Biomedical subjects

J Sakai

Publications and source records attributed to J Sakai.

At least 73 records · Page 4Linked to original sources

PubMed1998

Expression and characterization of a very low density lipoprotein receptor variant lacking the O-linked sugar region generated by alternative splicing.

The very low density lipoprotein receptor (VLDLR) gene contains an exon encoding a region of clustered serine and threonine residues immediately outside the membrane-spanning sequence, and this region has been proposed to be the site of clustered O-linked carbohydrate chains. Two forms of VLDLR transcripts, with and without the O-linked sugar region, are generated through alternative splicing. Reverse transcription polymerase chain reaction with RNAs from various rabbit tissues revealed that the VLDLR transcript with the O-linked sugar region (type-1 VLDLR) is the major transcript in heart and muscle, while the VLDLR transcript without the O-linked sugar region (type-2 VLDLR) predominates in non-muscle tissues, including cerebrum, cerebellum, kidney, spleen, adrenal gland, testis, ovary, and uterus. Hamster fibroblasts expressing type-2 VLDLR bound with relatively low affinity to beta-migrating very low density lipoprotein compared with type-1 VLDLR-transfected cells. In contrast, the internalization, dissociation, and degradation of the ligand were not significantly impaired in either type of VLDLR-transfected cell. The receptor proteins in type-2 VLDLR-transfected cells underwent rapid degradation and accumulated in the culture medium, while those in type-1 VLDLR-transfected cells were stable and resistant to proteolytic cleavage. Analysis of the O-linked sugars of both types of transfected cells suggested that the O-linked sugar region is the major site for O-glycosylation.

Alternative Splicing↗

PubMed1998

Pancreatic cancer associated with granulocyte-colony stimulating factor production confirmed by immunohistochemistry.

We report an 83-year-old man with pancreatic body cancer of 4.5 cm in diameter. Peripheral leukocyte count was 15,700/microl and the serum concentration of granulocyte-colony stimulating factor (G-CSF) was 123 pg/ml (normal, 6.0-21.9 pg/ml) on admission. Furthermore, not only K-ras codon 12 (GGT --> GAT) but also p53 at codon 247 (CGG --> CCG) mutations were identified in the pancreatic juice aspirated endoscopically. We performed chemotherapy with two courses of 5-fluorouracil, pirarubicin hydrochloride, and mitomycin-C, resulting in no beneficial effect. After the second course the patient developed interstitial pneumonia, probably caused by anticancer drugs, and died 4 months after the tumor was detected. In the autopsy tissue, the tumor macroscopically occupied the pancreas body and was 7 x 6 x 5 cm in size. Histopathologic diagnosis of the tumor was poorly differentiated adenosquamous carcinoma. Immunohistochemical staining of the autopsy tissue showed that pancreatic cancer cells were positive for G-CSF. This is the first case report of G-CSF-positive pancreatic cancer confirmed by immunohistochemistry.

Aged↗

PubMed1998

The risk of purified protein derivative positivity in homeless men with psychotic symptoms.

BACKGROUND: Homeless people with mental illness have relatively high rates of human immunodeficiency virus, comorbid antisocial personality disorder, and may be homeless more frequently and for greater lengths of time. All of these factors may increase the risk of tuberculosis. METHODS: Our study was done to ascertain if homeless men with psychotic disorders are at an increased risk for tuberculosis infection. One hundred fifty homeless men were interviewed and given purified protein derivatives (PPDs) at a downtown shelter in New Orleans, Louisiana, during a 3-month period. RESULTS: The findings show a strong relationship between psychotic disorders and positive PPDs, with a relative risk of 4.48. CONCLUSIONS: Homelessness and mental illness present barriers to seeking and completing treatment for medical illnesses such as tuberculosis. Use of services may be low even when available; therefore, homeless men with psychotic disorders may be serving as a reservoir for tuberculosis.

Adult↗

PubMed1998

Videofunduscopy and videoangiography using the scanning laser ophthalmoscope in Vogt-Koyanagi-Harada syndrome.

