Biomedical subjects

M Waki

Publications and source records attributed to M Waki.

At least 37 records · Page 2Linked to original sources

PubMed1997

Antisense src expression inhibits tyrosine phosphorylation of Shc and its association with Grb2 and Sos which leads to MAP kinase activation in U937 human leukemia cells.

We constructed a recombinant plasmid which expresses antisense src RNA after dexamethasone (Dexa) treatment, and transfected it into U937 human monoblastic leukemia cells (U937-ASRC). Induction of antisense src RNA expression diminished the amounts of c-Src and its protein tyrosine kinase (PTK) activity in U937-ASRC cells. The declines in c-Src and its PTK activity subsequently reduced the proliferation of U937-ASRC cells. To elucidate the growth signal transduction pathway downstream of c-Src, tyrosine phosphorylation of Shc was examined in U937-ASRC cells treated with Dexa. The decline in c-Src by induction of antisense src RNA expression decreased the level of tyrosine phosphorylation of Shc. Immunoprecipitated c-Src directly phosphorylated immunoprecipitated Shc on tyrosine residues in vitro. The amounts of Grb2 and Sos co-immunoprecipitated with Shc were decreased after Dexa treatment. However, the amount of Sos co-immunoprecipitated with Grb2 was apparently not affected by Dexa treatment. These results indicate that Grb2 and Sos constitutively associate with each other in U937 cells. Furthermore, the level of phosphorylation on tyrosine (204) essential for MAP kinase activation was decreased after Dexa treatment. Taken together with all these findings, it is suggested that c-Src directly phosphorylates Shc on tyrosine residues, which in turn binds to Grb2 constitutively associated with Sos to form a Shc-Grb2-Sos complex, and that the complex formation is coupled with MAP kinase activation mediated by Ras activation in U937 cells.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Altered interleukin-2 receptor alpha-chain is expressed in human T-cell leukaemia virus type-I-infected T-cell lines and human peripheral blood mononuclear cells of adult T-cell leukaemia patients through an alternative splicing mechanism.

A polymerase chain reaction (PCR) method was used to detect the interleukin-2 receptor alpha-chain (IL-2R alpha) chain which lacks the conventional transmembrane (TM) domain in mRNA from human T-cell leukaemia virus type-I (HTLV-I)-infected cell lines or peripheral blood mononuclear cells (PBMC) isolated from adult T-cell leukaemia (ATL) patients. Primer pairs encompassing the TM domain were selected to generate a 357-base pair (bp) fragment. A 146-bp PCR product was observed consistently in addition to the target 357-bp PCR product in mRNA from HTLV-I-infected cell lines, such as MT-1, MT-2, MT-4 and in PBMC isolated from ATL patients. However, this 146-bp PCR product was undetectable in HTLV-I-negative cell lines. The product was also detected in PBMC from normal individuals if activated in vitro with phytohaemagglutinin but not without stimulation. DNA sequence analyses revealed that exons from 5 to 7, which define a 211-bp region containing the conventional TM domain, were deleted in the 146-bp PCR product. The C-terminal amino acid sequence starting from Gly174 of the 211-bp-deleted molecule was distinct from that of conventional IL-2R alpha as a result of an altered reading frame. We identified a 45000 MW peptide generated from IL-2R alpha mRNA through this exon skip in cell lysate of MT-1 and MT-2 by Western blot analyses using an antibody raised against the peptides specific to an altered IL-2R alpha. Our results indicate that an altered IL-2R alpha chain is expressed in HTLV-I-infected T lymphocytic cell lines and in ATL patients.

Adult↗

PubMed1997

Cloning and characterization of the gene (farA) encoding the receptor for an extracellular regulatory factor (IM-2) from Streptomyces sp. strain FRI-5.

