Preliminary results of gene transfer to central nervous system by continuous injection of DNA-liposome complex.
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Analysis of Variance↗
Biomedical subjects
T Yasuda
Publications and source records attributed to T Yasuda.
Ribonuclease inhibitors in human blood: comparative studies on the inhibitors detected in erythrocytes, platelets, mononuclear leukocytes and granulocytes.
The erythrocyte ribonuclease inhibitor (RI) and platelet RI were separately purified to homogeneity and showed similar properties to those of human brain and placental RIs. The same type of RI seemed to be present in mononuclear leukocytes and granulocytes. The RI contents of these cells were detectable by immunoblot analysis using anti-human placental RI antibody. Neither RI activity nor the molecule cross-reactive with the anti-human placental RI antibody was detectable in any plasma sample. It is intriguing to detect active RI in mature erythrocytes, where no nucleus exists and RNA metabolism is unlikely to occur.
Amino Acid Sequence↗
Deoxyribonuclease I from rat urine: affinity purification, characterization, and immunochemical studies.
Deoxyribonuclease I (DNase I) from rat urine was purified about 3,000-fold to apparent homogeneity with a 14% yield by affinity chromatography utilizing polyguanylic acid-agarose and DNA-cellulose. The purified enzyme preparation was found to contain no other detectable nucleases. Isoelectric focusing electrophoresis revealed that all six isoelectric forms of the enzyme had been purified, and the resulting bands all contained DNase I activity. Quantitative amino acid analysis and N-terminal amino acid sequencing were performed on the purified DNase I. The N-terminal sequence up to the 15th residue of the enzyme was identical to that of rat parotid DNase I. The enzyme was found to be a glycoprotein, containing 1 fucose, 10 galactose, 17 mannose, 12 glucosamine, and at least 3 sialic acid residues per molecule. The isoelectric multiplicity of the enzyme was partly due to differences in the sialic acid content of the isoforms. Gel filtration on Superose 12 and electrophoresis on sodium dodecyl sulfate polyacrylamide gels indicated an approximate molecular mass for DNase I of 32 kDa. The enzyme had an optimum pH of 6.5 and required divalent cations such as Ca2+ for its activity. Its activity was inhibited by 1 mM EDTA and EGTA, but not G-actin. An antibody against the purified enzyme was found to be monospecific against rat urine and the pure antigen, and completely blocked the activity of the purified enzyme.
Amino Acid Sequence↗
Production of monoclonal antibodies directed to Hanganutziu-Deicher active gangliosides, N-glycolylneuraminic acid-containing gangliosides.
We have established three kinds of monoclonal antibodies against gangliosides containing N-glycolylneuraminic acid (NeuGc) by immunization of BALB/c mice with the purified gangliosides inserted into liposomes comprising Salmonella minnesota R595 lipopolysaccharides, and fusion of spleen cells with a mouse myeloma cell line. One monoclonal antibody, SHS-1, which was generated by immunizing mice with purified i-active ganglioside(NeuGc), reacted specifically with the i-active ganglioside(NeuGc) used as an immunogen. Structurally related gangliosides, such as GM3(NeuGc), sialosylparagloboside (SPG) (NeuGc), or I-active ganglioside(NeuGc), corresponding gangliosides [GM3 containing N-acetylneuraminic acid (NeuAc), SPG(NeuAc), i-active ganglioside(NeuAc), and I-active ganglioside(NeuAc)], other gangliosides, or neutral glycosphingolipid (GSL) were not recognized by the monoclonal antibody. These findings indicate that the SHS-1 monoclonal antibody may be specific for NeuGc-containing i-active ganglioside. On the other hand, the other two monoclonal antibodies, MSG-1 and SPS-20, which were generated by immunizing mice with purified ganglioside GM3(NeuGc) and SPG(NeuGc), respectively, showed cross-reactivity to structurally related gangliosides. The MSG-1 monoclonal antibody exhibited reactivity to ganglioside GM3(NeuAc). The SPS-20 monoclonal antibody also cross-reacted with SPG(NeuAc), i-active ganglioside(NeuGc), and i-active ganglioside(NeuAc). Neither MSG-1 nor SPS-20 reacted with corresponding gangliosides, other gangliosides, or neutral GSLs tested. Using the SHS-1 antibody specific for i-active ganglioside(NeuGc), we studied the expression of NeuGc-containing antigen in human colon cancer tissue. An NeuGc-containing glycoconjugate was detected in the colon cancer tissue.
