Biomedical subjects
Publications and source records attributed to S Fukuda.
We describe two common single-base polymorphisms of the N-acetylgalactosamine-6-sulfate sulfatase gene after StyI or StuI restriction sites. These polymorphisms were readily detected by single-strand conformation polymorphism (SSCP), using the polymerase chain reaction (PCR).
Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or non-carrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.
Tailgut cysts are rare congenital lesions. To date, only four cases have been reported in Japan, and the occurrence of a tailgut cyst with rectal cancer has never been documented. We describe here the case of a patient in whom a tailgut cyst in the retrorectal space was associated with rectal cancer. Preoperative computed tomography scans and endorectal ultrasonography failed to identify the lesion as cystic, instead suggesting an involved lymph node. This case emphasizes the necessity for careful diagnosis of masses in the retrorectal space in patients with rectal cancer.
Helicobacter pylori in the stomach is an etiological factor of gastritis and peptic ulcer. It is now considered that gastric cancer can be, at least in some cases, a late complication of H. pylori infection. In 123 consecutive endoscopic antral biopsies obtained from patients with the Okamoto Hospital, the specimens were subjected to the rapid urease test (RUT), histology (H&E stain), and culture, for the identification of H. pylori. The results of these methods were compared semi-quantitatively in order to evaluate these detection methods for identifying H. pylori. The results of these methods were found to agree well, with the Spearman's rank correlation coefficient between RUT and culture being 0.90 (P < 0.01) and that between histology and culture being 0.80 (P < 0.01). RUT is considered to be a very simple, sensitive, and highly specific test which enables the endoscopist to diagnose H. pylori infection.
The three-dimensional fibrillar arrangement of the basilar membrane in the mouse cochlea was studied using scanning electron microscopy. Fibrils of the basilar membrane were exposed by removing cellular elements of the cochlea using a sodium hydroxide maceration technique. The arrangement of fibrils in the basilar membrane was different between the pars arcuata and pars pectinata. In the pars arcuata, fibrils were arranged in radial and spiral direction, showing a woven pattern. In the pars pectinata, most of the fibrils ran in the radial direction. These findings suggest that the vibration pattern of the pars arcuata and pars pectinata is different when the basilar membrane vibrates.
BACKGROUND: The left thoracotomy approach to avoid injury of the patent old graft and the myocardium with mid sternal reentry at coronary artery reoperation. METHODS: The left thoracotomy approach was used in 13 patients. There were 11 men and 2 women with a mean age of 63 years, ranging from 39 to 75 years. Three patients were having their third coronary bypass operation. In 11 patients, distal anastomoses were performed under circulatory arrest with moderate hypothermia. In the other 2 patients, distal anastomoses were performed on a beating heart. No aortic cross-clamp was applied in all patients. The mean number of distal anastomoses was 1.8; the grafted vessels were 11 anterior descending, 3 diagonal, 8 circumflex, and 1 posterolateral coronary arteries. Used grafts were 17 saphenous veins, 4 left internal thoracic arteries, and 2 gastroepiploic arteries. Inflow sites of the free graft were descending aorta in 10 patients and left subclavian artery in 3 patients. RESULTS: All patients were alive and well at the mean follow-up of 16 months, and all grafts were patent. CONCLUSIONS: The left thoracotomy approach is safe and effective for reoperation on the left coronary artery system, and circulatory arrest is convenient and safe for performing distal anastomosis.
By using the reverse transcription polymerase chain reaction and Southern blot hybridization, we demonstrated that Fc epsilon RI alpha-chain (Fc epsilon RI alpha) messenger RNA was expressed in eosinophils purified from the peripheral blood of patients with allergic rhinitis and that this expression was enhanced by IL-4. However, studies in which flow cytometry or immunostaining was used did not reveal the expression of Fc epsilon RI alpha protein on eosinophils from peripheral blood. Neither IL-4 alone nor the combination of IL-4 and other cytokines could induce detectable Fc epsilon RI alpha protein; nevertheless, they do express Fc epsilon RI alpha mRNA. Double-labeling immunostaining on cryostat sections of nasal mucosa clearly demonstrated that some Fc epsilon RI alpha-positive cells were eosinophil cationic protein-positive, which confirms their eosinophilic nature. Not all the eosinophil cationic protein-positive cells express on Fc epsilon RI alpha signal. Considering that Fc epsilon RI alpha mRNA was detectable in four of five samples of eosinophils from those patients with nasal allergy and that only one of five eosinophil samples from normal subjects expressed Fc epsilon RI alpha mRNA, the level of Fc epsilon RI expression may be correlated with the activation of eosinophils. It seems very likely that some other unidentified factors are required for the process from the expression of Fc epsilon RI alpha mRNA to that of Fc epsilon RI as a protein.
