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1.
J Dent Res ; 99(4): 429-436, 2020 04.
Article in English | MEDLINE | ID: mdl-31986066

ABSTRACT

Although many variants of the parathyroid hormone 1 receptor (PTH1R) gene are known to be associated with primary failure of eruption (PFE), the mechanisms underlying the link remains poorly understood. We here performed functional analyses of PTH1R variants reported in PFE patients-namely, 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q)-using HeLa cells with a lentiviral vector-mediated genetic modification. Two particular variants, P119L and P132L, had severe reduction in a level of N-linked glycosylation when compared with wild-type PTH1R, whereas the other 2 showed modest alteration. PTH1R having P119L or P132L showed marked decrease in the affinity to PTH1-34, which likely led to severely impaired cAMP accumulation upon stimulation in cells expressing these mutants, highlighting the importance of these 2 amino acid residues for ligand-mediated proper functioning of PTH1R. To further gain insights into PTH1R functions, we established the induced pluripotent stem cell (iPSC) lines from a patient with PFE and the heterozygous P132L mutation. When differentiated into osteoblastic-lineage cells, PFE-iPSCs showed no abnormality in mineralization. The mRNA expression of RUNX2, SP7, and BGLAP, the osteoblastic differentiation-related genes, and that of PTH1R were augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active vitamin D3 induced the expression of RANKL, a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control and PFE-iPSCs. In sharp contrast, exposure to PTH1-34 resulted in no induction of RANKL mRNA expression in the cells expressing P132L variant PTH1R, consistent with the idea that a type of heterozygous PTH1R gene mutation would spoil PTH-dependent response in osteoblasts. Collectively, this study demonstrates a link between PFE-associated genetic alteration and causative functional impairment of PTH1R, as well as a utility of iPSC-based disease modeling for future elucidation of pathogenesis in genetic disorders, including PFE.


Subject(s)
Receptor, Parathyroid Hormone, Type 1/genetics , Tooth Diseases , Tooth Eruption , HeLa Cells , Humans , Mutation , Parathyroid Hormone
2.
J Dent Res ; 86(2): 163-8, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17251517

ABSTRACT

Dental preparation sometimes causes transient congestion, edema, and necrosis of the pulp. We hypothesized that nitric oxide (NO) is involved in the pathophysiological changes in pulp after preparation. The mRNA and protein expression of the inducible isoform of NO synthase (iNOS) was examined in murine pulp after dental preparation. The effects of NO on the proliferation, mineralization, and apoptosis of pulp cells were also studied in vitro. We found that not only iNOS, but also mRNAs for alkaline phosphatase and plasma membrane glycoprotein-1, were expressed in the pulp after preparation. NOC-18, an NO donor, suppressed the proliferation of pulp cells without inducing cell death, whereas it promoted the mineralization of cells cultured in the presence of beta-glycerophosphate, ascorbic acid, dexamethasone, and KH(2)PO(4). Under these conditions, NOC-18 induced the apoptosis of pulp cells. These results suggest that NO regulates the growth, apoptosis, and mineralization of pulp cells.


Subject(s)
Dental Cavity Preparation/adverse effects , Dental Pulp/metabolism , Nitric Oxide Synthase Type II/biosynthesis , Nitric Oxide/physiology , Alkaline Phosphatase/biosynthesis , Animals , Apoptosis , Cell Differentiation , Cell Growth Processes/drug effects , Cell Growth Processes/physiology , Cells, Cultured , Dental Pulp/cytology , Dental Pulp/physiopathology , Enzyme Induction , Male , Mice , Mice, Inbred C57BL , Nitric Oxide Donors/pharmacology , Nitroso Compounds/pharmacology , Phosphoric Diester Hydrolases/biosynthesis , Pulpitis/etiology , Pyrophosphatases/biosynthesis , Tooth Calcification
3.
J Control Release ; 79(1-3): 229-42, 2002 Feb 19.
Article in English | MEDLINE | ID: mdl-11853934