AIM: To observe pathophysiological changes in patients with Vogt-Koyanagi-Harada (VKH) syndrome using the scanning laser ophthalmoscope (SLO) to perform videofunduscopy, and indocyanine green (ICG) and fluorescein videoangiography. METHODS: 18 patients diagnosed with VKH syndrome were studied. 15 patients were examined in both acute and convalescent phases, and three patients were examined in the convalescent phase only. Retrospective review of charts was performed to obtain data on visual acuities, clinical findings, and results of cerebrospinal fluid cytology and histocompatibility leucocyte antigen D locus testing. RESULTS: Videofunduscopy revealed abnormalities in the central macula at the level of the retinal pigment epithelium or choroid in 40% of patients in the acute phase and 83% of patients in the convalescent phase. ICG videoangiography showed irregular hypofluorescence (80%), slow or patchy filling (67%), indistinct or fewer choroidal vessels (73%), filling defects (73%), and focal leakage (33%) in patients in the acute phase. These changes improved with systemic corticosteroid treatment, although at least one ICG videoangiographic abnormality persisted into the convalescent phase in 83% of patients in the absence of clinical inflammation. In contrast, fluorescein videoangiography revealed fewer and decreased severity of abnormalities, with greater resolution in the convalescent phase when compared with ICG videoangiography. CONCLUSIONS: SLO videofunduscopy and videoangiography revealed numerous abnormalities in patients with VKH syndrome, many of which persisted well after clinical recovery, suggesting the development of permanent morphological changes in the fundus. ICG videoangiography was more sensitive than fluorescein videoangiography in delineating abnormalities in layers deep to the sensory retina.

Acute Disease↗

PubMed1998

Magnesium as a regulator of thrombin formation in bovine ovarian follicular fluid.

In the ovarian follicular fluid (FF) of Holstein cows, calcium (Ca) and magnesium (Mg) levels and their roles on thrombin generation were examined and compared with the blood samples. Total Ca levels in FF increased while the total Mg levels decreased with follicular development from preantral to preovulatory stage of follicles. These changes resulted in Ca values being significantly (p < 0.05) higher in FF from the most developed follicles and the Mg values being significantly higher (p < 0.05) in the least developed follicles. To determine whether the high level of Mg might function to regulate thrombin generation in FF as occurs in plasma, the influence of Mg supplementation of FF from various types of follicles was examined. In FF from small size follicles, Mg accelerated the prothrombin time, an estimation of the overall rate of thrombin production, although a similar effects was not observed in FF from medium and large size follicles. The addition of Mg to FF from all sizes of follicles resulted an inhibition in factor X activation. Since activation of factor X is a precursor step for thrombin formation it is concluded that Mg can function as a slow accelerator of thrombin generation in FF from follicles at the antral stage of development. It is likely to have a more important role in regulating the rate of thrombin generation as the follicle develops.

Animals↗

PubMed1998

Distribution of Dopamine Transporter in the Rat: an Immunohistochemical Study.

OBJECTIVE: To investigate the organ distribution of dopamine transporter (DAT) in rats by immunohistochemical method. METHODS: Dopamine transporter (DAT) was identified immunohistochemically in the tissues using specific antipeptide antiserum raised in New Zealand white rabbits immunized with a conjugate of synthetic DAT peptide (29-45) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. RESULTS: DAT immunoreactivity was visualized in the neural perikarya, axons and dendrites of the central nervous system, retina, adrenal medulla, Auerbach's nervous branch and Meissner's nervous branch of the stomach, small intestine and colon, anterior pituitary, and lung. When using antiserum preincubated with synthetic DAT peptide (DAT, 29-45) or hypothalamus homogenate which contains DAT, no significant stain of neurons in the hypothalamus was detected. CONCLUSION: These findings suggest that DAT is widely distributed and that the method used is valuable in studying the distribution of DAT in rats.

Journal Article↗

PubMed1997

Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein.

SREBP cleavage-activating protein (SCAP) stimulates the proteolytic cleavage of membrane-bound SREBPs, thereby initiating the release of NH2-terminal fragments from cell membranes. The liberated fragments enter the nucleus and stimulate transcription of genes involved in synthesis and uptake of cholesterol and fatty acids. Sterols repress cleavage of SREBPs, apparently by interacting with the membrane attachment domain of SCAP. In the present studies we show that SCAP, like the SREBPs, is located in membranes of the endoplasmic reticulum and nuclear envelope. The COOH-terminal domain of SCAP, like that of the SREBPs, is located on the cytosolic face of the membranes. Co-immunoprecipitation experiments show that SCAP and SREBP-2 form a complex that can be precipitated with antibodies to either component. Complex formation occurs when cells express only the COOH-terminal domain of either SREBP-2 or SCAP, indicating that the complex forms between the two COOH-terminal domains. Truncation of SREBP-2 at its COOH terminus prevents the formation of complexes with SCAP and simultaneously reduces proteolytic cleavage. We conclude that proteolytic cleavage of SREBPs requires the formation of a complex with the COOH-terminal domain of SCAP and that SCAP is therefore a required element in the regulation of sterol and fatty acid metabolism in animal cells.

