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Biomedical subjects

H Higashi

Publications and source records attributed to H Higashi.

At least 19 recordsLinked to original sources
PubMed1996

The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.

Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G1/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS780PIPHIPR that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser780 in pRB was phosphorylated in the G1 phase in a cell cycle-dependent manner. Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin A/E Cdk2 phosphorylate different sites of pRB in vivo.

Amino Acid Sequence↗
PubMed1996

Imaging of Ca2+/calmodulin-dependent protein kinase II activity in hippocampal neurones.

Our aim was to visualize the dynamic features of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. In order to do so, we synthesized a new reagent by conjugating a fluoroprobe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), to syntide 2, a specific peptide substrate for CaMKII. In cell-free conditions, the conjugate was found to be an effective indicator of calmodulin activation by Ca2+ and the subsequent activation of CaMKII. The reagent is cell-permeable and can stain living cells when bath-applied. Using this technique we were able to obtain fluorescence images of stained cells and analyse the dynamic features of CaMKII inside the cells by means of image processing. Regional heterogeneity of CaMKII activation in cultured hippocampal neurones was seen following L-glutamate administration.

Animals↗
PubMed1996

Calcium-dependent potentiation of M-current in bullfrog sympathetic neurons.

Whole-cell voltage-clamp recordings were made from cultured bullfrog sympathetic neurons to measure the steady-state activation curve of M-type potassium current. When measured with a calcium-deficient (10 nM) pipette solution M-conductance was 4.8 nS at -35 mV having the 50%-activation voltage at-20 mV. Respective values were 17.2 nS at -35 mV with the 50%-activation voltage at -42 mV when measured with a calcium-rich (1 microM) solution, indicating the hyperpolarizing displacement of the activation curve with high internal calcium. It is suggested that intracellular calcium ions can modulate kinetics of M-current which thereby regulate the number of M-channels being open at given membrane potentials.

Animals↗
PubMed1996

Cyclin-dependent kinase-2 (Cdk2) forms an inactive complex with cyclin D1 since Cdk2 associated with cyclin D1 is not phosphorylated by Cdk7-cyclin-H.

Cyclin-dependent kinases (Cdks) form complexes with cyclins, and as a consequence they generally express kinase activities. One of these Cdks, Cdk2, is known to bind with cyclins A and E, and plays an important role in the progression of the cell cycle via phosphorylation of target proteins such as the product of the retinoblastoma tumor-suppressor gene (pRB). It has been suggested that Cdk2 bound with cyclin D1 and Cdk2-cyclin-D1 complex show neither H1 histone nor pRB kinase activity. However, it is not clear whether Cdk2-cyclin-D1 has unknown targets and why Cdk2 is not activated by binding with cyclin D1. We investigated these questions using Cdk, cyclin and Cdk-cyclin complexes produced in a baculovirus expression system. Cdk2 formed a complex with cyclin D1 in this system. After extensive purification, Cdk2 was still bound to cyclin D1. The Cdk2-cyclin-D1 complex did not phosphorylate any tested substrates, such as H1 histone, pRB, SV40 large T antigen, p53, E2F-1 or a preparation of nuclear proteins from HeLa cells; in contrast, Cdk2-cyclin-E and Cdk2-cyclin-A phosphorylated these proteins. Moreover, the Cdk2-cyclin-D1 complex was not activated by incubation with Cdk4 or cyclin E. Thus, Cdk2 and cyclin D1 formed a stable complex that was not activated. In order to determine why Cdk2-cyclin-D1 lacks kinase activity, we investigated the phosphorylation of Cdk2. Under-shifted Cdk2, the active form of Cdk2, was not detected in the Cdk2-cyclin-D1 complex in the baculovirus system. In human WI-38 cells, cyclin D1 began to form a complex with Cdk2 as well as with Cdk4 from the mid-G1 phase of the cell cycle. The Cdk2 bound to cyclin D1 in human cells was also the inactive form that was slowly migrated. Moreover, we found that Cdk2 bound to cyclin D1 was not phosphorylated by Cdk7-cyclin-H, while Cdk2 bound to cyclin E, as well as free Cdk2, was was phosphorylated by Cdk7-cyclin-H. Additionally, Cdk2 phosphorylated by Cdk7-cyclin-H did not bind to cyclin D1. These results strongly suggest that Cdk2 forms a stable complex with cyclin D1 but is not activated because the Cdk2 molecule in the complex is not phosphorylated by Cdk7-cyclin-H and the phosphorylated Cdk2, an active form, does not bind to cyclin D1.