OBJECTIVES: Several substances may be released from ischemic tissues with the declamping shock that occurs during abdominal aortic aneurysm surgery. To clarify the relationship between tissue ischemia and venous endothelin-1 (ET-1) level in humans, plasma ET-1 and oxygen content in the iliac vein were measured before anesthesia, after the induction of anesthesia, after the release of the proximal and first distal clamps, after the release of the second distal clamp, and 1 hour after the second clamping. DESIGN: Prospective study. SETTING: A University hospital. PARTICIPANTS: Seven patients who underwent abdominal aneurysmectomy and replacement with bifurcated graft. INTERVENTIONS: A 20G catheter was inserted into the radial artery for the direct measurement of blood pressure and for collecting arterial blood. An 18G, 20-cm catheter was inserted into the femoral vein for collecting venous blood from the lower extremities. MEASUREMENTS AND MAIN RESULTS: The arterial and venous ET-1 levels did not change after the induction of anesthesia. Immediately after the initial release of the proximal and distal clamps, venous oxygen content dramatically decreased from 11.3 to 3.6 mL/dL (vol%) with significant increases in venous ET-1 concentration from 2.3 to 4.9 pg/mL. Concomitant with the decrease in venous oxygen content, venous plasma pH and base excess decreased with increase in PCO2, suggesting that ischemic changes in tissues distal to the cross-clamp may occur during aortic clamping. Venous ET-1 levels were significantly correlated with venous oxygen content, pH, PO2, oxygen saturation, base excess, blood sodium concentration, and potassium concentration. One hour after the second declamping, the venous ET-1 level remained high in comparison with the preanesthetic level, whereas the venous oxygen content returned to the preanesthetic level. There was no correlation between venous plasma ET-1 and venous plasma norepinephrine or epinephrine concentration. CONCLUSIONS: Tissue ischemia may increase venous ET-1 levels in humans. Factor(s) other than tissue ischemia may provoke the increase in venous ET-1 that occurs after the release of the second distal clamp.
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The adhesion of cells to the microvascular endothelium is an essential step in the inflammatory response and metastasis. We have found that pretreatment of a human epidermoid carcinoma cell line, KB, with natural human interferon-alpha (IFN-alpha) inhibited the binding of the malignant cells to human umbilical vein endothelial cells (HUVEC) in a dose- and time-dependent manner. As one of several possible mechanisms for this inhibition, the expression of some revelant adhesion molecules on KB cell surfaces was examined after IFN-alpha treatment. Apart from a slight increase in the expression of integrin alpha 4 beta 1 (very late activation antigen 4, VLA-4), no changes in the expression of other adhesion molecules, such as sialyl Lewis X, CD44, and leukocyte function-associated antigen 1 (LFA-1), which is known to be a heterodimer of CD11a and CD18, were observed after treatment with IFN-alpha. In addition, the cell viability of KB was not affected by treatment of the cells with IFN-alpha, although the cell proliferation was markedly inhibited, indicating that the inhibitory effect of IFN-alpha on KB cell binding to vascular endothelium is not a result of a cytotoxic effect of IFN-alpha. Because the metastatic process requires not only the adhesion of tumor cells to vascular endothelium during their extravasation but also proliferation at distant sites, our findings from this in vitro experimental model suggest that IFN-alpha may have a potential inhibitory effect on tumor cell metastasis.
Mutations causing mucopolysaccharidosis IVA in 15 Japanese and one Caucasian patient were characterized. To screen these mutations, we used a combination of single strand conformation polymorphism analysis and heteroduplex analysis for PCR products of targeted cDNA or genomic DNA. Various small mutations were identified in 23 of 26 alleles, while the other six alleles had large rearrangements. Cycle sequencing of PCR products revealed 15 different mutations, including 12 missense, one nonsense, one frame shift (2 bp deletion) and one splice site mutation, in accord with the broad range of clinical phenotypes. Two alleles have different mutations in the same nucleotide position of exon 3 (R94C, CGC-->TGC; R94G, CGC-->GGC), diagnosed by sequencing and by allelic-specific oligohybridization (ASO). One allele had two amino acid changes, E450V in exon 12 and V488M in exon 13, thereby indicating a double point mutation. All 16 mutations reported were confirmed by restriction enzyme assay or by allelic-specific oligohybridization. Transfection of mutagenized cDNAs into patients' fibroblasts showed that all mutations caused completely deficient or markedly decreased N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity, thereby indicating that these mutations were responsible for the enzyme deficiency.
Mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, is a lysosomal storage disorder caused by a deficiency in the enzyme beta-glucuronidase. Various clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of human beta-glucuronidase cDNA and the genomic sequences facilitate analysis of molecular defects underlying the different phenotypes, and eight mutations in the beta-glucuronidase gene have been described. This report summarizes studies characterizing four new mutations in two Caucasian patients with a severe form of MPS VII. Three are point mutations, resulting in two missense and one nonsense change, and one is a 38 bp deletion. The first patient was a compound heterozygote having P148S and Y495C alleles. The second patient was a compound heterozygote of W507X and a 38 bp deletion at position 1642-1679 in exon 10(1642 delta 38nt). The 38 bp deletion was caused by a single base change mutation in exon 10 that generates a new, premature 5' splice site. Expression of mutant cDNAs encoding each of the four mutations showed that all four resulted in a severe reduction of beta-glucuronidase activity, indicating that these mutations are responsible for the reduced enzyme activity in patient cells. These four previously undescribed mutations provide further evidence for the broad molecular heterogeneity in Sly syndrome.
