Basal-plane birefringence and magnetic ordering in hexagonal antiferromagnets CsNiX3 (X=C1, Br).
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Journal Article↗
Biomedical subjects
K Nagata
Publications and source records attributed to K Nagata.
cDNA-directed expression of rat testosterone 7 alpha-hydroxylase using the modified vaccinia virus, T7-RNA-polymerase system and evidence for 6 alpha-hydroxylation and delta 6-testosterone formation.
The modified vaccinia virus, T7-RNA-polymerase cDNA-expression system was used to express rat cytochrome P-450a. Various parameters such as host-cell type and density, and duration of infection were tested to optimize the level of expression of cytochrome P-450a enzyme activity. Cytochrome P-450a expressed from the cDNA sequence was exclusively incorporated into the membrane-containing portions of the cell lysates, as expected from its normal association in the liver endoplasmic reticulum. The enzyme displayed a carbon-monoxide-reduced-cytochrome-P-450a difference spectrum with a Soret maximum of 450 nm. Activity measurements revealed that cytochrome P-450a produced three metabolites of testosterone; 7 alpha-hydroxytestosterone and 6 alpha-hydroxytestosterone and delta 6-testosterone at a ratio of about 38:1:1. Under the appropriate conditions, the vaccinia-virus, T7-RNA-polymerase system produces high levels of a single form of cytochrome P-450 in cells that are virtually devoid of endogenous cytochrome P-450. Analysis of the cytochrome P-450 in its natural membrane-bound state, as opposed to artificial-lipid reconstitution studies of purified enzymes, allows accurate and confident measurements of substrate specificities.
Animals↗
Low Mr GTP-binding proteins in human platelets: cyclic AMP-dependent protein kinase phosphorylates m22KG(I) in membrane but not c21KG in cytosol.
We have purified and characterized two kinds of GTP-binding proteins with Mr of 22,000 in human platelet membrane (main; m22KG(I), minor; m22KG(II)) (Nagata, K. and Nozawa, Y. (1988) FEBS Lett. 238, 90-94). In this study, the main GTP-binding protein (m22KG(I)) was found to be phosphorylated by cyclic AMP-dependent protein kinase (A-kinase), but not by protein kinase C. About 0.5 mol of phosphate was maximally incorporated into one mol of the protein and this phosphorylation was inhibited in the presence of A-kinase inhibitor. Phosphorylation of m22KG(I) did not alter either its GTP-binding or GTPase activity. When m22KG(I) was incubated alone or in the presence of 100 microM guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and then exposed to A-kinase, no significant changes in the level of phosphorylation were observed. On the other hand, the most abundant GTP-binding protein with Mr of 21,000 (c21KG) in human platelet cytosol, which was identified as a transformation suppressor gene product (rap 1 protein, smg p21 and Krev-1 protein), was not phosphorylated by A-kinase under the same condition. However, c21KG was phosphorylated by A-kinase after pretreatment with alkaline phosphatase.
Alkaline Phosphatase↗
Monoclonal antibody defining a molecule possibly identical to the p75 subunit of interleukin 2 receptor.
A mouse hybridoma cell line, TU27, producing an mAb was established. TU27 mAb reacted with various human and Gibbon ape T cell lines bearing the IL-2R p75 (IL-2Rp75), but not with cell lines expressing only Tac antigen, IL-2Rp55, and numbers of its binding sites on cell surfaces were similar to those of high-affinity IL-2R. Radioimmunoprecipitation with TU27 mAb defined a molecule with a molecular mass of 75 kD on the surface of IL-2Rp75 bearing cells. TU27 mAb completely blocked IL-2 binding to IL-2Rp75 and to the high-affinity IL-2R but not to IL-2Rp55 composing the low-affinity IL-2R. The IL-2-dependent growth of a human T cell line, ILT-Mat, was significantly inhibited by TU27 mAb only at low concentrations of IL-2, and combination of TU27 mAb and H-31 mAb specific for IL-2Rp55 completely inhibited the cell growth even at high concentrations of IL-2. These data strongly suggest that TU27 mAb is specific for the human IL-2Rp75.
Antibodies, Monoclonal↗
Electroporation: application to human lymphoid cell lines for stable introduction of a transactivator gene of human T-cell leukemia virus type I.
