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Biomedical subjects

J Sakai

Publications and source records attributed to J Sakai.

At least 19 recordsLinked to original sources
PubMed1992

Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity.

A cDNA that expresses a receptor for very low density lipoprotein (VLDL) was isolated from a rabbit heart cDNA library and characterized. The deduced amino acid sequence of the cDNA revealed that the cDNA encodes a protein with striking homology to the low density lipoprotein (LDL) receptor. Like the LDL receptor, the mature protein consists of the following five domains spanning 846 amino acids: 328 N-terminal amino acids including an 8-fold repeat of 40 amino acids homologous to the ligand binding repeat of the LDL receptor; 396 amino acid residues homologous to the epidermal growth factor precursor including three cysteine-rich repeats; a region immediately outside of the plasma membrane rich in serines and threonines; 22 amino acids traversing the plasma membrane; and 54 amino acids including the NPVY sequence that is required for clustering of the LDL receptor in coated pits and that projects into the cytoplasm. LDL-receptor-deficient Chinese hamster ovary cells transfected with the cDNA bound and internalized VLDL, beta-migrating VLDL, and intermediate density lipoprotein but did not bind LDL with high affinity. The 3.6- and 9.5-kilobase mRNAs for the VLDL receptor are highly abundant in heart, muscle, and adipose tissue. Barely detectable amounts of the mRNAs were present in liver. Based on the structural features, ligand specificity, and tissue expression of the mRNAs, we suggest that this VLDL receptor may mediate uptake of apolipoprotein E-containing lipoproteins enriched with triglyceride in nonhepatic tissues that are active in fatty acid metabolism.

Amino Acid Sequence↗
PubMed1992

Imbalance of deoxyribonucleoside triphosphates and DNA double-strand breaks in mouse mammary tumor FM3A cells treated in vitro with an antineoplastic tropolone derivative.

The mechanism by which alpha,alpha-bis(2-hydroxy-6-isopropyltropon-3-yl)-4-methoxytolu ene (JCI-3661) kills mouse mammary tumor FM3A (F28-7) cells was studied. When the cells were exposed to the drug at 3.7 microM, the intracellular dNTP pool became imbalanced because of decreases in dGTP and dATP and an increase in dTTP. The pattern of the dNTP imbalance was the same as that caused by hydroxyurea. When JCI-3661 was added to the culture medium, mature DNA strands broke, giving fragments of 100-200 kilobase pairs long as found by orthogonal-field-alternation gel electrophoresis. DNA strand breaks, detected by this technique, were observed in the cells at 12 h after the addition. The beginning of cell death was observed at about 14 h (trypan blue staining) or at about 12 h (colony-forming ability) after cultivation Breaks in the single and double strands of DNA, as measured by alkaline and neutral filter elution assay, became evident 24 h after treatment with 3.7 microM JCI-3661. Comparison of the ratio of single- and double-strand breaks caused by JCI-3661 to that following radiation suggested that JCI-3661 broke only double strands. Cycloheximide inhibited both the breakage of double strands and the cell death caused by JCI-3661. JCI-3661 decreased DNA synthesis more than RNA or protein synthesis. The breaks in double strands of DNA were probably important in the cell death caused by JCI-3661.

Animals↗
PubMed1992

Localization of collagen (I) and collagenase mRNA by in situ hybridization during corneal wound healing after epikeratophakia or alkali-burn.

The expression and localization of type I collagen and collagenase gene were studied by in situ hybridization using rabbit cornea during wound healing following epikeratophakia or alkali-burn. In corneas 24 days after epikeratophakia, alpha 1(I) collagen mRNA was detected in keratocytes which had migrated from the host cornea into the keratolens. In contrast, collagenase mRNA was detected in cells which seemed to be inflammatory cells around the suture between the host stroma and the keratolens. The increase of alpha 1(I) collagen mRNA in keratocytes was observed in corneas 94 days after epikeratophakia and in alkali-burned corneas 1-2 months after the burn. These results provide evidence that keratocytes synthesize collagen and that this synthesizing activity lasts for a long period during corneal wound healing.