ABSTRACT

A novel linker consisting of poly(ethylene glycol) (PEG) and dipeptide was used for conjugation of adriamycin with tumor-specific monoclonal antibody, NL-1, to confirm that the linker can be cleaved selectively with the tumor specific enzyme to express cytotoxicity of the anti-tumor agent. Initially, adriamycin-conjugated PEG linkers through different amino acid compositions, alanyl-valine (Ala-Val), alanyl-proline (Ala-Pro), and glycyl-proline (Gly-Pro) sequences, were prepared to confirm selective digestion with model enzymes. Adriamycin was released by particular model endoproteases, thermolysin and proline endopeptidase, from the linkers with different efficiency. When conjugates were prepared using these adriamycin-bound linkers, conjugates had no loss of binding affinity and specificity for common acute lymphoblastic leukemia antigen (CALLA) expressed on the Daudi cell surfaces as the target of NL-1 antibody. In addition, adriamycin release from the conjugates was also confirmed by incubating them with specific proteases. Tumor cell growth was inhibited dose-dependently for the conjugates carrying Ala-Val and Gly-Pro linkers, whereas significant inhibitory effect was abolished for the conjugate carrying Ala-Pro linker, indicating that cytotoxic effect can be controlled by specificity of antibody and composition of linker peptide. IC(50) for Ala-Val linked conjugate was approximately 3.5 microg/ml and that for Gly-Pro linked conjugate was 5.2 microg/ml. PEG-dipeptidyl linker demonstrated here will be an effective tool for the preparation of immunoconjugate, especially specific activation of anti-tumor agents at desired tumor tissues.


Subject(s)
Antibodies, Monoclonal/chemistry , Antineoplastic Agents/chemistry , Doxorubicin/chemistry , Immunotoxins/chemistry , Polyethylene Glycols/chemistry , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/toxicity , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/toxicity , Binding Sites , Chemistry, Pharmaceutical , Dose-Response Relationship, Drug , Doxorubicin/pharmacokinetics , Doxorubicin/toxicity , Drug Carriers/chemistry , Drug Carriers/isolation & purification , Drug Carriers/pharmacokinetics , Drug Carriers/toxicity , Drug Evaluation, Preclinical/methods , Drug Screening Assays, Antitumor/methods , HeLa Cells/drug effects , HeLa Cells/immunology , Humans , Immunotoxins/isolation & purification , Immunotoxins/pharmacokinetics , Immunotoxins/toxicity , Polyethylene Glycols/pharmacokinetics , Polyethylene Glycols/therapeutic use , Solvents , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/immunology
4.
J Hypertens ; 19(9): 1643-50, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11564985

ABSTRACT

OBJECTIVE: Increased Na+-H+ exchanger activity (NHE) has been reported as an intermediate phenotype in hypertensive subjects, particularly those with insulin resistance. To investigate whether NHE abnormality plays a role in hypertension, Wistar fatty rat (WFR) with overt obesity, hyperglycemia and marked hyperinsulinemia was examined. METHODS: WFR and Wistar lean rats (WLR) as a control (n = 12, each) were fed either with normal (0.38%) or high sodium (4% NaCl) diet for 12 weeks and then sacrificed to examine platelets NHE activity. RESULTS: Mean arterial pressure (MAP) was higher in WFR than in WLR (113 +/- 4 versus 96 +/- 7 mmHg, P < 0.05) under a normal chow. Vmax values of NHE activity were significantly higher in WFR than in WLR. WFR fed with a high sodium diet showed higher MAP than those with a normal chow (128 +/- 3 versus 113 +/- 4 mmHg, P < 0.05). Though Km values were not different between WFR and WLR under a normal chow, both maximal transport rate (Vmax) and half maximal transport (Km) values were significantly higher in WFR with a high salt diet than those with a control diet. Vmax showed significant correlation with MAP, whereas Km values correlated with immunoreactive insulin (IRI) levels. Significant interaction between dietary sodium intake and the strain differences was observed both on blood pressure and on IRI levels by two-way analysis of variance (ANOVA). CONCLUSION: WFR presented salt-sensitive blood pressure elevation. NHE activity was enhanced in WFR in correlation with the blood pressure. These results suggest that augmented NHE activity contributes to the development of salt-sensitive blood pressure elevation in WFR.


Subject(s)
Blood Pressure/drug effects , Insulin Resistance , Obesity/physiopathology , Sodium Chloride/pharmacology , Sodium-Hydrogen Exchangers/physiology , Animals , Biological Transport/drug effects , Diet , Dose-Response Relationship, Drug , Rats , Rats, Wistar , Sodium Chloride/administration & dosage , Sodium-Hydrogen Exchangers/blood , Thinness
5.
Clin Nephrol ; 54(5): 366-73, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11105797

