Biomedical subjects
Publications and source records attributed to M Okubo.
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Insulin induced glycogen synthase activity and decreased glycogen synthase mRNA concentrations in rat hepatoma H4 cells. Total enzyme activity measured with glucose 6-phosphate gradually increased during a 24-h insulin incubation. The time course of glycogen synthase activation measured by the activity ratio (low G-6-P/high G-6-P) in response to insulin was biphasic with the first peak at 15 min and the second peak at 4 to 6 h. When cells were incubated with insulin and cycloheximide, the first peak persisted while the second peak was abolished. These data suggest that the first activation peak derives from the classic effect of insulin via dephosphorylation and the second peak from an insulin-induced protein synthesis of a glycogen synthase activator. Ribonuclease protection assays with a cloned rat liver glycogen synthase cDNA were used to quantitate glycogen synthase mRNA. Insulin unexpectedly decreased glycogen synthase mRNA in a time- and a dose-dependent manner. After incubation with the RNA synthesis inhibitor, 5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) without and with insulin, the half time of glycogen synthase mRNA decreased from 6.0 +/- 0.80 to 3.9 +/- 0.75 h, respectively. Nuclear run-off experiments with isolated nuclei showed no change of transcription of glycogen synthase mRNA. These data suggest that insulin in this system affects glycogen synthase mRNA stability rather than transcription.
C-reactive protein (CRP) and serum amyloid P component (SAP), which are known to increase in sera from humans and many other animals with acute inflammation caused by infection, toxic drug administration or injury, were previously purified from bovine serum. These serum levels were determined by enzyme-linked immunosorbent assay (ELISA) using specific antiserum to bovine CRP or SAP which was prepared by immunizing rabbits and goats with each purified protein. Among 68 healthy Holstein cows, 45 non-lactating cows had levels of CRP and SAP of 20.6 +/- 1.4 and 27.6 +/- 1.3 micrograms/ml respectively; 23 lactating cows had higher levels of CRP and SAP (76.0 +/- 13.6 and 38.3 +/- 5.5 micrograms/ml respectively). In the latter group, there was a high correlation between milk yield and serum CRP levels (P less than 0.001). From these observations, it was assumed that lactation might stimulate CRP synthesis rather than SAP synthesis in bovine liver as an acute phase reaction, and that CRP might be called a lactation-associated protein.
The therapeutic effect of 15-deoxyspergualin (DSP) in old New Zealand Black/White F1 mice (B/W mice) with clinical nephropathy was studied and compared with cyclophosphamide (CY). The mice were treated with 0.05 ml phosphate-buffered saline, subcutaneously, four times/week, with DSP, 6 mg/kg body weight, s.c., four times/week, or with CY, 15 mg/kg, i.p., once a week, starting at the 28th week of age. They were serially semiquantitated for proteinuria, and serum IgG anti-dsDNA antibody was measured by ELISA. Spleen cell surface markers such as L3T4, Lyt2 and IgG were flow-cytometrically analyzed, and interleukin-2 (IL-2) activity in vitro was measured using CTLL cells. Kidney specimens were studied with light and immunofluorescence microscopy. The mice treated with either CY or DSP survived significantly longer than the control mice. L3T4+ cells in the DSP-treated mice at 40 weeks of age were significantly less than those in the 28-week-old control mice (p less than 0.05). In contrast, IL-2 generation in the three groups of mice showed no significant variations at 32-40 weeks of age. Serum anti-DNA antibody levels in both of the CY and DSP groups remained low and comparable with that in the 28-week-old mice, and the incidence of significant proteinuria decreased. Likewise, glomerular histology in the treated groups was improved compared with the 28-week-old control mice, and the deposition of IgG and C3 in the treated groups remained unchanged or further decreased. Accordingly, the renal (immuno)histological findings in the DSP group were quite comparable with or even better than those in the CY-treated mice. DSP may have suppressed the abnormal antibody production by modulating the T cell function(s), which is in contrast to the direct action against B cells due to CY.