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The glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen was treated with pepsin and glycopeptidase A to release asparagine-linked oligosaccharides. The reducing ends of the oligosaccharides were aminated with the fluorescent reagent 2-aminopyridine. The oligosaccharide derivatives were purified by gel permeation chromatography and reversed-phase HPLC. Their structures were determined by sequential exoglycosidase digestion and 500 MHz 1H-NMR spectroscopy. Four oligosaccharide structures, A, B, C, and D, were identified as the xylose-containing complex-type. They were present at a molar ratio of 8:1:6:1. By amino acid sequence analyses of the tryptic peptides, Asn-170 and Asn-333 of Cry j I were found to carry asparagine-linked oligosaccharides. [formula: see text]
This study attempted to determine the efficacy of lansoprazole plus clarithromycin therapy in the eradication of Helicobacter pylori in gastric ulcer patients. The influence of H. pylori eradication on healing and relapse of ulcers was also studied. Thirty-nine patients received either lansoprazole 30 mg daily for 8 weeks (group 1) or clarithromycin 200 mg twice daily for 2 weeks and lansoprazole 30 mg daily for 8 weeks (group 2). Before treatment, H. pylori status was evaluated by a rapid urease test and histologic examination. H. pylori clearance and eradication were evaluated by a rapid urease test, polymerase chain reaction, and a [13C]urea breath test. Clearance of H. pylori was 0% in group 1 and was 33% in group 2. Eradication of H. pylori was 0% in group 1 and 21% in group 2. Although all five ulcers were healed in patients with H. pylori eradication, ulcers were not healed in the five patients without eradication. Relapse of ulcer was observed in three patients in whom eradication had failed. In this study, for H. pylori-positive gastric ulcer patients, better results were obtained when lansoprazole plus clarithromycin therapy was used, and H. pylori eradication was achieved.
We found that mycoplasma-infected cells have a higher ability to metastasize in vivo than non-mycoplasma-infected cells. To investigate this phenomenon, we obtained a monoclonal antibody, MAb 243-5, by immunization with Mycoplasma arginini-infected RPMI 4788 cells. This MAb recognized a mycoplasmal protein with an MW of 47 kDa and completely inhibited the experimental metastasis of M. arginini-infected RPMI 4788 cells using a nude mouse model. Using this MAb, we purified a molecule called Ag 243-5 and determined the N-terminal amino acid sequence and clarified the entire nucleotide sequence of the Ag 243-5 gene. PCR analysis showed the existence of a homologous gene in Mycoplasma hyorhinis. Four sequential injections of Ag 243-5 (30 micrograms/shot) promoted the experimental metastasis of non-mycoplasma-infected RPMI 4788 cells more than 10-fold using a nude mouse model. Ag 243-5 also promoted the experimental metastasis of the non-mycoplasma-infected mouse colon cancer cell line colon 26. This metastasis-promoting effect was neutralized by MAb 243-5.
We examined Cry j 2, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, for polygalacturonase enzyme activity, since a nucleotide sequence of cDNA of Cry j 2 showed a significant homology with that of tomato polygalacturonase. Polygalacturonase is well known to depolymerize preferentially polygalacturonic acid (PGA) by hydrolysis. However, Cry j 2 did not act on PGA, but was found to depolymerize pectin and methylesterified PGA in a dose-dependent manner. The substrate specificity of Cry j 2 was different from that of polygalacturonase derived from Aspergillus niger. The depolymerizing activity of Cry j 2 reached a maximum at 50%-60% of methylesterification of PGA. In contrast, polygalacturonase showed its maximum activity of PGA, and the activity decreased as the degree of methylesterification increased. Interestingly, the pectin-depolymerizing activity of Cry j 2 was due to a hydrolysis, but not a lyase, activity which splits the glycosidic bonds by beta-elimination, since no unsaturated uronides were found by measurement of absorbance at 235 nm in the reaction mixture. The enzyme activity was markedly inhibited by anti-Cry j 2 antibodies. These results indicate that Cry j 2 probably has polymethylgalacturonase enzyme activity, as postulated by von Neukom in 1963, although existence of this activity has not yet been proven.
1. Because of low levels of serum calcium (Ca) in stroke-prone spontaneously hypertensive rats (SHRSP), intestinal Ca absorption and urinary Ca excretion were examined. 2. SHRSP were observed to have lower intestinal Ca absorption and higher urinary Ca excretion, but with a latent function of intestinal Ca absorption able to respond with calcium regulating hormones to Ca loading in the aged, especially to a new Ca agent as examined in this study.