Animals↗
Molecular analysis of the third allele of human deoxyribonuclease I polymorphism.
In addition to common phenotypes 1, 1-2 and 2 of human deoxyribonuclease I (DNase I), phenotypes 1-3 and 2-3, encoded by a third allele DNASE1*3, have been found by means of isoelectric focusing. The main objective of this study was to identify the mutation site(s) underlying phenotype 3. All eight exons covering the entire open reading frame of the human DNase I structural gene were amplified by the polymerase chain reaction (PCR) and subjected to direct DNA sequencing. When the entire 780-bp coding region and exon/intron junctions of the DNase I gene of two individuals with phenotypes 1-3 and 2-3 were sequenced, only one nucleotide substitution, a C-G transition (CCC-->GCC), in the codon for amino acid 132 of the mature enzyme located in exon VI was found that resulted in the replacement of proline with alanine (P132A). The mutation was confirmed by allele-specific amplification of genomic DNA. The replacement of the amino acid residue may reduce the hydrophobicity of the enzyme and thus increase the pI value of the type-3 isozyme compared with that of type 1, as increasing the hydrophobicity of a protein is known to decrease its pI value. The specific PCR-amplifications of exons and alleles developed in this study may provide a new tool suitable for rapid screening of DNase I variants.
Alleles↗
Structure of the human deoxyribonuclease I (DNase I) gene: identification of the nucleotide substitution that generates its classical genetic polymorphism.
The objectives of this study were to elucidate the structural organization of the gene for human deoxyribonuclease I (DNase I) and to identify the mutation site underlying its classical genetic polymorphism. In order to determine the organization of this gene, we utilized a combination of direct polymerase chain reaction (PCR)-amplification of human genomic DNA and isolation of the overlapping clones from a cosmid human genomic library. Restriction endonuclease mapping, Southern blotting and DNA sequencing showed that the DNase I gene was approximately 3.2 kilobases long, it comprised 9 (I-IX) exons separated by eight introns and its complete sequence was determined. The first exon contained only the non-translated sequences of mRNA. In addition to several putative regulatory elements, TATA-like and CAAT-like sequences were observed in the region upstream of the translation initiation codon. These results provide information that will help to understand the expression and regulation of DNase I. The isoelectric focusing patterns of human DNase I showed that it exhibits classical genetic polymorphism (Kishi et al. 1989, 1990). A comparison of the entire translated sequences of the DNase I gene from two pairs of individuals with common DNase I phenotypes 1 and 2 revealed only one nucleotide residue difference in exon VIII, A for phenotype 1 and G for phenotype 2, thus producing Gln and Arg amino acid substitutions respectively at position 222 from the NH2-terminus of the mature enzyme. The predicted charge changes attributable to these amino acid substitutions are entirely consistent with the isoelectric focusing profiles of these two DNase I isozymes. We conclude that this substitution is solely responsible for the classical polymorphism of DNase I protein.
Adult↗
The pcsA gene is identical to dinD in Escherichia coli.
The pcsA68 mutant of Escherichia coli is a cold-sensitive mutant which forms long filaments with a large nucleoid in the central region at 20 degrees C. We here show that (i) the coding region for the pcsA gene is identical with orfY located upstream of pyrE and can be deleted without loss of viability; (ii) pcsA is also identical to dinD, a DNA damage-inducible gene, whose expression is regulated by the LexA-RecA system; (iii) the cold-sensitive phenotype of the pcsA68 mutation is suppressed by delta recA or lexA1 (Ind-) mutation, but not by sulA inactivation; (iv) overproduction of PcsA68 leads to inhibition of cell growth in recA+ and delta recA strains at 20 and 37 degrees C, but PcsA+ does not show such an effect at any temperature; (v) SOS response is induced in the pcsA68 mutant cells at 20 degrees C. We discuss the possible function of the pcsA gene, comparing it with the sulA or the dif-xerCD function. We also describe a new method for gene disruption with positive and negative selection.
Bacterial Proteins↗
Unique cholesteryl glucosides in Helicobacter pylori: composition and structural analysis.
