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Biomedical subjects

Y Sakaki

Publications and source records attributed to Y Sakaki.

At least 163 records · Page 9Linked to original sources
PubMed1992

Accumulation of alpha B-crystallin in brains of patients with Alexander's disease is not due to an abnormality of the 5'-flanking and coding sequence of the genomic DNA.

alpha B-Crystallin is a major protein component of Rosenthal fibers, which massively accumulate in the brains of patients suffering from Alexander's disease. To examine whether or not accumulation of alpha B-crystallin is due to any abnormality of the gene structures, we determined the sequence of the alpha B-crystallin gene in two cases of pathologically confirmed Alexander's disease. Direct sequencing of the promoter and coding regions of the alpha B-crystallin gene in patients revealed them to have a normal sequence. Northern blotting showed a single alpha B-crystallin mRNA species expressed in the Alexander's disease brain.

Aged↗
PubMed1992

Gly145 to Arg substitution in HBs antigen of immune escape mutant of hepatitis B virus.

A Japanese child born to an HBeAg-positive carrier mother received anti-HBs immunoglobulins and a plasma-derived HBs vaccine with a poor anti-HBs-antibody response. The child, who is now 3 years old, is presently suffering from chronic hepatitis with unusual serological findings that are positive for HBsAg, anti-HBs and HBeAg, since being infected with a measles virus at 12 months of age. The nucleotide sequences of the S region of HBV DNA obtained from the patient, the mother and an HBeAg-positive brother were completely identical except for one nucleotide at position 587 (mother and brother: guanosine, patient: adenosine), giving an amino acid change: Gly - greater than Arg at position 145 of the major HBs protein.

Amino Acid Sequence↗
PubMed1992

Production of a unique multi-lamella structure in the nuclei of yeast expressing Drosophila copia gag precursor.

Drosophila retrotransposon copia produces virus-like particles (VLPs) in the nuclei of cultured Drosophila cells. The VLPs contain copia RNA and reverse transcriptase activity, and thus, play a major role in copia replication. Here we have expressed the copia gag polyprotein precursor in yeast. The precursor, which includes copia protease itself, showed correct autoprocessing to produce a unique multi-lamella structure in the nuclei of the yeast cells. This expression system should be useful for the analysis of nuclear localization of the major copia VLP protein, and furthermore, would provide important information concerning the mechanism of copia VLPs formation.

Animals↗
PubMed1992

Positive and negative regulatory elements for the expression of the Alzheimer's disease amyloid precursor-encoding gene in mouse.

We cloned, sequenced and characterized a promoter region of the mouse homologue of the Alzheimer's disease amyloid precursor protein (APP)-encoding gene. The promoter region is highly homologous to that of the human APP (hAPP) gene. It has a high G+C content, lacks typical 'TATA' and 'CAAT' boxes, and contains possible binding sites for AP-1, heat-shock factor, Sp1 and AP-4. The promoter region was fused with the cat reporter gene, and the fusion genes were transfected to both the NB41A3 (mouse neuroblastoma) and L-cell lines. The promoter activity was monitored by chloramphenicol acetyltransferase (CAT) activity in a transient expression assay. The promoter was equally active in both cell lines. The deletion analysis revealed that there existed a negative regulatory element(s) between -153 and -100 bp and a positive element(s) between -100 and -37 bp. The negative element was shown to suppress the transcriptional activity of heterologous simian virus 40 promoter. DNase I footprinting experiments revealed that three nuclear protein-binding sites existed in the regulatory region, one in the negative and two in the positive regulatory regions. Gel retardation assay showed that Sp1 was one of the factors binding to the positive regulatory region. A nuclear factor binding to the negative regulatory region seemed to be missing in brain.

Alzheimer Disease↗
PubMed1992

High-sensitive fluorescent DNA sequencing and its application for detection and mass-screening of point mutations.