Does chylomicronemia cause atherosclerosis? To directly address this clinically important problem, we conducted experiments in guinea pigs which lack apolipoprotein CII and develop chylomicronemia when they are fed a high-fat diet. After 6-12 months on a high-fat diet, guinea pigs were sacrificed, and subjected to histopathological and ultrastructural examination. Both the aorta and the coronary arteries revealed no atherosclerotic changes in all 4 animals examined: the endothelium was intact, and neither lipid-laden foam cells nor migrating cells were present within the arterial wall. Our studies indicate that chylomicronemia does not predispose to atherosclerosis.
Skeletal muscle blood flow (MBF) and intramuscular pressure (IMP) were measured using a heated thermocouple (HTC) and a solid-state catheter-tip transducer during electrical nerve stimulation in the gracilis muscle of anesthetized dogs. MBF values obtained simultaneously by the HTC method and the inhalated hydrogen gas (H2) clearance method showed an excellent linear regression with a correlation coefficient of 0.93. Electrical nerve stimulation at frequencies of 5 and 100 Hz increased total MBF, determined by an electromagnetic flowmeter, over control values 6.57 +/- 0.82 and 6.32 +/- 0.93 ml/min/100 g, respectively. Regional MBF at 5 Hz stimulation determined with HTC increased in the same magnitude both at the midpoint and the distal portion of the gracilis muscle. However, following 100 Hz stimulation, MBF at the midpoint was decreased from 8.15 +/- 1.55 to 4.09 +/- 1.54 ml/min/100 g, whereas at the distal portion flow was increased from 6.39 +/- 1.71 to 21.2 +/- 5.29 ml/min/100 g. IMP in both regions was low and approximately 2.0 mmHg at rest. IMP was remarkably increased to 27.9 +/- 8.43 mmHg at the midpoint but not in the distal portion during 100 Hz tetanic contraction, but showed no significant change in either region following 5 Hz stimulation. These data suggest that HTC can serve to measure MBF, and that following 100 Hz tetanic contraction, increased IMP may be associated with decreased regional MBF, irrespective of a total increase in flow in the gracilis muscle.
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To investigate the antigenicity of glomerular cell products for the induction of Heymann nephritis (HN), eight Lewis rats were immunized with an isolated glomerular protein (GLP) with only limited tubular cell contamination. Four out of four rats immunized with 240 micrograms GLP showed proteinuria at week 12. Their kidney specimens taken at week 16 showed contiguous granular deposits of IgG along the glomerular basement membrane by direct immunofluorescence and corresponding electron-dense deposits; the findings were identical to those in classical HN induced by the brush border protein. By immunoprecipitation, IgG eluted from isolated glomeruli of rats that received 240 micrograms GLP precipitated only a 700 kd glycoprotein (gp 700) in GLP, whereas 330, 440, and 700 kd glycoproteins were precipitated in the crude preparation (Fx1A), which is derived mainly from renal tubules. Two different monoclonal antibodies (anti-gp 330 and 14C1) against gp 330 precipitated the antigens in the same fashion as the glomerular eluate. These data reveal that gp 700, gp 440, and gp 330 share epitopes and that the gp 700 is demonstrable both in the glomerulus and in tubular brush border but that gp 440 and gp 330 are present only in the brush border. In addition, glomerular gp 700 was shown to induce Heymann-type glomerulonephritis just as gp 330 did.
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A hitherto undescribed intimate association of the nerve fibers with the central lacteal endothelium in the canine duodenum was demonstrated by transmission electron microscopy of ultrathin serial sections. Around the proximal half of the entire extent of the central lacteal, there exist three to ten unmyelinated nerve fibers 0.1-0.15 microns in diameter. Some of these exhibit bulbar swellings (0.7-1.0 microns in diameter) which contain small clear vesicles (30-35 nm in diameter) and/or rather large vesicles (60-90 nm) with electron-dense cores (40-60 nm). Most of the bulbar structures of the nerve fibers are in contact with or surrounded by the central lacteal endothelial cells. These results suggest the existence of a neuronal control over the activity of the lacteal endothelial cells.
