Search PubMed⌕ Search

Biomedical subjects

K Tsukuda

Publications and source records attributed to K Tsukuda.

At least 73 records · Page 4Linked to original sources
PubMed1991

The glucose transport activity of GLUT1 is markedly decreased by substitution of a single amino acid with a different charge at residue 415.

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of aspartic acid for asparagine 415, which is conserved among all facilitative glucose transporter isoforms. Although a significant amount of the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells by transfection with expression vector, almost no increase in glucose transport activity was observed. Analysis of glucose uptake with Lineweaver-Burk plot depicts that the mutation induced a marked decrease (more than 5-fold) in turnover number and a slight increase (1.5-fold) in Km compared with the wild-type GLUT1. Results obtained with cytochalasin B and ethylidene glucose suggested that the inner but not outer glucose binding site was modulated. These results suggest that asparagine 415 is located close to the inner glucose binding site and the putative inner gate of GLUT1 glucose transporter and that an ionic charge in this domain might play an important role in the rate of conformational change between an inward-facing form and an outward-facing form of glucose transporter.

Animals↗
PubMed1991

Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter.

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.

Affinity Labels↗
PubMed1991

Altered expression of glucose transporter isoforms with aging in rats--selective decrease in GluT4 in the fat tissue and skeletal muscle.

To elucidate the cellular mechanisms of glucose intolerance associated with aging, both the protein and mRNA levels of glucose transporter isoforms were studied in the various tissues of young (7-week-old) and aged (20-month-old) rats. GluT4 (adipose/muscle-type glucose transporter) protein, which is specifically expressed in insulin-responsive tissues, was selectively decreased per milligram of cellular membrane protein in both the epididymal fat tissues and the gastrocnemius muscle of the aged rats compared with the young rats. When the changes in total cellular membranes per gram of tissue are taken into account, a further decrease in GluT4 protein per gram of tissue was observed in the tissues of the aged rats compared with the young rats. The decreased amount of GluT4 protein in the fat tissues of the aged rats is probably due to the decreased protein synthesis rather than the stability, since GluT4 mRNA/micrograms of cellular total RNA was also decreased. In contrast, GluT4 mRNA in the gastrocnemius muscle was rather increased and a ratio of GluT4 protein/GluT4 mRNA was decreased by 70% in the aged rats, suggesting that the translational efficiency and/or stability of GluT4 protein is decreased in the skeletal muscle of the aged rats compared with the young rats. GluT2 (liver-type glucose transporter) protein and mRNA in the liver were also decreased in the aged rats, while no apparent decrease in GluT1 (HepG2/brain-type glucose transporter) protein/mg of cellular membrane protein was observed in the skeletal muscle and fat tissues of the aged rats compared with the young rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1991

Peptide-based radioimmunoassay specific for GLUT1 glucose transporter.

A radioimmunoassay for the GLUT1 glucose transporter was developed with a synthesized peptide based on the sequence of the cDNA for GLUT1. A peptide corresponding to the COOH-terminal domain of the GLUT1 glucose transporter (Thr-Pro-Glu-Glu-Leu-Phe-His-Pro-Leu-Gly-Ala-Asp-Ser-Gln-Val) was synthesized and conjugated to keyhole limpet hemocyanin through the NH2-terminal of the peptide. An antibody was raised against this complex and affinity purified with the immobilized peptide. A second peptide, with tyrosine residue added to the NH2-terminal of the above peptide, was synthesized and used as a standard and iodinated for preparation of the radioactive ligand. The assay is highly reproducible, sensitive, and specific for the COOH-terminal domain of the GLUT1 glucose transporter. It has no cross-reactivity with the other glucose-transporter isoforms GLUT2 and GLUT4. Furthermore, the results obtained with this radioimmunoassay on the number of glucose transporters in human erythrocytes were in good agreement with previous studies based on cytochalasin B binding, suggesting that this radioimmunoassay is able to quantify the number of glucose transporters. The assay is completed within 4 h and can be used for simultaneous measurement of GLUT1 in many samples. In addition, it can be applied to the measurement of GLUT1 in several types of tissue.

