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1.
Adv Med Sci ; 57(2): 244-50, 2012.
Article in English | MEDLINE | ID: mdl-23183766

ABSTRACT

PURPOSE: This study was aimed to investigate etiology and clinical profiles of recurrent acute pancreatitis (RAP), particularly from the morphology of the pancreaticobiliary duct system. MATERIAL AND METHODS: Pancreaticobiliary morphology was examined in 230 of 381 patients with acute pancreatitis (AP) using endoscopic retrograde cholangiopancreatography. We analyzed factors associated with RAP including the pancreaticobiliary duct system. RESULTS: RAP was diagnosed in 74 patients (19%). Major etiologies of RAP were alcoholic (38%), idiopathic (26%) and pancreaticobiliary malformation (22%). Patients with alcoholic RAP were significantly younger (47.2±11.6 years) than those with gallstone RAP (67.3±16.8; p<0.05). RAP with pancreaticobiliary malformation (male-to-female ratio: 1:4.3; p<0.01) and gallstone RAP (1:1.7; p<0.05) occurred predominantly in females in comparison with alcoholic RAP (1:0.2). Recurrence rate was 80% for AP with pancreaticobiliary malformation, significantly higher than for the others (p<0.01). Pancreas divisum was suspected as the etiology of mild RAP in 7 patients. Four RAP patients with pancreas divisum underwent endoscopic minor papilla sphincterotomy and improved. Pancreaticobiliary maljunction with biliary dilatation (choledochal cyst) was suspected as the etiology of mild RAP in 3 patients. The 3 RAP patients with choledochal cyst underwent prophylactic flow diversion surgery with complete resection of the dilated common bile duct, and achieved improvement. High confluence of pancreaticobiliary ducts was suspected as the etiology of mild RAP in 6 patients. CONCLUSION: Pancreaticobiliary malformation is one of the major causes of RAP. As some of them benefit from endoscopic or surgical treatment, morphology of the pancreaticobiliary duct system should be examined where possible in RAP patients.


Subject(s)
Bile Ducts/abnormalities , Pancreatic Ducts/abnormalities , Pancreatitis/etiology , Acute Disease , Adolescent , Adult , Aged , Aged, 80 and over , Child , Cholangiopancreatography, Endoscopic Retrograde , Choledochal Cyst/complications , Choledochal Cyst/surgery , Female , Humans , Male , Middle Aged , Pancreatitis/surgery , Recurrence , Sphincterotomy, Endoscopic , Young Adult
2.
Dis Esophagus ; 23(8): 641-5, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20545978

ABSTRACT

The chemotherapy regimen currently used for treating esophageal and gastric carcinoma has been either epirubicin, cisplatin, and fluorouracil (5-FU) or docetaxel, cisplatin, and 5-FU. Here, we report the efficacy and toxicity of doxorubicin, cisplatin, and 5-FU for only esophageal squamous cell carcinoma (ESCC). Between January 2000 and October 2008, a total of 41 ESCC patients with a distant metastasis were enrolled. The most common sites of metastasis were liver (26, 63.4%), lung (9, 22.0%), and bone (8, 19.5%). Doxorubicin was administered on day 1 at 30 mg/m(2) , cisplatin on days 1-5 at 14 mg/m(2)/day, and 5-FU on days 1-5 at 700 mg/m(2)/day. The median number of cycles was 2.0 (range 1-8). The dose intensities of doxorubicin, cisplatin, and 5-FU were 92.9, 92.4, and 92.5%, respectively. The overall response rate was 43.9%; one showed complete response, 17 showed partial response, 13 showed a stable disease, and 10 showed progressive disease (PD). The median survival time was 306 days (95% CI = 74-935) and the 1-year survival rate was 37.6%. Grade 3 neutropenia was seen in seven patients and grade 4 in one patient. Grade 3 fatigue, anorexia, mucositis, and diarrhea were observed in three, two, two, and one patient, respectively. This regimen is effective as a first-line therapy for ESCC with distant metastasis.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Bone Neoplasms/secondary , Carcinoma, Squamous Cell/secondary , Esophageal Neoplasms , Liver Neoplasms/secondary , Lung Neoplasms/secondary , Aged , Bone Neoplasms/drug therapy , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/mortality , Cisplatin/administration & dosage , Cisplatin/adverse effects , Disease Progression , Docetaxel , Drug Administration Schedule , Drug Interactions , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/mortality , Esophageal Neoplasms/pathology , Female , Fluorouracil/administration & dosage , Fluorouracil/adverse effects , Humans , Liver Neoplasms/drug therapy , Lung Neoplasms/drug therapy , Male , Middle Aged , Neutropenia/chemically induced , Survival Rate , Taxoids/administration & dosage , Taxoids/adverse effects , Treatment Outcome
3.
Water Sci Technol ; 58(5): 1129-35, 2008.
Article in English | MEDLINE | ID: mdl-18824814