We describe a rapid and sensitive DNA sequencing method for an automated fluorescent DNA sequencer (AFDS) and its application for detection of point mutations. The method is based on an improved cycle sequencing procedure in which only 10-50 fmol of template DNA is required. Furthermore, it is able to use crude DNA preparation as a template as well as the purified one. Thus, the improved method provided a simplified procedure for sequencing of various types of DNA, including cosmid DNA, in which purification steps were unnecessary. We also developed a novel system for detection of point mutations using AFDS. A set of four lanes is used for the parallel analysis of single-base profiles of four different samples, instead of for the four-base profile of a sample. The AFDS exhibits the base profiles of the samples with four different colors in the analyzed data, which enables us to identify a mutation as an additional peak with a color specific for the lane. The feasibility of our system was tested by analyzing polymerase chain reaction (PCR)-amplified genomic DNAs from four individuals including a carrier of a mutation of C to T. The mutation was clearly identified as an additional "T" peak of a color specific for the carrier. The mutation was also detectable even if 16 individuals including the carrier were simultaneously analyzed on a set of four lanes (four individual samples for each lane). Thus, the novel system is useful for simultaneous detection of mutations in a large number of individual samples.

Automation↗
PubMed1992

Antibody titers to HTLV-I-p40tax protein and gag-env hybrid protein in HTLV-I-associated myelopathy/tropical spastic paraparesis: correlation with increased HTLV-I proviral DNA load.

We studied the relationship between antibody titers to recombinant HTLV-I p40tax protein and gag-env hybrid protein in serum (by an enzyme-linked immunosorbent assay) and HTLV-I proviral DNA load in peripheral blood mononuclear cells (by a quantitative polymerase chain reaction method) in 18 patients with HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), 17 HTLV-I carriers without HAM/TSP and 16 HTLV-I uninfected controls. The IgG and IgA antibody titers to either of the proteins correlated significantly with the HTLV-I pX (coding p40tax protein) and pol DNA amounts in HTLV-I infected subjects. HAM/TSP patients had significantly higher titers of IgG and IgA antibodies to the HTLV-I proteins than did the HTLV-I carriers without HAM/TSP. While the IgM antibodies to the HTLV-I proteins were found in only 6% of HTLV-I carriers without HAM/TSP, they were found in 40% of HAM/TSP patients, especially those having both a high HTLV-I proviral DNA load and high titers of the IgG and IgA antibodies. HAM/TSP patients with the IgM antibodies had a tendency to deteriorate more frequently on the Kurtzke's disability status scale and magnetic resonance imaging of the brain (leukoencephalopathy) than did those without in the two-year follow-up. Thus, the presence of IgM antibody and high titers of IgG and IgA antibodies to the HTLV-I proteins, together with the increased HTLV-I proviral DNA load, appears to distinguish HAM/TSP patients from HTLV-I carriers without HAM/TSP.

Adult↗
PubMed1992

Postnatal development of mRNA specific for a metabotropic glutamate receptor in the rat brain.

We examined the ontogenesis of a subtype of metabotropic glutamate receptors, termed mGluR1, which is linked to phosphoinositide metabolism, in various regions of rat brain during neonatal development. Northern blot analyses of mGluR1 mRNA indicated that mRNA increased monotonously or remained at plateau levels during the first 5 weeks after birth. In situ hybridization analyses supported this conclusion. The result is in contrast with the reported development of the activity in excitatory amino acid-stimulated phosphoinositide turnover during the same period. The latter increases during the first few weeks and then decreases sharply.

Animals↗
PubMed1992

Amyloidogenic and non-amyloidogenic transthyretin Asn 90 variants.

Recently, a new transthyretin (TTR) variant was described in the normal Portuguese and German populations. The same substitution was found associated with familial amyloidotic polyneuropathy (FAP) in an American family of Italian origin. Comparative isoelectric focusing studies showed a difference in the mobility pattern between the non-pathogenic and pathogenic variants. However, comparative DNA sequencing between them did not reveal any additional mutation. Comparative isoelectric focusing between the variants and TTR Asn 90 produced by recombinant techniques indicated that the non-pathogenic variant has the electrophoretic behaviour expected for the mutation. We suggest that an as yet unknown post-translational modification may have occurred in the FAP-associated Asn 90 variant, turning it into an amyloidogenic molecule.

Amyloidosis↗
PubMed1992

Parental methylation patterns of a transgenic locus in adult somatic tissues are imprinted during gametogenesis.