Animals↗
PubMed1996

Development of postoperative delirium in relation to a room change in the general surgical unit.

To analyze the relationship between the development of postoperative delirium and a change of the patient's room, 1,006 cases of patients who had undergone surgery with general anesthesia were reviewed. Postoperative delirium developed in 84 (8.3%) cases. On the basis of symptomatic features, postoperative delirium was divided into four types: (1) excitement type, (2) excitement-hallucination type, (3) hallucination type, and (4) disorientation type. Of the 31 excitement-type cases, 21 developed within the 2nd postoperative day (POD) while 27 of 29 hallucination types developed after POD 2. Of 29 hallucination types, 22 developed after a room change while 20 of these 22 cases were transferred to a single room before POD 2. A quiet, dark, and isolated environment in a single room is suggest to contribute to the development of hallucinations. The development of postoperative delirium with hallucinations alone should thus be taken into consideration whenever a room change is decided.

Adult↗
PubMed1996

Extreme hyperbilirubinemia induced by endoscopic injection sclerotherapy in a patient with esophageal varices and thalassemia: report of a case.

We describe herein the case of a 57-year-old man with thalassemia who developed acute liver failure after undergoing endoscopic injection sclerotherapy (EIS) to control hemorrhage from a ruptured esophageal varix. The patient, who had been confirmed as having liver cirrhosis due to chronic hepatitis C with thalassemia in 1989, was admitted to our department to undergo EIS for esophageal varices, at which time his serum total bilirubin level was 5.5 mg/dl. As a small amount of hematemesis occurred just after a percutaneous transhepatic portography was performed, emergency EIS was carried out, following which the serum total bilirubin level markedly increased, mainly with a direct fraction, until it reached 70 mg/dl. The patient eventually died from acute liver failure with extreme hyperbilirubinemia on the 27th day after experiencing hematemesis despite all treatment. This unfortunate case demonstrates that sclerotherapy could be an inappropriate method of treatment for patients with hemolytic disease.

Biopsy↗
PubMed1996

The Epstein-Barr virus (EBV) alters the B cell glycolipid which is recognized by the human monoclonal antibody to i-blood group antigen.

To analyze the differentiation-related glycolipids, we have recently developed human monoclonal anti-i antibodies (mAbs) using a combination of EBV-transformation and bovine i-active glycolipid (NeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->1Cer)-containing liposome immune lysis assay (LILA). Using complement cytolysis with these mAbs, we found the occurrence of a surface antigen (Ag) in the EBV-negative but not in the EBV-positive cell lines. The fresh EBV infection reveals that the suppressed expression of the antigen was a result of the EBV infection. The Ag recognized with these mAbs appeared to have a very low density on the B cell lines. Unexpectedly, thin layer chromatogram (TLC)-immunostaining using the mAbs revealed that the major immunoreactive substance in the EBV-negative B cell lines was an extremely minor glycolipid that was distinct from the i-active glycolipid however, this was not the case in the EBV-positive B cell lines.

Antibodies, Monoclonal↗
PubMed1996

Interaction of ganglioside with specific peptide sequences as a mechanism for the modulation of calmodulin-dependent enzymes.

We examined the interaction between gangliosides and synthetic peptides of calmodulin (CaM)-dependent enzymes to confirm the hypothesis that interaction between gangliosides and the CaM-like site (CLS) of the enzyme is a mechanism for the modulation of the enzyme activity by gangliosides. Gangliosides, GD1b, GT1b, and GD1a, inhibited the activity of CaM-independently activated cAMP-phosphodiesterase and their inhibition was cancelled by a peptide consisting of 17 amino acid residues of a plasma membrane Ca(2+)-pump CLS, suggesting the involvement of the interaction between the peptide and the gangliosides. The peptide of an assumed CLS of phosphodiesterase also cancelled the inhibition. On the other hand, the gangliosides interacted with synthetic CaM-binding site (CBS) peptides of phosphodiesterase, calcineurin, Ca(2+)-pump, and Ca2+/calmodulin-dependent protein kinase II. Moreover, gangliosides GM3 and LM1, that activate but do not inhibit phosphodiesterase, interacted with the CBS peptides, whereas they did not bind to CLS peptides. On the basis of these new findings, we propose a revised model for the ganglioside-mediated modulation of CaM-dependent enzymes, i.e. without CaM, gangliosides bind to CBS and thus stimulate the enzyme activity, acting like CaM. At higher concentrations, they bind to CLS of the enzymes as well and inhibit the activity, acting like the CBS of the enzyme.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Coronary vasomotor response to acetylcholine late after angioplasty.

