Purification and characterization of a DNA binding protein in a nuclear scaffold fraction from rat ascites hepatoma cells

Our most recent work [Hibino et al. (1995) Cancer Lett., 88, 49-55] has shown that the selective binding affinities of highly repetitive DNA components for a nuclear scaffold protein from rat ascites hepatoma cells (P230) depend on the degree of sequence-directed bending of the helix axis. In the present experiment, this protein has been highly purified and isolated by a series of column chromatographic procedures to migrate as a single band to a molecular weight position of 230 kd on a SDS-polyacrylamide gel. A filter binding assay showed that the binding of a repetitive AT-rich component (369 bp XmnI fragment) from the hepatoma nucleus, which has a strongly bent overall structure, to isolated P230 is based on a cooperative mode of interaction. Distamycin A, which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding to this protein. These results suggest that AT-rich regions in highly repetitive DNA cause bending of the helix axis to be recognized by nuclear scaffold protein(s). Moreover, it has been shown that the nuclear scaffold fraction from rat liver or an actively growing hepatocyte cell line (Ac2F cells) does not contain P230, but does have a repetitive bent DNA binding protein (P130), which has an apparent molecular weight of 130 kd. In addition, the immunoblot analysis showed that mouse anti-P130 antiserum reacts with P230. Thus, the results in the present study imply that there is some difference in the higher order structure of the nuclear DNA attachment region between rat liver or actively growing hepatocytes and the hepatoma, although P230 appears to be immunochemically similar to P130.     

Fluorescent phalloidin enables visualization of actin without effects on myosin's actin filament sliding velocity and hydrolytic properties in vitro

Recent reports have demonstrated an activating effect of phalloidin in striated muscle. Furthermore, modeling of X-ray diffraction and crystallographic data suggests that phalloidin binding may induce conformational changes in actin. To determine whether phalloidin affects the mechanics of the actomyosin interaction, the velocity of actin filaments variably labeled with rhodamine-phalloidin was measured. In addition, solution actin-activated myosin subfragment-1 ATPase activity with phalloidin-labeled actin was compared to unlabeled actin. Here we found that phalloidin does not significantly effect actin filament velocity or parameters of ATPase, namely Vmax and K(m). Possible differences between muscle strip data and these in vitro results are discussed.     

Inhibitory effects of cyclosporin A on rat insulinoma cell proliferation, polyamine content and insulin secretion

RATIONALE AND OBJECTIVES: In percutaneous ethanol injection therapy, uncontrolled spread of ethanol sometimes causes complications. This study tested the accuracy of ultrasonography in showing the spread of ethanol during injection in normal postmortem pig livers. METHODS: Methylene blue-stained 96% (vol) ethanol (0.5-2 ml) was injected into pig livers under sonographic control. The volumes of the sonographically determined areas infiltrated by the injections and the corresponding pale lesions seen after dissecting the livers were measured and compared. The locations of the stained areas were determined. RESULTS: Uncontrolled spread of ethanol was often seen sonographically as hyperechoic lines spreading centrifugally from the injection site. After dissection, nearly all vessels and ducts were stained blue, indicating a major escape of ethanol from the injection sites. The volumes of the sonographically determined injection areas were significantly larger (P < 0.001) than the lesions found after dissection. CONCLUSIONS: Ultrasonography may overestimate the spread of ethanol, making the survival of peripheral tumor cells possible.     

Investigation of the effects of antisense oligodeoxynucleotides to islet amyloid polypeptide mRNA on insulin release, content and expression