A chloroform-methanol-extracted lipid of Helicobacter pylori was studied. Three kinds of glycolipids, accounting for about 25% (wt/wt) of the total lipid, were detected and identified to be cholesteryl glucosides. The structures of two of them were determined to be cholesteryl-alpha-D-glucopyranoside and cholesteryl-6-O-tetrade-canoyl-alpha-D-glucopyranoside, and the plausible structure of the third one was identified as cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside. Cholesteryl glucosides are very rare in animals and bacteria. Furthermore, those in H. pylori had an alpha-glycosidic linkage, which is rather unusual for natural glycosides, and a phosphate-linked cholesteryl glycoside like the cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside has not been reported previously. As the cholesterol glucosides were detected in strains obtained from diverse geographical locations, the presence of cholesteryl glucosides in H. pylori is a very unique and a characteristic feature of the species. These findings add a new facet to the physiology and biochemistry, especially the cholesterol and glucose metabolism, of H. pylori. Furthermore, the cholesteryl glucosides of H. pylori showed hemolytic activities.
Cholesterol↗
Multidrug resistance gene expression in childhood medulloblastoma: correlation with clinical outcome and DNA ploidy in 29 patients.
Twenty-nine children treated for medulloblastoma between 1987 and 1991 were reviewed. Thirteen patients with high-risk medulloblastoma characterized by incomplete resection, diploid tumor or subarachnoid dissemination received chemotherapy following radiation therapy. Three received postoperative chemotherapy. Eight patients who had been treated with postoperative radiation therapy also received chemotherapy for recurrent tumors. After a minimum 3-year follow-up period, 16 were alive but 13 had died from recurrent tumors. In order to evaluate the possible participation of P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) in medulloblastoma therapy and its correlation with prognosis, archival specimens were examined by immuno-histochemistry utilizing 3 monoclonal antibodies against Pgp and 6 cases by reverse-transcriptase polymerase chain reaction (RT-PCR) using MDR1-specific primers. Sixteen patients (55%) had MDR expression detected either by 1 of the 3 antibodies or by RT-PCR. DNA ploidy study was also performed on 18 specimens. We correlated patients' outcome with variable factors (extent of surgical resection, chemotherapy, DNA ploidy) and MDR expression. Patients who were treated with radiation therapy and adjuvant chemotherapy had a significantly better (p = 0.036) survival than those with radiation therapy alone, despite the fact that the former group of patients was considered to be high-risk. The extent of surgical resection and DNA ploidy did not correlate with prognosis. However, a statistically significant association was found between MDR expression and outcome (p = 0.007). Among the patients who received chemotherapy, positive MDR expression significantly correlated with poor outcome (p = 0.036). Our results showed that Pgp-mediated intrinsic MDR in medulloblastomas seems to correlate with an adverse outcome. This information may be used in designing new therapeutic protocols for medulloblastoma.
Adolescent↗
Chromosomal assignment of the human deoxyribonuclease I gene, DNASE 1 (DNL1), to band 16p13.3 using the polymerase chain reaction.
To localize the human deoxyribonuclease I (DNase I) gene, DNASE1 (DNL1), we performed a polymerase chain reaction (PCR) using DNA extracted from a panel of cloned human x rodent hybrid cell lines carrying different human chromosomes and screened for the presence of the expected PCR products. Two different sets of oligonucleotide primers specific for human DNase I cDNA sequences were used to amplify unique fragments in the human DNase I gene. Based on this work, DNL1 could be assigned to human chromosome 16. Furthermore, regional localization of the gene to 16p13.3 was performed by PCR analysis of a high-resolution mouse x human somatic cell hybrid panel that contained defined portions of human chromosome 16.
Animals↗
Normal post-race antimyosin myocardial scintigraphy in asymptomatic marathon runners with elevated serum creatine kinase MB isoenzyme and troponin T levels. Evidence against silent myocardial cell necrosis.
Recent epidemiologic studies confirm that heavy physical exertion can trigger myocardial infarction. Diagnosis of acute myocardial injury in marathon runners is complicated by elevations of serum creatine kinase MB isoenzyme activity in asymptomatic finishers with normal post-race infarct-avid myocardial scintigraphy. Such isoenzyme elevations can arise from exertional rhabdomyolysis of skeletal muscle biochemically altered by training, from silent injury to the myocardium or from a combined tissue source. To assess silent myocardial cell necrosis in marathon runners, we performed quantitative anti-myosin myocardial scintigraphy after competition with serum immunoassays for creatine kinase MB isoenzyme and troponin T. Therefore, 8 male marathon runners with a mean age of 52 years underwent quantitative antimyosin myocardial scintigraphy immediately following the 1988 and 1993 Boston Marathons. Serum immunoassays for creatine kinase MB isoenzyme by a chemiluminescent method (CLIA) and troponin T by an enzyme-linked immunosorbent assay were performed in 4 runners after the 1993 race. Quantitative antimyosin myocardial scintigraphy was normal in all runners including 3 who participated after both races 5 years apart. Post-race serum creatine kinase MB isoenzyme and/or troponin T levels were in a range otherwise diagnostic of acute myocardial infarction in 3 of 4 subjects. Normal quantitative antimyosin myocardial imaging in asymptomatic marathon runners excludes silent myocardial cell necrosis as the source of elevated serum protein markers. Such imaging may be the optimal diagnostic modality for detection of myocardial cell necrosis in symptomatic athletes when results of conventional testing are inconclusive.
