Search PubMed⌕ Search

Biomedical subjects

T Kitani

Publications and source records attributed to T Kitani.

At least 55 records · Page 3Linked to original sources
PubMed1995

Full activation of brain calmodulin-dependent protein kinase IV requires phosphorylation of the amino-terminal serine-rich region by calmodulin-dependent protein kinase IV kinase.

Rat brain calmodulin-dependent protein kinase IV (CaM-kinase IV) was activated approximately 20 to 30-fold by incubation with CaM-kinase IV kinase purified from rat brain under the Ca2+/calmodulin-dependent phosphorylation conditions. When CaM-kinase IV was incubated without CaM-kinase IV kinase, no significant activation was observed, indicating that the marked activation of CaM-kinase IV occurred as a result of the action of CaM-kinase IV kinase. More than 3 mol of phosphate were incorporated into 1 mol of the enzyme after incubation with CaM-kinase IV kinase at 30 degrees C for 20 min, but the activation occurred upon the initial incorporation of 1 mol of phosphate. The rate of the initial phosphorylation increased when the amount of CaM-kinase IV kinase added into the reaction mixture increased, but the rate of phosphorylation following the initial phosphorylation did not increase, suggesting that the initial phosphorylation was catalyzed by CaM-kinase IV kinase, and that subsequent phosphorylation was catalyzed by CaM-kinase IV itself activated by CaM-kinase IV kinase. The initial phosphorylation occurred in the amino-terminal serine-rich region of CaM-kinase IV. Kinetic analysis revealed that the increase in the activity upon phosphorylation was due mainly to an increase in the Vmax values.

Amino Acid Sequence↗
PubMed1995

Inactivation of Ca2+/calmodulin-dependent protein kinase IV by Ca2+/calmodulin and restoration of the activity by Mg2+/EGTA.

The activity of calmodulin-dependent protein kinase IV (CaM-kinase IV) was progressively decreased by incubation at 30 degrees C with calmodulin in the presence of Ca2+, becoming one-half to one-fifth of the original activity within several minutes. The amount of calmodulin necessary to produce the maximal inactivation was approximately 1 mol for 1 mol of the enzyme. The inactivation of CaM-kinase IV by Ca2+/calmodulin was prevented by ATP in the presence of Mg2+, but such protection was not observed with either of the two alone or with a peptide substrate such as syntide-2. The activity of the calmodulin-inactivated enzyme was increased by incubation at 30 degrees C with Mg2+ in the presence of EGTA, being completely restored to the original level within several minutes, indicating that the Ca2+/calmodulin-induced inactivation of the enzyme was not due to irreversible denaturation of the enzyme. Both the inactivation of CaM-kinase IV by Ca2+/calmodulin and the restoration of its activity by Mg2+/EGTA were time- and temperature-dependent reactions. Kinetic analysis revealed that the alterations of the enzyme activity were due mainly to changes in Vmax of the enzyme.

Animals↗
PubMed1995

Comparison of Ca2+/calmodulin-dependent protein kinase IV from rat brain, expressed in insect cells, and expressed in Escherichia coli.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) is thought to play crucial roles in the functioning of Ca2+ in the central nervous system and immune system, and the regulation of its activity is therefore very important. Recombinant CaM-kinase IV is invaluable for studies of its regulatory mechanism, because of its large-amount availability and ready site-specific mutagenesis. In the present study, rat CaM-kinase IV was expressed in Sf9 cells and Escherichia coli, and the kinetic properties were examined with syntide-2 and peptide-gamma as substrates. The recombinant enzymes were produced highly efficiently, comprising as much as about 15% of the total protein in Sf9 cells and 9% in E. coli. The brain enzyme shows two Km values for syntide-2 in the presence of Ca2+/calmodulin, but the recombinant enzymes showed normal kinetic behavior. The brain enzyme and Sf9 enzyme showed Km values for peptide-gamma of 53 and 82 microM, respectively, but the Km of the E. coli enzyme was as high as 1.7 mM, in the presence of Ca2+/calmodulin. Thus, the three enzymes differed in their kinetic properties, but all the three were markedly activated upon incubation with CaM-kinase IV kinase under the Ca2+/calmodulin-dependent protein phosphorylation conditions.

Amino Acid Sequence↗
PubMed1995

Decay-accelerating-factor-deficient erythrocytes during the long-term clinical course of patients with paroxysmal nocturnal hemoglobinuria.