We have investigated the effects of antisense oligodeoxynucleotides (oligos) to islet amyloid polypeptide (IAPP) mRNA on the expression and secretion of IAPP and insulin, in the clonal beta-cell line HIT-T15. Phosphorothioate-modified oligos were cytotoxic compared with phosphodiester (D)-oligos. Of the nine oligos tested using a lipofection reagent, O3, a 30-mer D-oligo complementary to a sequence downstream of the IAPP initiation codon, showed a significant dose-dependent suppression of IAPP mRNA, with a 42% decrease at 7.5 microM, compared with a scrambled (MSO3) control oligo (n = 3, P < 0.01). A subsequent 89% suppression of IAPP release was observed in the 4-h period following antisense treatment (1.78 +/- 0.13 (MSO3) vs 0.19 +/- 0.14 (O3) pmol/10(6) cells per 240 min, n = 7, P < 0.01). A significant increase in insulin mRNA (100 +/- 10% (MSO3) vs 124 +/- 8% (O3), n = 3, P < 0.05) and insulin content (13.0 +/- 0.9 (MSO3) vs 17.4 +/- 1.4 (O3) pmol/10(6) cells, n = 7, P = 0.028) was observed following treatment with O3 at 7.5 microM. O8, a 20-mer D-oligo directed to a region of IAPP mRNA further downstream than O3, also showed a decrease in IAPP mRNA and peptide release and an increase in insulin content. No significant changes were observed in the expression and release of the unrelated beta-cell peptide, neuropeptide Y. We thus show a suppression of synthesis and release of IAPP in HIT-T15 cells using antisense oligos. The associated increase in insulin mRNA and content in these cells after treatment with IAPP antisense oligos is in accord with an inhibitor action of IAPP on insulin availability.     

The additive effects of glucose and insulin on the proliferation of infragenicular vascular smooth muscle cells

PURPOSE: Peripheral vascular disease involving the infragenicular arterial tree is common in patients with diabetes mellitus (DM). Accelerated proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of atherosclerosis. Insulin and glucose stimulate VSMC proliferation and are elevated in patients with non-insulin-dependent DM. We have previously described the mitogenic effect of insulin on VSMCs in vitro; the effects of insulin and glucose separately and in combination on the proliferation of VSMCs grown in serum-free media were studied. METHODS: Human infragenicular VSMCs isolated from diabetic patients with end-stage peripheral vascular disease undergoing below-knee amputation were used. Cells from passages 3 to 5 were grown in serum-free media with varying glucose (0.05%, 0.1%, 0.2%, 0.4%, 0.6%, and 0.8%) and insulin (no added insulin, 100 ng/mL, and 1000 ng/mL) concentrations for 6 days. RESULTS: Insulin stimulated VSMC growth at glucose concentrations more than 0.2% (0.4% glucose with no added insulin resulted in 13,073 +/- 336 cells/mL, 0.4% glucose with 100 ng/mL insulin resulted in 16,536 +/- 1175 cells/mL, 0.4% glucose with 1000 ng/mL insulin resulted in 17,500 +/- 808 cells/mL, 0.6% glucose with no added insulin resulted in 14,167 +/- 1062 cells/mL, 0.6% glucose with 100 ng/mL insulin resulted in 18,984 +/- 1265 cells/mL, 0.6% glucose with 1000 ng/mL insulin resulted in 20,450 +/- 1523 cells/mL, 0.8% glucose with no added insulin resulted in 15, 853 +/- 1650 cells/mL, 0.8% glucose with 1000 ng/mL insulin resulted in 26,302 +/- 1919 cells/mL; P <.05 compared with glucose with no added insulin). Glucose stimulated VSMC proliferation up to a concentration of 0.2% (42% and 117% higher growth at 0.1% and 0.2% glucose, respectively, compared with the baseline, P <.05), regardless of the insulin concentration in the media. The greatest growth (26,302 +/- 1919 cells/mL) occurred in the group with the highest concentration of both insulin (1000 ng/mL) and glucose (0.8% glucose; P <.05). CONCLUSION: Both insulin and glucose stimulate the growth of diabetic infragenicular VSMCs. The mitogenic effects of insulin and glucose are additive and may contribute to the development of atherosclerosis in patients with DM.     

Retinoid-X receptors and the effects of 9-cis-retinoic acid on insulin secretion from RINm5F cells

OBJECTIVE: We evaluated the influence of chronic blockade of the renin-angiotensin system on hypertension induced by long-term thyroxin (T4) administration. To this end, we determined the effects of chronic treatment with captopril on blood pressure, cardiac hypertrophy and other renal and metabolic variables of hypertensive hyperthyroid rats. METHODS: T4 was administered s.c. at 0.38 mumol/kg per day and captopril was given in the drinking water (1.38 mmol/l). Both treatments were maintained for 6 weeks. Control rats received tap water. After the treatment period, the rats were placed in metabolic cages. Later, blood pressure was measured in conscious rats by intra-arterial determination. RESULTS: T4-treated rats showed an increased mean arterial pressure (MAP) whereas, in rats treated with T4 plus captopril, MAP was similar to that of the control group. Captopril did not affect the increased heart rate or ventricular weight/body weight ratio of hyperthyroid rats, but it improved the reduced creatinine clearance of these animals. CONCLUSIONS: The elevation in blood pressure produced by long-term T4 administration was prevented by chronic blockade of the renin-angiotensin system. Captopril improved the renal function of hyperthyroid rats, but did not affect the relative cardiac hypertrophy of these animals.     