Adult↗
Induction of heparin-binding epidermal growth factor-like growth factor mRNA in rat kidney after acute injury.
Previous studies have suggested that EGF or other members of the EGF family of mitogenic proteins are involved in proliferation of renal tubular epithelial cells occurring during recovery from injury to the kidney. The present studies examined whether expression of mRNA for the recently identified heparin-binding EGF-like growth factor (HB-EGF) is regulated in response to renal injury induced by either ischemia/reperfusion or mercuric chloride. Increased expression of HB-EGF mRNA was demonstrated in the post-ischemic kidney within 45 min of unilateral ischemia/reperfusion in the rat. Induction of HB-EGF mRNA occurred only when ischemia was followed by reperfusion, and was not eliminated by removal of blood cells from the post-ischemic kidney by saline perfusion. In situ hybridization with 35S-labeled antisense riboprobes of HB-EGF indicated that compared with control, there was increased HB-EGF mRNA expression in the 6 h post-ischemic kidney in the inner cortex and outer medulla in a patchy distribution, with the greatest expression in the inner stripe of the outer medulla. Expression occurred primarily in tubular epithelial cells. Recombinant human HB-EGF stimulated [3H]-thymidine incorporation in both primary cultures of rabbit proximal tubule cells and NRK 52E normal rat kidney epithelial cells, with potency similar to that of EGF. Induction of HB-EGF mRNA was observed in tubules freshly isolated from rat renal cortex or outer medulla when the tubules were subjected to reoxygenation after incubation in anoxic conditions. The nephrotoxin, mercuric chloride, also caused induction of HB-EGF mRNA both in vivo and in isolated rat cortical tubules. The anoxia/reoxygenation-induced expression of HB-EGF mRNA in isolated tubules was inhibited by the free radical scavengers, di- and tetra-methylthiourea, indicating involvement of reactive oxygen species. These findings indicate that HB-EGF mRNA is inducible in the kidney in vivo by acute tubular injury and suggest that HB-EGF may act as an autocrine/paracrine growth factor involved in proliferation of tubular epithelial cells and repair of the kidney.
Animals↗
MRI demonstrates dorsal column involvement of the spinal cord in Sjögren's syndrome-associated neuropathy.
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Aged↗
Fatty acid composition of ganglioside GM3 of renal glomerular epithelial SGE1 cells during spontaneous dome formation in vitro.
Ganglioside GM3, one of acidic components of membrane glycosphingolipids (GSL), has been known to change its content quantitatively during growth and differentiation of various cells in vitro. Detailed analysis of lipid portion of GM3 of rat renal glomerular SGE1 cells revealed that fatty acids with long carbon chains, especially that of C24:0 and C24:1 increased, while that of short C18:0 and C20:0 decreased after spontaneous dome formation. Since not only fatty acid composition of neutral GSL, sulfatide and phospholipid but also composition of long-chain bases (LCB) did not change, it was suggested that only C24 fatty acid of GM3 specifically increased in relation to dome formation. The spontaneous dome formation has been reported to be related with induction of cellular differentiation in many transporting epithelial cells. We thus assume that the change of fatty acid composition of GM3 is involved in cellular differentiation of SGE1 cells.
Animals↗
Urinary and biliary metabolites of puerarin in rats.
Examination was made of the urinary and biliary excretion of the metabolites of puerarin, the major component of the roots of Pueraria lobata OHWI (Leguminosae) in rats. The urine of rats administered puerarin orally contained puerarin and four major metabolites, daidzein 4',7-di-O-sulfate (M-I), daidzein 7-O-beta-D-glucuronide (M-II), daidzein 4'-O-sulfate (M-III), daidzein (M-IV), as determined from spectroscopic and chemical data. Total cumulative amounts of the puerarin and four metabolites excreted in the urine at 48 h following the oral administration of puerarin were approximately 3.6% the doses administered. The bile of rats administered puerarin orally contained puerarin and two major metabolites, which were identified as puerarin 4'-O-sulfate (PB1) and puerarin 7-O-beta-D-glucuronide (PB2) on the basis of chemical and spectroscopic data. These experimental data suggest that C-glycoside puerarin is partially hydrolyzed to aglycone in the body, but mainly excreted in the urine as unchanged puerarin.