ABSTRACT

Removal property of nine pharmaceuticals (clofibric acid, diclofenac, fenoprofen, gemfibrozil, ibuprofen, indomethacin, ketoprofen, naproxen and propyphenazone) by chlorination, coagulation-sedimentation and powdered activated carbon treatment was examined by laboratory-scale experiments under the conditions close to actual drinking water treatment processes. Indomethacin and propyphenazone were completely degraded by chlorination within 30 minutes, but others remained around 30% (naproxen and diclofenac) or more than 80% of the initial concentration after 24 hours. A couple of unidentified peaks in a chromatogram of the chlorinated samples suggested the formation of unknown chlorination by-products. Competitive adsorption was observed when the mixed solution of the target pharmaceuticals was subjected to batch adsorption test with powdered activated carbon. Clofibric acid and ibuprofen, which were relatively less hydrophobic among the nine compounds, persisted around 60% of the initial concentration after 3 hours of contact time. Removal performance in actual drinking water treatment would become lower due to existence of other competitive substances in raw water (e.g. natural organic matter). Coagulation-sedimentation using polyaluminium chloride hardly removed most of the pharmaceuticals even under its optimal dose for turbidity removal. It is suggested that the most part of pharmaceuticals in raw water might persist in the course of conventional drinking water treatments.


Subject(s)
Charcoal/chemistry , Water Pollutants, Chemical/isolation & purification , Water Purification/methods , Antipyrine/analogs & derivatives , Antipyrine/chemistry , Antipyrine/isolation & purification , Clofibric Acid/chemistry , Clofibric Acid/isolation & purification , Diclofenac/chemistry , Diclofenac/isolation & purification , Fenoprofen/chemistry , Fenoprofen/isolation & purification , Gemfibrozil/chemistry , Gemfibrozil/isolation & purification , Halogenation , Ibuprofen/chemistry , Ibuprofen/isolation & purification , Indomethacin/chemistry , Indomethacin/isolation & purification , Ketoprofen/chemistry , Ketoprofen/isolation & purification , Naproxen/chemistry , Naproxen/isolation & purification , Water Pollutants, Chemical/chemistry , Water Supply/analysis
4.
Neuropediatrics ; 35(5): 274-8, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15534759

ABSTRACT

We report on a 10-year-old girl with anhidrosis and insensibility to pain, but no severe mental retardation or self-mutilation, diagnosed as hereditary sensory and autonomic neuropathy type IV (HSAN IV). Genetic analysis of her TRKA gene, which is responsible for HSAN IV, revealed two novel missense mutations in the tyrosine kinase domain. Cardiovascular autonomic nervous system function tests showed normal muscle sympathetic nerve activity associated with arterial baroreflex, reduced skin sympathetic nerve activity in the second and fifth fingers and palms, and abnormal circadian rhythm of cardiovascular autonomic nervous system. These findings have never before been reported in HSAN IV and may provide a clue to the neurological pathophysiology of this disease.


