Biomedical subjects

S Okinaga

Publications and source records attributed to S Okinaga.

At least 55 records · Page 3Linked to original sources

PubMed1992

Purification and localization of a calcium-binding protein in human spermatozoa.

The purpose of this study was to purify a calcium-binding protein (CalBP) from human spermatozoa of 226 men and to determine the localization of this protein in spermatozoa. The sperm cells were extracted with 0.6 M KCl, and the KCl extract was then subjected to gel filtration and high performance liquid chromatography. Two CalBPs of Mr = 38 kDa and 52 kDa were found by 45Ca(2+)-binding on blotted proteins. Only a 52 kDa CalBP (CalBP-52) was purified to a final yield of 0.007%. The isoelectric point of this protein was 5.1. The human CalBP-52 reacted with rabbit antiserum directed against rat 52 kDa CalBP. An immunocytochemical study showed that this protein was localized on the sperm head. It is postulated that this protein may have important functions related to Ca(2+)-transport into sperm cells.

Animals↗

PubMed1992

Reconstruction of surgical defects using the gluteus maximus myocutaneous flap following radical vulvectomy.

Radical vulvectomy is often complicated by problems associated with insufficient closure of large skin defects involving postoperative necrosis of the suture line over the mons pubis and the inguinal areas. To resolve these problems, the present study compared radical vulvectomy (RV, 7 cases) with radical vulvectomy followed by reconstructive operation using the gluteus maximus myocutaneous flap (RVR, 5 cases). There was no significant difference in operation time and blood loss between the two groups. Three of the 7 RV patients had wound separations requiring reoperation, while only 1 patient in the RVR group did. The average hospital stay was 86 days in the RV group and 38 days in the RVR. Risks of postoperative infection and wound breakdown were reduced with the flap technique, and ambulation and rehabilitation could begin earlier in this group. Surgical wounds were stable and the quality of life after operation was improved dramatically using the flap technique.

Aged↗

PubMed1992

Analysis of urinary protein by immunoblot method using unconcentrated urine in preeclampsia.

In order to investigate the renal change in preeclampsia, molecular weight and specific protein analyses in unconcentrated urine were performed by the immunoblot method. Urine samples were taken from 34 preeclamptic cases (pure type), including 20 severe cases. Polypeptide profiles of urine consisted of four patterns: low MW (L) pattern (tubular damage), high MW (H) pattern (glomerular damage), high and low MW (HL) pattern, and middle MW (M) pattern. The incidences of the HL, H, L, and M patterns were 26.5%, 14.7%, 11.8%, and 47.1%, respectively. The HL pattern was found more frequently in severe proteinuria than in mild proteinuria. High incidences of the HL and H patterns were found in the hypertensive group. Larger amounts of IgM, fibronectin, IgG, and beta 2 microglobulin in urine were confirmed using specific antibodies. Our results suggest that the immunoblot method makes it possible to differentiate glomerular and tubular damages and to evaluate the severity of renal damage in preeclampsia using unconcentrated urine.

Female↗

PubMed1992

Molecular bases of tyrosinase-negative oculocutaneous albinism: a single base insertion or a missense point mutation in the tyrosinase gene.

We have identified two different mutations in the tyrosinase genes of Japanese patients with tyrosinase-negative oculocutaneous albinism (OCA). One is a single base insertion in the exon 2 of the tyrosinase gene that shifts the reading frame and introduces a premature termination codon (TGA) after the amino acid residue 298 (codon 316). The other is a G to A transition at residue 312, leading to a single amino acid substitution, arginine at position 59 (codon 77) to glutamine. The promoter activity of the patients' tyrosinase genes was evaluated in the cell-free transcription system prepared from pigmented melanoma cells, indicating that the patients' genes were accurately transcribed in vitro. It is therefore conceivable that the tyrosinase gene is expressed in their melanocytes. Furthermore, transient expression of the mutated genes indicates that the truncated tyrosinase or the tyrosinase containing glutamine 59 is unable to form melanin in melanocytes. We therefore propose that these mutations in the tyrosinase genes lead to a phenotype of tyrosinase-negative OCA.

