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Biomedical subjects

K Anzai

Publications and source records attributed to K Anzai.

At least 19 recordsLinked to original sources
PubMed1992

Purification of the cardiac 1,4-dihydropyridine receptor using immunoaffinity chromatography with a monoclonal antibody against the alpha 2 delta subunit of the skeletal muscle dihydropyridine receptor.

The 1,4-dihydropyridine receptor associated with L-type Ca2+ channels was purified about 1700-fold from porcine cardiac sarcolemmal membranes using a simple and rapid (ca. 8 h) two-step procedure: wheat germ agglutinin affinity chromatography followed by immunoaffinity chromatography with a monoclonal antibody (MCC-1) against the alpha 2 delta subunit of the skeletal muscle Ca2+ channel with a glycine elution buffer (pH 3). Gel electrophoresis of this purified sample under non-reducing conditions revealed a major polypeptide band with molecular weight of 190 kDa, which was separated under reducing conditions to a 155 kDa band and 2-3 bands with M(r) about 20 kDa, corresponding to alpha 2 and delta subunits, respectively. The peptide band corresponding to the alpha 1 subunit was not detected in this gel electrophoresis. However, the alpha 1 subunit without bound alpha 2 delta was selectively eluted from MCC-1 Sepharose with 1% Triton X-100. A 190 kDa band corresponding to the alpha 1 subunit was visualized by fluorography and by silver staining in the fraction eluted with Triton X-100. Electrophoretically, the amount of alpha 1 was smaller than that of the alpha 2 subunit in the purified sample obtained here.

Animals↗
PubMed1992

Developmental change in subcellular location of Bp-1 protein with an ability to interact with both identifier sequence and its brain-specific transcript, BC-1 RNA.

Identifier sequences are transcribed to generate a brain-specific BC-1 RNA present as a ribonucleoprotein particle in the dendrites and somata of neurons. This ribonucleoprotein particle contains an identifier sequence-binding protein (Bp-1 protein). We report here the purification of BC-1 RNA and demonstrate that Bp-1 protein interacts directly with the RNA. We also demonstrate an accumulation of Bp-1 protein in the nucleus of brain cells from mouse fetus and newborns that precedes the postnatal increase in BC-1 RNA. Cytoplasmic Bp-1 protein present in a complex with BC-1 RNA increases postnatally with a concomitant decrease in nuclear Bp-1 protein. These observations suggest that Bp-1 protein may play a role(s) in the synthesis and nuclear export of BC-1 RNA.

Aging↗
PubMed1992

The 10 S BC-1 ribonucleoprotein particle contains identifier sequence-binding proteins that interact with an array of GCAAG/CTTGC motifs between split promoter sequences for RNA polymerase III.

BC-1 RNA is a brain-specific small RNA transcript of identifier sequences present in the somas and dendrites of neurons. We recently reported that the RNA is complexed with a protein(s) to form a 10 S ribonucleoprotein particle (Kobayashi, S., Goto, S., and Anzai, K. (1991) J. Biol. Chem. 266, 4726-4730). We demonstrate here that this 10 S BC-1 ribonucleoprotein particle contains a DNA-binding protein(s) (Bp-1 protein) capable of interacting with a region between split promoter sequences for RNA polymerase III within the identifier sequences. The region has short inverted repeats: a perfect octanucleotide repeat (GCGCTTGCCTAGCAAGCGC) and an imperfect heptanucleotide repeat (GCCTAGCAAGCGCAAGGC), each of which contains a GCAAG/CTTGC motif. We also demonstrate that the binding of this protein either to the array of pentamer motifs or to BC-1 RNA is mutually exclusive. The molecular masses of photo-cross-linking adducts of Bp-1 protein to a 32P-labeled GCAAG/CTTGC motif-specific probe were estimated to be about 31 and 36 kDa, indicating that two species of Bp-1 proteins may be present in the brain.

