Biomedical subjects

R Sakakibara

Publications and source records attributed to R Sakakibara.

At least 127 records · Page 7Linked to original sources

PubMed1992

Biochemical studies on oral toxicity of ricin. IV. A fate of orally administered ricin in rats.

After oral administration of ricin in rats, its distribution in the gastrointestinal tract, body fluids and principal organs was determined by an enzyme immunoassay, and the immunoreactive ricin detected was identified by gel filtration followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, protein blotting and the immunobinding method. When ricin D (10 mg/kg rat) was given orally to a rat, which dose is equivalent to 1/3 LD50, about 75% of the ricin was found in the stomach and small intestine within 2 h, and most of it was transferred to the large intestine after 24 h. It was also demonstrated by an in vitro toxicity test of immunoreactive ricin in the blood and lymph obtained from the intoxicated rats that a part of the ricin was absorbed from the small intestine into the tissues and organs via the circulatory systems (lymphatic and blood vessels) as the active ricin. The participation of the blood vessels was greater in the absorption of ricin from the gastrointestinal tract than that of the lymphatic system. Ricin, after absorption, was detected in liver and spleen and ricin found in the liver was predominantly in the form of intact ricin, although an undetectable amount of ricin in other organs cannot be eliminated. These results infer that a small fraction of orally-given ricin was transferred to the circulating system and was responsible for rat's death as in the case of i.p. administration.

Administration, Oral↗

PubMed1992

Biochemical studies on oral toxicity of ricin. V. The role of lectin activity in the intestinal absorption of ricin.

In order to investigate a possible role of lectin activity of ricin in its absorption from the small intestine, we prepared two ricin derivatives. BMH-ricin, prepared by crosslinking A and B chains of ricin with 1,6-bismaleimidohexane, was nearly non-toxic but the lectin activity was unaltered. And, NBS-ricin, prepared by the oxidation of tryptophanyl residues of ricin with N-bromosuccinimide, was not only non-toxic but also non-lectinic. After the oral administration of ricin derivatives to rats, their interaction with the digestive tract and absorption into the circulatory systems have been compared with those of ricin, immunochemically and histologically. It was shown by immunostaining that ricin and BMH-ricin could bind to the intestinal mucosa, whereas NBS-ricin could not. No appreciable damage in the small intestine from rats treated with either BMH-ricin or NBS-ricin has been observed, in contrast to ricin treatment where severe impairment of the small intestinal tissues resulted after 5 h. Immunoreactive ricin in the liver has been determined with the ricin enzyme immunoassay (EIA). When compared at 48 h after oral administration, NBS-ricin was not detected, whereas BMH-ricin was found to be 38 micrograms/liver and ricin 100 micrograms/liver. From these results, it was inferred that the lectin activity of ricin plays an important role in the absorption of ricin from the small intestine and that the absorption of ricin protein was enhanced by its high toxicity.

Administration, Oral↗

PubMed1992

Interaction of toxic lectin ricin with epithelial cells of rat small intestine in vitro.

To clarify the mechanism of oral toxicity of ricin, the interaction of ricin with the epithelial cells isolated from rat small intestine was compared in vitro with those of other plant lectins by two different determinations, i.e., viability and cytotoxicity. After incubation of the cells for 1 h at 37 degrees C with ricin, B-chain, castor bean hemagglutinin (CBH), soybean agglutinin (SBA), wheat germ agglutinin (WGA), concanavalin A (Con A), and peanut agglutinin (PNA), respectively, followed by staining with trypan blue, ricin and ricin B-chain as well as CBH and SBA were found to have effectively reduced the number of viable cells. On the contrary, only ricin inhibited protein synthesis in the cells and the effect was blocked by D-galactose. Additional experiments employing [125I]-labeled ricin strongly suggested that ricin was first bound via its B-chain to the galactosyl residues on the cell surface followed by internalization into cells as the whole 62 kDa molecule. These results infer first that ricin, as well as other lectins mentioned above, was able to reduce viability of the epithelial cells of rat small intestine by direct binding to the cell surface. The second effect, specific to ricin, was the inhibition of cellular protein synthesis.

Animals↗

PubMed1991

A putative mouse oocyte maturation inhibitory protein from urine of pregnant women: N-terminal sequence homology with human nonsecretory ribonuclease.