ABSTRACT

BACKGROUND: Insulin resistance is associated with advanced and moderate chronic renal failure (CRF). However, insulin resistance in chronic glomerulonephritis (CGN) before onset of frank renal dysfunction is not fully evaluated. We attempted to investigate the association of insulin resistance with mild renal dysfunction and with abnormal calcium homeostasis. PATIENTS AND METHODS: Eighteen young, lean non-diabetic male patients with biopsy-proven CGN (age 30 +/- 7 years, body mass index 23.0 +/- 2.5 kg/m2) were enrolled. Insulin sensitivity was estimated by the glucose infusion rate (M value) during euglycemic hyperinsulinemic clamping for 60 to 120 min. Calcium-related parameters including intracellular calcium concentrations ([Ca2+]i) in platelets were also measured. Renal function was normal or slightly impaired (serum creatinine, 1.0 +/- 0.2 mg/dl; glomerular filtration rate (GFR), 68 to 131 ml/min/1.48 m2). We divided subjects into an insulin-sensitive (IS) group (M value > 7.3 mg/kg/min, the overall mean) and an insulin-resistant (IR) group (M value < 7.3 mg/kg/min). RESULTS: During a 75 g oral glucose tolerance test, the plasma glucose concentration at 120 min after glucose loading and the immunoreactive insulin concentration at 60 min were significantly higher in the IR group. GFR was notably lower in the IR group than in the IS group (p = 0.0003), and was significantly correlated with insulin sensitivity (p < 0.02, r = 0.58). The basal [Ca2+]i was significantly higher in the IR than in the IS group (39 +/- 9 vs. 30 +/- 9 nM, p < 0.05). CONCLUSION: Mild renal dysfunction and elevated basal [Ca2+]i are associated with insulin resistance in CGN.


Subject(s)
Glomerulonephritis/physiopathology , Insulin Resistance , Renal Insufficiency/physiopathology , Adult , Blood Platelets/chemistry , Calcium/metabolism , Chronic Disease , Glomerular Filtration Rate , Glomerulonephritis/complications , Glucose Clamp Technique , Glucose Tolerance Test , Homeostasis , Humans , Insulin/blood , Male , Renal Insufficiency/complications , Renal Plasma Flow
6.
J Control Release ; 69(1): 27-41, 2000 Oct 03.
Article in English | MEDLINE | ID: mdl-11018544

ABSTRACT

A model compound of anti-tumor agent, segment B of duocarmycin derivative DU-86, was conjugated to tumor-specific antibody via a cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine (Ala-Val), to confirm the feasibility of the linker for application to immunoconjugate. The release of segment B from the linker was evaluated by HPLC analysis. When segment B was derivatized to have an amino residue and then linked to PEG through a dipeptide, segment B was cleaved at the peptide bond by a particular enzyme, thermolysin (EC 3.4.24.4), but not by plasmin (EC 3.4.2 1.7.), indicating that certain protease specifically expressed at the tumor site would be capable of peptide-specific digestion and release of anti-tumor agent since a thermolysin-like enzyme has been reported to be expressed at many tumor cells. Furthermore, the results showing that cell extract from G361 human melanoma had an ability to digest the linker peptide while the linker was stable in normal human serum suggested the tumor-specific activation of the conjugated agent. Segment B was conjugated via the linker to murine monoclonal antibody KM641 reactive to GD3 ganglioside to form immunoconjugate and the quantitative release of segment B under the treatment with the enzyme was also confirmed. These results indicate the possibility of double targeting based on both the recognition ability of tumor specific antibody and tumor specific activation of the anti-tumor agents to enhance tumor treatment efficacy and to decrease unwanted side effects.


Subject(s)
Dipeptides/chemistry , Immunoconjugates/chemistry , Polyethylene Glycols/chemistry , Antibodies, Monoclonal/chemistry , Antineoplastic Agents/chemistry , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Duocarmycins , Enzyme-Linked Immunosorbent Assay , Magnetic Resonance Spectroscopy , Pyrrolidinones/chemistry
7.
Bioorg Med Chem ; 8(8): 2175-84, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11003162

ABSTRACT

Novel anti-tumor agent, duocarmycin derivative DU-257, was designed and synthesized to prepare immunoconjugate in order to confirm the feasibility of enzymatically cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine. Oxyethylamine arm was introduced at 4-methoxy position of segment B of DU-86 to form DU-257 and evaluated its property. DU-257 retained similar stability and potency with DU-86 while enhanced hydrophilicity suggested. DU-257 was condensed to the PEG-dipeptidyl linker through carboxyl terminal of dipeptide, and enzymatic release of DU-257 using a model enzyme, thermolysin, similar enzyme of which was shown to be overexpressed at various tumor sites, was evaluated by HPLC analysis. Cleavage between the linker amino acids by the model protease and release of DU-257 as valine conjugated form was confirmed. The enzymatically released form of DU-257 expressed its cytotoxicity without loss of the potency for HeLaS3 and SW1116 tumor cell lines, although the efficacy was different in individual cells. DU-257 was then conjugated through the linker to KM231 monoclonal antibody specifically reactive to GD3 antigen which was shown to be expressed on the surface of many malignant tumors such as SW1116. The conjugate retained its binding specificity for SW1116 cell with a similar activity with KM231. Furthermore, the conjugate showed significant growth inhibition on SW1116 cell at a concentration of 75 microg/mL while no effect on antigen negative cell, HeLaS3. These results suggest that the conjugate retained its anti-tumor effect only when it bound on and was activated at the target cell, simultaneously. DU-257 will be one of the candidate of anti-tumor agent for application to immunoconjugate and its conjugate with KM231 via PEG-dipeptidyl linker will be a useful entity for cancer therapy related to sLe(a) expression.