Animals↗
PubMed1992

rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain.

rab3A is a low molecular weight (LMW) GTP-binding protein specifically expressed in brain and localized to synaptic vesicles. rab3A has been proposed to play a role in neurotransmitter release by regulating membrane flow in the nerve terminal. In an attempt to define other LMW GTP-binding proteins that may regulate neurotransmitter release, seven cDNA clones encoding new members of the rab family of LMW GTP-binding proteins were isolated from a rat brain cDNA library. The rab proteins contain the four conserved structural domains essential for GTP binding in addition to domains required for membrane localization and effector protein interactions. One protein, rab16, is closely related to members of the rab3 subfamily, whereas two others are assigned as the rat homologs of canine rab8 and rab10. Four additional clones, rab12, rab13, rab14, and rab15, revealed unique sequences and are new members of the rab family of LMW GTP-binding proteins. The patterns of expression of rab15 and rab3A closely overlap but differ from that observed for all other known LMW GTP-binding proteins. This data suggests that rab15 may act in concert with rab3A in regulating aspects of synaptic vesicle membrane flow within the nerve terminal.

Amino Acid Sequence↗
PubMed1992

In vivo comparative therapeutic study of optimal administration of 5-fluorouracil and cisplatin using a newly established HST-1 human squamous-carcinoma cell line.

The efficacy and toxicity of 5-fluorouracil (5-FU) and cisplatin (CDDP) given in a sequential combination were evaluated in nude mice transplanted with HST-1, a newly established human squamous-carcinoma cell line. 5-FU and CDDP were given i.p. for 5 days and 1 day, respectively, either as single agents or in a sequential manner separated by a 24-h interval. The treatment was repeated every 30 days. Although inhibition of tumor growth was seen in all of the treated groups after two cycles, the sequence of 5-FU followed by CDDP significantly reduced the tumor burdens throughout all three courses and was more effective than the reverse sequence or either drug alone. Neither treatment-related death nor significant hematologic or nephrologic toxicities were seen, even following three cycles of therapy. Significant weight loss was observed only in mice treated with CDDP followed by 5-FU. This sequence dependence of the activity and toxicity of the 5-FU and CDDP combination should thus be incorporated into the design of a clinical trial.

Animals↗
PubMed1992

Increase of peripheral B lymphocytes committed to the production of monoreactive and high affinity antibodies to HTLV-1 in patients with HAM/TSP.

We quantitated the frequency of B lymphocytes capable of producing antibodies to HTLV-1 in the peripheral blood from patients with HAM/TSP, non-HAM/TSP HTLV-1 carriers and seronegative healthy subjects. Epstein-Barr virus (EBV) was used as a polyclonal activator of B lymphocytes in a limiting dilution condition. We found that B lymphocytes committed to the production of monoreactive-IgG and -IgA antibodies to recombinant HTLV-1 (gag + env) hybrid protein were significantly increased in a number in patients with HAM/TSP as compared to non-HAM/TSP HTLV-1 carriers and seronegative healthy subjects. By transforming these B lymphocytes with EBV and fusing them with human-mouse heteromyeloma (F3B6), a stable hybridoma producing IgG monoclonal antibody (mAb) to HTLV-1 (gag + env) protein was generated from a patient with HAM/TSP. This mAb (IgG1, kappa), designated F31.1, specifically bound to the amino acid residues from 235 to 254 of HTLV-1 envelope glycoproteins (gp46) with high affinity (Kd = 4.0 x 10(-9) mol/l). These data indicate that the antigen-driven process of B lymphocytes maturation by HTLV-1 antigens is markedly increased in patients with HAM/TSP.

Antibody Affinity↗
PubMed1991

Interaction with phospholipid bilayers, ion channel formation, and antimicrobial activity of basic amphipathic alpha-helical model peptides of various chain lengths.