Subject(s)
Autonomic Nervous System/physiopathology , Cardiovascular System/physiopathology , Hereditary Sensory and Autonomic Neuropathies/physiopathology , Child , Female , Hereditary Sensory and Autonomic Neuropathies/genetics , Hereditary Sensory and Autonomic Neuropathies/pathology , Humans , Mutation, Missense/genetics , Receptor, trkA/genetics , Sural Nerve/pathology
5.
Eur J Surg ; 167(8): 598-604, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11716446

ABSTRACT

OBJECTIVE: To find out whether the addition of 1-hexylcarbamoyl-5-fluorouracil to a postoperative regimen of oral cyclophosphamide and tamoxifen improved the prognosis of carcinoma of the breast. DESIGN: Randomised controlled clinical trial. SETTING: 127 Institutions in Japan. SUBJECTS: 785 Patients with stage II carcinoma (palpable axillary nodes) who had total mastectomy and axillary clearance. INTERVENTIONS: The control group were given oral cyclophosphamide 50 mg/day and tamoxifen 20 mg/day for 2 years; the experimental group were given these drugs plus 1-hexylcarbamoyl-5-fluorouracil 300 mg/day for 2 years. RESULTS: There was no survival advantage (and more toxicity) in the experimental group, except in a subgroup with 1-3 axillary nodes involved. CONCLUSION: The advantage of triple chemotherapy in the subgroup must be substantiated by a new randomised trial confined to patients with 1-3 axillary nodes involved.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Fluorouracil/analogs & derivatives , Mastectomy , Administration, Oral , Adult , Aged , Axilla , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Breast Neoplasms/surgery , Combined Modality Therapy , Cyclophosphamide/administration & dosage , Female , Fluorouracil/administration & dosage , Humans , Lymphatic Metastasis , Middle Aged , Neoplasm Metastasis , Prospective Studies , Survival Rate , Tamoxifen/administration & dosage
6.
Rinsho Byori ; 49(2): 189-92, 2001 Feb.
Article in Japanese | MEDLINE | ID: mdl-11307315

ABSTRACT

In this report, we describe a one-year-old girl of the Miller-Dieker syndrome(MDS) with lissencephaly, seizures, microcephaly and mental disorders. Cytogenetic studies of this patient confirmed the presence of a 46,XX, 17ps+ chromosome karyotype, but it could not find the microdeletion of 17p13.3. Fluorescence in situ hybridization(FISH) studies confirmed a terminal deletion in the patient using the LIS1 gene probe which mapped to 17p13.3. Further it was also found the satellite on 17p13(17ps) in the patient who was rare associated with MDS. These findings suggest that FISH analysis may be useful method to detect microdeletion of LIS1 gene as 17-specific probe in the investigation of MDS patients.


Subject(s)
Brain/abnormalities , Chromosomes, Human, Pair 17 , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Female , Humans , In Situ Hybridization, Fluorescence , Infant , Microtubule-Associated Proteins/analysis
7.
Scand J Urol Nephrol ; 35(5): 350-6, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11771860

ABSTRACT

OBJECTIVE: The purpose of this study was to evaluate the circadian rhythm of the autonomic nervous system functions of children with primary monosymptomatic nocturnal enuresis. MATERIAL AND METHODS: The subjects were allocated to three groups--enuresis group (n = 72), normal control group (n = 26), post-treated and cured enuresis group (n = 13). In the three groups. we evaluated the autonomic nervous system function by the power spectrum analysis of heart rate variability (HRV) using 24-hour Holter electrocardiograms. Frequency components of the power spectra of HRV (% power) were divided into three factors; the very low frequency (VLF), the low frequency (LF) and the high frequency (HF) by computer analysis using Fast Fourier Transformation (FFT). RESULTS: In the three groups, the HF component reflecting parasympathetic activity was significantly higher, compared with the LF during sleep. The HF in the enuresis group was significantly higher, compared with the HF in the control group during sleep. The HF in the control group was significantly lower, compared with the LF during waking. However, the HF in the enuresis group was significantly higher, compared with the LF during waking. It showed more increased parasympathetic tone in the enuresis group, compared with that of the control group during sleep and waking. In the post-treated enuresis group, the HF during sleep and waking were significantly decreased, compared with those before treatment. CONCLUSION: These findings suggest the parasympathetic hyperfunction through 24 hours and abnormal circadian rhythm of autonomic nervous system functions in the enuresis group. The post-treated enuresis group showed normal autonomic nervous system functions.