Conditions were developed for stable introduction of foreign DNA into human lymphoid cell lines by electroporation. To introduce stably the p40 gene of human T-cell leukemia virus type I (HTLV-I) into the human lymphoid cell line Jurkat, the p40 expressing plasmid, pMAXRHneo-1, which carries the neo resistant gene, was transfected into Jurkat cells at a voltage of 2500 V and capacitance of 21.7 microF, and stable transformants were screened for neo (G418) resistance. The frequency of transformants was more than one per 2 x 10(5) cells used initially. Clones that were resistant to G418 were shown to have the p40 gene integrated into the host genome and to express mRNA and protein from the introduced plasmid. Expression of p40 in the transformed Jurkat cells was also confirmed by testing the trans-activating effect of HTLV-I enhancer by p40. High frequencies of stable transformations of 10(-4) to 10(-6) were also reproducibly obtained by electroporation of the human T cell lines HSB-2 and TALL-1, a human B cell line Raji, a human monocytic cell line U937, and a human erythroleukemia cell line K562. These results demonstrate that electroporation is a very efficient method for introducing foreign DNA into human lymphoid cell lines.
Blotting, Northern↗
Purification and characterization of a novel calcium-dependent serine proteinase secreted from malignant hamster embryo fibroblast Nil2C2.
A novel serine proteinase was purified from the conditioned medium of malignant hamster embryo fibroblasts, Nil2C2. The molecular weight of the purified enzyme was estimated to be 88,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. The enzyme was split into two subunits (Mr 66,000 and 33,000) with a reducing agent. The enzyme hydrolyzed not only synthetic peptides which are susceptible to trypsin digestion but also extracellular matrix proteins such as type I and IV collagen, fibronectin and gelatin. For the digestion of these proteins, Ca2+ at millimolar concentrations was essential but Ca2+ or chelators did not affect the esterase activity for synthetic peptides. The proteolytic activity was inhibited by diisopropyl fluorophosphate (DFP) and also by phenylmethylsulfonyl fluoride. DFP was shown to bind to the 33 kDa subunit, indicating that the catalytic machinery of the enzyme is located in this subunit.
Animals↗
An interesting syndrome of hemolytic anemia, degeneration of the liver and diabetes associated with a high red cell Mg-ATPase, detected by 31P NMR spectroscopy.
31P NMR was used to study the erythrocytes of three patients who exhibited a familial multisystem disease characterized by fatty liver, diabetes and nonspherocytic hemolytic anemia of unknown etiology. 31P NMR measurements disclosed an abnormally high level of intracellular inorganic phosphate (Pi) and an abnormally low level of ATP in the erythrocytes 6 h after blood withdrawal from proband (I-1). This finding suggested that ATP was markedly decreased in the red cells of this proband, as compared with those of normal subjects. Time-dependent changes of 31P NMR spectra of the erythrocytes from the two daughters (II-1, II-2) of the proband demonstrated clearly an enhanced decomposition of ATP with a concomitant increment of Pi. Several ATP-consuming enzymes in erythrocytes, such as those in the Embden-Meyerhof system, pentose phosphate pathway enzymes, Na+, K(+)-ATPase and Ca2+, Mg2(+)-ATPase, were within normal limits of activity, but Mg2(+)-ATPase was drastically above the normal limit. The Mg2(+)-ATPase activity was 3 times higher in the red cell membranes of these patients than in those from normal subjects.
Adult↗
Monoclonal antibody analysis of influenza virus matrix protein epitopes involved in transcription inhibition.
Influenza virus M1 protein has been shown to inhibit viral RNA transcription, and in this study the epitopes on M1 critical for this function were localized. When a battery of 15 monoclonal anti-M1 antibodies were reacted with chemically cleaved fragments of M1 on a western blot, five distinct banding patterns were observed. A representative antibody was selected from each banding group, and its ability to reverse M1-effected transcription inhibition was measured. From these data, the sites on M1 critical for transcription inhibition were deduced. It appears now that the regions on M1 in the vicinity of amino acid residues #70 and #140 are critical for inhibition. Furthermore, by taking into account the hydropathicity and secondary structure, it is hypothesized that amino acids #70 and #140 are physically close together in the final three-dimensional conformation of M1 protein and that the residues in between form a loop and are thus removed from the functional site.
Antibodies, Monoclonal↗
Sequential PET studies in neuro-Behçet's syndrome.
A case of neuro-Behcet's syndrome is presented with sequential positron emission tomography (PET) studies. Regional cerebral blood flow (rCBF) and oxygen consumption (rCMRO2) were decreased in the brain lesion; however, on follow-up studies 3 months after steroid therapy rCBF and rCMRO2 had increased in the lesion, which demonstrated the reversibility of this disease. Such monitored improvement may accurately reflect the early stage of the disease and its response to steroid therapy.
Behcet Syndrome↗
Immunological functions and phenotypes of peripheral blood lymphocytes from human T-cell leukemia virus-I carriers.