Basic amphipathic alpha-helical peptides Ac-(Leu-Ala-Arg-Leu)3 or 4-NHCH3 (4(3) or 4(4)) and H-(Leu-Ala-Arg-Leu)3-(Leu-Arg-Ala-Leu)2 or 3-OH (4(5) or 4(6)) were synthesized and studied in terms of their interactions with phospholipid membranes, biological activity, and ion channel-forming ability. CD study of the peptides showed that they form alpha-helical structures in the presence of phospholipid liposomes and thus they have amphipathic distribution of the side chains along the axis of the helix. A leakage study of carboxyfluorescein encapsulated in phospholipid vesicles indicated that the peptides possess a highly potent ability to perturb the membrane structure. Membrane current measurements using the planar lipid bilayer technique revealed that the peptide 4(6), which was long enough to span the lipid bilayer in the alpha-helical structure, formed cation-selective ion channels at a concentration of 0.5 microM in a planar diphytanoylphosphatidylcholine bilayer. In contrast, other shorter peptides failed to form discrete and stable channels though they occasionally induced an increase in the membrane current with erratic conductance levels. The probability of detecting a conductance increase was in the order of 4(6) greater than 4(5) greater than 4(4) greater than 4(3), which corresponds to the order of the peptide chain lengths. Furthermore, 4(6) but not 4(5) showed an antimicrobial activity against both Gram-positive and -negative bacteria. The structure of ion channels formed by 4(6) and the relationship between the peptide chain length and biological activity of the synthetic peptides are discussed.

Amino Acid Sequence↗
PubMed1991

Establishment of Epstein-Barr virus-negative diffuse large cell lymphoma cell line with an 8;22 chromosomal translocation.

A new human B-cell lymphoma cell line was established from a pleural effusion of a patient with a diffuse large cell lymphoma which originated from an ileocecal tumor. The cell line, designated KAL-1, has been passaged 280 times over a period of 22 months. This cell line was successfully maintained in a chemically defined serum-free medium; its doubling time is approximately 24 h. Immunologically, the cells were demonstrated to express IgM lambda on the cell surface and to react with monoclonal antibodies to B-cell antigen including B1, B4, HLA-DR, and common acute lymphoblastic leukemic antigen but not with B2 and all the T-cell markers. Immunoglobulin gene analysis revealed rearrangements of both JH and C lambda. These data indicate that this cell line represents the B-cell lineage at the immature B-cell stage. This cell line was negative for Epstein-Barr virus nuclear antigen and had no detectable Epstein-Barr virus genome in cellular DNA. Chromosome analyses revealed that the cells carried an 8;22 chromosome translocation, reminiscent of variant type Burkitt's lymphoma. However, there was no histological evidence for Burkitt's lymphoma. Molecular studies showed that KAL-1 had deregulated high constitutive expression of c-myc. This cell line was demonstrated to be highly tumorigenic when injected into athymic nude mice. This tumor model should provide clues about the molecular mechanism involved in the pathogenesis of B-cell malignancy and appears to be a useful in vivo model for the study of molecular events during B-cell differentiation and therapeutic investigations.

Adult↗
PubMed1991

Formation of ion channels in planar lipid bilayer membranes by synthetic basic peptides.

We made use of a planar lipid bilayer system to examine the action of synthetic basic peptides which model the prepiece moiety of mitochondrial protein precursors and have antibacterial activity against Gram-positive bacteria. The sequences of the peptides used were as follows: Ac-(Ala-Arg-Leu)3-NHCH3 (3(3], Ac-(Leu-Ala-Arg-Leu)2-NHCH3 (4(2], Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3], Ac-(Leu-Leu-Ala-Arg-Leu)2-NHCH3 (5(2]. These peptides interacted differently with planar lipid bilayer membranes and membrane conductance increased by the formation of ion channels. The effects of the peptides on the macroscopic current-increase and on the probability of channel formation, at the single channel level were in the order of 4(3) greater than 4(2) approximately 5(2) much greater than 3(3), a finding which correlates with the antibacterial activity of these peptides. The micromolar (microM) order concentration at which the channel was formed resembles that causing antibacterial activity. Thus, the peptide antibacterial activity may occur through an increase in ion permeability of the bacterial membrane. The single-channel properties were investigated in detail using 4(3), the peptide with the highest ion channel-forming activity. Many types of channels were observed with respect to conductance (2-750 pS) and voltage dependency of gating. However, the channels were all cation-selective. These results suggest that the ion channels formed by peptide 4(3) may be able to take on a variety of conformations and/or assembly.