Subject(s)
Antibiotics, Antineoplastic/chemical synthesis , Immunotoxins/chemistry , Indoles/chemical synthesis , Pyrrolidinones/chemistry , Pyrrolidinones/chemical synthesis , Antibiotics, Antineoplastic/administration & dosage , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/immunology , Dose-Response Relationship, Drug , Drug Delivery Systems , Duocarmycins , Flow Cytometry , HeLa Cells , Humans , Immunotoxins/administration & dosage , Immunotoxins/metabolism , Indoles/administration & dosage , Indoles/chemistry , Indoles/immunology , Molecular Structure , Polyethylene Glycols/chemistry , Pyrrolidinones/administration & dosage , Pyrrolidinones/immunology , Tumor Cells, Cultured
8.
J Hypertens ; 18(7): 911-7, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10930189

ABSTRACT

OBJECTIVE: To investigate the effects of oestrogen supplementation after ovariectomy on systolic blood pressure and platelet aggregation on different sodium content diet in the female Dahl salt-sensitive rats. METHODS: At 12 weeks of age, rats were ovariectomized or sham-operated and were fed either a high NaCl (8%) or low NaCl (0.3%) diet Ovariectomized rats were treated with either 17beta-oestradiol or placebo for 8 weeks, whereas sham-operated rats received placebo alone. After 8 weeks, the systolic blood pressure and platelet aggregation were measured and analysed by two-way analysis of variance. RESULTS: The systolic blood pressure of ovariectomized rats was significantly higher than that of sham-operated rats, and this increase in systolic blood pressure was suppressed by oestrogen supplementation. Systolic blood pressure was inversely correlated with plasma 17beta-oestradiol levels (r= -0.77, P< 0.01) and with the uterus weight to body weight ratio (r = -0.47, P < 0.01). Platelet aggregation was significantly enhanced by salt loading. Salt loading and female hormonal manipulation significantly interacted on platelet aggregation. Only in Dahl salt-sensitive rats fed a low sodium diet, ovariectomy increased platelet aggregation, whereas hormone replacement did not improve it. In Dahl salt-sensitive rats fed a high sodium diet, hormone replacement reduced platelet aggregation. CONCLUSIONS: Oestrogen replacement suppresses the development of hypertension and attenuates platelet aggregatory function in the salt-loaded ovariectomized Dahl salt-sensitive rats. It has a potential to inhibit the atherosclerotic process in postmenopausal hypertension.


Subject(s)
Blood Pressure/drug effects , Estradiol/administration & dosage , Hormone Replacement Therapy , Hypertension/prevention & control , Ovariectomy , Platelet Aggregation/drug effects , Sodium, Dietary/toxicity , Administration, Oral , Animals , Female , Hypertension/blood , Hypertension/etiology , Hypertension/physiopathology , NG-Nitroarginine Methyl Ester/pharmacology , Platelet Aggregation/physiology , Rats , Rats, Inbred Dahl
9.
Genetics ; 155(2): 785-92, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10835399

ABSTRACT

Epistasis used to be considered an obstacle in mapping quantitative trait loci (QTL) despite its significance. Numerous epistases have proved to be involved in quantitative genetics. We established a backcross model that demonstrates a major QTL for hypertension (Ht). Seventy-eight backcrossed rats (BC), derived from spontaneously hypertensive rats (SHR) and normotensive Fischer 344 rats, showed bimodal distribution of systolic blood pressure (BP) values and a phenotypic segregation ratio consistent with 1:1. In this backcross analysis, sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase (Serca) II heterozygotes showed widespread bimodality in frequency distribution of BP values and obviously demonstrated Ht. First, in genome-wide screening, Mapmaker/QTL analysis mapped Ht at a locus between D1Mgh8 and D1Mit4 near Sa in all 78 BC. The peak logarithm of the odds (LOD) score reached 5.3. Second, Serca II heterozygous and homozygous BC were analyzed separately using Mapmaker/QTL. In the 35 Serca II heterozygous BC, the peak LOD score was 3.8 at the same locus whereas it did not reach statistical significance in the 43 Serca II homozygotes. Third, to map Ht efficiently, we selected 18 Serca II heterozygous BC with 9 highest and 9 lowest BP values. In these 18 BC, the peak LOD score reached 8.1. In 17 of the 18, D1Mgh8 genotypes (homo or hetero) qualitatively cosegregated with BP phenotypes (high or low) (P < 0.0001, by chi-square analysis). In conclusion, selective genotyping with epistasis can be utilized for a major QTL mapping near Sa on chromosome 1 in SHR.