To elucidate the changes in coronary vasomotion in a previously balloon-dilated segment, we examined the vasoconstricting response of previously balloon-dilated and non-dilated segments to acetylcholine. Acetylcholine was administered into coronary arteries cumulatively (left and right coronary artery: 10-100 micrograms) in 15 patients (age: 60 +/- 3 years, 12 males and 3 females) at 7.4 +/- 1.5 months after successful percutaneous transluminal coronary angioplasty (PTCA). In PTCA segments with no restenosis, does-dependent constriction in response to acetylcholine was observed in only 1 patient. In non-PTCA segments of PTCA and non-PTCA arteries, 12 patients showed dose-dependent vasoconstriction in response to acetylcholine. Coronary spasm, which was defined as a more than 75% reduction in coronary diameter compared with that after isosorbide dinitrate, was provoked in one PTCA segment (7%). In non-PTCA segments of PTCA and non-PTCA arteries, 15 of 44 arteries (34%) demonstrated coronary spasm in 9 of 15 patients (60%, p < 0.005 vs PTCA segment). In conclusion, PTCA segments free of restenosis showed no hyper-reactivity to acetylcholine, while non-PTCA segments showed hypercontractility in response to acetylcholine. Coronary balloon angioplasty may alter the coronary vasomotor reaction to acetylcholine several months after angioplasty.

Acetylcholine↗
PubMed1996

Laparoscopic splenectomy for idiopathic thrombocytopenic purpura: comparison of laparoscopic surgery and conventional open surgery.

In chronic idiopathic thrombocytopenic purpura (ITP), the two main therapeutic choices are steroid treatment or splenectomy. The adult form of ITP is described as a disease found primarily in young adults, with a female predominance. Treatment with steroids effects a complete response in less than 30% of patients, whereas splenectomy is successful in more than 60% of patients who undergo it. The minimal access afforded by laparoscopic splenectomy is considered highly desirable for these patients. The purpose of this study was to compare the clinical benefits of a laparoscopic splenectomy with those of conventional open surgery for patients with ITP. From 1968 to 1993, splenectomy was performed on 51 patients: 10 operations done laparoscopically and 41 performed conventionally. Complications, postoperative pain, recovery, and hospital charges were then compared. Laparoscopic splenectomy involved minimal incisions, and a significantly lower frequency of analgesia was required for postoperative abdominal pain (1.3 vs. 3.3); hospital stay was shorter (8.2 vs. 20.1 days) (p < 0.005). Operative time was significantly longer for the laparoscopic surgery (249.2 vs. 99.8 min) (p < 0.0001), but blood loss was less (176.0 vs. 511.7 g) (p < 0.01). No intraoperative or postoperative major complications occurred with the laparoscopic procedures, compared with 46.3% with conventional surgery. Finally, the total hospital costs were lower with laparoscopic splenectomy, especially for postoperative care (p < 0.05). A laparoscopic splenectomy may well be considered the surgical treatment of choice for patients requiring a splenectomy in view of both quality of life and economy.

Adult↗
PubMed1996

[Effects of Gosha-Jinki-Gan on the function of the urinary bladder in anesthetized dogs].

Gosha-Jinki-Gan is clinically used for the treatment of pollakiuria in patients with benign prostatic hyperplasia. The effect of Gosha-Jinki-Gan on the function of the urinary bladder in anesthetized dogs was examined. Gosha-Jinki-Gan (100 mg/kg) inhibited the rhythmic bladder contractions (RBC). However, the frequency of RBC increased resembling the effect of atropine (0.1 mg/body), and the amplitude decreased. On the cystometrogram, Gosha-Jinki-Gan caused an increase in maximum vesical volume (not significant). These findings indicated that Gosha-Jinki-Gan is a useful drug for the treatment of pollakiuria.

Anesthesia↗
PubMed1995

Differences in substrate specificity between Cdk2-cyclin A and Cdk2-cyclin E in vitro.