The effects of insulin on 3 beta-hydroxysteroid dehydrogenase expression in human luteinized granulosa cells

OBJECTIVE: We determined the relative effects of insulin and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human luteinized granulosa cells. METHODS: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treated with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3 beta HSD were evaluated. RESULTS: Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/microL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3 beta HSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02). Insulin (greater than 100 nmol/L) alone increased 3 beta HSD protein content as measured by Western analysis 1.8-2-fold over basal levels, whereas FSH alone increased protein content 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3 beta HSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3 beta HSD 3.2-fold. In the presence of insulin plus FSH, 3 beta HSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3 beta HSD. CONCLUSION: Insulin is a regulator of both 3 beta HSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.     

Studies on the formation and stability of aminoacyl-tRNA synthetase complexes from Ehrlich ascites cells

Nine aminoacyl-tRNA synthetases from Ehrlich ascites cells were examined with respect to their ability to be isolated as high molecular weight complexes, soluble enzymes, and ribosome-bound enzymes. Several different methods were employed for cell homogenization and enzyme isolation, with particular attention paid to the effects of hypotonic, isotonic, and hypertonic buffers on enzyme isolation. The binding of all synthetases to ribosomes was eliminated if the low ionic strength of the isolation buffer was raised to isotonic levels. In contrast, neither the ionic strength or composition of the buffers, nor the procedures used for cell homogenization or enzyme isolation had any significant effect on the isolation of the high molecular weight synthetase complex. Certain enzymes (lysyl-, methionyl- and isoleucyl-tRNA synthetases) formed very stable complexes and high molecular weight species were the predominant forms of these enzymes under all conditions of cell homogenization and enzyme isolation. Other enzymes (glycyl-, tyrosinyl- and threonyl-tRNA synthetases) formed complexes very weakly, if at all, and always appeared predominately in the soluble enzyme fraction. Isolated soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases did not associate to form significant amounts of complex upon re-isolATION, SUGGESTING THAT A COMPONENT NECESSARY FOR COMPLEX FORMATION WAS MISSING FROM THE SOLUBLE ENZYME FRACTION. However, the soluble forms of these enzymes, but not the glycyl-, tyrosinyl- and threonyl-tRNA synthetases, did for complexes when mixed with ribosomal RNA or polyuridylic acid. Preliminary experiments showed no significant differences between the complexed and soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases with respect to Km values or ability to charge different isoaccepting tRNAs.     

The effects of propofol, isoflurane, and sevoflurane on oxygenation and shunt fraction during one-lung ventilation

Propofol's effect on hypoxic pulmonary vasoconstriction during one-lung ventilation (OLV) has not been determined. Twenty patients who had long-term OLV for esophageal surgery were allocated randomly to one of two study groups; one in which isoflurane administration preceded propofol, and another in which sevoflurane administration preceded propofol. Arterial and mixed venous blood samples and hemodynamics were measured as follows: before OLV, during OLV, OLV at 4 cm of positive end-expiratory pressure (PEEP), OLV after conversion from volatile anesthetics to propofol, OLV at 4 cm of PEEP, and after OLV. After the application of 4 cm of PEEP during propofol anesthesia, PaO2 increased significantly in both groups. The shunt fraction (Qs/Qt) increased significantly after the initiation of OLV in both groups and decreased significantly after the conversion from volatile anesthetics to propofol in both groups. Propofol can be used safely during OLV because PaO2 increased after the application of 4 cm of PEEP during propofol anesthesia, and Qs/Qt decreased significantly after the conversion from inhaled anesthetics to propofol anesthesia. IMPLICATIONS: During one-lung ventilation, the arterial partial pressure of oxygen values with propofol were greater than those with isoflurane and sevoflurane, and shunt fraction values with propofol were lower than those with both volatile anesthetics. Propofol improved oxygenation and shunt fraction during one-lung ventilation compared with volatile anesthetics.     