Albinism, Oculocutaneous↗

PubMed1991

A new membrane-associated Ca(2+)-binding protein of rat spermatogenic cells: its purification and characterization.

A Ca(2+)-binding protein of Mr = 52000, estimated by SDS-PAGE, was purified to a final yield of 0.04% from rat spermatogenic cells. Purification steps included gel filtration, ammonium sulfate precipitation and HPLC. Amino acid analysis showed the content of 34% acidic residues and 15% basic residues. The isoelectric point of this protein was 4.7. Dot-blot analysis indicated that the Ca(2+)-binding protein bound 2 mol of calcium per mol of protein. This protein had two binding sites with dissociation constants of 4.8 microM and 0.2 microM. No appreciable amount of hexose was observed (less than 1 microgram of hexose/70 micrograms of protein). This protein may play an important role such as the Ca(2+)-transport in the plasma membrane of spermatogenic cells.

Amino Acids↗

PubMed1991

Effect of an acute maternal stress on the fetal hypothalamo-pituitary-adrenal system in late gestational life of the rat.

We investigated in this study the response of the fetal hypothalamo-pituitary-adrenal (HPA) system during an acute maternal stress in rats, in order to find out a possible role of developing fetal hypothalamus and to correlate its function to the androgen unbalance during the critical period of sex-specific brain differentiation. Pregnant rats of days 18-22 of gestation were subjected to an acute forced immobilization, and plasma levels of corticosterone (B) and ACTH were measured in mothers and fetuses. Hypothalamic contents of CRH and beta-endorphin (EP), pituitary content of ACTH, and plasma levels of B and ACTH were measured in mothers and fetuses under the maternal stress on day 20 of gestation. By an acute exposure to the 20 minutes' stress, plasma levels of B and ACTH elevated significantly in mothers on each day of gestation. A significant increase of fetal plasma ACTH was detected from day 18 in males, and from day 20 in females. During the maternal stress on day 20 of gestation, hypothalamic contents of CRH and EP decreased significantly in male and female fetuses, when plasma levels of B and ACTH elevated significantly. These results indicate that fetal HPA axis seems to actually respond to the maternal stress during the late gestational period. Further, a release of CRH under the stress together with an activation of EP system in the fetal hypothalamus suggests a possible mechanism regulating the androgen secretion by the fetal hypothalamus via changes of the LH levels.

Adrenocorticotropic Hormone↗

PubMed1991

Presence of a 16 kd protein in human seminal plasma counteracts the effects of the anti-fertility agent, gossypol.

Gossypol inhibited lactate dehydrogenase (LDH) noncompetitively in human spermatozoa. The inhibitory effect of gossypol on LDH was cancelled by the addition of human serum albumin, human gamma-globulin, bovine serum albumin or human seminal plasma. Seminal plasma was at least 10 times more effective than the other three proteins, when expressed on a per mg protein basis. Attempts were made to purify the active fraction from human seminal plasma. The purification steps included gel filtration, ammonium sulphate precipitation, centrifugal microconcentration and fast-performance liquid chromatography. A single active protein of Mr = 16,000 was purified to a final yield of 0.18%. The 16 kd protein was not observed in male blood plasma. The protein was found to be heat-stable and leucine-rich (16% of the molecule), and has been designated 'gossact'. The inhibitory effect of gossypol on the LDH reaction was completely blocked by the addition of gossact (5 micrograms/ml); human blood plasma (25 micrograms/ml) and human serum albumin (200 micrograms/ml) were far less potent in this assay. In addition, gossact bound 1.4 mol of gossypol/mol of protein with the dissociation constant (Kd) = 3.06 x 10(-5) M. The role of gossact in the protection of LDH from gossypol is discussed.

Binding, Competitive↗

PubMed1991

The monoclonal antibodies TMH-1 and TMH-2 specifically bind to a protein encoded at the murine b-locus, not to the authentic tyrosinase encoded at the c-locus.