Animals↗
Gangliosides as a possible receptor on the bovine erythrocytes for Theileria sergenti.
To elucidate whether or not gangliosides on the bovine erythrocytes serve as a receptor for Theileria sergenti merozoites, the reactivities of the T. sergenti piroplasms with gangliosides were studied by the liposome agglutination test. The parasites reacted weakly with I-active ganglioside containing N-acetylneuraminic acid (NeuAc) and strongly with I-active ganglioside containing N-glycolylneuraminic acid (NeuGc). However, none of the other gangliosides expressed on the bovine erythrocytes, such as GM3 (NeuAc), GM3 (NeuGc), sialosylparagloboside (SPG) (NeuAc), SPG (NeuGc), i-active ganglioside (NeuAc), and i-active ganglioside (NeuGc), were recognized. After infection with T. sergenti, furthermore, the content of I-active ganglioside (NeuAc) was less (p < 0.05), and I-active ganglioside (NeuGc) content was much less in the erythrocytes (p < 0.01), though the contents of other NeuAc- and NeuGc-containing gangliosides did not so vary with T. sergenti infection. These results suggest that the parasites recognize the I-active ganglioside as their receptor and bind preferentially to NeuGc-carrying I-active ganglioside rather than to NeuAc-type in the target cell membranes, and that the reduction of the contents of I-active gangliosides (NeuAc and NeuGc) on the erythrocytes was related to T. sergenti infection.
Agglutination Tests↗
Transient pseudohypoparathyroidism of the neonate.
We report three neonates with transient hypoparathyroidism with elevated parathyroid hormone (PTH) levels to clarify further the pathogenesis of late neonatal hypocalcemia and calcium homeostasis. Clinical signs were seizures starting at age of 10 and 11 days. The biochemical features were characterized by transient hypocalcemia and hyperphosphatemia due to a high transport maximum of the phosphate/glomerular filtration rate, despite high PTH levels. All had normal magnesium and calcidiol levels (at least 5 micrograms/l) for their age, and this precludes hypoparathyroidism due to low magnesium levels and hyperparathyroidism due to overt vitamin D deficiency. To diagnose pseudohypoparathyroidism type I, intravenous human PTH (1-34) infusions were performed; however, they showed brisk responses of plasma and/or urine cyclic AMP in response to the PTH infusion, but the phosphaturic response to the PTH was sluggish compared to the controls. All three showed an increase in serum alkaline phosphatase activity, suggesting PTH stimulation of osteoblasts. They were treated initially with calcium lactate or (1 alpha)-hydroxycalciol/calcitriol. Their hypoparathyroid condition, however, was transient; they maintained normal serum calcium and PTH levels without medication before the age of 6 months. The etiology, possibly intracellular signal transduction distal to cyclic AMP and/or distinct from adenylate cyclase in the kidney, is developmental and the condition was resolved completely within 6 months of age. We have termed this condition "transient pseudohypoparathyroidism of the neonate".
Alkaline Phosphatase↗
Increased urinary excretion of non-albumin antigen detected with YO-2, a novel monoclonal antibody, in diabetic patients.
Monoclonal antibodies were raised against urine proteins from diabetic patients. An antibody, YO-2, stained three protein bands with apparent molecular weights of 66, 49, and 36 kDa. These bands were not reactive with an anti-human albumin antibody. The urine levels of YO-2-reactive antigen in the normal control were 0.97 +/- 0.37 U/g-Cr (units per gram of urine creatinine) (mean +/- SD). Those of the normo-, micro-, and macroalbuminuric diabetic patients, respectively, were 1.38 +/- 1.36, 2.87 +/- 2.07, and 3.92 +/- 3.33 U/g-Cr. They were significantly higher in the micro- and macroalbuminuric patients. The urine levels of YO-2-reactive antigen had no significant correlation with the urine albumin levels and hemoglobin A1c. We concluded that; a) monoclonal antibody YO-2 recognized a non-albumin urine antigen increasingly excreted in diabetic patients with nephropathy, b) recent glycemic control of diabetes would not significantly affect the urinary excretion rate of YO-2-reactive antigen, and c) the excretion rate and probably the mechanism of YO-2-reactive protein differed from those of albumin. The urine levels of YO-2-reactive antigen could be a clinical marker of diabetic nephropathy.
Aged↗