Effect of insulin on farnesyltransferase. Specificity of insulin action and potentiation of nuclear effects of insulin-like growth factor-1, epidermal growth factor, and platelet-derived growth factor

We have previously demonstrated that insulin activates farnesyltransferase (FTase) and augments the amounts of farnesylated p21 (Goalstone, M. L., and Draznin, B. (1996) J. Biol. Chem. 271, 27585-27589). We postulated that this aspect of insulin action might explain the "priming effect" of insulin on the cellular response to other growth factors. In the present study, we show the specificity of the effect of insulin on FTase. Insulin, but not insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), or platelet-derived growth factor (PDGF), stimulated the phosphorylation of the alpha-subunit of FTase and the amounts of farnesylated p21. Even though all four growth factors utilized the Ras pathway to stimulate DNA synthesis, only insulin used this pathway to influence FTase. Insulin failed to stimulate FTase in cells expressing the chimeric insulin/IGF-1 receptor and in cells derived from the insulin receptor knock-out animals. Insulin potentiated the effects of IGF-1, EGF, and PDGF on DNA synthesis in cells expressing the wild type insulin receptor, but this potentiation was inhibited in the presence of the FTase inhibitor, alpha-hydroxyfarnesylphosphonic acid. We conclude that the effect of insulin on FTase is specific, requires the presence of an intact insulin receptor, and serves as a conduit for the "priming" influence of insulin on the nuclear effects of other growth factors.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.     

Hepatotrophic effects of insulin on glucose, glycogen, and adenine nucleotides in hepatocytes isolated from fed adult rats

In vivo observations have suggested that there is an hepatotrophic effect of insulin. By contrast, subsequent in vitro work, using the isolated perfused liver system, showed no effect or indeterminate effects of insulin on the transport of glucose into the hepatocyte. However because this system may not have endured long enough to show such an influence we explored the transport of glucose using a 48-h suspension culture of hepatocytes isolated from young adult fed rats, the suspension being infused continuously with insulin at a rate approximating the maximum entering portal blood in the fed state. (In a separate study phloridzin was added after 2 h of incubation.) DNA, intracellular glucose and its inward transport, glycogen, and the adenine nucleotides were measured at intervals. By comparison with control or untreated cells, insulin-treated cells showed significantly more DNA and intracellular glucose, and the differences were abolished by phloridzin. Glucose transport rates fell to low values in untreated controls and still lower with insulin plus phloridzin, but the initial rate was maintained to the end (48 h) by insulin alone. Results for glycogen were similar to those for intracellular glucose. There was a close correlation (r = 0.96) between these two. The total adenine nucleotide pool and the concentration of ATP were maintained for about 24 h and fell to half their initial values by 48 h. Insulin had increased these concentrations significantly by 6 h. Although concentrations of ADP and AMP decreased gradually in all groups of cells, insulin enhanced the level of ADP by 12 h but had no measurable effect on that of AMP. The energy charge increased slightly throughout incubation but more so (by 6 h) in the presence of insulin. In conclusion the data support the concept that in the longer term (greater than 12 h) insulin in the portal circulation maintains the characteristic free permeability of the hepatocyte to glucose and this permits a variety of effects related to glucose entry into the hepatocyte.     

The effects of insulin on transport and metabolism of glucose in skeletal muscle from hyperthyroid and hypothyroid rats