Subject(s)
Autonomic Nervous System/physiopathology , Enuresis/physiopathology , Child , Circadian Rhythm , Electrocardiography, Ambulatory , Evaluation Studies as Topic , Female , Humans , Male
8.
Fertil Steril ; 74(4): 753-9, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11020519

ABSTRACT

OBJECTIVE: To quantify messenger RNA (mRNA) levels of the two estrogen receptor isoforms, estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) in the eutopic endometrium and ovarian endometriotic cysts. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Patients with endometriosis and patients with uterine leiomyoma or carcinoma in situ. INTERVENTION(S): Gonadotropin-releasing hormone agonist (GnRH-a)-treated (n = 12) or untreated (n = 24) endometriotic cysts were obtained from 36 patients during laparoscopic cystectomy. Eutopic endometrium tissues were obtained from 24 patients during or immediately after surgery. MAIN OUTCOME MEASURE(S): ER-alpha and ER-beta mRNA levels, using a real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay, TaqMan RT-PCR. RESULT(S): Eutopic endometrium and ovarian endometriotic cysts showed predominantly higher levels of ER-alpha mRNA than ER-beta mRNA. Although ER-alpha and ER-beta mRNA levels in the eutopic endometrium were affected by a cyclic change in ovarian hormones, ovarian endometriotic cysts were less affected. Moreover, a long-term hypoestrogenic state induced by GnRH-a especially decreased ER-alpha mRNA levels in endometriotic cysts. Consequently, the relative ratios of ER-alpha to ER-beta mRNA levels in both GnRH-a-treated and untreated endometriotic cysts were significantly lower than those in the eutopic endometrium. CONCLUSION(S): The results suggest that the principal and regulatory effects of estrogens may be mediated mainly via ER-alpha rather than ER-beta in both the eutopic endometrium and endometriotic cysts.


Subject(s)
Endometriosis/metabolism , Endometrium/metabolism , Ovary/metabolism , RNA, Messenger/metabolism , Receptors, Estrogen/analysis , Adult , Delayed-Action Preparations , Endometriosis/drug therapy , Estrogen Receptor alpha , Estrogen Receptor beta , Female , Humans , Leuprolide/administration & dosage , Leuprolide/therapeutic use , Prospective Studies , Receptors, Estrogen/genetics , Reverse Transcriptase Polymerase Chain Reaction
9.
Biosci Biotechnol Biochem ; 64(5): 1084-7, 2000 May.
Article in English | MEDLINE | ID: mdl-10879489

ABSTRACT

Functional analysis of regulatory elements in the human tissue plasminogen activator (tPA) gene showed that its promoter (-119/+169) is activated by UV irradiation in HeLa cells. We demonstrated here that the AP-2 like CCCCACCC sequence is involved in the UV-mediated activation and Sp1 binds to the sequence.