Alkalies↗
PubMed1992

High frequency of mitochondrial ND4 gene mutation in Japanese pedigrees with Leber hereditary optic neuropathy.

The association of the ND4 gene mutation (mutation) at nucleotide position 11778 of mitochondrial DNA (mtDNA) was investigated in 14 definitive Japanese pedigrees with Leber hereditary optic neuropathy (LHON). The mutation was detected by SfaNI and MaeIII restriction fragment length polymorphisms of mtDNA amplified by polymerase chain reaction. All 14 LHON pedigrees exhibited the mutation, whereas 10 controls did not. The association of this mutation with LHON was revealed to be significantly higher in Japanese (91.7%) than in 27 reported Caucasian (51.9%) LHON pedigrees, implying genetic heterogeneity. In the tested 14 pedigrees, 28 cases with the mutation comprised 19 affected (17 male and 2 female) and 9 asymptomatic (all female except for one) individuals. Such a predominance of males in the incidence of LHON suggested probable participation of additional pathogenetic factor(s) in the development of optic atrophy in LHON patients.

Adolescent↗
PubMed1991

Purification and characterization of three forms of differently glycosylated recombinant human granulocyte-macrophage colony-stimulating factor.

We have purified recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) produced in human lymphoblastoid Namalwa cells. From the results of tunicamycin treatment and N-glycosidase F digestion, it was demonstrated that Namalwa-derived hGM-CSF was highly glycosylated at two potential N-glycosylation sites and several O-glycosylation sites as previously shown for naturally occurring hGM-CSF. We classified the hGM-CSF molecules into three groups according to the molecular weight corresponding to the degree of N-glycosylation: the molecules with two N-glycosylation sites occupied (designated 2N), the molecules with either site glycosylated (1N), and the molecules lacking N-glycosylation (0N). Despite such varied degrees of N-glycosylation, almost all molecules were O-glycosylated. To investigate the role of carbohydrate moieties of hGM-CSF, we isolated each form of hGM-CSF and examined its biological properties. The 2N-type showed 200-fold less in vitro specific activity compared with unglycosylated Escherichia coli-derived hGM-CSF, although the activity of the 0N-type was equivalent to that of the E. coli-derived material. The 1N-type showed an intermediate level of activity. However, in terms of clearance from blood circulation in the rat, the 2N-type showed a half-life five times longer than that of the 0N-type and E. coli-derived hGM-CSF. From these findings, we concluded that N-linked carbohydrate moieties of hGM-CSF play conflicting physiological roles in the efficacy of the protein in vivo but that O-linked carbohydrate moieties do not have such effects.

Animals↗
PubMed1991

Collagen metabolism during healing of lacerated rabbit corneas.

We previously demonstrated that there are two waves of increased collagen synthesis following corneal laceration in rabbits. In the present study, we have examined whether increases in collagen synthesis and degradation result from increased amounts of mRNAs for collagen and collagenase, respectively. Rabbits were anesthetized by combined administration of ketamine (intramuscular) and pentobarbital (intravenous). A penetrating 8-mm incision was made at the center of each cornea. The lacerated corneas were allowed to heal for 0-49 days. The rabbits were then killed and the corneas excised. The total RNA was extracted from the tissue and subjected to slot-blot hybridization using 32P-labeled alpha 1(I) cDNA. The results indicate that there is a two-phase increase in the amount of alpha 1(I) mRNA in injured corneas and that the collagenase mRNA is elevated at most times throughout the healing period. However, the increase is collagenase mRNA may not fully account for the accelerated collagen degradation during corneal wound-healing. Thus, we propose that cells in the wound area may be directly involved in collagen degradation by phagocytosis. To examine our hypothesis, we cultured injured rabbit corneas in the presence or absence of leupeptin, a proteinase inhibitor. The tissues were then examined by electron microscopy. In the presence of leupeptin, lysosomes within fibroblasts or fibroblast-like cells in the wound area of the lacerated corneas healed for 2 and 3 weeks, contain collagen fibrils. In the absence of leupeptin no identifiable collagen was seen in the lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Three cases of EB virus-associated uveitis.