We studied immunological functions of peripheral blood lymphocytes (PBL) from human T-cell leukemia virus type I (HTLV-I)-seropositive healthy carriers in vitro. Proliferative responses of PBL to T-cell and B-cell mitogens such as concanavalin A (Con A), pokeweed mitogen (PWM), and Staphylococcus aureus Cowan I (SAC) were moderately impaired in HTLV-I carriers compared with normal controls. Immunoglobulin (Ig)-producing activity of PBL stimulated with B-cell mitogens were also impaired in HTLV-I carriers. However, cytotoxic T-cell activity induced by in vitro culture was not impaired but slightly increased in HTLV-I carriers. Natural killer-cell activity was only slightly decreased. By a flow cytofluorometric analysis of the cell surface phenotypes of PBL, the percentage and the mean fluorescence intensity (MFI) of CD 3-positive cells and CD 4-positive cells were significantly decreased in HTLV-I carriers. The percentage and the MFI of CD 8-positive cells was not changed. The percentage and the MFI of CD 25-positive cells were increased. These results suggest that some immunological abnormalities are already present in HTLV-I carriers and such abnormalities have some roles for the leukemogenesis from the infection of the HTLV-I into adult T-cell leukemia (ATL).
Adolescent↗
Electroencephalographic correlates of blood flow and oxygen metabolism provided by positron emission tomography in patients with cerebral infarction.
Quantitative EEG data were analyzed statistically with respect to cortical cerebral blood flow (CBF) and oxygen metabolism (CMRO2) measured by positron emission tomography in 47 patients with unilateral cerebral infarction. Relative value of the square root of average power was used as a percentage power fraction (PPF) for each frequency category. Power ratio index (PRI) was calculated by dividing the combined delta-PPF and theta-PPF by the combined alpha-PPF and beta-PPF. Delta-PPF, theta-PPF and PRI correlated negatively with regional CBF (rCBF) and CMRO2 (rCMRO2) whereas alpha-PPF and beta-PPF correlated positively. In the acute stage, delta-PPF, alpha-PPF and PRI correlated with rCBF at all but the frontopolar region whereas the correlation with rCMRO2 was poor. Alpha-PPF and PRI correlated also with rCMRO2 in the frontal, central, parietal and occipital regions while delta-PPF correlated with rCBF only in the parietal and occipital regions in the subacute stage. In the chronic stage, all EEG quotients correlated significantly with both rCBF and CMRO2 in the central and parietal regions. In the frontopolar region, only the theta-PPF correlated with rCBF throughout. In the comparison of hemispheric mean values, the correlations were always closer for the affected hemisphere than for the contralateral hemisphere. Although hemispheric mean CBF and CMRO2 were significantly lower in patients with cortical infarcts on CT than in those with small subcortical infarcts, there was no significant difference in the EEG data between the 2 groups.
Brain↗
Primary empty sella syndrome caused by an abnormal dilatation of the optic recess.
A new variety of primary empty sella syndrome is reported. A partial herniation of the optic chiasm into an enlarged empty sella compressed by a dilated optic recess mimicking a cystic tumor was confirmed at operation. The pathogenesis and the treatment of this type of empty sella syndrome are discussed.
Adult↗
Specific binding of radioiodinated human GM-CSF to the blast cells of acute myeloblastic leukemia.
We describe here the presence of two classes of binding sites for GM-CSF expressed on blasts freshly isolated from five AML patients and one patient with CML in blastic phase: one of high-affinity (38-177 per cell, KD 8-150 pM) and one of low-affinity (121-806 per cell, KD 503-2683 pM). No correlation is observed between the receptor number, receptor affinity, and the growth stimulatory effect of GM-CSF on leukemic blast progenitors. Blasts from two cases showed no or negligible response to GM-CSF but expressed comparable numbers of receptors when compared with the numbers expressed by the sensitive blasts. Our data suggest that significant proliferative effects of GM-CSF can occur at low levels of high-affinity receptor occupancy. Lack of responsiveness to GM-CSF in some AML patients is not correlated to the absence of GM-CSF receptors on leukemic cells. Reduction in the growth of blast progenitors at high concentrations of GM-CSF may be attributed to the differentiating activity of GM-CSF via low-affinity receptors on leukemic cells.
Binding, Competitive↗
Phosphoinositide-specific phospholipase C of a cloned mast cell line, mastocytoma P-815: purification and some characterization.
1. Multiple forms of phosphoinositide-phospholipase C (PLC) were isolated from mastocytoma; two cytosolic forms (cPLC-I, Mr 150,000; cPLC-II, Mr 110,000) and two membrane-associated forms (mPLC-I, Mr 85,000; mPLC-II, Mr 85,000). 2. Four PLC forms differently behaved in substrate specificity and effect of GTP-binding proteins.
Animals↗
Effect of thyrotropin-releasing hormone on cerebral blood flow in conscious rat.