Amino Acid Sequence↗
PubMed1991

Brain-specific small RNA transcript of the identifier sequences is present as a 10 S ribonucleoprotein particle.

BC-1 RNA is a small RNA transcript of the identifier repetitive sequences present in rodent genomes. The RNA has been reported to be specific to the brain and confined to the cytoplasm. The RNA level increases during the 1st month after birth. To understand its cytoplasmic function, it seems important to examine whether BC-1 RNA is present as an RNP. It is believed that the protein component may govern the functions of BC-1 RNA in the brain cells. In the present report, we have demonstrated that BC-1 RNA is not free but complexed with proteins to form a 10 S RNP in the cytoplasm. We have also shown that the 10 S RNP is not associated with cytoplasmic structures such as polysomes/ribosomes or microsomes. The buoyant density of the RNP was 1.26 g/cm3 in metrizamide. Furthermore, some of the protein components were shown to be in direct contact with RNA, since photo-cross-linking adducts of protein to BC-1 RNA were identified upon UV irradiation of the 10 S BC-1 RNP.

Animals↗
PubMed1991

Elevated antibodies to synthetic peptides of HTLV-1 envelope transmembrane glycoproteins in patients with HAM/TSP.

We studied the antibody response to various kinds of well-characterized synthetic peptides of human T lymphotropic virus type 1 (HTLV-1) envelope glycoproteins in patients with HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis (TSP) and non-HAM/TSP HTLV-1 carriers. The serum antibody titers to most of the synthetic peptides were significantly higher in patients with HAM/TSP than those in non-HAM/TSP HTLV-1 carriers. However, the degree of the increase of antibody titers to the synthetic peptides corresponding to the transmembrane portions of HTLV-1 envelope glycoproteins (env-p20E), such as p20E 332-352, 374-392, 426-448 and 458-488, was greater than those to synthetic peptides of exterior portions of HTLV-1 envelope glycoproteins (env-gp46) in sera from patients with HAM/TSP. Antibodies to env-p20E 332-352 and 374-392 were elevated in the cerebrospinal fluid (CSF) only from patients with HAM/TSP but not from non-HAM/TSP HTLV-1 carriers. These data indicate that the increase of antibody titers to transmembrane portions of HTLV-1 envelope glycoproteins in sera and CSF is a characteristic feature of antibody response in patients with HAM/TSP and may be closely associated with the development of HAM/TSP from non-HAM/TSP HTLV-1 carriers.

Adult↗
PubMed1990

[Study on the clinical usefulness of NCC-ST-439 in cases of digestive tract cancer].

NCC-ST-439 is a monoclonal antibody established from human stomach cancer xenografted nude mice. The values of NCC-ST-439 were measured in 139 cases with various digestive tract cancers and 294 cases with benign digestive tract diseases with the NCC-ST-439 EIA kit (Nihon Kayaku Co., Ltd.), and its clinical usefulness was compared with those of CA19-9 and CEA. The positive rates of NCC-ST-439 in cases of digestive tract cancer were high, i.e., 66.7% for cancer of the bile duct, 58.3% for pancreatic cancer and 52.9% for colorectal cancer. In the benign digestive tract diseases, the overall positive rate seen in case of cholelithiasis and cholangitis, chronic gastritis, benign colorectal diseases and hepatitis, was only 3.7%. The positive rate of NCC-ST-439 was lower than those for CA19-9 and CEA in cases of stomach cancer, colorectal cancer and liver cancer, but it was the same as that of CA19-9 and higher than that of CEA in cases of biliary tract cancer and pancreatic cancer. The false positive rate of NCC-ST-439 in benign diseases of the digestive tract was the lowest among the three markers. With respect to sensitivity, specificity and efficiency, CA19-9 showed the highest sensitivity, but NCC-ST-439 and CEA showed better specificity than CA19-9, and NCC-ST-439 showed the highest efficiency. In combination assays using combinations of NCC-ST-439, CA19-9 and CEA, the positive rates for ST-439 alone were 22.1% for stomach cancer, 52.9% for colorectal cancer, 15.0% for liver cancer and 58.3% for pancreatic cancer, while the combined rates increased to 51.9%, 70.6%, 75.0% and 66.7%, respectively. In an investigation of changes with time in NCC-ST-439 values during chemotherapy of various types of digestive tract cancer, there was a decrease in PR cases, no change in NC cases and a tendency to increase in PD cases. These results suggested that it was possible to apply NCC-ST-439 clinically.