Subject(s)
Gene Expression Regulation , Response Elements/radiation effects , Tissue Plasminogen Activator/genetics , Ultraviolet Rays , Base Sequence , DNA Damage , Genes, Reporter , HeLa Cells/radiation effects , Humans , Melanoma/genetics , Molecular Sequence Data , Mutation , Recombinant Fusion Proteins , Response Elements/genetics , Sp1 Transcription Factor/metabolism , Tumor Cells, Cultured
10.
Rinsho Byori ; 48(2): 162-6, 2000 Feb.
Article in Japanese | MEDLINE | ID: mdl-10804820

ABSTRACT

The failure of chemotherapy to eradicate tumor cells is often due to the development of drug resistance. MDR(multidrug resistance) whose one form of resistance results from a decreased intracellular accumulation of the drugs, most often mediated by the overexpression of P-glycoprotein. MRP also related to pump function of cell membrane in acute leukemia. We have developed the new quantitative assay based on real-time PCR to measure expression of drug-resistance related genes such as MDR-1 and MRP in clinical samples. These results indicates that real-time PCR system is a reliable method to quantitatively determine drug resistant genes expression, it may be to predict responsiveness to chemotherapy by using this technique.


Subject(s)
Drug Resistance, Multiple/genetics , Neoplasms/genetics , Gene Expression , Humans , Neoplasms/drug therapy , Polymerase Chain Reaction/methods
11.
Rinsho Byori ; 48(1): 54-9, 2000 Jan.
Article in Japanese | MEDLINE | ID: mdl-10756676

ABSTRACT

We constructed the standard RNA synthesized for the chimeric AML1-MTG8 transcripts and the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase(GAPDH) transcripts in real-time quantitative RT-PCR system. AML-MTG8 transcripts was detectable in 10 fg of synthetic RNA(3.5 x 10(3) copies). Linearity was from 3.5 x 10(3) to 3.5 x 10(9) copies. Threshold cycle(CT) is defined as the fractional cycle number at which the reporter fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. The standard curve, where the known amounts of RNAs were used, showed a good correlation between the copies of AML1-MTG8 RNA and CT(r = -0.995). The within-run and day-to-day coefficients of variation(CV) in AML1-MTG8 RNA by this system were 9.5-24.7%(n = 10) and 21.7-42.2% (n = 8), respectively. GAPDH transcripts was detectable in 10 fg of synthetic RNA(6.1 x 10(4) copies). Linearity was from 6.1 x 10(4) to 6.1 x 10(8) copies. The standard curve, where the known amounts of RNAs were used, showed a good correlation between the copies of GAPDH RNA and CT(r = -0.993). The within-run and day-to-day CV in GAPDH RNA by this system were 9.3-14.6%(n = 10) and 14.7-15.8% (n = 10), respectively. Thus, we suggested that synthesized RNA as a standard RNA may be useful in quantitative RT-PCR for clinical application.


Subject(s)
Polymerase Chain Reaction , RNA/standards , Core Binding Factor Alpha 2 Subunit , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Humans , Oncogene Proteins, Fusion/genetics , RUNX1 Translocation Partner 1 Protein , Transcription Factors/genetics
12.
Rinsho Byori ; 48(10): 887-91, 2000 Oct.
Article in Japanese | MEDLINE | ID: mdl-11215098

ABSTRACT

Advancement molecular diagnostics in hematological malignancy has provided us with a broad menu of new assays and techniques. By integrating the data gleaned from these techniques we can formulate a more rational broad-based diagnosis. Hematological malignancies have traditionally been classified by morphological criteria. However, molecular advances have provided new insights in the genetic backbone of chimeric genes. These malignancies have shown chromosome abnormalities of translocation with chimeric genes, and revealed the rearrangement of chimeric genes by PCR analysis and quantitative PCR system. This review summarized the recent technology for detecting chimeric genes along with concepts of laboratory performance.


