Biomedical subjects

R Sakakibara

Publications and source records attributed to R Sakakibara.

At least 91 records · Page 5Linked to original sources

PubMed1997

Inhibition of fructose-6-phosphate,2-kinase by N-bromoacetylethanolamine phosphate in vitro and in vivo.

Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-BPase), a bifunctional enzyme, catalyzes the synthesis and degradation of a potent activator, fructose-2,6-bisphosphate (Fru-2,6-P2), of phosphofructokinase, and has been postulated to be an important enzyme in the regulation of glycolysis in mammalian tissues. The purpose of this study was to determine whether or not N-bromoacetylethanolamine phosphate (BrAcNHEtOP), a specific active site-directed inactivator of Fru-6-P,2-kinase, is useful for studies on the role of Fru-6-P,2-kinase in the regulation of glycolysis in vivo. BrAcNHEtOP inactivated purified recombinant rat testis-type Fru-6-P,2-kinase as well as Fru-6-P,2-kinase in a rat liver extract, with half maximum inactivation concentrations of 2 and 15 mM, respectively, on 30 min incubation at 30 degrees C. The increases in Fru-6-P,2-kinase activity and the Fru-2,6-P2 concentration in livers, prepared from fasted rats, induced by high glucose (50 mM) perfusion were suppressed in parallel after pre-perfusion with 1 to 10 mM BrAcNHEtOP, dose-dependently. Five hours after intraperitoneal injection of BrAcNHEtOP (50 to 150 mg/kg) into mice, the Fru-6-P,2-kinase activity and Fru-2,6-P2 concentration in livers had decreased in parallel, dose-dependently. These effects continued for 24 h and were accompanied by decreases in the fructose-1,6-bisphosphate, triose phosphates, and lactate contents, although the contents of glucose-6-phosphate and fructose-6-phosphate did not change. These results suggested that BrAcNHEtOP inactivates Fru-6-P, 2-kinase, resulting in a decrease in the Fru-2,6-P2 level, which causes inactivation of phosphofructokinase and consequently inhibition of glycolysis in liver. Furthermore, the suppressed levels of Fru-6-P,2-kinase activity and metabolites in mice livers were sustained by daily injection of BrAcNHEtOP for 4 days, and body weight gain was also suppressed during the administration of BrAcNHEtOP. These results suggested that BrAcNHEtOP will be a useful reagent for studying the role of Fru-6-P,2-kinase in vivo.

Animals↗

PubMed1997

Characterization of a human placental fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase.

A full-length cDNA, which encodes a human placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a molecular weight of 58,000 by gel electrophoresis under denaturing conditions, compared to the deduced molecular weight of 59,410. The N-terminal sequence of 15 amino acids coincided with that of the deduced sequence. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with Vmax values of 142 and 0.2 milliunits/mg for the kinase and phosphatase activities, respectively. The phosphatase activity was extremely low, because one phosphatase active site residue was mutated, and consequently the kinase/phosphatase ratio was the highest among the known isozymes. Furthermore, the enzyme was phosphorylated by cAMP-dependent protein kinase, protein kinase C and also by [2-32P]fructose-2,6-bisphosphate. Phosphorylation by cAMP-dependent protein kinase and protein kinase C increased the maximal Fru-6-P,2-kinase activities by 1.8- and 1.1-fold, respectively. These results suggested that placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase is important in maintaining and regulating a relatively high rate of glycolysis in placenta.

Cyclic AMP-Dependent Protein Kinases↗

PubMed1997

Micturitional disturbance in patients with Guillain-Barré syndrome.

OBJECTIVES: To examine the frequency and pathophysiology of micturitional disturbance in patients with Guillain-Barré syndrome. METHODS: Micturitional symptoms were noted and neurological examinations made repeatedly during admission to hospital of patients with clinical and neurophysiologically definite Guillain-Barré syndrome. Urodynamic studies consisted of uroflowmetry, measurement of residual urine, urethral pressure profilometry, medium fill water cystometry, and external sphincter EMG. RESULTS: Seven of 28 (25%) patients with Guillain-Barré syndrome showed micturitional disturbance. The symptoms included voiding difficulty in six, urinary retention in three, nocturnal urinary frequency in three, and urge incontinence in two. These micturitional symptoms appeared after weakness occurred, and improved gradually along with the neurological signs. All three patients who showed retention became able to urinate. Urodynamic studies were made on four symptomatic patients two of whom underwent repeated study. Disturbed bladder sensation was noted in one patient, bladder areflexia in one, and absence of the bulbocavernosus reflex in one. Cystometry showed decreased bladder volume in two and bladder overactivity in two, one of whom had urge urinary incontinence and the other urinary retention. CONCLUSIONS: A quarter of the patients with Guillain-Barré syndrome tend to have micturitional disturbance. The patients studied had evacuation and storage disorders, as well as bladder areflexia and disturbed bladder sensation indicative of peripheral types of parasympathetic and somatic nerve dysfunction. Decreased bladder volume with bladder overactivity but no evidence of CNS involvement was also found, evidence that bladder overactivity also occurs in peripheral nerve lesions with probable pelvic nerve irritation.