Aged↗
PubMed1990

Measurement of steady-state Ca2+ pump current caused by purified Ca2(+)-ATPase of sarcoplasmic reticulum incorporated into a planar bilayer lipid membrane.

The electrogenicity and some molecular properties of the sarcoplasmic reticulum Ca2+ pump protein were studied by measuring steady-state Ca2+ pump currents. Ca2(+)-ATPase protein was solubilized from rabbit skeletal muscle sarcoplasmic reticulum membrane preparations and purified by liquid chromatography. The purified Ca(+)-ATPase molecules were reconstituted into proteoliposomes and then incorporated by fusion into a planar bilayer lipid membrane. Short circuit currents across the planar membrane were detected when the ATPase molecules were activated by addition of ATP under optimal ionic conditions. Thus, the electrogenicity of the Ca2+ pump molecules was directly demonstrated. The amplitude of the pump current was dependent on the ATP concentration, and the relation was described by a Michaelis-Menten-type equation. The Michaelis constant was calculated to be 0.69 +/- 0.16 mM, which agrees well with the dissociation constant for a low affinity ATP-binding site deduced previously from the kinetics of ATP hydrolysis and from ATP binding.

Adenosine Diphosphate↗
PubMed1990

Sonographic diagnosis of venocclusive disease of the liver and danazol therapy for autoimmune thrombocytopenia in an autologous marrow transplant patient.

A 16-Year-old boy with lymphoblastic lymphoma underwent an autologous bone marrow transplantation (ABMT) after conditioning with high-dose busulfan and cyclophosphamide. On day 39 post-transplant, right upper quadrant pain occurred with an increase in the size of the liver. Liver function tests showed a subsequent deterioration. Ultrasonographic studies of the abdomen disclosed hepatosplenomegaly, ascites, thickening of the gall bladder wall and a failure to visualize the major hepatic veins. The venocclusive disease of the liver (VOD) diagnosis was confirmed from these findings. Ultrasonographic monitoring reflected the disease status well and demonstrated a complete recovery from the VOD. We emphasize, thus, that abdominal ultrasonography can be applied easily, being a non-invasive procedure, and is useful in diagnosing VOD. Furthermore, the procedure can be repeated serially for evaluating the severity of VOD. Although engraftment was confirmed with granulocytes exceeding 500/microliters, platelet recovery was delayed; the megakaryocytes had not decreased in bone marrow aspirates and platelet-associated IgG was significantly elevated. Since autoimmune thrombocytopenia was highly suspected, to prevent immunosuppression danazol was given as an immune modulator instead of prednisolone, and a complete recovery was obtained. Accordingly, danazol can be used as an alternative to prednisolone for the treatment of autoimmune thrombocytopenia after bone marrow transplantation.

Adolescent↗
PubMed1989

Regional distribution of metabotropic glutamate response in the rat brain using Xenopus oocytes.

The regional distribution of metabotropic L-glutamate responses was investigated in Xenopus oocytes injected with poly(A)+-RNA from a rat brain which was separated into 3 parts: cerebrum, cerebellum and brainstem. Under voltage-clamp, oscillatory current responses were induced more in cerebellum or brainstem poly (A)+-RNA-injected oocytes, and less in cerebrum poly(A)+-RNA-injected oocytes. These results suggest that the metabotropic glutamate receptor is distributed mostly in cerebellum and brainstem.

Animals↗