Subject(s)
Clinical Laboratory Techniques , Hematologic Neoplasms/diagnosis , Laboratories, Hospital , Adult , Chromosome Aberrations , Chromosome Disorders , Female , Gene Rearrangement , Hematologic Neoplasms/genetics , Humans , In Situ Hybridization, Fluorescence , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Recombinant Fusion Proteins/genetics
13.
Rinsho Byori ; 47(11): 1059-63, 1999 Nov.
Article in Japanese | MEDLINE | ID: mdl-10590684

ABSTRACT

The mutations of K-ras gene have been demonstrated at frequencies of about 40% in human colorectal cancer. We applied a developed PCR-preferential homoduplex formation assay (PCR-PHFA) to detect a point mutation of K-ras gene in the surgical specimens from thirty patients with colorectal cancer. This method is based on the strand competition during hybridization between a double labeled amplicon, prepared from biotin and DNP labeled primers, and an unlabeled amplicon. The procedure of this method is simple and speedy, and suitable to detect mutations in a small number of samples. By using this method, the mutations were found in 37% (11/30) and confirmed by sequencing analysis. The results suggest that the PCR-PHFA system may be useful for detecting low frequent mutation of K-ras gene even in the case of an early cancer.


Subject(s)
Colorectal Neoplasms/genetics , Genes, ras/genetics , Polymerase Chain Reaction/methods , Humans , Point Mutation
14.
Tohoku J Exp Med ; 187(3): 263-72, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10458483

ABSTRACT

We analyzed the androgen receptor (AR) gene in five Japanese patients diagnosed with androgen insensitivity syndrome (AIS). All AR genes from the five patients had single-nucleotide substitutions, which introduced a premature termination codon in three patients (Gln640, Arg752, and Gln640 and Trp751), and a single amino acid substitution in two patients (Arg831 to Gln, and Leu812 to Phe). All the mutations occurred in the steroid-binding domain, comprising exons D through G. The three patients with the premature termination codon(s) and the one patient with Arg831Gln were clinically diagnosed as having complete AIS, while the patient with Leu812Phe had a partial form of AIS. Pubic skin fibroblasts from four of the five patients did not show detectable androgen binding. These data on mutations that have not been reported previously, provide valuable information for the further characterization of structural and functional relationships in the steroid-binding domain of the AR protein.


Subject(s)
Androgen-Insensitivity Syndrome/genetics , Point Mutation , Receptors, Androgen/genetics , Adult , Asian People , Binding Sites/genetics , Humans , Japan , Male , Middle Aged
15.
Arzneimittelforschung ; 49(4): 304-10, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10337448

ABSTRACT

The abilities of 2-(2-methylphenyl)-5,7-dimethoxy-4-quinolyl carbonylguanidine dihydrochloride (CAS 181048-29-3, MS-31-050) and 2-phenyl-8-(2-methoxyethoxy)-4-quinolyl carbonylguanidine bismethanesulfonate (CAS 181048-36-2, MS-31-038) in inhibiting Na(+)-H+ exchange, ischemia- and reperfusion-induced injury were determined and compared with those of 4-isopropyl-3-methylsulfonylbenzoyl guanidine methanesulfonate (CAS 159138-81-5, IMGM), a selective inhibitor of Na(+)-H+ exchange. MS-31-050 and IMGM exhibited comparable inhibitory effects on Na(+)-dependent pH recovery and antiarrhythmic effects during ischemia in anesthetized rats. In rats subjected to ischemia and reperfusion, MS-31-050 (10 mg/kg i.v.) significantly reduced the infarct size when given prior to the onset of ischemia. However, postischemic treatment with either MS-31-050 or IMGM failed to protect reperfused hearts. In contrast, MS-31-038 reduced the infarct size dramatically from 65.4 +/- 7.4% in control to 29.9 +/- 11.6% at 3 mg/kg and 9.8 +/- 3.4% at 10 mg/kg even when administered before the onset of reperfusion. These results suggest the beneficial effects of Na(+)-H+ exchange inhibitors on myocardial ischemia/reperfusion injury.