Adolescent↗

PubMed1997

Human placental fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase: its isozymic form, expression and characterization.

The nucleotide sequence of 1981 bp cDNA containing the entire coding region of a human placental fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase was determined. The sequence encodes 469 amino acids and, based on homology to the rat testis enzyme, appears to be the testis-type isozyme expressed in placenta. The enzyme was expressed in Escherichia coli BL21 (DE3) by using a T7 RNA polymerase-based expression system and purified to homogeneity. The expressed enzyme was bifunctional with specific activities of 75 and 80 mU/mg of kinase and phosphatase, respectively. Kinetic parameters of the expressed enzyme are similar to those of the rat testis enzyme.

Amino Acid Sequence↗

PubMed1996

Micturitional disturbance and the pontine tegmental lesion: urodynamic and MRI analyses of vascular cases.

Micturitional histories were taken from 39 patients with acute brainstem stroke. Within 3 months from onset, 49% had irritative as well as obstructive urinary symptoms, the most common being voiding difficulty and nocturnal urinary frequency in 28%, followed by urinary retention in 21%. Urodynamic studies of 11 symptomatic patients revealed detrusor hyperreflexia in 73%, low compliance bladder in 9%, atonic cystometrogram in 27%, detrusor-sphincter dyssynergia in 45% and uninhibited sphincter relaxation in 27%. Three asymptomatic patients had normal urodynamic findings. Brain magnetic resonance images of the lesions of the symptomatic patients were concentrated in the dorsolateral pons including pontine reticular nucleus and the reticular formation adjacent to the medial parabrachial nucleus and the locus coeruleus. These regions seem to be mainly responsible for supranuclear types of pelvic and pudendal nerve dysfunction in our patients with brainstem stroke, corresponding to the pontine urinary storage and micturation center reported in animal studies.

Adult↗

PubMed1996

Micturitional disturbance in acute disseminated encephalomyelitis (ADEM).

In eleven patients with acute disseminated encephalomyelitis (ADEM), micturitional histories taken during the acute stage revealed that nine were in a state of urinary retention, and the other two had urinary frequency and urge incontinence together with difficulty in voiding. After the follow-up period of 3 to 38 months seven of nine patients with retention became able to urinate, but five of them had difficulty in voiding and four of them developed urinary frequency or urge incontinence. Two patients had urinary retention even after 26 and 38 months from the onset. Ten patients underwent urodynamic studies and disclosed detrusor hyperreflexia in six, low compliance bladder in two, atonic cystometrogram in one and detrusor-sphincter dyssynergia in two patients. Motor unit analysis of the external sphincter revealed polyphasic neurogenic changes is one of four patients. The results were compared with our previous findings in multiple sclerosis (MS), and we found that micturitional disturbance in ADEM seemed to be as common and as severe as in MS. Supranuclear as well as nuclear types of pelvic and pudendal nerve dysfunction seemed to be responsible for micturitional disturbance in our patients with ADEM. Micturitional disturbance seemed to be related to the pyramidal tract involvement, and probably reflecting the severity of spinal cord lesions.

Adult↗

PubMed1996

Treatment of primary erythromelalgia with cyproheptadine.

Erythromelalgia is a rare disorder characterized by burning pain of the extremities associated with red discoloration and increased temperature of the skin. We observed the effects of several drugs on two patients with primary erythromelalgia. Anti-inflammatory drugs such as aspirin had no effects for their symptoms. The alpha-adrenergic agonist midodrine slightly reduced skin temperature and redness of the extremities, and minimally reduced the pain in one patient. The partial alpha-adrenergic agonist and 5-hydroxytryptamine (5-HT) agonist/antagonist dihydroergotamine transiently reduced skin temperature and pain to some degree in one patient. The 5-HT and histamine antagonist cyproheptadine effectively relieved the burning pain and increased skin temperature.

Adult↗

PubMed1996

Micturitional disturbance after acute hemispheric stroke: analysis of the lesion site by CT and MRI.