Animals↗

PubMed1997

Cleavage site for sterol-regulated protease localized to a leu-Ser bond in the lumenal loop of sterol regulatory element-binding protein-2.

A sterol-regulated protease initiates release of the NH2-terminal segments of sterol regulatory element-binding proteins (SREBPs) from cell membranes, thereby allowing them to enter the nucleus and to stimulate transcription of genes involved in the uptake and synthesis of cholesterol and fatty acids. Using SREBP-2 as a prototype, we here identify the site of sterol-regulated cleavage as the Leu522-Ser523 bond in the middle of the 31-residue hydrophilic loop that projects into the lumen of the endoplasmic reticulum and nuclear envelope. This site was identified through use of a vector encoding an SREBP-2/Ras fusion protein with a triple epitope tag that allowed immunoprecipitation of the cleaved COOH-terminal fragment. The NH2 terminus of this fragment was pinpointed by radiochemical sequencing after replacement of selected codons with methionine codons and labeling the cells with [35S]methionine. Alanine scanning mutagenesis revealed that only two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage site (leucine 522), and 2) the arginine at the P4 position (arginine 519). These define a tetrapeptide sequence, RXXL, that is necessary for cleavage. Cleavage was not affected when the second transmembrane helix of SREBP-2 was replaced with the membrane-spanning region of the low density lipoprotein receptor, indicating that this sequence is not required for regulation. Glycosylation-site insertion experiments confirmed that leucine 522 is located in the lumen of the endoplasmic reticulum. We conclude that the sterol-regulated protease is a novel enzyme whose active site faces the lumen of the nuclear envelope, endoplasmic reticulum, or another membrane organelle to which the SREBPs may be transported before cleavage.

Amino Acid Sequence↗

PubMed1997

Association between early-onset alcoholism and the dopamine D2 receptor gene.

We examined the allelic association between the dopamine D2 receptor (DRD2) gene and alcoholism in 100 biologically unrelated Japanese alcoholics and 93 unrelated controls. Genomic DNA was prepared from peripheral white blood cells using the phenol-chloroform method. A 310-bp region surrounding the TaqA site at the DRD2 locus was amplified by polymerase chain reaction (PCR), and the PCR product was incubated with TaqI. The A1 allele remained intact while the A2 allele was cut. The frequency of the A1/A1 genotype and the frequency of the A1 allele were higher in early-onset alcoholics than in controls, P < 0.05 and P < 0.01, respectively. Moreover, the frequency of the A1/A1 genotype and the frequency of the A1 allele were higher in early-onset alcoholics with family histories of alcohol dependence than in controls, P < 0.01 and P < 0.01, respectively. The results indicate that the DRD2 gene is associated with susceptibility to early-onset alcoholism, and that each additional A1 allele shifts onset of alcoholism to an earlier age.

Adult↗

PubMed1997

The Tn10 synaptic complex can capture a target DNA only after transposon excision.

Tn10 transposes nonreplicatively. Staged in vitro reactions demonstrate that a Tn10 synaptic complex can become committed to a particular target DNA molecule via a noncovalent interaction in the absence of strand transfer. Commitment occurs only after double-strand cleavage at both transposon ends (in "double-end break" [DEB] complexes). Stable noncovalent DEB-target DNA cocomplexes can be detected, but no cocomplexes occur with synaptic complexes containing uncleaved ends. Preincubation of DEB complexes with target DNA accelerates the rate of strand transfer. Postcleavage target capture is remarkable for Tn10; Mu and Tn7 select a target site prior to cleavage. Promiscuous target selection may favor evolution of IS-based composite elements while being suicidal for other types of transposons.

Calcium Chloride↗

PubMed1997

Prevention of experimental autoimmune uveoretinitis by monoclonal antibody to interleukin-12.