We report our experience with three cases of Epstein-Barr virus (EBV)-associated uveitis. These patients had the following common clinical features: 1) a common cold-like prodrome, 2) bilateral involvement, 3) severe acute anterior uveitis with fibrinous exudate, 4) granulomatous anterior uveitis in the chronic stage, 5) little vitreous inflammation, 6) marked hyperemia and edema of the disc, 7) sunset glow fundus developed very early, 8) a good response to steroids but recurrence was common, 9) good final visual acuity, 10) no systemic changes in the course of the uveitis. High EBV IgG VCA antibody titers were found in the aqueous humor in all three cases (case 1, 1: 40; case 2, 1: 160; case 3, 1: 20). As controls, 37 cataract patients and 14 patients with various types of uveitis were examined for EBV antibodies in the aqueous humor. The result was negative in every case. On the basis of these data, the uveitis in these three patients appears to represent a new clinical entity: uveitis caused by EBV.

Acute Disease↗
PubMed1990

[An application of electrochemical detector to dental pharmacology].

The electrochemical detection (ECD) is highly sensitive and can distinguish selectively electrochemically active materials. A sensitive and selective ECD is widely used for high-performance liquid chromatography (HPLC) of phenolic compounds, such as catecholamines, indoleamines and phenolic steroids in biological materials. In the present study, the HPLC/ECD system was applied for the simultaneous determination of various dental drugs, such as phenol, guaiacol, m-cresol, o-chlorophenol, p-chlorophenol, m-chlorophenol, eugenol, pyrogallol and resorcin. As a compromise between sensitivity and stability, the applied voltage was set at 1.2 V vs the reference electrode. These drugs were rapidly and selectively separated on a reversed-phase column using 10 mM acetate buffer (pH 4.0)-methanol (7:3, v/v) as a mobile phase. The detection limits of these drugs were determined to be 0.25 pmol per injection. These results imply that the HPLC/ECD method should be capable of determining these dental drugs with high sensitivity. The ECD was also applied to determine the enzyme activity. A highly sensitive and specific assay for alkaline and acid phosphatases and N-acetyl-beta-glucosaminidase in biological materials, such as plasma, saliva and bone, has been established. Phenol, formed enzymatically from the substrate, was determined by HPLC/ECD. The retention time of phenol was 7 min and no other peaks were observed. The method is rapid and sensitive with a detection limit for phenol of as little as 0.5 pmol. Thus, as little as 0.5 microliters of rat plasma or 10 microliters of human saliva is all that is required for both alkaline and acid phosphatase assays. The assay is accurate and reproducible. Using this assay, alkaline and acid phosphatase activities in human saliva and in rat saliva elicited by pilocarpine and isoproterenol were determined. It will be possible to apply this new assay method to extremely small biological samples. Thus, the application of the HPLC/ECD system to determine dental drugs and enzyme activity with high sensitivity should prove to be of great value in the field of dental pharmacology.

Acid Phosphatase↗
PubMed1990

[The possible association of Epstein-Barr virus and uveitis].