Subject(s)
Chromosome Mapping , Epistasis, Genetic , Hypertension/genetics , Quantitative Trait, Heritable , Animals , Genotype , Phenotype , Rats , Rats, Inbred F344 , Rats, Inbred SHR
10.
J Biol Chem ; 275(26): 19819-23, 2000 Jun 30.
Article in English | MEDLINE | ID: mdl-10749873

ABSTRACT

Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP-2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4-knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor.


Subject(s)
Bone Resorption/genetics , Dinoprostone/metabolism , Receptors, Prostaglandin E/genetics , Receptors, Prostaglandin E/metabolism , Animals , Animals, Newborn , Blotting, Northern , Blotting, Western , Bucladesine/metabolism , Cells, Cultured , Collagenases/metabolism , Dose-Response Relationship, Drug , Gelatinases/metabolism , Genotype , Mice , Mice, Knockout , Polymerase Chain Reaction , Receptors, Prostaglandin E/physiology , Receptors, Prostaglandin E, EP1 Subtype , Receptors, Prostaglandin E, EP4 Subtype , Signal Transduction/genetics , Skull/metabolism
11.
Endocrinology ; 141(4): 1554-9, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10746663

ABSTRACT

PGE2 functions as a potent stimulator of bone resorption. The action of PGE2 is thought to be mediated by some PGE receptor subtypes present in osteoblastic cells. In this study, we examined the involvement of PGE receptor subtypes, EP1, EP2, EP3, and EP4, in PGE2-induced bone resorption using specific agonists for the respective EPs. In mouse calvaria cultures, EP4 agonist markedly stimulated bone resorption, but its maximal stimulation was less than that induced by PGE2. EP2 agonist also stimulated bone resorption, but only slightly. EP1 and EP3 agonists did not stimulate it at all. RT-PCR showed that osteoblastic cells isolated from newborn mouse calvaria expressed all of the EPs messenger RNA (mRNA). Both EP2 agonist and EP4 agonist induced cAMP production and the expression of osteoclast differentiation factor (ODF) mRNA in osteoblastic cells. Simultaneous addition of EP2 and EP4 agonists cooperatively induced cAMP production and ODF mRNA expression. In mouse bone marrow cultures, EP2 and EP4 agonists moderately induced osteoclast formation, but the simultaneous addition of the two agonists cooperatively induced it, similar to that by PGE2. In calvaria culture from EP4 knockout mice, a marked reduction in bone resorption to PGE2 was found. In EP4 knockout mice, EP4 agonist failed to induce bone resorption, but EP2 agonist slightly, but significantly, induced bone resorption. These findings suggest that PGE2 stimulates bone resorption by a mechanism involving cAMP and ODF, which is mediated mainly by EP4 and partially by EP2.


Subject(s)
Receptors, Prostaglandin E/physiology , Animals , Bone Resorption/chemically induced , Bone Resorption/physiopathology , Carrier Proteins/genetics , Cells, Cultured , Cyclic AMP/biosynthesis , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout/genetics , Mice, Knockout/physiology , Osteoblasts/metabolism , Osteoblasts/pathology , Osteoclasts/pathology , Protein Isoforms/physiology , RANK Ligand , RNA, Messenger/metabolism , Receptor Activator of Nuclear Factor-kappa B , Receptors, Prostaglandin E/agonists , Receptors, Prostaglandin E/genetics , Receptors, Prostaglandin E, EP4 Subtype
12.
Bioorg Med Chem ; 6(12): 2459-67, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9925302

ABSTRACT

Hybrid peptides were constructed from endothelin B receptor (ET(B)) selective antagonist RES-701-1 (1) and endothelin (ET-1). They have N-terminal 10 amino acids derived from 1 and C-terminal 10 amino acids derived from ET-1. RES-701-1(1-10)-[Ala15]ET-1(12-21) and its analogues substituted or truncated at the residues derived from RES-701-1 had proved to possess high receptor binding activity selective for ETB as well as 1. Substitutions at the residues derived from ET-1 had produced some analogues that possessed high affinity not only for ETB but for ETA. Although all analogues had antagonistic effects on ETA, some analogues had proved to function as agonist on ETB confirmed by the changes in intracellular calcium concentrations of ET receptor-transfected COS-7 cells. We have found four types of ET receptor-binding peptides: (1) ETB-selective agonist with weak ETA antagonism (3, KT7421); (2) ETB-selective antagonist with weak ETA antagonism (29, KT7539); (3) ETB agonist with potent ETA antagonism (27, KT7538); and (4) non-selective ETA/ETB antagonist (26, KT7540).