Amino Acid Sequence↗
PubMed1990

C-terminal truncated glucose transporter is locked into an inward-facing form without transport activity.

The facilitated glucose transporters comprise a structurally related family of proteins predicted to have 12 membrane-spanning domains, with the amino terminus, a relatively large middle loop and the carboxy-terminus all oriented towards the cytoplasm. An alternating conformation model has been proposed to explain the mechanism of facilitated glucose transport. To understand the structure-function relationships, especially the role of the intracellular C-terminal domain, we have modified the rabbit equivalent of the erythroid-type transporter, GLUT1 (ref. 18), using complementary DNA to code for a deletion mutant that lacks most (37 out of 42 amino acids) of the intracellular C-terminal domain. This deletion mutant is expressed at the cell surface of Chinese hamster ovary (CHO) cells, but is functionally inactive, probably because it has lost its capacity to alternate in conformation and so is locked into an inward-facing form.

Animals↗
PubMed1990

Increased liver glucose-transporter protein and mRNA in streptozocin-induced diabetic rats.

The effect of insulin-deficient diabetic states on the rat liver glucose-transporter (L-transporter isoform) protein and mRNA levels were studied. Rats were injected with 65 mg/kg streptozocin to induce diabetes and were maintained for 10 days and then treated with or without insulin for the next 5 days. The L-transporter isoform with apparent Mr of 55,000 was observed to be increased approximately twofold in the membranes from liver homogenates of diabetic rats compared with control rats when assessed by Western blot analysis with an anti-peptide antibody directed against rat L-transporter isoform. Insulin treatment of diabetic rats decreased the amount of L-transporter isoform protein toward levels observed in nondiabetic rats. Northern blot analysis demonstrated similar alterations in the rat L-transporter isoform mRNA that paralleled the changes observed in the L-transporter isoform protein. The increased levels of the L-transporter isoform protein and mRNA in diabetic rats are in marked contrast to the effects of insulin deficiency in rat adipocytes, which specifically decrease the amount of the adipocyte glucose-transporter isoform protein and mRNA. These results suggest that glucose-transporter isoforms in rat liver and adipocytes are regulated by different mechanisms and that an increased synthesis of the L-transporter isoform may contribute to the increased glucose output that occurs from the liver during insulin deficiency.

Amino Acid Sequence↗
PubMed1986

Elevations of cathepsin B and cathepsin L activities in forelimb and hind limb muscles of genetically dystrophic mice.

The combined activities of cathepsin B and cathepsin L were studied in the forelimb and hind limb muscles of dystrophic mice. The activities of these proteases in the forelimb and hind limb muscles of young and adult dystrophic mice were significantly higher than those in normal mice. However, clinical involvement of dystrophy appeared in the hind limbs but not in the forelimbs. We therefore suggest that the increase in protease activity begins at a very early age and that the clinical involvement is not linked with the increase in cathepsin B and L.

Animals↗
PubMed1986

Augmentation of lymphokine-activated killer cell activity in vitro by aloctin A.

Culturing of mouse spleen cells with IL2-containing MLA144 conditioned medium (MLA144 CM) resulted in the induction of cells cytotoxic to syngeneic and allogeneic tumor cells in vitro. The cytotoxicity was markedly augmented by the presence of aloctin A (Alo A), a lectin having anti-tumor activity, during the assay for the cytotoxicity. The results of cytotoxicity assay after treatment with antisera and complement indicated that the killer cells mainly consisted of Thy 1+, Lyt 1-2-, asialo GM1-T cells.

Animals↗
PubMed1985

Induction of K562 lysis in autologous LGL and dense T cell cocultures.