A putative mouse oocyte maturation inhibitory protein was purified from a urine preparation from pregnant women by Sephadex G-100 gel filtration and reverse-phase chromatography on the basis of inhibitory activity of polar body formation of denuded mouse oocytes in culture. Amino terminal sequence analyses showed that residues 5 to 15 of this protein were identical to residues 1 to 11 of human nonsecretory ribonuclease. Furthermore, residues 1 to 4 of this protein were identical to residues -4 to -1, corresponding to part of a signal peptide region of eosinophil-derived neurotoxin, whose mature sequence is identical to nonsecretory ribonuclease. These results indicate that the protein purified as a putative mouse oocyte maturation inhibitory protein from the urine of pregnant women may be a product of an peculiar processing of a nonsecretory ribonuclease precursor.

Amino Acid Sequence↗

PubMed1991

Steroidogenic activity of synthetic hybrid molecules composed of human chorionic gonadotropin and either the A or B chain of lectin ricin or horseradish peroxidase.

Human chorionic gonadotropin (hCG) is a glycoprotein consisting of noncovalently bound alpha- and beta-subunits which shows hormonal activity (stimulatory effect on testosterone production) toward rat Leydig cells. To modify the hormonal activity of hCG, hybrid molecules composed of hCG and other glycoproteins, either the A or B chain of lectin ricin (hCG-A and hCG-B) through disulfide bridges and horseradish peroxidase (hCG-HRP) through Schiff's base, were synthesized. Hormonal activity and the effect of these hybrids on [125I]hCG binding to rat Leydig cells were compared to those of native hCG. Modification of hCG resulted in a significant decrease in hormonal activity for hCG-HRP but not for hCG-A to approximately 1/100 and 1/10 that of native hCG, respectively. On the other hand, hCG-B unexpectedly showed hormonal activity similar to that of native hCG. These hybrids inhibited the binding of 125I-labeled hCG to rat Leydig cells with potencies of 1/10, 1/100 and 1/500 that of hCG for hCG-B, hCG-A and hCG-HRP, respectively. These results indicate that the B chain of ricin, the active component hCG-B, participated in stimulating testosterone production according to its own nature. Data which indicate that hybrids consisting of hCG subunits and the B chain of ricin (alpha-B and beta-B) stimulated testosterone significantly more than hybrids consisting of hCG subunits and A chain (alpha-A and beta-A) support the above finding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

[Striatal involvement on MRI in adrenomyeloneuropathy].

Adrenomyeloneuropathy (AMN), a clinical variant of child adrenoleukodystrophy (ALD), is an adult-onset progressive disorder which presents spastic paraparesis with peripheral nerve involvement and affects mainly the pyramidal tracts from the brainstem to the spinal cord. We report a case of AMN in which serial MRI showed unusual development of areas of high signal in the right striatum. The patient was in good health until the age of 12, when he began to lose his hair. At age 25 he started to have progressive gait disturbance and erectile impotence. In his first admission to our hospital at age 33, he showed diffuse baldness. He was intelligent but childish. His cranial nerves were normal. Muscle strength was weak (3-4/5) in the lower extremities. Deep tendon reflexes were hyperactive in the lower extremities while normal in the upper extremities. Babinski signs were elicited bilaterally. Pinprick and vibratory sensation was impaired in the lower legs. Proprioceptive sensations were normal. Co-ordination was intact. There were urinary incontinence and impairment of erection with preserved libido and ejaculation. Routine laboratory data including hematological studies, serum chemistry and urinalysis were all normal except for mild hyperlipidemia. Serum cortisol response to ACTH was low and serum levels of very long chain fatty acids were increased. Nerve conduction studies were abnormal and consistent with peripheral polyneuropathy. A biopsy specimen of left sural nerve revealed a mild loss of myelinated fibers with thinning of the myelin. These findings and the clinical features confirmed the diagnosis of AMN. MRI in SE2000/40 scans at age 34 disclosed areas of high signal in the bilateral internal capsules.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenoleukodystrophy↗

PubMed1991

Intracellular immature subunits of human chorionic gonadotropin in first trimester placental cells: purification and characterization.