We performed long-term follow-up studies of 5 patients with paroxysmal nocturnal hemoglobinuria (PNH). The percentages of decay-accelerating-factor (DAF)-deficient erythrocytes were almost stable or slowly increased during the 2-4 years' observation periods when the effects of treatment with drugs or blood transfusion and pregnancy were excluded. However, a tendency of increase in the percentages of DAF-deficient erythrocytes was observed along with the duration of the disease. Our findings suggest that the growth advantage of PNH clones beyond the normal hematopoiesis may be different from the other clonal stem cell disorders.

Adult↗
PubMed1995

[The expression pattern of adhesion molecules and their role of the tumor cells in patients with multiple myeloma].

The expression pattern of beta 1 integrin on the surface of tumor cells in patients with multiple myeloma (MM) is reviewed and compared with that of other B cell malignancies. The expression pattern of beta 1 integrin of plasma cells of healthy individuals was also compared with that of malignancies of plasma cells. Normal immature CD10+ B cell precursors in bone marrow are alpha 4+ and alpha 5+, while mature peripheral B cells are alpha 4+ and alpha 5+. In contrast to mature peripheral B cells, it is reported that plasma cells are alpha 4+ and alpha 5+. There are three points following in the phenotype characteristic of MM cells; (i) MM cells are alpha 4 strong positive, (ii) MM cells are beta 1 strong positive and (iii) MM cells are alpha 6strong positive. Because alpha 6+ cell lines of malignant plasma cells showed spread and chemotaxis on stimulation with laminin, alpha 6 beta 1 integrin might contribute of MM cells to transit laminin rich basement membrane of blood vessel to exit to the extravascular space. The difference of the phenotype between plasma cells and MM cells is the expression of alpha 5; plasma cells express alpha 5, but MM cells are lost in one half of the cases. The functional role of VLA-5 in MM cells is reviewed.

Amino Acid Sequence↗
PubMed1995

[Establishment and usage of human multiple myeloma cell line].

It was known that malignancies of plasma cell (MPC) consists of three categories, one is multiple myeloma (MM), second is plasmacytoma, third is plasma cell leukemia. One of the aim of our review is that we listed MPC cell lines reported. It became clear from the list that the success of establishment of cell lines may expected only when trials are done concerning MM cells shown extra-medullary invasion cells from patients. In contrast, IL-6 dependent cell lines may established by chance and it may not depend on cell origin. The second aim of the article is to review the usage of cell lines of MPC. We chose 7 issues; one is the correlation between the proliferation and IL-6, second is the contribution of other cytokines to MPC lines, third is how to make human MM model in SCID mouse, fourth is the chromosomal abnormalities and oncogenesis, fifth is the contribution of fibronectin and MPC lines, sixth is the issue of osteoblast activating factor (OAF), finally, we discussed the tumour aberration of MPC lines. We expect that the accumulation of these acknowledges concerning MPC cell lines would contribute to the development of plasmacytology in the future.

Cell Line↗
PubMed1995

[Multiple myeloma--diagnosis, prognostic factors and treatment].

The problem of diagnosis, prognostic factors and the efficacy of therapies were investigated in 330 patients with multiple myeloma (MM) and 51 patients with benign monoclonal gammopathy (BMG)/monoclonal gammopathy of undetermined significance (MGUS). Seven out of 51 patients with BMG/MGUS were transformed into MM. The mean time to the transformation was 61.6 months. M protein level in these patients had been gradually and constantly increasing until the transformation in contrast with stable level in non-transformed patients. In MM there was one year difference between median survival from the time of diagnosis and start of chemotherapies. It depended on the deferral of treatment in patients with stage I myeloma. No difference of survival time was found between initial and differed therapy for stage I myeloma. Earlier therapy is not advantageous in this stage. Stages and immunoglobulin classes of MM were prognostic factors. Stage I or IgG myeloma had the longest survival and stage III or BJP myeloma had the shortest one. The new protocol, DMVM plus natural interferon alpha therapy induced high complete remission rate of 37.1% in initial treatment patients. The survival rate at three years from the treatment was 70%.

Adult↗
PubMed1995

Effects of interferon-alpha on L-BCGF- and TNF-alpha-induced proliferation of hairy cell leukemia in Japan.