Three hybridomas, TMH-1, TMH-2, and TMH-3, were previously reported by Tomita et al to produce monoclonal antibodies against murine and human T4-tyrosinase localized in melanosome for the formation of melanin pigment. However, TMH antibodies were unable to react with K1735 cells transfected with the authentic tyrosinase-cDNA construct, but did react with those transfected with the pMT4-cDNA construct. The cDNA pMT4 was initially cloned as a putative tyrosinase cDNA by Shibahara et al, but it is now known to encode mouse brown (b) locus protein, which was named "tyrosinase-related protein" by Jackson or "b protein" by Hearing and Jimenez. Furthermore, TMH antibodies recognize hair bulbs of C57BL/6J-c2J/c2J mouse (B/B, c/c) lacking tyrosinase activity, but do not recognize hair bulbs of b-locus mutated DBA/2 mouse (b/b, C/C), which have authentic tyrosinase. Considering these observations, we conclude that TMH antibodies specifically recognize the protein encoded at b-locus.

Animals↗

PubMed1991

Ontogeny of the response of the hypothalamo-pituitary-adrenal axis to maternal immobilization stress in rats.

In this study we investigated the response of the rat fetal hypothalamo-pituitary-adrenal (HPA) axis to an acute maternal stress in late gestation. On day 20 of gestation, pregnant rats were exposed to forced immobilization stress for up to 60 min. In mothers, a significant increase in plasma ACTH and corticosterone(B) was observed at 20 and 60 min. The ACTH content in the maternal pituitary decreased significantly at 60 min. Fetal blood pH was decreased by the maternal stress, showing a hypoxic condition of the fetus. Fetal plasma ACTH increased transiently at 20 min. Fetal plasma B increased at 20 and 60 min. ACTH in the fetal pituitary and the placenta did not show marked changes due to the maternal stress. Pregnant rats on day 18-21 of gestation were subjected to a 20 min maternal stress. In the basal condition without stress, fetal plasma ACTH and B showed parallel ontogenic patterns, having a peak value on day 19 of gestation. Fetal plasma ACTH as well as plasma B were increased significantly by the maternal stress at all points evaluated. These results indicate that fetal hypoxia is important in stress transmission to the fetal HPA axis in this type of maternal stress, and the fetal HPA axis responds to the stress as early as day 18 of gestation.

Adrenocorticotropic Hormone↗

PubMed1991

The effect of an acute stress in late pregnancy on hypothalamic catecholamines of the rat fetus.

Prenatal stress in the last trimester of pregnancy induces permanent disorders in sex-specific differentiation of the brain in rats. In order to determine the neuroendocrine response of the fetal hypothalamus to the maternal stress, we investigated the temporal change in fetal hypothalamic catecholamines when their mothers were exposed to an acute stress. On day 20 of gestation, pregnant rats were subjected to forced immobilization and sacrificed at 0, 30, 60, 120 and 180 minutes. Plasma corticosterone reached a peak at 60 minutes of stress in mothers and fetuses. Hypothalamic norepinephrine and epinephrine decreased at 120 and 180 minutes of stress in mothers, whereas in fetuses, hypothalamic norepinephrine decreased at 120 and 180 minutes of stress, and dopamine also showed a tendency to decrease. These results suggest that maternal stress in late pregnancy decreases fetal hypothalamic norepinephrine which is involved in androgen-dependent sex differentiation of the brain.

Androgens↗

PubMed1991

Electron microscopic studies on duck hepatitis B virus particles in hepatocytes and sequential changes in various patterns of infection.

To investigate the critical factors involved in the elimination of the duck hepatitis B virus (DHBV) in acute infection, the sequential changes in the number of DHBV particles in hepatocytes were studied electron microscopically in ducks experimentally infected by DHBV. Twenty Japanese white Peking ducks were infected with DHBV on the day of hatching, and on the 7th day and 14th day after hatching. Inoculation of DHBV on the day of hatching, and on the 7th and 14th day after hatching resulted in persistent viremia, transient viremia and no viremia, respectively in ducks as tested by spot hybridization assay. The number of DHBV particles in the liver correlated well with the amount of serum DHBV-DNA, DHBV particles decreased in hepatocytes without any interaction of inflammatory cells over the observation period, and the number of particles was not associated with the degree of hepatic inflammation. From these results, the elimination of the virus was thought to be induced by a reduction of viral replication in the hepatocytes and not by destruction of their host cells. There must be an age-dependent factor which strongly suppresses the viral replication.