Subject(s)
Enzyme Inhibitors/therapeutic use , Myocardial Infarction/drug therapy , Quinolines/therapeutic use , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Anesthesia , Animals , Hydrogen-Ion Concentration , In Vitro Techniques , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/metabolism , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myocardial Infarction/pathology , Myocardial Ischemia/complications , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/prevention & control , Rats , Rats, Sprague-Dawley , Tachycardia/etiology , Tachycardia/prevention & control , Ventricular Fibrillation/etiology , Ventricular Fibrillation/prevention & control
16.
Nucleic Acids Res ; 26(24): 5728-33, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9838005

ABSTRACT

We report a novel procedure, which can be applied to probing of specific DNA, for covalently attaching probe DNA to complementary sequences in double-stranded target DNA. Employing hairpin-like oligonucleotide probes in combination with successive use of recA protein and DNA ligase, probes can be attached directly to target DNA molecules without dissociation of the DNA. The hairpin-like structure of the probes was designed so that the terminus of the probe oligonucleotide can be brought into close stereochemical proximity to the terminus of the complementary strand of target DNA for ligation. Because of the elimination of the DNA dissociation and subsequent hybridization (and washing) steps in the currently employed method, the probing process has become greatly simplified and more efficient and may lead to development of fully automated probing systems.


Subject(s)
DNA Probes , DNA/analysis , Nucleic Acid Hybridization/methods , Animals , Base Sequence , DNA/genetics , Escherichia coli , Humans , Molecular Sequence Data
17.
Nucleic Acids Res ; 26(24): 5734-7, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9838006

ABSTRACT

A novel cloning procedure which makes use of covalent attachment of probe DNA to specific target DNA is reported. We show that specific gene fragments found in complex genomes such as the human genome can be cloned directly from a pool of genomic DNA with very high efficiency. This direct cloning method totally eliminates certain steps in current cloning procedures such as construction of DNA libraries and colony (plaque) hybridization. The resulting process has made cloning methods simpler and more time efficient, while achieving high cloning efficiency due to the stable nature of the probe-target DNA complex through covalent bonding. Most importantly, since clones are directly obtained from a pool of genomic DNA, the isolated clones are considered to be faithful copies of the original genes. This has apparently solved the problem of isolating clones with misincorporated bases or chimeric DNA, both of which are often encountered in cloning processes using PCR or other methods involving in vitro DNA synthesis.


Subject(s)
Cloning, Molecular/methods , DNA Probes , DNA/analysis , Animals , Base Sequence , DNA/genetics , Humans , Molecular Sequence Data
18.
Immunology ; 95(2): 200-7, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9824476

ABSTRACT

IgA antibodies in the mucosal immune system are produced specifically to environmental antigens such as virus and bacteria, and possibly to some food components, which will provide a potential luminal antigen, DNA. To study the immune response to DNA in the gut, we established B-cell hybridomas producing IgA monoclonal antibodies (mAb) from Peyer's patches (PP) of non-immunized, non-autoimmune, specific pathogen-free BALB/c mice, and examined their specificity by enzyme-linked immunosorbent assay (ELISA). Three mAb out of 18 bound strongly to self, bacterial and synthetic DNA, with Kd of about 10-7 m. One of the three mAb also reacted with the histone component and another reacted with some mouse food component. The VH genes of these three mAb have not previously been reported to have anti-DNA specificity, and carry putative somatically mutated sites favouring DNA binding in CDR. The features resemble those of anti-DNA antibodies found in human and murine models of systemic lupus erythmatosus (SLE), and are indicative of an antigen-driven selection process. Our findings suggest that even in normal healthy animals, anti-DNA antibodies of IgA isotype can be produced in certain peripheral environments such as in PP by spontaneous antigenic stimulation.