IM-2 is a butyrolactone autoregulator that controls production of blue pigment and nucleoside antibiotics in Streptomyces sp. strain FRI-5. An IM-2-specific receptor gene, farA, was cloned from strain FRI-5, and nucleotide sequencing revealed that the farA gene consists of 666 bp encoding a 221-amino-acid protein of 24.3 kDa with an NH2-terminal amino acid sequence identical to that of purified native receptor. Another gene, farX, encoding a homolog of AfsA of Streptomyces griseus, was present upstream of farA. The monocistronic nature of the farA transcript was shown by Northern blot hybridization, and the transcript level increased upon addition of IM-2. Recombinant FarA expressed in and purified from E. coli showed clear ligand specificity toward IM-2, with a dissociation constant (Kd) for [3H]IM-2-C5 of 18.2 nM. FarA showed high overall homology to BarA (virginiae butanolide receptor from S. virginiae) and ArpA (A-factor receptor from S. griseus). Sequence alignment of the three receptor proteins revealed that the NH2-terminal region containing a helix-turn-helix DNA binding motif was highly conserved. The DNA binding motif is common in procaryotic repressors of the TetR family, suggesting that all the Streptomyces autoregulator receptors may act as transcriptional repressors.

4-Butyrolactone↗

PubMed1997

A case of membranous glomerulonephritis associated with gastric cancer.

We describe a patient with gastric cancer and membranous glomerulonephritis (MGN). The patient, a 61-year-old male, was admitted to our Hospital in May, 1996, because of proteinuria and hyperlipidemia persisting for a year. Laboratory examination filled the criteria of nephrotic syndrome and renal biopsy revealed MGN of stage II. Prednisolone therapy (40 mg/day p.o.) was started, followed by a gradual decrease in proteinuria from 4.5 g/day to 0.1 g/day. Endoscopic examination was performed because of stomach-ache revealed advanced gastric cancer of Borrmann 4. Desiring for a conservative therapy, he was discharged and moved to a hospice. In literature review, MGN is the most frequent lesion among various glomerular diseases associated with malignancy, such as the lung, stomach, and colon, particularly at an elderly ages, and sometimes antedates the detection of malignancy, as in the present case. In several cases with MGN, immune-complexes composed of tumor antigens, such as carcino-embryonic antigen, and antibodies have been reported to deposit in basement membrane of glomeruli, causing MGN. In the renal and gastric cancer tissues of the present case, the presence of three novel tumor-associated antigens, Span-1, Thomsen-Friedenreich antigen (T antigen) and F1 alpha antigen, was examined, using a immuno-peroxidase method. Although none of these three antigens were immuno-stained in the renal tissue, clinical course and literature review suggest that MGN in this patient seems to be associated with gastric cancer, which may have produced MGN-causing tumor antigens other than the three antigens. It should be emphasized that malignancy should be carefully and routinely examined in patients with MGN, particularly at elderly ages.

Glomerulonephritis, Membranous↗

PubMed1996

An anti-HIV peptide, T22, forms a highly active complex with Zn(II).

T22 ([Tyr5,12, Lys7]-polyphemusin II) has been shown to have strong anti-human immunodeficiency virus (HIV) activity, comparable to that of 3'-azide-2', 3'-dideoxythymidine (AZT). T22 takes an antiparallel beta-sheet structure maintained by two disulfide bridges and contains two antiparallel repeats of Cys-Tyr-Arg-Lys-Cys. As reported herein, fully reduced T22 was found by HPLC and ion spray mass spectrometric analyses to form a complex in a molar ratio of 1:1 with Zn(II) ion at neutral pH in aqueous solution. Complexation of Zn(II) ion to this peptide appears to result in tetracoordinate bonding to sulfur atoms of four Cys residues. We also found that the anti-HIV activity of the T22-Zn(II) complex was fourfold stronger than that of T22.

Amino Acid Sequence↗

PubMed1996

Analysis of the interaction of an anti-HIV peptide, T22 ([Tyr5, 12, Lys7]-polyphemusin II), with gp120 and CD4 by surface plasmon resonance.

We have previously found that T22 ([Tyr5, 12, Lys7]-polyphemusin II) exhibits strong anti-human immunodeficiency virus (HIV) activity comparable to that of 3'-azido-2', 3'-dideoxythymidine (AZT). The inhibition mechanism of T22 on HIV-replication has not been elucidated precisely yet, and hence the target molecules of T22 have not been identified. However, our recent research suggested that T22 exerts its effect by blocking virus-cell fusion at an early stage of HIV infection and that T22 might interact with an HIV envelope protein and/or a T-cell surface protein, both of which are critical for HIV infection. In this paper we demonstrated that T22 binds specifically to both gp120 (an envelope protein of HIV) and CD4 (a T-cell surface protein) and that both bindings can be inhibited by an anti-T22 antibody, using biosensor technology (BIAcoreTM) based on the principles of surface plasmon resonance. Linearization by the BIAcoreTM system (BIAlogue software) and nonlinear least squares analysis by curve fitting with exponential equations showed that both interactions have close dissociation constants (approximately 10(-7) M). The present study suggests that T22 inhibits the virus-cell fusion process through binding to both gp120 and CD4.