Animals↗

PubMed1990

Luteolytic effect of the antiprogestin and antiglucocorticoid agent RU486 in rats.

Ovarian cells of pregnant rats were cultured with synthetic progestins (R5020, R2323), dexamethasone and RU486. Progesterone and 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) in the medium were measured by specific radioimmunoassay. Both R5020 and R2323 increased concentrations of these intrinsic progestins. RU486 decreased concentrations of progesterone, however, the addition of R5020 or R2323 counteracted this action. Immature hypophysectomized rats treated with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) were administered with RU486; the serum levels of progesterone and 20 alpha-dihydroprogesterone tended to decrease. R5020 and R2323 inhibited the effect of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), whereas RU486 did not. Inhibition of the cholesterol side chain cleavage enzyme (CSCC) by RU486 was more marked than that by R5020 or R2323. These results show that RU486 decreases progesterone synthesis in cultured ovarian cells. A part of the mechanism may involve an inhibition of CSCC.

Algestone↗

PubMed1990

A point mutation in the tyrosinase gene of BALB/c albino mouse causing the cysteine----serine substitution at position 85.

Murine albinism is characterized by complete lack of melanin pigments in skin and retina. In order to study the molecular basis of albinism, we have cloned and characterized the tyrosinase gene of BALB/c mice (c/c). Sequence analysis of this gene reveals a point mutation at nucleotide residue 387 (G----C transversion) causing a Cys----Ser substitution at position 85 in one of the cysteine-rich domains of the tyrosinase molecule. Since this G----C transversion creates an additional DdeI site, we were able to confirm that this mutation is actually present in BALB/c genomic DNA using DNA amplification techniques. In contrast, both C57BL/6 (C/C) and DBA/2 (C/C) mouse strains carry the G residue at the same position, suggesting that this point mutation is specific for the albino mutation at the c locus. Moreover, we were able to show that the tyrosinase containing Ser-85 is not functional in transient expression of its cDNA. We therefore suggest that a G----C transversion at nucleotide residue 387 of the tyrosinase gene could lead to the albino phenotype of BALB/c mouse.

Albinism↗

PubMed1990

Modified rotational acetabular osteotomy (RAO) for advanced osteoarthritis of the hip joint in the middle-aged person. First report.

Classical methods for pelvic osteotomy, such as those of Salter, Pemberton, Chiari, and Wagner, have been developed for reconstruction of the subluxed hip joint in children and young adults. Regarding pelvic osteotomy involving a middle-aged patient, however, there are not as many operation methods to consider, and it is difficult to choose the most suitable technique for alleviating advanced osteoarthritis. Based on current practice, total hip replacement (THR) seems the accepted method, though it presents problems such as loosening, sinking, and infections; because of these factors physicians hesitate to recommend THR surgery, particularly if the patient is otherwise healthy and appears to have many good years ahead of him. As an alternative, we have been developing and improving the acetabular osteotomy, based on Tagawa's rotational acetabular osteotomy (RAO) and Wagner's acetabular osteotomy (type II). In this paper we present the results of a modified RAO operation performed on 50 middle-aged patients with an average age of 42 years and 2 months (31-61). The average follow-up was 3 years and 3 months (1-9 years). In 82% of patients the result was satisfactory (41 of 50 cases). A similar osteotomy technique has been used by Eppright and Wagner. We feel that our method achieves a more favorable result for an older patient with severe osteoarthritis, since both the surgery and the follow-up rehabilitation are more comprehensive. A modified acetabular osteotomy should not be regarded as merely an alternative to total hip replacement, but as the preferred choice for hip-joint reconstruction.

Adult↗

PubMed1990

Subrenal capsule assay as a test to predict the effectiveness of chemotherapy in choriocarcinoma.