As we previously reported [Sakakibara et al. (1986) Biochem. Biophys. Res. Commun. 137, 443-452; and Tominaga et al. (1989) J. Biochem. 105, 992-997], subunits of human chorionic gonadotropin (hCG) containing immature N-linked sugar chains (immature subunits), i.e., the 21 kDa form of alpha-subunit and the 23 and 19 kDa forms of beta-subunit, are present predominantly in first trimester placental cells. The molecular mass of intracellular hCG consisting of these subunits, based on gel filtration, was approximately 200 kDa, suggesting homo- or hetero-oligomerization of intracellular hCG. In the present study, we purified the 21 kDa form of alpha-subunit as well as the 23 and 19 kDa forms of beta-subunit from fresh normal first trimester placental tissues by gel filtration and reverse-phase high-performance liquid chromatography. Purified subunits were hydrolyzed (with a decrease in their molecular weighs) by endoglycosidase H and alpha-mannosidase but not by sialidase or sialidase followed by O-glycanase, indicating that those forms have presumably only high-mannose-type N-linked sugar chains but not O-linked sugar chains of the type present in mature beta-subunit. Fifteen cycles of Edman degradation of the purified forms of the subunits were performed. Only one phenylthiohydantoin amino acid, which was the same amino acid as in the urinary beta-subunit, was detected at each step for the mixture of 23 and 19 kDa forms of beta-subunit, indicating that the protein backbones of both forms are identical to each other as well as to the urinary beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Synthesis and secretion of human chorionic gonadotropin and its subunits in choriocarcinoma cells: a comparative study with normal placental cells.

The human choriocarcinoma cell line, BeWo, synthesizes the glycoprotein hormone, human chorionic gonadotropin (hCG). We have undertaken this study to compare the synthesized and secreted forms of hCG and their alpha- and beta-subunits in cell cultures of BeWo cells to those forms of normal placental cells by immunobinding techniques. BeWo cells appeared to synthesize and secrete one species of the respective hCG subunit. The immature alpha- and beta-subunits, synthesized in BeWo cells as well as those of placental cells, were digested by endoglycosidase H indicating N-linked sugar chain(s) to be the high-mannose type. The mature alpha- and beta-subunits, secreted by BeWo cells as well as subunits of urinary hCG which are usually used as a standard hCG secreted by normal placental cells, were sensitive to neuraminidase treatment indicating that these subunits have terminal sialic acid(s). Contrary to placental cells, mature subunits of BeWo hCG could not be found in any subcellular fraction indicating a rapid secretion rate or supporting the hypothesis that BeWo cells secrete hCG subunits without the formation of secretory granules. The alpha-subunit synthesized in BeWo cells had a slightly lower molecular weight than that of placental cells; however, the alpha-subunit secreted by BeWo cells had a slightly higher molecular weight than the alpha-subunit of urinary hCG. The beta-subunits synthesized and secreted by BeWo cells had slightly higher molecular weights than beta-subunits of both placental cells and urinary hCG. Even after digestion by N-glycanase as well as endoglycosidase H, molecular weights were still different between the respective subunits of BeWo and placental cells indicating that the apoprotein structures of BeWo hCG subunits may differ from those of placental cells. Moreover, urinary beta-subunit was sensitive to endo-alpha-N-acetylgalactosaminidase treatment but the beta-subunit secreted by BeWo cells was not, indicating that the structure of O-linked sugar chain(s), if present, may be unusual. Analysis of assembled and free forms of subunits of BeWo cell cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by immunobinding methods revealed that subunits are associated intracellularly and then secreted to the media as hCG. Moreover, only free beta-subunits, but not alpha-subunits, of BeWo hCG were found intra- and extracellularly.

Acetylglucosaminidase↗

PubMed1990

A nicked beta-subunit of human chorionic gonadotropin purified from pregnancy urine.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits (alpha and beta) and normally excreted in urine of pregnant women. An uncommon beta-subunit of hCG was purified from fresh early normal pregnancy urine by Sepralyte C8, resin adsorption. Sephadex G-100 column chromatography, and reverse-phase HPLC. SDS-PAGE under non-reducing conditions showed that the apparent molecular weight (39,000) of this beta-subunit was extremely similar to that of the native beta-subunit, which is known to consist of 145 amino acid residues and carbohydrates. However, SDS-PAGE, under reducing conditions, resulted in two bands with apparent molecular weights of 22,000 and 18,000, indicating that it consisted of two peptide fragments connected with disulfide bridge(s). These two peptide fragments, separated and purified from the reduced and carboxymethylated protein, were subjected to amino acid and N-terminal sequence analyses. It was found that this beta-subunit consisted of two polypeptide chains composed of residues 1-47 disulfide-bridged to residues 48-145 of the beta-subunit, which may be produced by nicking of the beta-subunit at the one site (Gly47-Val48). This beta-subunit was termed a nicked beta-subunit of hCG (N-hCG beta). It was also found that N-hCG beta was present in urine as an alpha beta dimer, indicating that an intrachain nicking of this site in the beta-subunit does not inhibit alpha beta dimer formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Effective purification of human chorionic gonadotropin and its subunits from pregnant women's urinary peptides absorbed on reverse-phase resin.