Amino Acid Sequence↗

PubMed1996

Interaction of an anti-HIV peptide, T22, with gp120 and CD4.

T22 ([Tyr5,12, Lys7]-polyphemusin II) has been shown to have strong anti-human immunodeficiency virus (HIV) activity. The precise mechanism of action of T22 on HIV-replication has not been elucidated yet, nor have the targets of T22 been identified. However, our previous research suggested that T22 exerts its effect by blocking virus-cell fusion and that T22 might interact with an HIV envelope protein and/or a T-cell surface protein. Herein we use a novel biosensor based on the principles of surface plasmon resonance (BIAcore) to demonstrate that T22 binds specifically to both gp120 (an envelope protein of HIV) and CD4 (a T-cell surface protein) and that both bindings can be inhibited by an anti-T22 antibody. The data obtained suggest that T22 inhibits virus-cell fusion through the double binding to the above two proteins.

Amino Acid Sequence↗

PubMed1996

Small cell carcinoma of the uterine cervix with syndrome of inappropriate antidiuretic hormone secretion.

In a 59-year-old woman suffering from the syndrome of inappropriate antidiuretic hormone secretion, a small cell carcinoma of the uterine cervix was detected. The tumor was immunoreactive for antidiuretic hormone as well as for neuron specific enolase, chromogranin A, and Leu-7, but not vimentin. Electron microscopic examination of the tumor revealed neurosecretory granules. To our knowledge, this is only the second report of the syndrome of inappropriate antidiuretic hormone secretion with small cell carcinoma of the uterine cervix and the first one confirmed immunohistopathologically.

CD57 Antigens↗

PubMed1995

Absorption characteristics of chemically modified-insulin derivatives with various fatty acids in the small and large intestine.

Absorption characteristics of insulin derivatives chemically modified with various fatty acids in the intestine were determined by in situ loop and in vitro modified Ussing chamber methods. The pharmacological activities of these acyl derivatives, as assessed by their hypoglycemic effects after intravenous administration, were reduced upon increasing the carbon number of the fatty acid(s) chemically attached to native insulin. However, high pharmacological activities were seen when mono-and dicaproyl derivatives were administered intravenously. The absorption of insulin after its small intestinal administration could be hardly improved by acylation. In contrast, its absorption after the large intestinal administration was increased by increasing the number of caproic acid molecules attached to insulin. Furthermore, by an in vitro modified Ussing chamber method, it was revealed that the permeability of insulin across both the duodenal and colonic mucous membranes was also improved by increasing the number of caproic acid molecules. These in situ and in vitro results indicated that the chemical modification of insulin with fatty acids was a useful approach for improving insulin absorption from the large intestine.

Animals↗

PubMed1995

Solution-phase synthesis of an anti-human immunodeficiency virus peptide, T22 ([Tyr5,12,Lys7]-polyphemusin II), and the modification of Trp by the p-methoxybenzyl group of Cys during trimethylsilyl trifluoromethanesulfonate deprotection.

T22 ([Tyr5,12,Lys7]-polyphemusin II) was previously synthesized by a solid-phase method and was found to have a strong anti-human immunodeficiency virus (HIV) activity, comparable to that of 3'-azido-2',3'-dideoxy-thymidine (AZT). In the present study, the solution-phase synthesis of T22 was attempted in order to produce this peptide on a large scale. An 18-residue peptide amide corresponding to the entire amino acid sequence of T22 was synthesized by assembling four peptide fragments and two amino acid derivatives, followed by thioanisole-mediated deprotection with 1 M trimethylsilyl trifluoromethanesulfonate (TMSOTf) in trifluoroacetic acid followed by air-oxidation. During this deprotection, a significant by-product derived from the transfer of the p-methoxybenzyl (MBzI) group from the sulfhydryl group of the cysteine residue to the side chain of the tryptophan residue was formed. This side reaction was found to be efficiently suppressed by adopting a two-step deprotection procedure using silver trifluoromethanesulfonate (AgOTf)-TMSOTf or trimethylsilyl bromide (TMSBr)-TMSOTf.