Subject(s)
Endothelin Receptor Antagonists , Endothelin-1/chemistry , Endothelin-1/pharmacology , Muscle, Smooth, Vascular/drug effects , Peptides, Cyclic/chemistry , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Peptides/chemical synthesis , Vasodilator Agents/chemical synthesis , Amino Acid Sequence , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiology , Endothelin-1/chemical synthesis , In Vitro Techniques , Indicators and Reagents , Mesenteric Artery, Superior/drug effects , Mesenteric Artery, Superior/physiology , Molecular Sequence Data , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle, Smooth, Vascular/physiology , Peptides/chemistry , Peptides/pharmacology , Phenylephrine/pharmacology , Rats , Receptor, Endothelin B , Receptors, Endothelin/agonists , Structure-Activity Relationship , Vasodilator Agents/chemistry , Vasodilator Agents/pharmacology
13.
Am J Hypertens ; 10(12 Pt 1): 1396-403, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9443776

ABSTRACT

Our purpose was to determine the effect of ovariectomy on intracellular Ca2+ mobilization and platelet aggregation in sodium induced hypertension. At the age of 12 weeks ovariectomy or sham operation was performed in female Dahl-Iwai salt sensitive rats on a 0.3% NaCl diet. Four weeks later we assessed the effects of ovariectomy and an 8% NaCl diet on agonist induced intracellular Ca2+ mobilization in fura-2 loaded platelets and platelet aggregation. Ovariectomy enhanced the increase of systolic blood pressure and heart to body weight ratio on an 8% NaCl diet. However, thrombin evoked intracellular Ca2+ was not correlated with systolic blood pressure (r = -0.338, P = .17), and was lowered by sodium loading and ovariectomy (360+/-23 to 285+/-9, 296+/-10 nmol/L, P < .05). Furthermore, the ionomycin induced intracellular calcium fraction in the absence of external Ca2+ that reflected internal Ca2+ discharge capacity was reduced in ovariectomized rats compared with sham operated rats on an 8% NaCl diet (648+/-15 v 768+/-35 nmol/L, P < .05). The internal Ca2+ discharge capacity was inversely correlated with systolic blood pressure (r = -0.506, P = .03). In addition to the decreased internal Ca2+ discharge capacity, intracellular Ca2+-independent platelet aggregation by phorbol 12-myristate 13-acetate, a protein kinase C activator, was significantly enhanced in hypertensive rats. We concluded that ovariectomy enhanced sodium induced hypertension associated with the decreased internal Ca2+ discharge capacity and increased platelet aggregation in Dahl-Iwai salt-sensitive rats.


Subject(s)
Blood Platelets/metabolism , Calcium/metabolism , Hypertension/etiology , Ovariectomy , Sodium, Dietary/administration & dosage , Animals , Female , Hypertension/blood , Hypertension/metabolism , Platelet Aggregation , Rats
14.
Intern Med ; 36(10): 700-4, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9372330

ABSTRACT

A 40-year-old male, with a past history of hypertension but receiving no medical treatment, was referred. He manifested malignant hypertension (190/130 mmHg; Keith-Wagener III), renal dysfunction (serum creatinine, 3.8 mg/dl), and elevated plasma aldosterone (450 pg/ml) and active renin concentration (ARC, 104 pg/ml). His blood pressure was controlled with multiple antihypertensive agents and ARC thus decreased (4.3 pg/ml), but aldosterone remained elevated. Abdominal magnetic resonance imaging (MRI) revealed a right adrenal adenoma, and aldosterone-producing adenoma was confirmed by adrenal venous sampling. Primary aldosteronism very rarely develops to malignant hypertension, and even in that case ARC is suppressed. Therefore this is a rare case of primary aldosteronism complicated with malignant hypertension and high ARC.


Subject(s)
Adrenal Cortex Neoplasms/complications , Adrenocortical Adenoma/complications , Hyperaldosteronism/complications , Hypertension, Malignant/etiology , Renin/blood , Adrenal Cortex Neoplasms/blood , Adrenal Cortex Neoplasms/surgery , Adrenalectomy , Adrenocortical Adenoma/blood , Adrenocortical Adenoma/surgery , Adult , Aldosterone/blood , Antihypertensive Agents/therapeutic use , Blood Pressure , Creatinine/blood , Humans , Hyperaldosteronism/blood , Hyperaldosteronism/surgery , Hypertension, Malignant/blood , Hypertension, Malignant/drug therapy , Magnetic Resonance Imaging , Male
15.
Anal Chem ; 66(22): 3889-94, 1994 Nov 15.
Article in English | MEDLINE | ID: mdl-7810897