Experimental autoimmune uveoretinitis (EAU) induced by immunization with interphotoreceptor retinoid-binding protein (IRBP), a retinal self antigen, has been regarded to be a typical T helper type 1 (Th1)-mediated inflammatory disease. In this study, we examined the effect of a neutralizing monoclonal antibody (mAb) to interleukin-12 (IL-12), which has been known to play a critical role in the Th1 differentiation, on the development of EAU. While 9 of 13 control mice developed EAU by the immunization with IRBP, none of 12 mice developed EAU when given anti-IL-12 mAb 1 day before immunization. These mice did not develop EAU even after a rechallenge with IRBP on day 30, indicating that a protective mechanism had been established by the anti-IL-12 treatment. The proliferative response of splenocytes to IRBP in vitro was not significantly impaired, but the production of IL-2 and IFN-gamma was greatly reduced by the anti-IL-12 treatment. Moreover, production of IL-5 and expression of IL-4 mRNA were increased by the anti-IL-12 treatment. Consistently, IgG2a anti-IRBP serum antibodies were decreased and IgG1 were increased. Administration of a neutralizing anti-IL-4 mAb at the time of IRBP rechallenge reversed the protection established by the anti-IL-12 treatment at the primary immunization. These results indicate that the anti-IL-12 treatment at the IRBP priming not only prevented the development of pathogenic Th1 cells, but also induced suppressive Th2 cells that protect the animals from further challenge with the same antigen.

Animals↗

PubMed1997

Sclerosing pancreatitis showing rapidly progressive changes with recurrent mass formation.

CONCLUSION: Sclerosing pancreatitis might develop repeatedly or might rapidly extend to the whole pancreas with recurrent mass formation. BACKGROUND: Nothing is known concerning course or development of sclerosing pancreatitis. METHODS: A 63-yr-old male was followed up for 2.5 yr. RESULTS: The patient was admitted because of a tumor in the body and tail of the pancreas. Serum pancreatic enzymes were transiently elevated, but tumor markers were all negative. Imaging studies showed a tumor 7 cm in size. The main pancreatic duct was normal in the head and obstructed at the body on endoscopic retrograde pancreatography (ERCP). The K-ras oncogene mutation was positive in pure pancreatic juice. Distal pancreatectomy was performed because pancreatic cancer was highly suspected. Pathological findings showed that the tumor was a densely fibrotic mass without malignant cells. Inflammatory cell infiltration was observed in the stroma. One year later, another mass 3 cm in size was noted in the remnant pancreatic head. ERCP revealed diffuse irregular narrowing of the main pancreatic duct, its branches, and the common bile duct. Liver dysfunction improved and an elevation of serum pancreatic enzymes subsided without any specific treatment, and the mass diminished in size. The patterns of various imaging studies on the second tumor were the same as those of the previous resected mass. Corticosteroid was not administered.

Genes, ras↗

PubMed1997

Complete nucleotide sequence and genome organization of sweet potato feathery mottle virus (S strain) genomic RNA: the large coding region of the P1 gene.

The complete nucleotide sequence of a sweet potato feathery mottle virus severe strain (SPFMV-S) genomic RNA was determined from overlapping cDNA clones and by directly sequencing viral RNA. The viral RNA genome is 10,820 nucleotides long, excluding the poly(A) tail and contains one open reading frame (ORF) starting at nucleotide 118 and ending at 10,599, potentially encoding a polyprotein of 3,493 amino acids (Mr 393,800). The ORF was followed by a 3' untranslated region of 221 nucleotides. The deduced polyprotein includes P1 (74K), HC-Pro (52K), P3 (46K), 6K1, CI (72K), 6K2, NIa-VPg (22K), NIa-Pro (28K), NIb (60K) and coat (35K) proteins, after an analysis of protein cleavage sites analogous to other potyvirus polyproteins. The polyprotein had a high level of amino acid identity with those of other potyviruses, except in the regions of P1 and P3. The P1 of SPFMV-S RNA has 664 amino acid residues, and is the largest and least similar to those of other potyviruses. HC-Pro and CI show high identity with those of other potyviruses. P3 has relatively low identity, however, the length of P3 was within the range of variability among other potyviruses. The 6K1 protein between P3 and C1 is also highly similar to those of other potyviruses. This is the first report on the complete nucleotide sequence of the sweet potato-infecting virus.

Genome, Viral↗

PubMed1997

Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs.

We report the cloning of a gene, S2P, that encodes a putative metalloprotease required for intramembrane proteolysis of sterol-regulatory element-binding proteins (SREBPs) at Site-2. SREBPs are membrane-bound transcription factors that activate genes regulating cholesterol metabolism. The active NH2-terminal domains of SREBPs are released from membranes by sequential cleavage at two sites: Site-1, within the lumen of the endoplasmic reticulum; and Site-2, within a transmembrane segment. The human S2P gene was cloned by complementation of mutant CHO cells that cannot cleave SREBPs at Site-2 and are cholesterol auxotrophs. S2P defines a new family of polytopic membrane proteins that contain an HEXXH sequence characteristic of zinc metalloproteases. Mutation of the putative zinc-binding residues abolishes S2P activity. S2P encodes an unusual metalloprotease that cleaves proteins within transmembrane segments.