We report the development of an animal model of multiple necrotizing enteritis (MNE) in rats. When rats were injected directly with a culture supernatant of lipopolysaccharide (LPS)-stimulated rat peritoneal macrophages into the abdominal aorta, the overt pathologic lesions of MNE developed within 30 minutes after injection. The rats showed an elevated level of blood fibrinogen degradation product content even 30 minutes after injection. Furthermore the rats that were pretreated intravenously with heparin sulfate did not develop MNE, indicating the acute disturbances of blood microcirculation in the intestine. Multiple necrotizing enteritis was developed also by the injection with recombinant tumor necrosis factor (rTNF) but rarely was observed with even a high dose of recombinant interleukin-1 (rIL-1) or platelet-activating factor (PAF). The supernatant was cytotoxic in vitro to TNF-susceptible LM and many other cells but was less cytotoxic to the TNF-resistant LR line. Partial purification of the supernatant suggested that the supernatant contained a cytokine that has biochemical features of TNF. Furthermore polyclonal anti-TNF antibody could inhibit not only the cytotoxicity in vitro but also MNE development in vivo by this factor. These data strongly indicate that MNE possibly could be caused by a TNF-like cytokine produced by macrophages that are stimulated by the endotoxin.
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Although epidural catheterization has many advantages in anesthesia and in the treatment of acute pain, spinal epidural abscess is a serious complication after the procedure. Since it is presumed that the epidural space is contaminated by bacteria on the skin via the space around the catheter, it seems important to clarify bacterial re-growth after application of skin disinfectant. Therefore, bacterial growths on human back 1, 2 days, and 1 week after application of disinfectants were studied in summer and winter to elucidate whether there are differences between the two seasons. Four disinfectants, 0.5% chlorhexidine in 80% ethyl alcohol (CA), 0.2% benzalkonium in 80% ethyl alcohol (BA), 10% povidone iodine (PI), and 80% ethyl alcohol (EA) were applied on the back of 76 adult healthy volunteers, and the specimens were taken by agar-contact method. The frequencies of positive cultures for bacteria were higher in summer than in winter. The frequencies of positive culture in summer after the applications of CA, BA, PI, and EA were as follows, respectively: 50%, 20%, 5%, and 40% after 1 day; 47%, 50%, 60%, and 50% after 2 days; and 82%, 82%, 70%, and 64% after 1 week. In winter, these frequencies after the application of CA, BA, PI, and EA were as follows, respectively: 0%, 0%, 18%, and 18% after 1 day; 5%, 26%, 32%, and 58% after 2 days; and 21%, 21%, 32%, and 42% after 1 week. We conclude that when an epidural catheter is in situ, more frequent skin disinfection has to be carried out, preferably by CA, in summer than in winter, since the presence of sweat on the back seems to hasten the re-growth of bacteria.
A WKA rat fetus fibroblast (WFB) was transfected by several oncogenes including EJras (activated H-ras), polyoma middle T (PyMT), v-src, c-myc, and adenovirus type 12 E1A-E1B. We analyzed the expression of the transformation-associated cell surface antigens on WFB by developing monoclonal antibodies. One of the WFB transformation-associated cell surface antigens, recognized by monoclonal antibody 067, was constitutively expressed only on two (W31 and W70) of ten WFB-EJras-transformed clones. This antigen could not be detected on parental WFB cells as well as ten WFB-PyMT transformant clones. Furthermore, it was not expressed on several clones of partially transformed WFB-v-src and WFB-adeno E1 transfectants or nontransformed WFB-c-myc transfectants. Monoclonal antibody 067 could form an immunoprecipitate with an approximate molecular weight of 67,000 which was composed of a single polypeptide chain. It was also shown that the expression of this antigen could be enhanced by cyclic AMP or cholera toxin treatment of W31; treated cells also showed a phenotypic reversion to the nonmalignant growth characteristics of the parental WFB. Moreover, the expression of this antigen could be induced on the WFB-EJras transformants such as W14, which do not constitutively express this antigen, by treatment of these agents. Furthermore, the expression of antigen was enhanced by heat and superoxide treatment on W31. These data suggest that the monoclonal antibody 067-defined molecule is a novel transformation-associated cell surface antigen that could be induced by heat shock or other physiological stress.