ABSTRACT

A new homogeneous electroenzymatic immunoassay was developed to determine antibody concentration using glucose oxidase and ferrocene as enzymatic and electrochemical amplifier, respectively. Digoxin (Dig)-conjugated glucose oxidase (GOx) was modified with ferrocene (Fec) to form Fec-GOx-Dig conjugate. After immunocomplex formation between the Fec-GOx-Dig conjugate and the anti-digoxin antibody, the complex underwent less electrochemical reaction due to the steric hindrance of the antibody. The ferrocene multilabeled conjugate was provided for the determination of anti-digoxin antibody. Since the strategy taken here is based on a combined effect of GOx and ferrocene, i.e., enzymatic amplification by GOx and electrochemical amplification by multilabeled ferrocenes, the antibody concentration was determined in the range from 1/50 to 1/500 dilution.


Subject(s)
Antibodies, Monoclonal/analysis , Digoxin/metabolism , Ferrous Compounds/metabolism , Glucose Oxidase/metabolism , Immunoenzyme Techniques , Organometallic Compounds/metabolism , Animals , Antibodies, Monoclonal/metabolism , Digoxin/chemistry , Digoxin/immunology , Electrochemistry , Ferrous Compounds/chemistry , Glucose Oxidase/chemistry , Humans , Immunoglobulin G/chemistry , Metallocenes , Mice , Organometallic Compounds/chemistry
16.
Adv Colloid Interface Sci ; 35: 139-72, 1991 Mar.
Article in English | MEDLINE | ID: mdl-2043288

ABSTRACT

In this article we review the adsorption of plasma proteins onto polymer latices on the basis of our experimental data. First, the surface characteristics of the latices were examined. Hydrophilic polymer layers (water-soluble polymer layers) were found to exist on the surfaces of copolymer latices, e.g., a polyacrylamide (polyAAm) layer existed on the surface of the styrene/acrylamide copolymer [P(St/AAm)] latex. These diffuse layers strongly affected the protein adsorption, that is, the amount of plasma proteins adsorbed onto copolymer latices (viz. P(St/AAm) and styrene/2-hydroxyethyl methacrylate copolymer [P(St/HEMA)] latices), particularly in the alkaline pH region, was much smaller than that onto a hydrophobic polystyrene (PS) latex. The protein adsorption was also studied as a function of pH, ionic strength and electrolyte concentration. Further, the adsorbability of heat- and urea-denatured albumins was investigated. A higher affinity of denatured components for polymer latices was observed compared with that of the native components.


Subject(s)
Blood Proteins , Adsorption , Blood Proteins/chemistry , Electrochemistry , In Vitro Techniques , Polymers , Protein Denaturation , Surface Properties
17.
Immunopharmacology ; 10(3): 157-62, 1985 Dec.
Article in English | MEDLINE | ID: mdl-3833854

ABSTRACT

Exogenous adenine strongly inhibited mitogen-stimulated transformation, cytoplasmic immunoglobulin production, and natural killer activity of human mononuclear leukocytes at the high concentration of 1.0 mM. These inhibitions by adenine were not due to cytotoxicity, because the viability of cultured cells was not affected by adenine up to 1.0 mM. As the magnitude of inhibition by adenine of these in vitro immunological functions was similar in normal and adenine phosphoribosyltransferase-deficient cells, its inhibition was not mediated by corresponding nucleotides. Adenine at the concentration of 0.1 mM caused 50% inhibition of cytoplasmic immunoglobulin production without alternating cell proliferation or viability. This suggests that an appropriate concentration of adenine may inhibit the differentiation of B cells to plasma cells rather than affecting cell proliferation. Understanding the mechanisms of adenine inhibition may lead to new approaches for the regulation of immune responses.


Subject(s)
Adenine Phosphoribosyltransferase/deficiency , Adenine/pharmacology , Lymphocytes/immunology , Pentosyltransferases/deficiency , Adenine Phosphoribosyltransferase/blood , Humans , Killer Cells, Natural/drug effects , Lymphocytes/enzymology
18.
Clin Exp Immunol ; 60(3): 509-17, 1985 Jun.
Article in English | MEDLINE | ID: mdl-3874728

