[Abnormalities of lymphocytes in autoimmune-prone mice].
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Animals↗
Biomedical subjects
M Inaba
Publications and source records attributed to M Inaba.
Histogenesis of hemopoietic bone marrow in adult mice.
Based on the concept that the hierarchy of regulatory genes may specify mammalian development, we attempted to evaluate how morphological and functional phenotypes of hemopoietic bone marrow develop in murine models. From studies of local irradiation, s.c. implantation of bone with or without marrow, and renal subcapsular implantation of marrow plugs or bone without marrow, we identified four sequential (necrotic, clearing, stromal proliferating, and hemopoiesis-recovering) responses in the early phase of the reconstruction of hemopoietic marrow that spatially and temporally proceeded in this restricted order. In addition, characteristic hexagonal sinuses were found to participate in the development of hemopoietic marrow in the early phase. The development of hexagonally arranged sinuses seems to coincide with the formation of abundant osseous trabeculae and to precede the appearance of colony-forming units in culture (CFU-C) and spleen colony-forming units (CFU-S). These findings are discussed from the point of the hierarchy of regulation.
Animals↗
O-N-acetyl-D-glucosamine moiety on discrete peptide of multiple protein 4.1 isoforms regulated by alternative pathways.
Erythrocyte protein 4.1 is a cytoplasmic protein that possesses a protein-saccharide modification structure, an O-N-acetyl-D-glucosamine (GlcNAc) moiety. We determined the amino acid sequence of the proteolytic fragment containing the O-GlcNAc moiety after labeling the saccharide with [3H]galactose in the presence of bovine milk galactosyltransferase. Glycosylation appears to occur on one or more serine or threonine residues in the following sequence: Thr-Ala-Gln-Thr-Ile-Thr-Ser-Glu-Thr-Pro-Ser-Ser-Thr-Thr-Thr-Thr-Gln-Ile-Thr-Lys . This sequence corresponds to the carboxyl-terminal half of the 34-amino acid peptide in the 22/24-kDa carboxyl-terminal domain of protein 4.1, which is one of the discrete peptides regulated by alternative RNA splicing. Multiple protein 4.1 isoforms in erythroid and nonerythroid cells including major components of erythrocyte membrane proteins, 4.1a and 4.1b, appear to contain this sequence since most immunochemically reactive proteins were labeled with [3H]galactose, with the exception of several variant polypeptides. These results appear to suggest the functional or biological significance of the O-GlcNAc linkage in protein 4.1.
Acetylglucosamine↗
Evaluation of antitumor activity in a human breast tumor/nude mouse model with a special emphasis on treatment dose.
Eight lines of human breast tumors implanted in nude mice were treated with various antitumor agents at two different doses, maximum tolerated doses (MTD) and rational doses (RD) that were pharmacokinetically equivalent to the clinical doses; the response rates to both doses were compared. With MTD, the response rates to mitomycin C and vinblastine were 100%, and those to other agents including cyclophosphamide, nimustine (a water-soluble nitrosourea), vincristine, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), 5-fluorouracil (5-FU), and methotrexate were 30%-50%, indicating high responsiveness to the former two agents. In contrast, when the RD were used, the response rates to the majority of these agents were 25%-40%, and those to vincristine and nimustine were 13% and 0%, respectively. These results agree with the reported clinical results compared with those with MTD, suggesting the importance of the use of clinically equivalent doses in the evaluation of antitumor efficacy in a human tumor/nude mouse system.
Animals↗
A new hypoglycemic agent, midaglizole, blunts diabetic platelet aggregation: a possible role of alpha 2 blockade.
A newly developed alpha 2 blocker, midaglizole (DG-5128, 2-[2-(4,5-dihydro-1H-imidazol-2-yl)-1-phenylethyl] pyridine dihydrochloride sesquihydrate) has been shown to have a hypoglycemic action in healthy controls as well as in diabetics. Since human platelets are rich in alpha 2 receptors, the effects of midaglizole on platelet aggregation were investigated. In normal controls, ADP- or epinephrine-induced platelet aggregation was significantly inhibited 2 h after oral administration of 300 mg midaglizole. Midaglizole also suppressed diabetic platelet aggregation stimulated by 10 or 100 microM epinephrine and delayed the initiation of collagen-induced aggregation at 30 micrograms/ml. In vitro addition of midaglizole at 9 or 90 microM significantly inhibited epinephrine-induced platelet aggregation. Furthermore, long-term administration of midaglizole suppressed diabetic platelet aggregation induced by 0.5-1 microM ADP or 1 microM epinephrine. These results suggest that alpha 2 blockade not only blunts diabetic epinephrine-induced platelet aggregation but also affects ADP- or collagen-stimulated platelet aggregation, indicating that this alpha 2 blocker may offer a new approach to the treatment of diabetic microangiopathy.