Subject(s)
Antibodies, Antinuclear/biosynthesis , B-Lymphocytes/immunology , Immunoglobulin A/immunology , Peyer's Patches/immunology , Amino Acid Sequence , Animals , Antibodies, Antinuclear/genetics , Antibodies, Antinuclear/immunology , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Base Sequence , DNA/immunology , Enzyme-Linked Immunosorbent Assay/methods , Female , Hybridomas/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mutation , Specific Pathogen-Free Organisms
19.
Biochemistry ; 37(40): 13958-67, 1998 Oct 06.
Article in English | MEDLINE | ID: mdl-9760230

ABSTRACT

The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equilibrium yielded a molecular mass of 96 +/- 2 kDa for the enzyme. Carbohydrate analysis revealed that both polypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleoside diphosphate kinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphate nucleotides. Immunogold labeling of bovine retina revealed that the enzyme is localized on both the membranes and in the cytoplasm. Screening of a retinal cDNA library yielded full-length clones encoding two distinct isoforms (NBR-A and NBR-B). Both isoforms were overexpressed in Escherichia coli and their biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-B were determined by X-ray crystallography in the presence of guanine nucleotide(s). Both isoforms are hexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures. The NBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while the NBR-B active site contained only cGMP.


Subject(s)
Nucleoside-Diphosphate Kinase/isolation & purification , Nucleoside-Diphosphate Kinase/metabolism , Protein Conformation , Retina/enzymology , Amino Acid Sequence , Animals , Binding Sites , Carbohydrates/analysis , Cattle , Cloning, Molecular , Crystallography, X-Ray , Cyclic GMP/metabolism , Guanosine Diphosphate/metabolism , Molecular Sequence Data , Nucleoside-Diphosphate Kinase/chemistry , Nucleoside-Diphosphate Kinase/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Retina/chemistry , Retina/ultrastructure , Subcellular Fractions/enzymology
20.
Childs Nerv Syst ; 12(2): 69-75, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8674084

ABSTRACT

Moyamoya disease is a cerebrovascular obstructive disease of unknown etiology. The rebuild-up phenomenon, slowing of waves on electroencephalogram (EEG) seen after cessation of hyperventilation (HV), is one of the characteristic phenomena of the disease and is thought to be related to a development of its symptoms. Therefore, we investigated the mechanism involved in the rebuild-up phenomenon to clarify the mechanism of development of transient ischemic attack (TIA) in moyamoya disease. Ten patients with moyamoya disease were studied; they ranged in age from 7 to 17 years. The power spectra of the EEGs in the occipital region were obtained with a Berg Fourier EEG analyzer for quantitative analysis. Arterial blood gas change (pH, PaO2, PaCO2), respiratory pattern (abdominal and nasal), tidal volume and respiratory rate were analyzed simultaneously every 30 s-1 min before, during, and after HV. The slow wave power spectrum (rebuild-up) increased and symptoms of TIA developed as a result of the sharp decrease in PaO2 (PaO2 60.5 +/- 15.4 mmHg) after cessation of HV. Based on the fact that hypoxemia was playing a main role, 100% oxygen was administered at a rate of 0.5 l/min in 4 cases where the rebuild-up phenomenon was clear. The EEG power spectra and arterial blood gas were analyzed during rebuild-up with and without O2 administration. The effectiveness of O2 administration at the beginning of rebuild-up as measure to prevent the symptoms was checked by a recovery rate of slow wave power percentage, a recovery time of slow wave power percent and by clinical observation. The recovery rates were 11.8 +/- 4.2%/min and 5.5 +/- 4.0%/min with and without O2 inhalation, respectively (P < 0.001). Recovery times of slow wave power percentage were 4.3 +/- 1.8 min and 8.1 +/- 1.2 min with and without O2 inhalation, respectively (P < 0.01). Thus, oxygen administration soon after the cessation of HV was shown to be effective in eliminating the rebuild-up phenomenon and hence in abolishing its symptoms.


Subject(s)
Hypoxia/therapy , Ischemic Attack, Transient/complications , Ischemic Attack, Transient/therapy , Moyamoya Disease/complications , Oxygen Inhalation Therapy , Adolescent , Blood Gas Analysis , Cerebral Angiography , Child , Electroencephalography , Female , Humans , Hyperventilation/complications , Hyperventilation/diagnosis , Hypoxia/etiology , Ischemic Attack, Transient/diagnosis , Male , Tomography, X-Ray Computed
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