Interferon-alpha (IFN-alpha) is very effective in patients with hairy cell leukemia (HCL), although its mechanism of action is still unknown. To investigate this issue, we studied the in vitro response to IFN-alpha of a variant type of HCL, recently reported by us as the Japanese variant. Their clinical response to IFN-alpha (remission rate 35.7% in the multicenter study in Japan) was inferior to that of typical HCL (remission rate 80%; mean of previous reports). We found that both low molecular weight B-cell growth factor (L-BCGF) and tumor necrosis factor-alpha (TNF-alpha) induced the proliferation of HC from patients with the Japanese variant, as well as those with typical HCL. While, in typical HCL, IFN-alpha strongly inhibited the in vitro proliferation of HC induced by L-BCGF and TNF-alpha, the inhibitory effect of IFN-alpha on L-BCGF and TNF-alpha-induced proliferation was low in most Japanese variant patients. These in vitro findings may be related to the extent of clinical efficacy of IFN-alpha in the Japanese variant, obtained in the multicenter study. Since the degree of inhibition was parallel in L-BCGF- and TNF-alpha-induced proliferation in three patients examined simultaneously, it appeared that the antiproliferative effect of IFN-alpha is not specific to individual growth factors. Rather, IFN-alpha might affect fundamental growth mechanisms triggered by these factors.

Cell Division↗
PubMed1994

Characterization of genomic PIG-A gene: a gene for glycosylphosphatidylinositol-anchor biosynthesis and paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by the presence of abnormal subpopulations of blood cells that are deficient in surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins. Recent studies showed that the gene termed PIG-A, which participates in the first step of GPI-anchor biosynthesis, is mutated in the abnormal blood cells from patients with PNH. In this study the genomic PIG-A gene was cloned and characterized to obtain nucleotide sequence information for analyzing somatic mutations of PIG-A in patients with PNH. The PIG-A gene is at least 17 kb long and has six exons. The exon-intron boundaries and 583 bp of the 5' flanking region were sequenced. The 5' flanking region has no TATA-like sequence, but includes four CAAT boxes, two AP-2 sequences, and a CRE sequence, some of which are present in regions necessary for the promoter activity. We report pairs of oligonucleotide primers for polymerase chain reaction that should be useful to amplify and analyze various regions of the PIG-A gene in patients with PNH.

Base Sequence↗
PubMed1994

Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder in which there is a deficiency in the synthesis by hematopoietic cells of the glycosyl-phosphatidylinositol molecules that anchor proteins to the cell membrane. Recently, we demonstrated that a gene termed PIG-A (for phosphatidylinositol glycan class A), a component of glycosyl-phosphatidylinositol biosynthesis, was responsible for PNH in two patients. The present study was undertaken to elucidate whether PIG-A is the gene responsible for all cases of PNH and to characterize further the somatically acquired abnormalities of this gene. METHODS: We studied granulocytes from 15 patients with PNH. The cell content of CD55 and CD59 was assessed by fluorescence-activated flow cytometry. PIG-A transcripts were reverse-transcribed, amplified by the polymerase chain reaction, and cloned into plasmids. The structure of the cloned complementary DNA was analyzed by nucleotide sequencing, and its function was assessed on the basis of its ability to restore to normal the abnormal phenotype of a PIG-A-deficient cell line after transfection. RESULTS: Three patients had size abnormalities of PIG-A transcripts with different patterns, and in one patient a very low level of the PIG-A transcript was found. Eleven patients had transcripts of normal size, but the transfection assay revealed that in each patient some of them were nonfunctional. The percentage of nonfunctional PIG-A transcripts was correlated with the percentage of affected granulocytes (P < 0.001). Sequence analysis demonstrated somatic mutations in two of the patients. CONCLUSIONS: PIG-A is the gene responsible for PNH in all patients studied to date.

Base Sequence↗
PubMed1994

Acylcarnitine deficiency in chronic fatigue syndrome.

One of the characteristic complaints of patients with chronic fatigue syndrome (CFS) is the skeletal muscle-related symptom. However, the abnormalities in the skeletal muscle that explain the symptom are not clear. Herein, we show that our patients with CFS had a deficiency of serum acylcarnitine. As carnitine has an important role in energy production and modulation of the intramitochondrial coenzyme A (CoA)/acyl-CoA ratio in the skeletal muscle, this deficiency might induce an energy deficit and/or abnormality of the intramitochondrial condition in the skeletal muscle, thus resulting in general fatigue, myalgia, muscle weakness, and postexertional malaise in patients with CFS. Furthermore, the concentration of serum acylcarnitine in patients with CFS tended to increase to the normal level with the recovery of general fatigue. Therefore, the measurement of acylcarnitine would be a useful tool for the diagnosis and assessment of the degree of clinical manifestation in patients with CFS.

Acyl Coenzyme A↗
PubMed1994

cDNA cloning and expression of human calmodulin-dependent protein kinase IV.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) is a Ca(2+)-responsive multifunctional protein kinase which occurs abundantly in the brain and thymus. A human cDNA clone encoding CaM-kinase IV was isolated from a Jurkat cell cDNA library and its nucleotide sequence was determined. The cDNA sequence encoded a protein consisting of 473 amino acids with a molecular weight of 51,925. The nucleotide sequence for the coding region and the deduced amino acid sequence showed 81 and 80% identities with those of the rat enzyme, respectively. Western blot analysis, using a polyclonal antibody raised against the recombinant human CaM-kinase IV, which was expressed in Escherichia coli, revealed two bands corresponding in mobility to molecular weights of 60,000 and 61,000, respectively, in a Jurkat cell extract. The antibody also cross-reacted with both isoforms of CaM-kinase IV from rat cerebellum, the apparent molecular weights being 62,000 and 64,000, respectively.