Adrenergic alpha-Antagonists↗
Immunological identification of 1,25-dihydroxyvitamin D3 receptors in human promyelocytic leukemic cells (HL-60) during homologous regulation.
Immunological techniques were utilized to detect 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptor levels and to characterize physical/chemical changes in receptors in human promyelocytic leukemic cells (HL-60) during continuous exposure to hormone. The monoclonal antibody (IVG8C11) raised against the porcine intestinal 1,25-(OH)2D3 receptor immunoprecipitated quantitatively 1,25-(OH)2D3 receptors in nuclear extracts from HL-60 cells. The highly enriched immunoprecipitated receptors were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes, which were probed with 125I-labeled IVG8C11. The basal receptor from the cells treated with 1,25-(OH)2D3 for 2 h was detected as a single form at 53 kDa. Moreover, receptors were shown to be up-regulated at 12 h and down-regulated at 48 and 72 h in the continuous presence of hormone as evidenced by the ratio of density of the bands, 1.0 (2 h):4.2 (12 h):1.2 (48 h):0.9 (72 h), as measured by laser scanning densitometry. The up- and down-regulated receptors were also detected as single forms and had the same molecular mass as the basal receptor. Therefore, the data presented here strongly support the hypothesis of homologous regulation of 1,25-(OH)2D3 receptors in intact human target cells.
Antibodies, Monoclonal↗
Corneal endothelial changes in pseudoexfoliation syndrome.
Using specular microscopy and computer-assisted morphometry, we studied 27 eyes with pseudoexfoliation syndrome, 17 apparently normal fellow eyes, and 15 eyes of matched normal subjects with no ocular disease other than senile cataract. Endothelium of the eyes with pseudoexfoliation syndrome showed significantly lower cell density than did the endothelium from control eyes. Endothelium of both eyes of patients with unilateral pseudoexfoliation syndrome showed significant morphologic changes in cell size (polymegethism) and shape variability (pleomorphism); these changes were essentially the same in overtly affected and apparently normal fellow eyes. The results suggested that the corneal endothelial changes are consistent and might serve as an early sign of the disorder.
Cell Count↗
Kinetic analysis of cell killing effect induced by cytosine arabinoside and cisplatin in relation to cell cycle phase specificity in human colon cancer and Chinese hamster cells.
We presented a cell kill pharmacodynamic model for cell cycle phase-specific antitumor agents as well as that for cell cycle phase-nonspecific agents. The former was based upon assumptions that a cell population could be divided into a drug-sensitive population having sensitive (or specific) cell cycle phase(s) and a drug-resistant population in resistant cell cycle phases. This model revealed that cell killing action of antitumor agents with sufficient cell cycle phase specificity was time dependent. This time dependence was actually observed with cytosine arabinoside by colony-forming inhibition assays using Chinese hamster V79 cells. On the other hand, the present analysis indicated that cell killing effects of drugs lacking cell cycle phase specificity are dependent on C x T (concentration-time product). We analyzed cell kill kinetics of cisplatin and showed C x T dependence of cell killing action of the drug using human colon cancer WiDr cells. These results indicate that cell killing action of antitumor agents can be kinetically analyzed in terms of cell cycle phase specificity.
Animals↗
In vivo circumvention of vincristine resistance in mice with P388 leukemia using a novel compound, AHC-52.