Micturitional histories and urodynamic studies were performed in 72 acute hemispheric stroke patients. Within 3 months from the onset, 53% of the patients had urinary symptoms including irritative as well as obstructive, and the most common symptom was nocturnal urinary frequency in 36%, which was followed by urge urinary incontinence in 29% and difficulty of voiding in 25% of the patients. We found a correlation between micturitional disturbance with hemiparesis (p <0.05) and not with hemianopsia (p <0.05). Micturitional disturbance was more common in lesions of the frontal lobe (p <0.05) than in those of the occipital lobe. Brain CT or MRI in symptomatic patients showed lesions of anterior and medial surface of the frontal lobe, anterior edge of the paraventricular white matter, genu of the internal capsule and large lesions of putamen or thalamus. Urodynamic studies of 22 symptomatic patients revealed various findings in 91% of them, including detrusor hyperreflexia in 68%, detrusor-sphincter dyssynergia (DSD) in 14% and uninhibited sphincter relaxation in 36%. Patients with urinary retention had atonic cystometrogram and DSD. Detrusor hyperreflexia was noted in lesions of the frontal lobe as well as the basal ganglia, uninhibited sphincter relaxation in the frontal lobe, and detrusor-sphincter dyssynergia common in the basal ganglia. Above findings seem to indicate that anteromedial frontal lobe and its descending pathway, and the basal ganglia seem to be mainly responsible for supranuclear types of pelvic and pudendal nerve dysfunction in our patients with stroke.

Adult↗

PubMed1996

Micturitional disturbance in syringomyelia.

Micturitional histories and urodynamic studies were performed in 14 patients with syringomyelia. Eleven patients were revealed to have urinary symptoms including difficulty of voiding in 8, urinary retention in 3, nocturnal and diurnal urinary frequency in 3, urinary incontinence in 2, and sense of urgency and enuresis in one. These urinary symptoms appeared after 5.3 years (ranging from 2 months to 13 years) from the occurrence of the neurological symptoms. Urodynamic studies revealed detrusor hyperreflexia in 7, detrusor areflexia in 4, detrusor-sphincter dyssynergia in 4 and uninhibited sphincter relaxation in 2 patients. Analysis of the motor unit potentials of the external sphincter revealed 5 of 6 patients had high amplitude or polyphasic neurogenic changes. Supranuclear as well as nuclear types of parasympathetic and somatic nerve dysfunctions seemed to be responsible for micturitional disturbance in our patients with syringomyelia. During the follow-up period of 2 to 63 months, urinary symptoms gradually improved in 4 of 6 patients after syringosubarachnoid shunts and in 3 of 4 after alpha-adrenoreceptor blocking agents.

Adult↗

PubMed1996

Micturition disturbance in acute transverse myelitis.

In ten patients with acute transverse myelitis (ATM), seven patients had urinary retention, and the other three patients had difficulty in voiding within 1 month from the onset of the disease. Five of the patients with retention became able to urinate. After the mean follow-up period of 40 months, nine still had urinary symptoms including difficulty in voiding in five and urinary frequency, urgency and incontinence in four patients. Four patients had urinary disturbance as the sole sequel of ATM. Urodynamic studies performed on nine patients revealed that all of the three patients with the urgent incontinence had detrusor hyperreflexia, all of the four patients with retention had an areflexic cystometrogram as well as sphincter hyperreflexia, and three of five patients with voiding difficulty had detrusor-sphincter dyssynergia. An areflexic cystometrogram tended to change to a low compliance bladder, followed by detrusor hyperreflexia or a normal cystometrogram. Analysis of the motor unit potentials of the external sphincter revealed that two of the three patients had high amplitude or polyphasic neurogenic changes. Supranuclear as well as nuclear types of parasympathetic and somatic nerve dysfunctions seemed to be responsible for micturition disturbance in our patients with ATM.

Acute Disease↗

PubMed1996

Late-onset hereditary ataxia with global thermoanalgesia and absence of fungiform papillae on the tongue in a Japanese family.

Two Japanese male siblings, aged 68 and 59 years, affected by late-onset progressive ataxia distinguished by extensive sensory and mild autonomic disturbances are described. They had global thermoanalgesia, positive Romberg signs, sensorineural deafness, canal paresis and ageusia. Their autonomic disturbances consisted of absence of overflow tears with usual stimuli, dysphagia, blood pressure and vasomotor instability, diarrhoea/constipation, and urinary frequency. Sensory nerve action potentials were completely absent, whereas motor conduction velocity was slightly reduced only in the lower extremities. Sural nerve biopsy on the younger brother demonstrated a marked loss of myelinated fibres and a reduction in the number of unmyelinated axons. Tongue histology revealed absence of fungiform papillae and taste buds. Autonomic function tests showed widespread but mild sympathetic and parasympathetic failures. Neuro-imaging studies revealed atrophy of the spinal cord, cerebellum, brainstem and corpus callosum, and enlargement of the lateral, third and fourth ventricles. These siblings represent a previously unrecognized variant of late-onset hereditary spinocerebellar degeneration with global thermoanalgesia and absence of fungiform papillae on the tongue.