We have already established a pair of human autologous clones, tumor-specific cytotoxic T-lymphocyte clone TcHMC-1 and tumor target clone HMC-1-8, that were derived from the metastatic pleural effusion of a patient with mammary carcinoma. In this paper, we describe the target antigen that was defined by monoclonal antibody 3A2. This monoclonal antibody selectively inhibited the cytotoxic action of TcHMC-1 against HMC1-8 autologous tumor target cells, but not the cytotoxicity of lymphokine-activated killer and possibly natural killer cells against HMC-1-8 cells. Western blot analysis using the 3A2 monoclonal antibody identified a molecule with an approximate molecular weight of 92,000. This antigen was highly expressed on autologous primary cancer cells of breast carcinoma tissue, but not on the normal mammary gland in the same patient. Moreover, this antigen can be detected on approximately 50% of human allogeneic breast carcinomas, but not on other neoplastic tissues such as gastric and colonic carcinomas except for one out of 10 prostatic carcinomas. Nonneoplastic normal cells did not express this antigen. It was also suggested that the antigen is not murine mammary tumor virus-related products. These data suggest that 3A2-defined antigen could participate in the cytotoxicity by human autologous cytotoxic T-lymphocytes as the target molecule expressed on tumor cells.
A rapid electrophoretic method detecting serum lipoprotein(a) (Lp(a)) was described. When serum lipoproteins were electrophoresed using an agarose gel film containing 0.6% agarose and 3% sucrose, a distinct extra-band was frequently detected between beta- and prebeta-bands. This lipoprotein band was identified as Lp(a) by immunoblotting technique using anti-Lp(a) serum. Using this electrophoretic method, we studied 1,353 subjects with a variety of diseases. The Lp(a)-positive subjects accounted for 8.4% of all. Neither sex nor age influenced the frequency of Lp(a) positivity. Frequencies of definite myocardial infarction in Lp(a)-positive subjects did not differ significantly from those in Lp(a)-negative ones. Those of cerebral infarction in Lp(a)-positive subjects were significantly higher than those in Lp(a)-negative ones. An electrophoretic method described herein is very useful for the rapid detection and screening of Lp(a), and hence for large-scale clinical studies of atherosclerotic risk assessment.
Pleural exudative lymphocytes (PLEL) from a 60-year-old female patient showed high cytotoxicity against the autologous mammary tumor line, HMC-2, and NK-susceptible K562 cells, although PLEL demonstrated only weak cytotoxic potentials against several allogeneic tumor lines. We successfully obtained seven cytotoxic T cell clones from PLEL bulk populations, and assessed the possibility that these lymphocytes are simply natural killer (NK)-like cells or have the dual cytotoxic activity of cytotoxic T lymphocytes (CTL) and NK-like cells. These clones, designated as TcHMC-2, showed strong cytotoxicity against both HMC-2 and K562 cells. In contrast, allogeneic human peripheral blood-derived NK cells could not kill HMC-2 targets. Furthermore, a blocking study of TcHMC-2 cytotoxicity using monoclonal antibodies against CD3, CD8 and human MHC class I products showed that all of these antigen molecules were involved in the cytotoxicity of TcHMC-2 clone against autologous HMC-2 cells, indicating MHC class I recognitive cytotoxicity. These data indicate that the TcHMC-2 clone may have dual cytotoxicity with CTL- and NK-like activity against autologous HMC-2 mammary tumor and K562 cells, respectively.
We have previously reported that the increase in glycogen synthase activity in human muscle during a euglycemic clamp was not associated with a measured increase in glycogen synthase phosphatase activity after a 200-min insulin administration. To investigate further the mechanism of the regulation of human muscle glycogen synthase by insulin, we measured the activity of cAMP-dependent protein kinase before and after a 200-min hyperinsulinemic euglycemic clamp in Southwest American Indians. Insulin infusion resulted in a decreased cAMP-dependent protein kinase activity assayed at physiological cAMP concentration with increased glycogen synthase activity in all subjects (n = 5; P less than 0.01). No significant change was observed in cAMP-independent protein kinase activity. These results suggest that 200 min of insulin administration during a euglycemic clamp may regulate human muscle glycogen synthase activity by mechanisms other than the stimulation of phosphatase; one probable mechanism is by decreasing the activity of cAMP-dependent protein kinase.