A novel compound partially analogous to nifedipine, AHC-52, was found to sensitize multidrug-resistant tumor cells. AHC-52 at 0.5 microgram/ml completely reversed the in vitro resistance to vincristine (VCR) in VCR-resistant P388 cells (P388/VCR). Of various regimens examined for the in vivo treatment of P388/VCR-bearing mice, the combination of 0.05 mg/kg of VCR with 100 mg/kg twice a day of AHC-52 daily demonstrated the best result with a 206% increase in life prolongation. This result was comparable with that observed in parental P388-bearing mice treated with the optimal dose of VCR alone, indicating almost complete circumvention of resistance by combination VCR-AHC-52 therapy. In addition, the combination of both agents exhibited therapeutic effects in the treatment of P388-bearing mice with some long term survivors. This result was presumably due to the elimination of heterogeneity of VCR sensitivity in this cell population. These results suggest that combination chemotherapy using a sensitizing agent such as AHC-52 will be effective in not only circumvention of multidrug resistance but also retardation of its occurrence.
Animals↗
The effect of hard contact lens wear on the keratoconic corneal endothelium after penetrating keratoplasty.
Using wide-field specular microscopy, we evaluated the morphologic features of the corneal endothelium in 14 eyes of 14 patients with keratoconus who had worn a polymethylmethacrylate hard contact lens for at least four years (mean, 71 months) after penetrating keratoplasty. Comparisons were made with 14 non-lens-wearing eyes of 14 patients with keratoconus matched for age, length of postoperative period, and other variables. Examination of the endothelium of the contact lens wearers showed a significantly higher coefficient of variation in cell size (polymegethism) and a marked decrease in the percentage of hexagonal cells (pleomorphism), with a significant decrease in cell density (P less than .05). The cause of these morphologic abnormalities remains unclear but may be related to lens-induced hypoxic stress to the corneal endothelium causing ongoing cell loss.
Cell Count↗
Stabilization of 1,25-dihydroxyvitamin D-3 receptor in the human leukemia cell line, HL-60, with diisopropylfluorophosphate.
We have investigated the reason for the lack of specific 1,25-dihydroxyvitamin D-3 binding activity in extracts of ATCC HL-60 cells. Although intact ATCC HL-60 cells specifically and saturably take up 1,25-dihydroxy[3H]vitamin D-3, whole cell extracts have little or no specific binding of 1,25-dihydroxyvitamin D-3. The absence of specific binding can now be explained by the action of a serine proteinase in these cells. When diisopropylfluorophosphate (DFP), a potent inhibitor of serine proteinase, is added to the buffer used for extraction, specific binding of 1,25-dihydroxy[3H]vitamin D-3 in the extract is observed. The loss of specific binding could not be prevented by hydrolyzed DFP or other serine proteinase inhibitors, such as phenylmethylsulfonylfluoride, benzamidine and aprotinin. The proteolytic activity from ATCC cells also destroyed specific 1,25-dihydroxy[3H]vitamin D-3 binding in high-salt extracts from pig intestinal nuclei or from another HL-60 cell line (LG HL-60 cells). However, the proteinase did not affect the levels of the specific binding in these preparations if the receptor was occupied with 1,25-dihydroxy[3H]vitamin D-3 prior to exposure to the proteinase. The binding and sedimentation characteristics of the receptors from various sources were not changed by the presence of DFP. The Kd of the receptor in ATCC HL-60 cells is 1.2.10(-10) M, which is identical to that in the LG HL-60 cells. The 1,25-dihydroxy[3H]vitamin D-3 receptor complex from the ATCC cells sediments as a single 3.5 S component and elutes from DNA-Sephadex column in two peaks at 0.09 and 0.15 M KCl. The material eluting at 0.15 M KCl has the same DNA-binding activity as preparations from pig intestine or LG HL-60 cells. Immunoprecipitation studies demonstrated that monoclonal antibodies to the pig receptor, IVG8C11, quantitatively precipitate the 1,25-dihydroxy[3H]vitamin D-3-binding activity from ATCC HL-60 cells as well as that from LG HL-60 cells or pig intestinal nuclei. Therefore, the previous failure to demonstrate the 1,25-dihydroxyvitamin D-3 receptor in ATCC HL-60 cells is because of the presence of a potent serine proteinase and not because of an abnormal or absent receptor.
Calcitriol↗
Kinetic analysis of the in vitro cell-killing action of neocarzinostatin.