Biopsy↗

PubMed1996

Cloning of cDNA encoding for a novel isozyme of fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase from human placenta.

Two independent cDNA clones encoding fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase were isolated from a human placental cDNA library. The deduced amino acid sequences showed that one of the clones, 2K-1, was almost identical to the rat testis isozyme and the other, 2K-3, was different from any known isozymes expressed in mammalian tissues. The results of Southern blot analysis suggested that clones 2K-1 and 2K-3 were encoded as single copy genes and located in different parts of the genome. Since open reading frames of the cDNA clones were not complete, we obtained the 5'-end of the clone 2K-3 cDNA using the 5'-rapid amplification of cDNA end method. The entire cDNA (HP; 1,756 bp) had a coding capacity of 519 amino acids (M(r) = 59,410), and putative phosphorylation sites for protein kinases A and C on the C terminus. Northern blot analysis using a fragment of the HP as a probe showed that a major band of 5.4 kb, significantly different in size from known isozyme mRNAs such as liver (2.1 kb), muscle (1.9 kb), heart (4.0 kb), and testis (2.0 kb), was present in poly(A)+RNA preparations of human first trimester and term placentae. These results strongly suggested that this 5.4 kb mRNA codes a novel isozyme of fructose 6-phosphate,2-kinase/fructose 2,6-bisphosphatase.

Amino Acid Sequence↗

PubMed1995

The location of the paths subserving micturition: studies in patients with cervical myelopathy.

Urodynamic studies and neurological examinations were performed on 128 patients with cervical myelopathies including 82 with spondylitic myelopathy and 46 with ossification of the posterior longitudinal ligament (OPLL), investigating the location of the paths subserving micturition in the spinal cord. Accurate history taking revealed micturitional symptoms in 95 patients, including irritative symptoms in 61 and obstructive symptoms in 71. Urinary incontinence was noted in 25 and residual urine of over 100 ml or retention was found in 22. Neurological examination revealed disturbed deep sensation in 55, disturbed superficial sensation in 63 and pyramidal signs (weakness, hyperreflexia of legs and Babinski sign) in 96 patients. Urodynamic studies revealed uninhibited contraction in 61 and detrusor-sphincter dyssynergia in 22 patients. Bladder capacity was smaller in patients with Babinski sign (P < 0.05) and in patients with uninhibited contraction (P < 0.001). Uninhibited contraction was more frequent in patients with all three of the above-mentioned pyramidal signs (P < 0.05). Detrusor-sphincter dyssynergia was more frequent in patients with disturbed deep sensation (P < 0.05) and pyramidal signs (P < 0.05, P < 0.0005). From above results and our previous findings in patients with anterior spinal artery syndrome and with tabes dorsalis, it is concluded that the pathway subserving detrusor function seems to be located mainly in the lateral column of the spinal cord. The descending pathway subserving coordination of bladder and urethral sphincter seems to be located mainly in the lateral column, and its ascending pathway seems to be located in the dorsal column. The path subserving urinary sensation seems to be located mainly in the dorsal column of the spinal cord.

Adult↗

PubMed1995

Oligomannose-coated liposomes as an adjuvant for the induction of cell-mediated immunity.

The effect of the coating of ovalbumin-reconstituted liposomes with various oligosaccharides on their immunogenicity was investigated in mice. The coating of liposomes with oligomannose or yeast mannan drastically enhanced their ability to induce an ovalbumin-specific delayed-type footpad swelling response with a peak at 24 to 48 h post-challenge. Among various oligosaccharides tested, only those with mannose residue at the nonreducing termini manifested the activity when applied to liposomes. Since such oligosaccharides are ubiquitously found in the body, these results suggested the usefulness of oligomannose-coated liposomes as a safe adjuvant for the induction of cell-mediated immunity.

Adjuvants, Immunologic↗

PubMed1995

Micturitional disturbance in myotonic dystrophy.

Micturitional disturbance has attracted little attention in myotonic dystrophy, but detailed micturitional histories revealed that two out of six patients (33%) had micturitional symptoms. One had difficulty urinating and the other had urinary frequency, urgency and stress incontinence. Urodynamic studies were performed in all patients and the results were as follows: Two had low maximum urethral closure pressure, two had large and three had small bladder capacities, one had detrusor hyperreflexia and one had atonic cystometrogram. Urethral sphincter electromyography revealed a decreased bulbocavernosus reflex in one, and an absent anal reflex in two. Motor unit analysis of external sphincter was performed with one patient and showed polyphasic potentials. Dystrophic changes of the lower urinary tract muscles, as well as supranuclear type of pelvic nerve dysfunction, could cause micturitional disturbance in patients with myotonic dystrophy.