The effects of insulin on the rates of glucose disposal were studied in soleus muscles isolated from hyper- or hypothyroid rats. Treatment with triiodothyronine for 5 or 10 days decreased the sensitivity of glycogen synthesis but increased the sensitivity of lactate formation to insulin. The sensitivity of 3-O methylglucose to insulin was increased only after 10 days of treatment and was accompanied by an increase in the sensitivity of 2-deoxyglucose phosphorylation; however, 2-deoxyglucose and glucose 6-phosphate in response to insulin remained unaltered. In hypothyroidism, insulin-stimulated rates of 3-O-methylglucose transport and 2-deoxyglucose phosphorylation were decreased; however, at basal levels of insulin, 3-O-methylglucose transport was increased, while 2-deoxyglucose phosphorylation was normal. In these muscles, the sensitivity of lactate formation to insulin was decreased; this defect was improved after incubation of the muscles with prostaglandin E2. The results suggest: (a) in hyperthyroidism, insulin-stimulated rates of glucose utilization in muscle to form lactate are increased mainly because of a decrease in glycogen synthesis; when hyperthyroidism progresses in severity, increases in the sensitivity of glucose transport to insulin and in the activity of hexokinase may also be involved; (b) in hypothyroidism, the decrease in insulin-stimulated rates of glucose utilization is caused by decreased rates of glycolysis; (c) prostaglandins may be involved in the changes in sensitivity of glucose utilization to insulin observed in muscle in altered thyroid states.     

Elements in the long terminal repeat of HIV-1 that interact with nuclear extracts from Jurkat cells persistently infected with vaccinia virus

Previous reports showed transactivation of the long terminal repeat (LTR) of HIV-1 in Jurkat cells persistently infected with vaccinia virus. In this communication, electrophoretic mobility shift assays were used to characterize the elements in HIV-1 LTR which might be responsible for the mechanism of transactivation. The results indicated that two elements, those for binding NF-kB and NFAT-1, were able to interact with nuclear extracts derived from Jurkat cells persistently infected with vaccinia virus, suggesting that they may play a role in the transactivation of HIV-1 LTR.     

The effects of oestrogen and progesterone on insulin sensitivity in female rats

Several reactive oxygen species, including singlet oxygen (1O2) and hydroxyl free radical (.OH), may potentially be involved in the photoinactivation of viruses by agents such as methylene blue (MB) and rose bengal (RB). Both 1O2 and .OH also mediate the formation of 8-oxoguanine (8-oxoGua) in DNA and RNA. Evidence that MB-or RB-induced bacteriophage (R17 or Q beta) inactivation and 8-oxoGua formation in RNA result from 1O2 rather than .OH was obtained utilizing complementary experimental approaches which show that: (i) the rate of phage photoinactivation by MB was unchanged by the presence of iron chelators or by different temperatures in the 13-37 degrees C range; (ii) MB- and RB-mediated rates of 8-oxoGua formation in isolated RNA have very little, if any, temperature dependence, in contrast to a significant temperature dependence of 8-oxoGua formation by a .OH generating system, the ultraviolet light irradiation of H2O2; and (iii) deuterium oxide (D2O) enhanced the RB-mediated rate of phage photoinactivation and 8-oxoGua formation in isolated RNA. The presence of superoxide dismutase in the RB photoinactivation reaction did not alter the rate of phage inactivation. The data suggest that 8-oxoGua serves as a marker that correlates qualitatively with 1O2-mediated lethal lesions in RNA bacteriophages.     

The effects of aluminum content,temperature and impurities on the electrical conductivity of synthetic bayer liquors

The electrical conductivity of pure sodium aluminate solutions with compositions in the range 93 to 128 gl⁻¹ free soda and O to 125 gl⁻¹’ alumina was investigated over the temperature range 40 to 70 °C. Under these conditions, the solution electrical conductivity was found to vary as a linear function of temperature. For a given free soda content, the temperature coefficient of conductivity varied as an approximate guadratic function of the alumina content. A single equation was developed relating solution conductivity to free soda content in the range 105 to 128 gl⁻¹ alumina, 40 to 100 gl⁻¹ over the temperature range 40 to 70 °C. The addition of sodium oxalate and sodium succinate at levels up to 20 gl⁻¹ was found to have no measurable effect on solution conductivity. However, the addition of sodium carbonate at levels of 40 gl⁻¹ had a significant effect, generally decreasing the conductivity. The magnitude of the effect is a complex function of solution composition and temperature. The technique was used to follow the precipitation of alumina from synthetic Bayer liquor continuously for 4 h. Possible applications to industrial practice, of the relationship determined, are briefly discussed. G. R. Browne, formerly Post-Graduate Student, School of Mining and Mineral Technology, Kalgoorlie, Western Australia C. W. P. Finn, formerly Alcoa Foundation Lecturer in Thermodynamics, School of Mining and Mineral Technology, Kalgoorlie, Western Australia