The relationship between the drug concentration and exposure time of neocarzinostatin (NCS) for a definite cell-killing effect was kinetically analyzed, taking into consideration its loss in biological activity during incubation. Its cell-killing activity was determined by a colony-forming inhibition assay, which was conducted at room temperature (25 degrees C) for 0.5-30 min exposure and at 37 degrees C for 5 min - 96 h exposure. Drug degradation at both temperatures was also investigated by bioassay. NCS lost its biological activity much faster at 37 degrees C than at 25 degrees C and the rate of loss in activity was higher at the lower initial concentration. Thus, the initial NCS concentrations necessary for 90% cell kill corresponding to each exposure time and a drug degradation constant were applied to a mathematical equation for the cell-killing effect of cell-cycle-phase-nonspecific agents. As a result, the curves for IC90-exposure time relationships predicted from drug degradation constants for 37 degrees C and 25 degrees C were fairly well fitted to the respective experimental data. These results indicate that the cell-killing action of NCS can be expressed by this mathematical equation with scrutiny of drug degradation and is dependent on the concentration-time product (C x T).
Animals↗
Responsiveness of human lung cancer/nude mouse to antitumor agents in a model using clinically equivalent doses.
The responses of 14 lines of human lung cancer xenografts in BALB/c-nu/nu mice to eight known antitumor agents were investigated. These xenografts consisted of four small-cell carcinomas (SCLC) and ten non-small-cell carcinomas (four large cell, three squamous cell, and three adenocarcinomas; NSCLC). The doses used in the experiments were the maximum tolerated dose (MTD) in nude mice and the "rational dose" (RD), the latter considered to be pharmacokinetically equivalent to the clinical dose. When given at MTDs, all drugs except 5-fluorouracil (5-FU) and methotrexate (MTX) were extremely effective against NSCLC as well as SCLC. The response rates of drug-sensitive SCLC to mitomycin C (MMC), ACNU, and vinblastine (VLB) were 100%, and those to Adriamycin (ADR) and vincristine (VCR) were 75%. In addition, the response rates of even drug-resistant NSCLC to MMC and VLB were 70% and 90%, respectively. In contrast, the response rates of NSCLC to RDs of the drugs were reduced to less than 40% and corresponded well to the respective clinical rates. In SCLC, a good correlation of experimental and clinical response rates was observed with four drugs [cyclophosphamide (CPM), ACNU, VLB, and 5-FU]. As a result, we emphasize that a more reasonable prediction of the clinical effectiveness of antitumor agents can be made by a protocol using clinically equivalent doses.
Adenocarcinoma↗
DNA binding property of vitamin D3 receptors associated with 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3.
Using [3H]-26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (F6-1,25-(OH)2D3), we have examined its ability to bind to the 1,25-(OH)2D3 receptor, and the ability of the resulting complex to bind DNA. The binding sites for [3H]F6-1,25-(OH)2D3 in the chick intestinal receptor represented a limited number of saturable sites for which 1,25-(OH)2D3 competes. 1,25-Dihydroxyvitamin D3 is three times more active than F6-1,25-(OH)2D3 in displacing [3H]F6-1,25-(OH)2D3. By affinity chromatography using DNA-Sephadex, the [3H]F6-1,25-(OH)2D3 receptor complex eluted from the column in a single peak at 0.14 M KCl, while [3H]-1,25-(OH)2D3 receptor complex eluted at 0.13 M KCl. These results indicate that F6-1,25-(OH)2D3 and 1,25-(OH)2D3 recognize the same binding site of the receptor and that the F6-1,25-(OH)2D3 receptor complex binds DNA more tightly than the 1,25-(OH)2D3 receptor complex. We suggest that the higher binding affinity for DNA may contribute to the greater biological activity of F6-1,25-(OH)2D3.
Animals↗
Effect of captopril or enalapril on renal prostaglandin E2.