The effect of thyrotropin-releasing hormone (TRH) was studied on local CBF (LCBF) in normal conscious rats. LCBF was measured by the autoradiographic [14C]iodoantipyrine method 5 min after TRH (5 mg/kg, i.v.) administration. TRH significantly increased LCBF in 22 of 33 brain regions. This increase of LCBF exceeded 100% of the control values in the cerebral cortices, whereas there was no significant increase in white matter or in some gray matter structures. The increase of CBF following TRH administration was abolished by pretreatment with indomethacin (5 mg/kg, i.v.). The mechanisms underlying the increase of CBF following TRH administration are discussed in relation to prostaglandin metabolism.
Animals↗
A case of associative visual agnosia: neuropsychological findings and theoretical considerations.
A 61-year-old right-handed man fell downstairs and was found unconscious. There was no abnormality in the general physical examinations. Neurological examinations revealed a quadrantanopia. Neuropsychological examination revealed an impairment in recent verbal memory, alexia, agraphia, object agnosia, color naming difficulty, prosopagnosia, and visuospatial constructional disability. CT scan demonstrated subcortical hematomas in the temporo-occipital regions of both hemispheres. MRI demonstrated extensive low-intensity lesions in the lingual, fusiform and posterior inferior temporal gyri on both hemispheres. Both inferior longitudinal fasciculi were also affected. His neuropsychological deficits seem best described as a typical form of associative visual agnosia. From the results of neuroradiological findings, the authors emphasize that associative visual agnosia might be produced by an intrahemispheric disconnection between the visual cortices and the temporal lobes which are supposed to be the storage site of the engrams of visual memories. Moreover, the neuropsychological findings suggest that the visuo-constructional ability to convert the two-dimensional input to the three-dimensional construction and the capacity of three-dimensional imagination were severely impaired in our patient. It was considered that these neuropsychological features play an important role in the recognition difficulties of associative visual agnosia.
Agnosia↗
Tissue-specific DNA binding of nuclear proteins that bind to the adenovirus inverted terminal repeat.
Adenovirus (Ad) DNA replication requires nuclear factor I (NFI), a cellular sequence-specific DNA binding protein which binds to the Ad DNA inverted terminal repeat (ITR). The NFI binding consensus sequence, TGGN6-7GCCAA, possibly includes the CCAAT-box which is often observed in regulatory elements of transcription. Adjacent to the NFI binding site on the Ad ITR is the binding site for nuclear factor III (NFIII), another cellular factor that stimulates Ad DNA replication in a cell-free system. Using gel retardation assay, we have examined the tissue specificity of these DNA binding activities in mouse. High NFI activity was detected in nuclear extracts from mouse liver, kidney, and spleen. Competition gel retardation assay with double-stranded oligonucleotides containing herpes simplex virus thymidine kinase (HSV-TK) gene or hsp70 gene CCAAT-box showed no effect on mouse NFI binding to its binding site. In the course of competitive DNA-binding assay, we detected a novel DNA binding protein in mouse kidney nuclear extracts, designated nuclear factor K (NFK). The NFK binding site is included in the NFIII binding site on the Ad ITR. NFK seems to be different from NFIII, in mode of binding to its binding site.
Adenoviridae↗
In vitro synthesis of influenza viral RNA: biochemical complementation assay of factors required for influenza virus replication.
Using isolated nuclei prepared from influenza virus-infected HeLa cells, factors affecting the synthesis of two species of positive-sense RNA transcripts, i.e., mRNA and cRNA (complementary RNA to vRNA) were analyzed. In the presence of low concentrations of salt, both mRNA and cRNA were synthesized, whereas in the presence of high concentrations of salt, mRNA was synthesized predominantly. Salt-extracts of nuclei (NE) mainly produced cRNA while mRNA was a major product synthesized by salt-treated nuclei (delta N). In the presence of high concentrations of salt, the NE produced mRNA instead of cRNA. After centrifugation of the NE, the precipitates (NEP) predominantly produced mRNA while the supernatant (NES) alone exhibited a low level of cRNA synthesis activity. With the addition of the NES fraction, mRNA synthesis by the NEP was switched to cRNA synthesis. Glycerol gradient centrifugation of the NES fraction in the presence of high salt yielded vRNA-RNA polymerase complexes that catalyzed mRNA synthesis. These observations indicate that a regulatory factor(s) that can be dissociated from vRNA-RNA polymerase complexes upon exposure to high ionic strength is involved in the switch from mRNA to cRNA synthesis. This activity was not detected in nuclear extracts prepared from uninfected cells, suggesting that such a factor(s) is either encoded by the virus genome or induced by virus infection.
Cations↗