The methylation status of a mouse metallothionein-I/human transthyretin fusion gene was studied during gametogenesis in transgenic mice. In the adult tissues of this mouse line, the promoter region of the transgene on chromosome 11 is methylated when it is maternally inherited and undermethylated when it is paternally inherited. Germ cells from various developmental stages of gametogenesis were isolated, and their DNAs were assayed using methylation-sensitive restriction endonucleases and the polymerase chain reaction. Only low to nonexistent levels of transgene methylation were detected in germ cells from 14.5-day-old male and female fetuses irrespective of the parental origin of the transgene. This undermethylated state persisted in oocytes from newborn females as well as in testicular spermatogenic cells and sperm. By contrast, the transgene promoter was completely methylated in fully grown oocytes arrested at the first meiotic prophase. The endogenous metallothionein-I gene promoter, located on a different chromosome, remained undermethylated at all stages examined, consistent with previous findings reported for a typical CpG island. Taken together, the results suggest that parental-specific adult patterns of transgene methylation are established during gametogenesis.

Animals↗
PubMed1991

CJD discrepancy.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed1991

The 717Val----Ile substitution in amyloid precursor protein is associated with familial Alzheimer's disease regardless of ethnic groups.

Alzheimer's disease (AD) is a devastating neurological disorder and the leading cause of dementia among aged individuals. The human amyloid beta protein, which is a cleavage product of amyloid precursor protein (APP), is a major component of the amyloid deposited in the brain of patients with AD. By using PCR direct sequencing of exon 17 (encoding part of the beta protein) of the APP gene, we have found that a Japanese AD patient harbours a C to T substitution, responsible for a valine to isoleucine change at position 717, heterogeneously. The mutation is exactly the same as that found in a Caucasian AD family by Goate et al. (1). Furthermore, the mutation was shown to co-segregate with AD in his family. These results suggest that the Val----Ile change in the APP causes AD, regardless of ethnic background.

Alzheimer Disease↗
PubMed1991

Selective processing of proalbumin determined by site-specific mutagenesis.

Rat proalbumin is cleaved at the dibasic pair Arg-Arg and converted into a mature form with Glu at the NH2 terminus. In the present study site-directed mutagenesis of the albumin cDNA was designed to generate proalbumin variants in which Glu1 was substituted with various amino acid residues. The expression plasmids constructed were transfected into COS-1 cells, and the intracellular processing of proalbumins expressed was examined by labeling experiments. Substitution of Glu1----Ser allowed the expressed proalbumin to be processed as observed for the wild-type precursor. However, replacement of Glu1 with a hydrophobic residue (Val, Leu or Ile) resulted in no processing of proalbumin, despite retaining the same cleavage signal Arg-Arg as above. The results indicate that the residue at position 1 adjacent to the dibasic pair is also important for recognition by the proalbumin-processing enzyme.

Amino Acid Sequence↗
PubMed1991

Production of recombinant human transthyretin with biological activities toward the understanding of the molecular basis of familial amyloidotic polyneuropathy (FAP).

Transthyretin (TTR) is a plasma protein interacting with thyroxine T4 and retinol binding protein (RBP). Several variants of TTR with single amino acid substitutions have been identified as the major components of the amyloid fibrils of familial amyloidotic polyneuropathy (FAP), a fetal, autosomal dominant genetic disease. The elucidation of the molecular nature of the variants distinct from that of the wild-type TTR is crucial for understanding the amyloidogenesis in FAP, but our understanding is very poor mainly because of the unavailability of pure variant TTRs. In the present study, we used an Escherichia coli OmpA secretion vector (Ghrayeb et al., 1984) and achieved an effective production of the variant TTRs related to FAP including Met-30, Ile-33, Ala-60, Tyr-77, Met-111, and Ile-122 types. The variant TTRs produced in this system were efficiently secreted to the culture media. The chemical analysis showed that the secreted TTR (Met-30 type) has the same N-terminus as the native one. IEF analyses also indicated that the secreted product is properly processed as assessed by its pI. Furthermore, the secreted TTR was shown to have biological activities, namely, the thyroxin binding activity and the ability to associate with retinol binding protein, indicating that the secreted TTR polypeptide is properly folded. The present work also demonstrated that the processing/secretion of the recombinant TTR molecules in E. coli was strongly affected by single amino acid substitutions.

Amino Acid Sequence↗
PubMed1991

Structural role of serine 127 in the NADH-binding site of human NADH-cytochrome b5 reductase.