ABSTRACT

We demonstrated that a primary exposure to the lymphocytosis promoting factor (LPF) of Bordetella pertussis-induced T cell colony formation. Colony formation was observed when mononuclear cells (MNC) were cultured at concentrations of more than 1 X 10(6)/ml, and reached a peak on day 8. However, the number of colonies generated with LPF was about one-third induced with phytohaemagglutinin (PHA). Removal of monocytes from MNC or T cells resulted in the failure of colony formation, but colony growth could be restored by the addition of monocytes or B enriched cells, indicating that they were required for the optimal colony growth induced by LPF. In the absence of accessory cells, optimal colony growth from monocyte depleted T cells could be obtained when an appropriate concentration of phorbol myristate acetate (PMA) or interleukin-2 (IL-2) was added in the cultures with LPF. PMA did not enhance LPF-induced colony formation in the cultures containing a sufficient amount of exogeneous IL-2. These findings suggest that IL-2 is essential to LPF-induced colony formation. Surface marker analysis showed that most of LPF-induced colony cells were T cells. The percentages of T4+ and T gamma cells of LPF-induced colony cells were more, and T8+ cells less, than those of PHA-induced colony cells. Ia1, T9 and Tac antigens were detected on many colony cells induced by LPF or PHA. These results indicate that the phenotype of LPF-induced colony cells differs from those of PHA, but the sequential antigen expression on lymphocytes triggered by IL-2 might be similar in both LPF- and PHA-induced colony formation.


Subject(s)
Bacterial Toxins/pharmacology , Interleukin-2/immunology , Interleukin-2/pharmacology , Phorbols/pharmacology , T-Lymphocytes/immunology , Tetradecanoylphorbol Acetate/pharmacology , Antigens, Surface/analysis , B-Lymphocytes/immunology , Clone Cells/drug effects , Humans , In Vitro Techniques , Leukocyte Count , Monocytes/immunology , Pertussis Toxin , Phenotype , Virulence Factors, Bordetella
19.
Clin Exp Immunol ; 58(2): 436-43, 1984 Nov.
Article in English | MEDLINE | ID: mdl-6094059

ABSTRACT

We studied the effect of lymphocytosis promoting factor (LPF), derived from the supernatant fluid of a culture of phase I Bordetella pertussis strain Tohama, on human lymphocyte proliferation. LPF was a potent mitogen for human mononuclear cells, specifically T cells. LPF failed to induce cytoplasmic immunoglobulin production by B cells. Removal of the monocytes from the T cell fraction diminished responses to LPF, but the response could be restored completely by the addition of 5.0% monocytes. These results suggest that LPF-induced cell proliferation is at least partially dependent on monocytes. In contrast to PHA, LPF stimulated T gamma cells to a greater extent than non-T gamma cells, but the magnitude of the T gamma or non-T gamma cell response was less than that of T cells, indicating that synergistic interactions between T gamma and non-T gamma cells are required for maximal response.


Subject(s)
Bacterial Toxins/pharmacology , Bordetella pertussis/immunology , Interleukin-2/administration & dosage , T-Lymphocytes/immunology , Cell Communication , Cell Separation , Cells, Cultured , Humans , Leukocytes/immunology , Lymphocyte Activation , Mitosis , Monocytes/immunology , Pertussis Toxin , Virulence Factors, Bordetella
20.
Immunology ; 53(3): 499-505, 1984 Nov.
Article in English | MEDLINE | ID: mdl-6333386

ABSTRACT

Human T lymphocyte colonies were grown in methylcellulose semi-solid cultures in the presence of phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA). Surface marker analysis showed lower percentages of OKT3- and OKT4-positive cells in PMA-induced colonies than those in PHA-induced colonies. The percentage of OKIa1-positive cells in PMA-induced colonies was approximately twice that in PHA-induced colonies. The percentage of OKT9-positive cells in PMA-induced colonies was significantly lower than that in PHA-induced colonies. These data suggest that the subsets of PMA-induced colony cells express a more immature phenotype than that of PHA-induced colony cells and that, among PMA-induced colony cells, there are fewer T cells in the proliferative status at the time tested. When 3 X 10(5)/ml monocyte-depleted T cells, at which concentration of seeded cells neither PHA nor PMA could induce colony growth, were cultured in the presence of both PHA and PMA, T cell colony growth was observed. In T cell colonies induced by a combination of PHA and PMA, the percentages of OKT3-, OKT4- and OKT8-positive cells were different from those in colonies induced by either PHA or PMA alone. These results suggest that PMA acts not only as a substitute for monocytes and/or interleukin-1, but may directly affect lymphocyte proliferation induced by a combination of PHA and PMA.


Subject(s)
Antigens, Surface/analysis , Lymphocyte Activation/drug effects , Phorbols/pharmacology , Phytohemagglutinins/pharmacology , T-Lymphocytes/immunology , Tetradecanoylphorbol Acetate/pharmacology , Antibodies, Monoclonal/immunology , Cell Division/drug effects , Clone Cells/drug effects , Clone Cells/immunology , Humans , Monocytes/immunology , T-Lymphocytes/cytology
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