Human chorionic gonadotropin (hCG) was extracted and purified by reverse-phase resin (Sepralyte C8 resin) adsorption and Sephadex G-100 column chromatography from urine of pregnant women. Approximately 15000 IU of hCG was recovered from 1000 ml of urine by C8 resin adsorption. hCG from the gel-filtration step stimulated testosterone production in rat Leydig cells and had a specific activity of approximately 8000 IU/mg, a value which was higher than those of commercial hCGs. Furthermore, hCG purified to near-homogeneity was separated effectively into its subunits by reverse-phase high-performance liquid chromatography using an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid with no special pretreatment for dissociation of subunits. The separated subunits were able to re-associate. The techniques used are simple and may be suitable for not only laboratory but also industrial production of hCG and its subunits.

Animals↗

PubMed1990

[Psycho-neurological symptoms and raised angiotensin-converting enzyme in cerebrospinal fluid in a patient with sarcoidosis beginning in the central nervous system].

A 30-year-old man with sarcoidosis was reported. The initial presentation of sarcoidosis was psychiatric symptoms which included abnormal behavior, hallucination and delirious state. After complete remission of these symptoms, eight months later, organic psychosis reappeared with corectopia, bilateral sensory deafness and staggering gait of bilateral vestibular origin. On admission there was no hilar lymphnode enlargement in chest roentgenogram nor raised angiotensin-converting enzyme (ACE) in serum and it was difficult to make diagnosis. Fourteen months after the onset of psychotic symptoms, bilateral facial palsy appeared and they led us to correct diagnosis of sarcoidosis by reinvestigation of the lesions of the chest. It seemed that corectopia indicated lesion of midbrain tegmentum and deafness and staggering gait indicated bilateral VIIIth nerve dysfunction. ACE and IgG-synthesis rate in the cerebrospinal fluid was raised against normal values of ACE and IgG in the serum. These findings seemed to contribute to the early diagnosis of sarcoidosis which had a selective activity in the central nervous system.

Adult↗

PubMed1990

Micturitional disturbance in cervical spondylotic myelopathy.

Neurourological studies were performed on 37 patients with cervical spondylotic myelopathy. Micturitional history revealed that 30 patients (81%) had voiding symptoms: obstructive in 14, irritative in 5, and both symptoms in 11. Urodynamic studies revealed that 9 of 20 had abnormal uroflowmetrogram, 17 of 37 had residual urine, 14 of 37 had detrusor hyperreflexia, and 6 of 37 had detrusor-sphincter dyssynergia. There were no statistical differences in lower extremity neurological signs in patients with or without residual urine.

Adult↗

PubMed1990

Micturitional disturbance in ossification of the posterior longitudinal ligament in the cervical spine.

Neurourological studies were performed on 39 patients with cervical myelopathy due to ossification of the posterior longitudinal ligament. Micturitional history revealed that 30 patients (77%) had voiding symptoms: obstructive in 8, irritative in 11, and both obstructive and irritative symptoms in 11. Ten patients had urge incontinence and 4 had urinary retention. Urodynamic studies revealed that 7 of 19 had abnormal uroflowmetrogram, 16 of 35 had residual urine, 18 of 39 had detrusor hyperreflexia, and 10 of 39 had detrusor-sphincter dyssynergia. There were no statistical differences in lower extremity neurological signs in patients with incontinence, and patients with residual urine or urinary retention.

Calcinosis↗

PubMed1989

Human chorionic gonadotropin-ricin A chain hybrid protein: a hormone analog for the study of signal transduction.

We have prepared a disulfide cross-linked hybrid consisting of human chorionic gonadotropin and the toxic ricin A chain, utilizing N-succinimidyl-3-(2-pyridyldithio)-propionate. The purified hybrid exhibited cytotoxicity toward rat testis Leydig cells due to inhibition of protein synthesis within the cells, although it inhibited protein synthesis in a cell-free system of a rabbit reticulocyte lysate only after treatment of the hCG-A hybrid with dithiothreitol. On the other hand, pretreatment of the hCG-A hybrid with dithiothreitol completely abolished its cytotoxicity. The hCG-A hybrid stimulated the testosterone production by rat testis Leydig cells to a similar degree to hCG. The hCG-A hybrid could elicit full testosterone production in the presence of about 1/3-1/2 less accumulated cAMP compared with in the case of hCG stimulation. The steroidogenetic effect of the hCG-A hybrid was largely inhibited by anti-hCG antibody but not by anti-ricin A chain antibody. These results suggest that the hCG-A hybrid first binds to Leydig cells via an hCG receptor and then is internalized into the cells, and finally the inhibition of cellular protein synthesis occurs on the release of the ricin A chain from the hybrid. Furthermore, it was suggested that hCG may stimulate testosterone production via another second messenger system as well as via the cAMP system, since the hCG-A hybrid stimulated full testosterone production without maximal cAMP accumulation.