Human large granular lymphocytes (LGL) were cultured with autologous dense T lymphocytes for 0-8 days. The mixed LGL dense T cell cultures had enhanced lytic activity against K562 on day 4, as compared to LGL cultured alone. Dense T cells cultured alone had no K562 lytic activity. The mixed LGL dense T cell cultures were reseparated on day 4 into low dense SRBC- and SRBC+ and into high dense subsets. Both low dense subsets did lyse K562 whereas the high dense subset did not lyse K562. Since the LGL population was the low dense SRBC- cells, the results suggested that the day 4 low dense SRBC+ cytotoxic population in the mixed LGL dense T cell culture originated from noncytotoxic dense T cells. Dense T cells were also separated into OKT8- and OKT8+ subsets and cultured together with LGL for 0-8 days. The LGL dense OKT8- T cell coculture had enhanced K562 lytic activity while the LGL dense OKT8+ T cell coculture had no enhanced lytic activity, as compared to LGL cultured alone. These results indicated that LGL interacted with the dense OKT8- T cells to produce the enhanced K562 lysis.

Antibodies, Monoclonal↗
PubMed1984

Virologic, immunologic, and clinical observations on a patient during the incubation, acute, and convalescent phases of infectious mononucleosis.

One patient with infectious mononucleosis (IM) was studied from the probable time of Epstein-Barr virus (EBV) infection (38 days before the onset of clinically overt disease), during the incubation and acute phases, until 6 months after clinical remission. Analysis of spontaneous outgrowth of EBV-carrying lymphoblastoid cells, by limiting dilution on feeder layer cultures, showed that virus containing B lymphocytes are already present early during the incubation period. Also low interferon serum levels were detected early after infection, and only before the onset of clinical disease. All other studied clinical laboratory and virus-associated variables were within normal range during the incubation phase, but changed to a pattern characteristic of IM in parallel to the clinical symptoms. During the acute disease EBV-associated nuclear antigen (EBNA)-positive cells could be directly detected among the lymphocytes, and antibodies to EBV antigens appeared. Lymphocytes stained by monoclonal antibodies, detecting Ia-like determinants (activated cells) and suppressor cells, increased dramatically, in parallel to a strong increase of functional suppressor cell activity, measured by inhibition of blastogenesis and PWM-induced immunoglobulin production. During the acute phase there was also a decrease of spontaneous cytotoxicity against the NK-sensitive cell line K562, while cytotoxicity (spontaneous) against an autologous EBV-positive lymphoblastoid cell line (LCL) was detected only during this phase. These reactions correlated to the presence of blasts, and the autologous reaction was exerted mainly by Fc-receptor-negative cells. Lymphokine production in response to EBV antigens was also initiated during the acute phase. During the convalescence period the serological and cellular immune parameters adjusted to the pattern of a normal EBV-seropositive person.

Adolescent↗
PubMed1984

Physiological identification of midbrain neurons related to lens accommodation in cats.

Single units were sought in the medial midbrain, whereas lens accommodation of the eye was monitored by using an infrared high-speed optometer. Nineteen of 135 units isolated were identified as accommodation-related units by two criteria: 1) they discharged in synchrony with spontaneously occurring accommodation, and b) the units were driven by stimulating the interpositus nucleus of the cerebellum. In eight other experiments, units were sought that were antidromically activated by stimulating parasympathetic preganglionic fibers to the ciliary ganglion. Eleven of 46 antidromically activated units were identified as accommodation-related units (accommodation-related oculomotor units). Accommodation-related oculomotor units were found in the medial midbrain within 1 mm of the midline. The majority of them were located lateral and dorsal to the Edinger-Westphal nucleus.

Accommodation, Ocular↗
PubMed1984

Immunological characterization of Hodgkin's and non-Hodgkin's lymphoma patients with high antibody titers against Epstein-Barr virus-associated antigens.

