human deciduous teeth.

For clarification of the mechanisms by which odontoclasts resorb deciduous teeth during physiological root resorption, cysteine-proteinases such as cathepsins B and G were immunocytochemically localized in odontoclasts at the ultrastructural level. Extracted human deciduous teeth undergoing root resorption were fixed with a mixture of formaldehyde and glutaraldehyde and processed for immunocytochemical detection of these enzymes. Sheep antisera, raised against either human cathepsin B or G, were used as primary antibodies. In odontoclasts, specific immunogold labeling of both anti-cathepsin B and G was clearly localized in lysosomes and pale vacuoles of various sizes, and in a portion of the extracellular canals of odontoclastic ruffled borders. In the presence of either antibody, the cytoplasmic matrix, mitochondria, and nuclei were minimally labeled by immunogold particles. The presence of these proteolytic enzymes in odontoclasts suggests that, during odontoclastic root resorption, these enzymes are involved in the formation of resorption lacunae by means of intra/extracellular degradation of collagen and other non-collagenous matrix proteins of deciduous teeth.