Animals↗

PubMed1989

Existence of associated, non-associated, and oligomeric forms of human chorionic gonadotropin subunits in placental extracts.

The molecular sizes of human chorionic gonadotropin (hCG) subunits in the native state in normal first trimester placental extracts were determined by gel filtration on Sephacryl S-300, followed by SDS-polyacrylamide gel electrophoresis, protein blotting, and immunobinding analysis using anti-alpha and - beta antibodies. Mature forms of hCG subunits in the extracts were only found in the same fraction as that which contained standard urinary hCG, indicating an alpha beta dimer. On the other hand, immature forms were detected with a wide range of molecular weights, which were higher than that of standard hCG, suggesting oligomerization of associated or non-associated immature subunits. In order to determine the associated state of these subunits, various forms of associated subunits (hCG alpha beta) in placental extracts were immunoprecipitated with anti-hCG antiserum, which only recognized hCG alpha beta, and Protein A-Sepharose. They were then analyzed by SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions, followed by immunobinding assaying. It has been suggested that there are three kinds of hCG alpha beta S (one mature and two immature). To confirm the above results and to clarify the existence of free subunits, placental extracts were subjected to two-dimensional SDS-polyacrylamide gel electrophoresis. With this technique, high molecular weight forms of immature hCG subunits were found to be present in placental cells as an oligomer of not only the alpha beta dimer but of each subunit as well.

Chorionic Gonadotropin↗

PubMed1989

[Orthostatically induced epileptoid attack].

An epileptoid attack induced by orthostatic hypotension seen in a 72-year-old man was reported. The patient had been suffering from progressive autonomic failure with parkinsonism for six years and he had severe orthostatic hypotension, syncope and generalized convulsion when he stood up. The convulsion sometimes associated with urinary incontinence, ceased immediately when he lay down although he remained drowsy for a while. Occasionally the seizure ceased spontaneously and he regained consciousness even while he was kept standing. On lying position his blood pressure was 167/88 mmHg and no abnormality was seen in electroencephalogram. When he was tilted up to 50 degrees his blood pressure fell to 70/46 mmHg, and he became unconscious followed by jaw twitching and generalized clonic seizure. Electroencephalogram during seizure showed sharp wave and rhythmic spikes. Other laboratory examination revealed diffuse and severe autonomic dysfunction and slight cerebral atrophy on brain CT scanning. He was treated with diphenylhydantoin 300 mg/day and the seizure responded partially. The reasons why the patient's seizure was thought to be epileptic rather than the convulsive syncope were as follows: the type of the seizure was similar to an epileptic generalized convulsion, the seizure and unconsciousness ceased spontaneously even during standing position, the seizure and impaired consciousness partially responded to diphenylhydantoin administration. The underlying pathophysiology of the seizure was thought to be transient cerebral hypoperfusion induced by orthostatic hypotension.

Aged↗

PubMed1988

Induction of cytosolic aspartate aminotransferase by a high-protein diet.

Induction of cytosolic aspartate aminotransferase (glutamic oxaloacetic transaminase) was observed in rat liver on administration of a high-protein diet. The enzyme activity in the liver of rats given 60% and 80% protein diet increased to 1.8- and 1.9-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of functional sGOT mRNA, measured by in vitro translation. Whereas the activity of mitochondrial aspartate aminotransferase did not increase. Induction of cytosolic aspartate aminotransferase was also detected immunocytochemically.

Animals↗

PubMed1987

An antibody and anti-idiotypic antibody against the extra signal peptide of pre-aspartate aminotransferase.

A peptide (extra signal peptide) comprising amino acids 1-29 of pig liver pre-mitochondrial aspartate aminotransferase (p-mAAT) was synthesized chemically. The peptide was found to block the import of rat liver p-mAAT into rat liver mitochondria. An antibody raised against the peptide immunoprecipitated rat liver p-mAAT synthesized in a rabbit reticulocyte cell-free translation system. These results suggested that the extra signal peptide sequence of p-mAAT is essential for import of p-mAAT into the mitochondria and that there is structural homology between the extra signal peptides of pig and rat liver p-mAAT. An anti-idiotypic antibody against the peptide was also prepared and purified by affinity chromatography on an Affi-Gel 10 anti-peptide IgG column and was then characterized.

Animals↗