We have studied nine Hodgkin's lymphoma (HD) and ten non-Hodgkin's lymphoma (NHL) patients with extraordinarily high anti-viral capsid antigen (VCA) titers (greater than 5120). Controls were 13 HD and 23 NHL patients with anti-VCA titers between 40 and 2560. High anti-VCA titers were present in NHL patients at the time of diagnosis or within 16 months, whereas the rise of anti-VCA titers in HD patients appeared to be a late event during the clinical course of the disease (mean time from diagnosis, 68 months). In particular, we have asked whether the exceptionally high anti-Epstein-Barr virus (EBV) titers in some HD and NHL patients can be correlated to some of the EBV-specific and -nonspecific parameters of cell-mediated immunity. The battery of non-EBV-specific immunological tests included the assessment of natural killer cell activity and the analysis of T-lymphocyte subclasses according to surface markers, together with spontaneous and mitogen-induced DNA synthesis and their helper or suppressor activity on PWM-generated immunoglobulin synthesis. Outgrowth inhibition (Ol) and leukocyte migration inhibition were used to assess EBV-specific cell-mediated immunity. The majority of the high-titer HD and NHL patients showed a drastically reduced OKT4:OKT8 ratio in their peripheral lymphocyte population. Low-titer HD and NHL patients showed no such reduction. There was no strict correlation between the number of OKT8-positive cells and suppressor activity in the functional PWM-induced immunoglobulin production test. Part of the high-titer HD patients showed defective cellular responses in the outgrowth inhibition test, directed against the proliferation of EBV-transformed (EBV-determined nuclear antigen-positive) cells. Some of them showed also a deficient leukocyte migration inhibition response to EBV-determined nuclear antigen but, interestingly, not to early antigen-VCA. In the NHL group, only one of the high-titer patients showed a similar defect. None of the low-titer HD and NHL patients showed such defects.

Adult↗
PubMed1982

Epstein-Barr virus (EBV) receptor implantation onto human B lymphocytes changes immunoglobulin secretion patterns induced by EBV infection.

Mosaic membrane vesicles containing both Epstein-Barr virus (EBV) receptors and Sendai virus envelope proteins were allowed to form by the previously described membrane solubilization and co-reconstitution technique. The vesicles were allowed to fuse with the membranes of normal human B lymphocytes, whereafter the cells were infected with transforming EBV (B95-8 substrain). Compared to similarly infected but otherwise unmanipulated cells, the receptor-implanted lymphocytes responded with a larger number of EBV-determined nuclear antigen positive and immunoglobulin-secreting plaque-forming cells (PFC). Moreover, there was a clear increase of the IgG/IgM PFC ratio in the receptor-implanted B lymphocytes. These results show that not all human B lymphocytes that can potentially be activated by EBV express functional EBV receptors. B lymphocytes programmed to secrete IgG appear to be more defective in this respect than IgM secretors.

Antibody-Producing Cells↗
PubMed1981

Cortical neurons in and around the Clare-Bishop area related with lens accommodation in the cat.

Single-unit discharges in the cat Clare-Bishop area were correlated with spontaneous accommodation responses. No appreciable change was found in accommodation responses evoked by stimulating the Clare-Bishop area, when cerebellar outflow was blocked reversibly by cooling the superior cerebellar peduncle. It is suggested, therefore, that the Clare-Bishop area plays an important role in the lens accommodation system through a pathway independent from that of the cerebellum.

Accommodation, Ocular↗
PubMed1981

Mesencephalic neurons controlling lens accommodation in the cat.

Thirty units were found in the midbrain of the anesthetized cat which discharged in correlation with spontaneously occurring lens accommodation. The frequency of spike potentials increased before the onset of the accommodation response. Increased discharges were followed by a silent period. These units were driven orthodromically by stimulating the interpositus nucleus of the cerebellum and the posterior commissure. Eleven of these units were identified antidromically as parasympathetic oculomotor neurons.

Animals↗
PubMed1981

Human B lymphocytes activated by Epstein-Barr virus (EBV) or by mitogens suppress mitogen-induced immunoglobulin production.

Polyclonal activation of human B lymphocytes by LPS or protein A, alone or in combination or by Epstein-Barr virus (EBV), generates suppressive conditions that inhibit the response of human B lymphocytes to pokeweed mitogen (PWM), measured by the induction of immunoglobulin-secreting cells (PFC). Moreover, EBV-transformed B cell lines of normal or neoplastic (Burkitt lymphoma) origin also suppressed the PWM-induced immunoglobulin production of normal B cells. Cell separation experiments have shown that mitogen activated autologous B cells stimulate suppressor T cells in a similar way as B cell-derived lymphoblastoid cell lines. The significance of this phenomenon is considered in relation to the escape of the activating microorganism or virus from immune control and the occurrence of network interactions within the immune system.

Antibody-Producing Cells↗