Animals↗

PubMed1997

The amino acid sequence of the light chain of Acanthamoeba myosin IC.

The amino acid sequence of the light chain of Acanthamoeba myosin IC deduced from the cDNA sequence comprises 149 amino acids with a calculated molecular weight of 16,739. All but the 3 N-terminal residues were also determined by amino acid sequencing of the purified protein, which also showed the N-terminus to be blocked. Phylogenetic analysis shows Acanthamoeba myosin IC light chain to be more similar to the calmodulin subfamily of EF-hand calcium-modulated proteins than to the myosin II essential light chain or regulatory light chain subfamilies. In pairwise comparisons, the myosin IC light chain sequence is most similar to sequences of calmodulins (approximately 50% identical) and a squid calcium-binding protein (approximately 43% identical); the sequence is approximately 37% identical to the calcium-binding essential light chain of Physarum myosin II and approximately 30% identical to the essential light chain of Acanthamoeba myosin II, and the essential light chain and regulatory light chain of Dictyostelium myosin II. The sequence predicts four helix-loop-helix domains with possible calcium-binding sites in domains I and III, suggesting that calcium may affect the activity of this unconventional myosin. This is the first report of the sequence of an unconventional myosin light chain other than calmodulin.

Acanthamoeba↗

PubMed1997

Effect of antipsychotic drugs on the gene expression of NMDA receptor subunits in rats.

It has been hypothesized that glutamatergic neurons play an important role in clinical manifestations of schizophrenia and that the therapeutic effect of antipsychotic drugs is related to glutamatergic neurotransmission. To elucidate the effect of antipsychotic drugs on glutamatergic transmission, we examined gene expressions of NMDA receptor subunits R1, R2A, R2B and R2C in the whole brains of rats after acute and chronic administrations of haloperidol and sulpiride, using the Northern blot technique. The levels of NMDAR2B mRNAs decreased after the acute administration of haloperidol, but showed no change after the chronic administration. The levels of NMDAR2A and R2B mRNAs decreased after the acute administration of sulpiride, whereas the levels of R2A and R2B increased following the chronic administration. Neither haloperidol nor sulpiride influenced NMDAR1 mRNA levels. These data support differential expression of NMDA receptor subunits in rats upon treatment with haloperidol and sulpiride. The results imply that NMDAR2 subunits may be crucial in the regulation and modification of antipsychotic drugs.

Animals↗

PubMed1997

Paracentric inversion of chromosome 9 with schizoaffective disorder.

A 36-year-old man with schizoaffective disorder was found to have a de novo 46 XY inv (9) (q31.2q34.3). Phenotypical abnormalities are short stature, depressed nasal bridge, hypertelorism and slender shoulders. Paracentric inversion of chromosome 9 is a rare chromosome aberration. This case suggests a new candidate region related to schizoaffective disorder.

Adult↗

PubMed1997

L-NAME-sensitive and -insensitive nonadrenergic noncholinergic relaxation of cat airway in vivo and in vitro.

The neurotransmitters responsible for neurogenic airway relaxation are still unknown. We investigated the effects of N omega-nitro-L-arginine methylester (L-NAME) on nonadrenergic and noncholinergic (NANC) relaxation evoked by electrical stimulation of vagus nerves in vivo and in vitro in cat. To that end, we measured pulmonary resistance during vagal nerve stimulation (VS) in vivo, and isometric tension of small bronchi (1-3 mm outer diameter) during electrical field stimulation (EFS) in vitro. During infusion of 5-hydroxytryptamine (5-HT), VS transiently decreased total pulmonary resistance in the presence of atropine and propranolol, with peak relaxation at several seconds after the VS and a gradual return to baseline within 2-3 min. L-NAME abolished the initial peak relaxation and reduced the peak amplitude, but did not affect the duration of the NANC relaxation. In small bronchi obtained from control cats, EFS evoked a biphasic NANC relaxation, comprising an initial fast component followed by a second slow component, and L-NAME (10(-5) M) selectively abolished the first component without affecting the second. Whilst in the small bronchi obtained from L-NAME pretreated cats, EFS elicited only the slow component of NANC relaxation, which was insensitive to L-NAME but sensitive to tetrodotoxin. These results indicate that nonadrenergic noncholinergic relaxation induced by vagal nerve stimulation during infusion of 5-hydroxytryptamine can be classified into two components, and that at least two neurotransmitters, including nitric oxide, are involved in the relaxation.

Airway Resistance↗