The possible association of Epstein-Barr Virus (EBV) with uveitis was investigated in 28 subjects with various types of uveitis. Serum or aqueous humor (AH) EB viral antibodies were measured in 28 uveitis patients and in 28 cataract patients as controls. Significant rises in EBV antibody levels were seen in 3 cases from the uveitis group. There was an 8-fold increase of the serum antibody titer in an infant subject, a 16-fold increase in the AH EB viral antibody titer in an adult subject, and a higher antibody quotient (10.2) compared with the serum and AH antibody titers in another adult case. No antibodies to EBV were detected in the AH in the control group. It appeared that EBV might have been involved in the pathogenesis of these 3 cases of uveitis. The clinical features of these cases were as follows. (1) A common cold-like prodrome was noted. (2) Severe acute anterior uveitis with fibrinous exudate was present at the onset. (3) A transition to chronic granulomatous anterior uveitis was seen in the 2 adult cases. (4) Little vitreous inflammation was present. (5) Marked hyperemia and swelling of the optic discs was noted. (6) At the onset there were no abnormal chorioretinal findings, except slight retinal vasculitis in the infant case. (7) Sunset-glow fundus appeared earlier in comparison with Harada disease in the adults and a retinal pigment epithelium disorder developed in the infant. (8) Bilateral involvement was seen in adults. (9) Good final visual acuity was recognized. (10) No systemic changes occurred in the course of the uveitis. This constellation of clinical features appears to comprise a new entity, i.e., EBV-associated uveitis.

Adolescent↗
PubMed1987

Highly sensitive assay for alkaline and acid phosphatase activities by high-performance liquid chromatography with electrochemical detection.

A highly sensitive and specific assay for alkaline and acid phosphatases in biological materials, such as plasma and saliva, has been established. Phenol, formed enzymatically from the substrate phenylphosphate, was determined by high-performance liquid chromatography with electrochemical detection. The retention time of phenol was 7 min and no other peaks were observed. The method is rapid and sensitive with a detection limit for phenol of as little as 5 pmol. Thus, as little as 0.5 microliter of rat plasma or 10 microliters of human saliva is required for both alkaline and acid phosphatase assays. The assay is accurate and reproducible. Using this assay, alkaline and acid phosphatase activities in saliva were found to be 1.12 +/- 0.12 nmol/min/ml and 9.79 +/- 1.23 nmol/min/ml, respectively. This new assay method should be applicable to extremely small biological samples.

Acid Phosphatase↗
PubMed1987

Synthesis and antitumor activity of tropolone derivatives. 4.

Modifications of monotropolone 2 having poor potency against P388 in mice were studied. The alpha-ethoxy group of 2, prepared from hinokitiol and benzaldehyde diethyl acetal, was replaced with a phenolic or heteroaromatic compound by heating 2 with the appropriate nucleophile. Structure-activity relationships indicated that an acidic hydroxyl and a proton-accepting group situated in the neighboring position, which permits the formation of a chelate with a metal ion, contributed to enhanced activity. Among the compounds studied, the 8-hydroxyquinoline analogue 10f was the most favorable compound.

Animals↗
PubMed1987

Synthesis and antitumor activity of tropolone derivatives. 5.

As part of a study on the structure-activity relationship of antitumor-active tropolone derivatives, a series of bistropone analogues, related to potently active bistropolone 1a, were synthesized and tested for their antitumor activity in in vitro (KB cell) and in vivo (leukemia P388 in mice) systems. The methoxytropones 3 and 5, hydroxytropothione 10, and (N-methylamino)tropones 12 and 13 were inactive in both systems. Methoxytropone 6 exhibited weak activity, whose potency was equal to that of monotropolone 2a.

Animals↗
PubMed1987

Synthesis and antitumor activity of tropolone derivatives. 6. Structure-activity relationships of antitumor-active tropolone and 8-hydroxyquinoline derivatives.

The bis derivative 6 of 8-hydroxyquinoline, which, like tropolones, readily forms a chelate, was synthesized and found to have high potency (dose = 12.5 mg/kg, T/C % = 164) against leukemia P388 in mice approximately equivalent to that of the bistropolone 1b. 8-Hydroxyquinoline analogues with broad structural variation were synthesized and their structure-activity relationships followed the same pattern as in the tropolone series. In addition, the bistropolones 1a-e were tested for their ability to bind to tubulin and found to have no such property. The results of this study suggested that bistropolone and bis(8-hydroxyquinoline) derivatives must form a chelate with the metal necessary for the enzyme, such as ribonucleotide reductase, which catalyzes the DNA biosynthetic pathways.

Animals↗