Amino Acid Sequence↗
PubMed1994

Inactivation of Ca2+/calmodulin-dependent protein kinase II by Ca2+/calmodulin.

Incubation of calmodulin-dependent protein kinase II with Ca2+ and calmodulin resulted in a marked inactivation of the enzyme. Chelation of Ca2+ by EGTA or addition of calmodulin antagonists, W-7 or trifluoperazine, completely blocked this inactivation. The concentration required for the half-maximal inactivation, 127 microM, is three to four orders of magnitude higher than that for the half-maximal activation of the enzyme. The Ca2+/calmodulin-independent activity of the proteolytic fragment of the enzyme, whose calmodulin-binding site involved in the enzyme activation was deleted, was also decreased by incubation with Ca2+ and calmodulin. These results suggest that calmodulin-dependent protein kinase II possesses a second, low-affinity calmodulin-binding site, which is distinct from the calmodulin-binding site involved in the activation of the enzyme, and that the binding of calmodulin to the second binding site causes the inactivation of the enzyme. The inactivation by Ca2+/calmodulin was temperature-dependent. The addition of both 500 microM ADP and 10 mM MgCl2 markedly protected the enzyme against the inactivation, while such a marked protection was not observed after the addition of either of the two alone. The addition of 5 microM autocamtide-2, a synthetic substrate peptide containing the amino acid sequence of the autophosphorylation site (Thr286/Thr287 in alpha/beta, gamma, and delta isoforms) lying within the autoinhibitory domain, also protected the enzyme against the inactivation by Ca2+/calmodulin, while syntide-2, another synthetic substrate peptide corresponding to a phosphorylation site of glycogen synthase, did not protect it even at a concentration as high as 304 microM.

Animals↗
PubMed1994

Purification and characterization of Ca2+/calmodulin-dependent protein kinase IV kinase from rat brain.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) kinase was recently discovered in the rat brain by its activity to activate the inactive recombinant CaM-kinase IV expressed in Escherichia coli [Okuno, S. and Fujisawa, H. (1993) J. Biochem. 114, 167-170]. In the present study, CaM-kinase IV kinase was purified approximately 2,000-fold from rat cerebral cortex by purification procedures including calmodulin affinity chromatography, and its properties were examined. The highly purified CaM-kinase IV kinase gave one major protein band corresponding to a molecular weight of about 66,000 upon SDS-PAGE. The purified CaM-kinase IV kinase phosphorylated and concomitantly activated CaM-kinase IV purified from rat brain as well as the recombinant kinase expressed in Escherichia coli in a Ca2+/calmodulin-dependent manner. The phosphorylation of CaM-kinase IV by CaM-kinase IV kinase occurred on only serine residue(s). Among a number of proteins, including several known to be phosphorylated by the various protein kinases tested, CaM-kinase IV was the best substrate for CaM-kinase IV kinase. Since syntide-2, a synthetic peptide known to be a good peptide substrate for calmodulin-dependent protein kinase II (CaM-kinase II), was a fairly good substrate for CaM-kinase IV kinase, some kinetic properties of CaM-kinase IV kinase were examined using syntide-2 as a substrate. The Km value for the peptide substrate in the presence of Ca2+/calmodulin was almost two orders of magnitude lower than that in its absence, although the Vmax value was almost the same in the presence and absence of Ca2+/calmodulin.

Amino Acid Sequence↗
PubMed1994

Prevalence of human herpesvirus 6 variants A and B in patients with chronic fatigue syndrome.

Peripheral blood mononuclear cells collected from 13 patients with chronic fatigue syndrome and 13 healthy controls were analyzed for the presence of human herpesvirus 6 (HHV-6) DNA by variant-specific polymerase chain reaction and dot blot hybridization. HHV-6 DNA was detected in 7 of 13 (53%) patients, and of those 7 patients, 4 were positive for HHV-6 variant A DNA and 3 were for variant B. No HHV-6 DNA was detected in the controls. Serum antibody titers to the late antigen and antibody prevalence to the early antigen of HHV-6 were significantly higher in the patient group. These results suggest active replication of HHV-6 in patients with chronic fatigue syndrome.

Antibodies, Viral↗