Amino Acid Sequence↗

PubMed1995

Synthesis of protegrin-related peptides and their antibacterial and anti-human immunodeficiency virus activity.

All disulfide analogs (types I, II and III) of protegrin (PG)-1, an 18-residue antimicrobial peptide having two intramolecular disulfide bonds, were synthesized using regioselective disulfide bond formation. Random air-oxidation of the fully reduced PG-1 formed the type III PG-1. In addition, a type III analog containing an amidated carboxy-terminal residue was also prepared. Each analog showed significant and different antibacterial and anti-human immunodeficiency virus (HIV) activity. Deletion of two disulfide bridges caused a significant decrease in activity.

Amino Acid Sequence↗

PubMed1994

Structure-activity relationships of an anti-HIV peptide, T22.

T22 ([Tyr5,12,Lys7]-polyphemusin II) has been shown to have a strong anti-human immunodeficiency virus (HIV) activity comparable to that of 3'-azido-2',3'-dideoxythymidine (AZT). We studied the structure-anti-HIV activity relationships of T22 and determined the following information. The number of Arg residues in the N-terminal and C-terminal regions of T22 is closely related with anti-HIV activity. Disulfide rings, especially the major disulfide ring, are indispensable for anti-HIV activity and maintenance of the secondary structure. Between two repeats of Tyr-Arg-Lys, which are a characteristic structure contained in T22, Tyr-Arg-Lys in the N-terminal portion is more closely related with anti-HIV activity. We found some compounds having a higher selectivity index (50% cytotoxic concentration/50% effective concentration) than that of T22.

Amino Acid Sequence↗

PubMed1994

Antisense src expression inhibits proliferation and erythropoietin-induced erythroid differentiation of K562 human leukemia cells.

We constructed a recombinant plasmid which expresses antisense src RNA in human cells and used it as a tool for investigating the role of pp60c-src in proliferation and differentiation of K562 human leukemia cells. In erythropoietin (EPO)-responsive cells, EPO induces rapid tyrosine phosphorylation of several cellular proteins including EPO receptor (EPOR) although EPOR has no tyrosine kinase domain. Here we show that antisense src RNA expression suppresses pp60c-src synthesis in the recombinant plasmid-transfected K562 cells, reduces the proliferation and inhibits hemoglobin synthesis and glycophorin A expression promoted by EPO in K562 cells. These findings suggest that pp60c-src plays crucial roles in the proliferation and EPO-induced erythroid differentiation of K562 cells.

Cell Differentiation↗

PubMed1994

Antisense src expression inhibits U937 human leukemia cell proliferation in conjunction with reduction of c-myb expression.

To elucidate the role of pp60c-src in U937 human monoblastoid leukemia cell proliferation, recombinant plasmids containing the src gene or myb gene, which could produce antisense src or antisense myb RNA after dexamethasone treatment, were constructed and transfected into U937 cells (U937-ASRC, U937-AMYB). pp60c-src synthesis in U937-ASRC was diminished by the third day after induction of antisense src RNA and the cell proliferation was reduced, furthermore, the amount of p75c-myb was significantly decreased by the third day. p75c-myb synthesis in U937-AMYB was diminished by the second day after induction of antisense myb RNA and the cell growth was significantly inhibited but the amount of pp60c-src in U937-AMYB was not reduced. These results suggest that a decrease in the amount of pp60c-src leads to an inhibition of p75c-myb expression and subsequent reduction in the U937 cell proliferation.

Cell Division↗

PubMed1994

Soluble interleukin-6 receptors released from T cell or granulocyte/macrophage cell lines and human peripheral blood mononuclear cells are generated through an alternative splicing mechanism.

To detect transcripts encoding the interleukin-6 receptor (IL-6R) molecule lacking the transmembrane (TM) domain, in various cell lines and peripheral blood mononuclear cells (PBMC), we used the polymerase chain reaction (PCR) with primer pairs that flank the TM domain and which were selected to generate a 398-bp fragment. We detected 398-bp and 304-bp DNA molecules in the PCR products of the U1, J22HL60, MT-2, MT-4, U937 and HL60 cell lines and of PBMC isolated from several individuals. The sequencing analysis of both DNA molecules showed that a 94-bp region consisting of the TM domain of IL-6R was deleted in the 304-bp molecule. Moreover, we detected a soluble (s) IL-6R protein of 45 kDa in culture supernatants of the MT-2, MT-4 and U937 cell lines by radioimmunoprecipitation using specific antibodies against sIL-6R. Our results indicate that active deletion of the TM domain by alternative splicing of mRNA represents one mechanism for release of sIL-6R into the culture supernatants of cells, or into serum or urine.