Serine 127 of human NADH-cytochrome b5 reductase was replaced by proline and alanine by site-directed mutagenesis. The former mutation has been found in the genes of patients with hereditary deficiency of the enzyme. Both the mutant enzymes (Ser-127----Pro mutant and Ser-127----Ala mutant) were overproduced in Escherichia coli and purified to homogeneity. The two purified mutant enzymes showed indistinguishable spectral properties which differed from those of the wild-type enzyme. The mutant enzymes showed higher molecular extinction coefficients at 462 nm than that of the wild-type enzyme. Quenching of FAD fluorescence in these mutant enzymes was significantly less than that in the wild-type enzyme. Furthermore, circular dichroism spectra of the mutant enzymes were different, in both the visible and ultraviolet regions, from that of the wild-type enzyme. The spectra of the mutant enzymes in the visible region were restored to almost the same spectrum as the wild type upon reduction with NADH. Ser-127----Pro mutant and Ser-127----Ala mutant showed very low Kcat/Km (NADH) values (5 x 10(7) and 3.5 x 10(7) s-1 M-1, respectively) with cytochrome b5 as an electron acceptor, than that of the wild-type enzyme (Kcat/Km (NADH) = 179 x 10(7) s-1 M-1), while the Kcat/Km (cytochrome b5) value for each enzyme was similar. The mutant enzymes were less thermostable than the wild-type enzyme. These results indicate that serine 127 plays an important role to maintain the structure of the NADH-binding site in the enzyme.

Base Sequence↗
PubMed1991

Increased HTLV-I proviral DNA in HTLV-I-associated myelopathy: a quantitative polymerase chain reaction study.

Using the polymerase chain reaction, we quantitated the amount of human T-lymphotropic virus type I (HTLV-I) proviral DNA in peripheral blood mononuclear cells from 18 patients with HTLV-I--associated myelopathy/tropical spastic paraparesis; 17 HTLV-I carriers without HTLV-I--associated myelopathy/tropical spastic paraparesis, with or without other autoimmune or inflammatory diseases; and 19 seronegative control subjects. The HTLV-I proviral DNA was 10- to 100-fold higher in the patients and in the HTLV-I carriers without HAM/TSP who had autoimmune or inflammatory diseases than in the carriers without autoimmune or inflammatory diseases. The patients who had had onset of myelopathy at a younger age (15 to 39 years) had an extremely high level of HTLV-I proviral DNA in the early phase, as compared with findings in those with a late onset of myelopathy (at 44 to 61 years). The large increase in HTLV-I proviral DNA in peripheral blood mononuclear cells is presumably closely related to the development of autoimmune or inflammatory processes in HTLV-I carriers, including HTLV-I--associated myelopathy/tropical spastic paraparesis.

Adult↗
PubMed1991

An improved pulsed-field polyacrylamide gel electrophoresis system for physical selection of linking clones: isolation of SfiI linking clones from a chromosome 21-specific library.

We had previously developed an efficient procedure for selective cloning of rare-cutter linking fragments that is based on physical separation of linking clone DNAs by pulsed-field polyacrylamide gel electrophoresis (PF-PAGE). An advantage of the physical selection procedure over the conventional cloning-based ones utilizing the insertion of selection marker or vector sequences into the rare-cutter sites is that it can be readily applied to the selection of linking fragments for rare-cutters, generating ambiguous cohesive end sequences such as SfiI (GGCCNNNN/NGGCC). In the present work, the physical separation procedure was improved by introducing a discontinuous buffer system into PF-PAGE, and its feasibility was exemplified by the selective isolation of SfiI linking clones from a human chromosome 21-specific library. This simple and efficient procedure will provide a useful tool for genome analysis.

Base Sequence↗
PubMed1991

Expression of Alzheimer amyloid beta-protein precursor gene in neuronal cells.

Five forms of Alzheimer amyloid beta-protein precursors (APP or BPP) have been found. Among them, APP695 is predominantly found in neuronal cells and may be involved in some neuronal functions. In the present work, we investigated the relationship between the APP expression and the formation of neurites, a typical neuronal morphology, by measuring the amounts of three major APP mRNAs in PC12 cells, using the polymerase chain reaction. In PC12 cells, nerve growth factor which induced a neurite formation, enhanced the expression of APP695. The ratio of APP695 to APP770 increased 2.6-fold after 6 days of treatment. Cyclic AMP also induced a similar morphological change, but did not affect the APP expression pattern. Thus, the neuronal expression of the APP gene is not directly related with neurite formation. We have also identified cis-elements for the neuronal expression of APP gene.

Alzheimer Disease↗