Cyclin-dependent kinase 2 (Cdk2), when bound to either cyclin A or cyclin E, recognizes the Ser/Thr-Pro-X-basic amino acid (motif A) as a phosphorylation site. In this study, we designed several peptides based on motif A and examined the substrate specificity of Cdk2-cyclin A and Cdk2-cyclin E using these peptides. Peptides containing a proline residue in the sequence Pro-X-Thr-Pro-X-basic amino acid (motif B) had higher affinity for both Cdk2 complexes than peptides containing motif A. Furthermore, differences in substrate affinity between the two Cdk2 complexes were caused by a proline residue adjacent to or three positions before the threonine residue. Similarly, the presence of different basic amino acids in motif B also had different effects on affinity for each complex. We demonstrate the possibility that the substrate specificity of Cdk2 bound to cyclin might be regulated by the species of cyclin.

Amino Acid Sequence↗
PubMed1995

Role of A delta afferent fibers in modulation of primary afferent input to the adult rat spinal cord.

To address the question of whether fine myelinated and unmyelinated primary afferent fibers contribute to the mechanism of presynaptic inhibition in the spinal cord, we studied dorsal root-evoked dorsal root potentials (DR-DRPs) using a newly developed longitudinal spinal cord slice preparations in the adult rat. Single stimuli applied to the L6 dorsal root elicited a DR-DRP in the L5 dorsal root which had an amplitude of 50-150 microV and had a half decay time of 20-66 ms. The DR-DRP was depressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-20 microM), while DL-2-amino-5-phosphonovaleric acid (APV, 50-100 microM) had no significant effect. DR-DRP was markedly depressed by bicuculline or picrotoxin. The evoked DR-DRP was unchanged in rats treated with capsaicin which eliminated the majority of unmyelinated C afferent fibers. Taken together with the higher voltages (> or = 1.9 V) required to elicit DR-DRP, this observation strongly suggests that the A delta afferent fibers are primarily responsible for producing and receiving the DR-DRP. The present study shows that the DR-DRP mediated by the A delta fibers in the slice preparation is analogous to those described for larger myelinated fibers in vivo. This pathway may contribute importantly to synaptic modulation of somatosensory information, including nociception at the superficial dorsal horn through an interneuronal connection which are mediated by the non-NMDA and GABAA receptors.

Afferent Pathways↗
PubMed1995

The interactions of E2F with pRB and with p107 are regulated via the phosphorylation of pRB and p107 by a cyclin-dependent kinase.

It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evident for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk. Therefore, we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays. We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as well as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.

Base Sequence↗
PubMed1995

A variant form of cyclin-dependent kinase 2 (Cdk2) in a malignantly transformed rat thyroid (FRTL-Tc) cell line.

Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.

Amino Acid Sequence↗
PubMed1995

Phosphorylation of E2F-1 by cyclin A-cdk2.

Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.

Baculoviridae↗
PubMed1995

Pineal region tumours treated with interstitial brachytherapy with low activity sources (192-iridium).

Three patients with tumours of the pineal region underwent interstitial Ir-192 brachytherapy. Histological diagnoses were obtained in all patients, by stereotactic biopsy and included one germinoma, one mixed pineoblastoma/pineocytoma, and one astrocytoma grade III. Our approach to pineal region neoplasms is first to decide whether stereotactic biopsy or surgery should be performed. When a pineal lesion is thought to be benign on the basis of imaging, such as benign teratoma, surgery is performed to resect the entire lesion. When a definitive diagnosis is not possible, stereotactic biopsy is performed to obtain a histological diagnosis for treatment planning, using a Brown-Roberts-Wells (BRW) stereotactic apparatus with computed tomography (CT) or magnetic resonance imaging (MRI). When a lesion is malignant and localized, stereotactic implantation of catheters for interstitial brachytherapy is performed simultaneously. Radioactive Ir-192 seeds are inserted into the catheters and maintained for 5-10 days to give 36 Gy of irradiation at the tumour periphery. Sequential CT scans and MRI after treatment revealed tumour disappearance in two patients with germinoma and high grade astrocytoma and tumour reduction in the patient with mixed pineoblastoma/pineocytoma. No significant morbidity or mortality occurred in any of these patients after stereotactic biopsy and brachytherapy. The technique and the advantages of this therapeutic approach to selected pineal region tumours are described and discussed.

Adult↗