The value of the 6-day subrenal capsule assay (SRCA) in predicting the effectiveness of chemotherapy was tested using three human choriocarcinomas. The SRCA method proved to be a valuable chemopredictive test for choriocarcinoma. The results of experimental chemotherapy in nude mice and the results of the SRCA in two types of choriocarcinoma correlated well. Two of three choriocarcinomas showed sensitivity to MAC combination chemotherapy [methotrexate, actinomycin D, cyclophosphamide), and all three showed greater sensitivity to VP-16. The administration of VP-16 for treatment of all three types of choriocarcinoma significantly reduced tumor size in a dose-dependent fashion. From these observations, we speculate that the SRCA may be applied to assess the effectiveness of chemotherapy against human choriocarcinoma.

Animals↗

PubMed1990

The effect of epidermal growth factor (EGF) on progestin secretion and cyclic AMP synthesis in cultured luteal cells from pregnant rats.

It is reported that steroid synthesis in ovarian cells is affected by epidermal growth factor (EGF). We cultured luteal cells from pregnant rats for 2 days with or without EGF, followed by incubation of the cells with or without stimulants (hCG, forskolin and dibutyryl cyclic AMP) for 5 hours. The levels of progesterone, 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) and cyclic AMP (cAMP) in the media were assayed. EGF had no effect on the basal levels of progesterone, 20 alpha-dihydroprogesterone and cAMP, but it suppressed these levels which were increased by the stimulants. We investigated binding capacity of [125I]-EGF to ovarian tissue of pregnant rats. Ovarian tissue had specific binding sites for EGF. The maximum number of binding sites was 2.38 fmol/mg tissue and the Kd value was 0.547 nM. It was indicated that EGF modified the reactivity of luteal cells to stimulants; counteracting the tropic effect of gonadotropins. It was shown that this effect of EGF might be exerted through its receptor in luteal cells.

Animals↗

PubMed1990

[Fundamental studies on the subrenal capsule assay as chemosensitivity test for nonsolid tumors].

Fundamental evaluation of the subrenal capsule assay (SRCA) method in nonsolid tumors was made, using two types of murine malignant ascites. Malignant ascites were obtained from mice bearing M-5076 ovarian reticular cell sarcoma or MH-134 hepatoma. These tumor cells were allowed to settle by standing at 4 degrees C to form a jelly-like clot. This clot was cut into fragments about 1mm3 in size and one of these fragments was mashed in trypan blue to estimate the viability grade of the implanted tumor cells. The rest of the fragments were implanted beneath the renal capsule of the mice. On the 6th day after implantation, the assay mice were killed, the increase in the size of the tumor was determined and histological examination was carried out. The results were as follows: (1) The clot was formed reproductively by allowing ascites to settle for one or two days and there was a high viability rate for the tumor cells: 79.9 +/- 11.0% of M-5076 and 90.1 +/- 5.9% of MH-134. (2) The ascites clot thus implanted grew rapidly in the control groups but growth was inhibited by chemotherapy: Tumors were reduced significantly (p less than 0.05-0.005) in the group treated with a single agent. This trend towards a suppressive effect of carcinocidal agents on the tumor growth was more conspicuous as a combination regimen was utilized, a combination of three agents producing the maximum effect. (3) The clot grew more quickly than the solid tumor in both the control and the treated groups. There was a high correlation (r = 0.93 in M-5076, and r = 0.64 in MH-134) between the growth rates of ascites and solid tumor in SRCA. (4) Histological examination revealed that viable tumor cells infiltrated widely under the renal capsule in both types of tumors. These results suggest that ascites and solid tumor are useful materials for the subrenal capsule assay method.

Animals↗

PubMed1989

Human oculocutaneous albinism caused by single base insertion in the tyrosinase gene.

Tyrosinase-negative oculocutaneous albinism (OCA) is an inborn error of metabolism, characterized by a complete lack of melanin pigments in the eyes and skin. We have isolated and characterized the tyrosinase gene of one affected child (S.S.) with tyrosinase-negative OCA. Sequence analysis reveals a single-base insertion in the exon 2 that shifts the reading frame and introduces a premature termination signal (TGA codon) after the amino acid residue 298. Functional analysis of the mutated gene indicates that such a truncated tyrosinase lacking one potential copper-binding region is catalytically inactive. We therefore conclude that the albino phenotype of the patient S.S. is a consequence of the inactive tyrosinase caused by the nonsense mutation in the tyrosinase gene.

Albinism↗