Since one of the hypotensive mechanisms of angiotensin-converting enzyme inhibitor (ACEI) has been suggested to be mediated through the renal kinin-prostaglandin (PG) axis, the present study was designed to investigate the effect of captopril (C) or enalapril (E) on renal PGE2 excretion or synthesis. Wistar male rats (BW 200-250 g) were given orally captopril at 30 mg/kg/day or enalapril at 10 or 30 mg/kg for one week. Before and after ACEI, blood pressure (tail cuff method) as well as PRA and urinary PGE2 excretion was determined. Renopapillary slices were obtained from some of the rats including controls and incubated to determine PGE2 synthesis. C or E administration resulted in a blood pressure decrease of 21 to 36 mm Hg with an increase in PRA. Urine volume and sodium excretion increased after daily treatment with C or E at 30 mg/kg. Urinary PGE2 excretion increased 1.4-fold in response to C, but not to E. Papillary PGE2 synthesis demonstrated a marked decrease 2 h after in vivo administration of either ACEI compared to controls. However, when C or enalaprilat was added in vitro to renal slices obtained from controls, only C at 10(-5) M showed a significant 2-fold increase in renal PGE2 synthesis. These results suggest that (1) renal PGE2 synthesis may be dependent on circulating angiotensin II. (2) C, but not enalaprilat, has a direct stimulatory effect on renal PGE2 synthesis and (3) renal PGE2 may not be involved very much in the hypotensive effect of ACEI.
Animals↗
Inherited persistence of immature type pyruvate kinase and hexokinase isozymes in dog erythrocytes.
1. Red cell pyruvate kinase (EC 2.7.1.40) and hexokinase (EC 2.7.1.1) in high and low potassium (K) dogs were shown to exist as multiple forms which were separable by electrophoresis and ion-exchange chromatography. The R2-type pyruvate kinase, which was determined to be a young type enzyme in canine red cells, was shown to be the predominant form of pyruvate kinase in high K cells. 2. The M2-type pyruvate kinase, a prototype isozyme in erythroid cells, existed in high K dog erythrocytes as well as in high K and low K dog reticulocytes. 3. Isozyme analysis of high K red cell hexokinase also showed a profile similar to that obtained for low K reticulocytes. 4. These results seem to reflect the immaturity of high K erythrocytes, which suggest that an abnormal cell differentiation or maturation may occur at an early stage of erythroid cell proliferation in high K dogs.
Animals↗
Saturable process involved in active efflux of vincristine as a mechanism of multidrug resistance in P388 leukemia cells.
Kinetic analysis of vincristine (VCR) efflux in multidrug-resistant and parental P388 leukemia cells was performed to investigate the difference in activity between the two cell lines. Efflux velocities of VCR were directly determined from the slope of the initial release of drug induced by resuspending the preloaded cells in VCR-free medium, representing unidirectional efflux from intracellular free or loosely bound drug pools. Further, the equilibrium binding of VCR to whole-cell homogenates was analyzed by ultrafiltration to estimate intracellular unbound drug concentrations. A two-site binding model was found to fit the data best for both cell lines, and depletion of ATP by the addition of apyrase decreased binding. The binding parameters were similar between the two cell lines. A Hofstee plot of efflux demonstrated the existence of both linear and saturable transport of VCR in both cell lines. The greater maximum velocity observed with VCR efflux in the resistant cells suggests that an increased number of transporters causes greater activity of this process in the resistant cells.
Animals↗
Long-term observations of autoimmune-prone mice treated for autoimmune disease by allogeneic bone marrow transplantation.
Long-term effects of allogeneic bone marrow transplantation (ABMT) across major histocompatibility complex barriers were studied in (NZB x NZW)F1 (B/W), BXSB, and MRL/Mr-lpr-lpr (MRL/lpr) mice with established autoimmune disease at the time of ABMT. In the BXSB or B/W mice, ABMT cured all aspects of autoimmune disease. Glomerular damage, revealed by histological study was dramatically improved. Serological abnormalities and immunologic functions also were normalized. Correction of autoimmune disease and advanced renal disease in BXSB and B/W mice regularly lasted greater than 5-6 mo and even 1 yr after ABMT. In the MRL/lpr mice, however, autoimmune and renal disease at first improved but then recurred after ABMT, apparently because of intolerance of mice for high doses of irradiation and a high degree of resistance of recipient stem cells to irradiation. In this model, H-2 typing revealed that by the time of relapse, immunocompetent cells of the chimeric mice had been replaced by host (MRL/lpr; H-2k) cells. B220+ Ly-1+ cells, present in increased numbers in untreated MRL/lpr mice, initially returned to normal levels after ABMT but then reappeared in the MRL/lpr mice that had received marrow from donors having few such lymphocytes. Thus, our results show that MRL/lpr mice possess abnormal radioresistant stem cells and provide impressive evidence that the origin of autoimmune diseases in this strain, as in the several other strains studied, residues in abnormalities present in stem cells.
Animals↗