Adult↗

PubMed1995

Inhibition of polar body formation in mouse denuded oocytes cultured in vitro by protein synthesis inhibitors.

Denuded oocytes obtained from the ovaries of ddY mice underwent spontaneous germinal vesicle breakdown (GVBD) and polar body formation (PBF) by cultivation for 3 and 16 h, successively. To study the significance of protein synthesis in oocytes regarding their maturation, denuded oocytes were cultured in the presence of reversible (cycloheximide and puromycin) and irreversible (ricin) protein synthesis inhibitors. When denuded oocytes were cultivated in the continual presence of any inhibitor, only PBF but not GVBD was found to be inhibited. These inhibitions were dose dependent, ranging from 10(-9) to 10(-6) M, and were relative to the protein synthesis inhibitions by them. The PBF inhibition was relieved by withdrawing cycloheximide for first 2, 3 and 4 h in the course of the culture. Further, the PBF inhibition occurred in the presence of ricin for only the first 2 or 3 h in the course of the culture. These results suggested that proteins synthesized during 2 to 4 h in the course of culture may play important role in PBF.

Animals↗

PubMed1995

Purification and characterization of high molecular weight hCG from human first trimester placenta.

Human chorionic gonadotropin (hCG) in first trimester placental cells is composed of immature alpha- and beta-subunits containing only N-linked high-mannose sugar chains. Intracellular immature intermediates are accumulated in rough endoplasmic reticulum in much greater quantity than mature hCG composed of mature subunits. We have previously shown that this immature hCG might be bound to other protein(s), including an ATP-binding protein, forming high molecular weight-hCG (HMW-hCG), which is not aggregate of immature hCG alone. To identify the ATP-binding protein forming the HMW-hCG in detail, proteins in HMW-hCG preparation were photoaffinity-labeled with 8-azido-[alpha-32P]ATP. Autoradiography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the labeled protein with M(r) = 78000 was immunoprecipitated with any antibody against alpha-subunit, beta-subunit and hCG, indicating that this protein is bound to immature hCG. Furthermore, to determine whether some other proteins associate to form HMW-hCG, we purified HMW-hCG without breakdown to its components using columns of DE52, Heparin-Sepharose and Sephacryl S-300. As the final step of the purification, HMW-hCG was allowed to adsorb on a column of ATP-agarose and anti-hCG IgG-agarose, respectively. SDS-PAGE analysis of eluted proteins from the columns bound to the respective column via the constituent of HMW-hCG, such as ATP-binding protein or immature hCG, showed four common protein bands with molecular weights of 78000, 43000, 28000 and 20000. The protein with M(r) = 43000 was stained with any antibody against alpha-subunit, beta-subunit and hCG, indicating it to be immature hCG. The protein band with M(r) = 78000, which might correspond to the ATP-binding protein described above, was stained with anti-heat shock protein 70 (HSP70) monoclonal antibody. To confirm the association of immature hCG and HSP70-like protein, immature hCG preparation was incubated with HSP70-like protein purified from placental extracts. The molecular weight change of immature hCG appeared to increase by this incubation and was close to HMW-hCG, but not exactly the same. These results suggest that immature hCG intermediate exists as HMW-hCG containing HSP70-like protein, which has ATP-binding capacity, and two other proteins in first trimester placental cells.

Chorionic Gonadotropin↗

PubMed1995

The binding of ricin to its receptor is not required for the expression of its toxicity.

Ricin toxin is a toxic glycoprotein comprising two polypeptide chains, A and B, joined by a disulfide bond. The binding of its B-chain to the cell surface glycoconjugate having non-reducing terminal galactose (ricin receptors) has been assumed to allow the internalization of ricin into the cell, followed by the release of the free A-chain into cytosol, which then inhibits cellular protein synthesis in eukaryotic cells (cytotoxic effect). In order to investigate whether the binding of ricin to its receptors is essential to the expression of its toxicity, ricin was allowed to be incorporated into the cells using liposome encapsulated ricin (ricin-encapsulated liposomes). Protein synthesis in cultured Hela cells was inhibited by incubation not only with intact ricin but also with ricin-encapsulated liposomes, indicating that the binding of ricin to its receptor is not required for the expression of its toxicity.

Dose-Response Relationship, Drug↗