Alternative Splicing↗

PubMed1994

Acute effect of low-calorie and low-carbohydrate diet on serum triiodothyronine-response to glucose ingestion and its relation to glucose tolerance.

This study was conducted to elucidate the mechanism of glucose intolerance due to carbohydrate restriction. 15 non-diabetic patients, aged 51 +/- 3 yr, were placed under dietary condition-I (2,012 kcal/day; carbohydrate 299 g) for more than 3 days after admission and then condition-I was substituted by condition-II (1,156 kcal/day; carbohydrate 139 g) for 2 weeks. At the end of condition-I and -II, 75 g OGTT was performed. After the comparison between condition-I and -II, patients were subdivided into two groups, namely, glucose tolerance-improved (n = 8) and -impaired (n = 7) groups. In the former, sigma glucose (mg/dl) during OGTT decreased from 898.1 +/- 28.0 to 738.5 +/- 36.0 (p < 0.05) and sigma IRI (microU/ml) decreased from 418.8 +/- 60.1 to 300.7 +/- 33.5 (p < 0.05) but sigma dT3 (ng/dl) increased from 11.3 +/- 14.7 to 70.3 +/- 15.1 (p < 0.05). In the latter, sigma glucose (mg/dl) increased from 774.6 +/- 46.6 to 867.1 +/- 50.1 (p < 0.05) and sigma IRI (microU/ml) increased from 323.4 +/- 47.8 to 400.8 +/- 47.8 (p < 0.05) but sigma dT3 (ng/dl) decreased from 45.3 +/- 7.1 to 20.0 +/- 14.7 although it did not reach a level of statistical significance. These results suggest that blunted serum T3-response to glucose ingestion is linked to the mechanism of glucose intolerance due to carbohydrate restriction.

Blood Glucose↗

PubMed1994

Noninvasive quantitative evaluation of early atherosclerosis and the effect of monatepil, a new antihypertensive agent. An interim report.

In two studies on the same group of patients we evaluated noninvasive methods of assessing atherosclerosis and determined the effect of the new calcium channel-blocking agent monatepil on the progression of early atherosclerosis in humans. Computed tomography (CT) of the lower abdominal aorta and ultrasonography of the carotid arteries were used as noninvasive methods to determine the extent of atherosclerosis. To evaluate the CT images, we developed a new medical image analysis program. This enabled aortic calcification volume (ACV) to be quantified using plain CT images, and aortic wall volume (AWV) and aortic wall and calcification volume (AWCV) to be quantified from contrast CT images. Interobserver coefficients of variation of ACV, AWV, and AWCV (n = 8) were 4.7, 2.4, and 5.0%, respectively. In the monatepil study, the effect of the drug on serum lipid profiles was evaluated. Preliminary results show that shortly after monatepil administration, total serum cholesterol levels decreased significantly from 253.8 +/- 35.6 to 244.8 +/- 38.6 mg/dL (P < .009) and triglyceride levels tended to decrease. A positive correlation between the change in total cholesterol and changes in mean platelet volume was found (P = .028). Fasting immunoreactive insulin levels decreased in the four patients in which they were determined. Although this is a preliminary study, results indicate that CT of the lower abdominal aorta in combination with our new analysis program may be a precise, reproducible means of assessing early atherosclerosis. We have also shown that monatepil significantly decreases total cholesterol levels. However, the long-term effects of monatepil on the progression of atherosclerosis remain to be determined.

Aged↗

PubMed1994

Tissue residues of sulfadimethoxine following dietary administration to broiler-chickens.

Broilers were divided into four groups and the first group served as the control. The second, third and fourth groups were given feed containing 25, 50 and 100 mg/kg of sulfadimethoxine (SDM), respectively, for 21 days, and thereafter each group received the SDM free feed. On certain days during the experiment period, three broilers in each group were sacrificed and tissues, including blood, heart, liver, spleen, gizzard, thigh muscle, breast muscle and fat, were collected and residual SDM were determined by HPLC. Two days after withdrawal, SDM in each tissue had decreased to below the detection limit of 0.01 microgram/g.

Animal Feed↗