Retardation of human drug-resistant HL-60 cell in G1 phase and induction of sensitive cell to apoptosis by cyclosporine A

To further study the relationship between resistance to apoptosis and drug resistance in harringtonine-resistant HL-60 cells (HR20), cyclosporine A (CsA) 20, 10 micrograms.ml-1 was shown to induce the sensitive HL-60 cells to apoptosis, showing a typical DNA "ladder" band. But the same concentrations of CsA retarded the HR20 cells in G1 phase and could not induce the cells to apoptosis. The cellular daunorubicin accumulation increased when HR20 cells were treated with low concentration of CsA and the reversal of drug resistance by CsA was unrelated to the retardation of cell cycle progression. High phosphorylation of about 50 kDa protein occured when HR20 cells were treated with CsA 10 micrograms.ml-1. The results domonstrate that cyclosporine A retarded the harringtonine-resistant HL-60 cells in G1 phase but induced HL-60 cells to apoptosis, and the retardation was unrelated to drug resistance.     

Autoantibodies to the insulin receptor. Effect on the insulin-receptor interaction in IM-9 lymphocytes

The serum of some patients with insulin-resistant "diabetes" contains antibodies that bind to and block the cell membrane receptors for insulin. In this report, we have characterized the effects of the antireceptor antibodies on the interaction of (125)I-insulin with its receptor on the human lymphoblastoid cell line IM-9. Up to 95% of specific insulin binding can be inhibited by pretreatment of the cells with these immunoglobulins. The onset of the inhibitory effect is time- and temperature-dependent, and the effect is reversed extremely slowly if the cells are suspended in a large excess of antibody-free buffer. These features of antibody binding can be easily distinguished from those for insulin binding to its receptor. The inhibitory effect of the antibodies can be reversed by exposure of the cells to conditions known to elute surface immunoglobulins. The three antireceptor sera studied appear to alter the insulin-receptor interaction in different ways. Two antisera markedly reduce receptor affinity through combined effects on the insulin association and dissociation rates, and, additionally, have smaller effects on available receptor number. A third antiserum primarily affects available receptor number and has little effect on receptor affinity. All three antisera inhibit the capacity of insulin to promote negatively cooperative site-site interactions among insulin receptors. The data suggest that these autoantibodies to the insulin receptor bind to different determinants on the receptor and may therefore be useful as unique probes of insulin receptor structure and function.     

Uncouplers can shuttle between localized energy-coupling sites during photophosphorylation by chromatophores of Rhodopseudomonas capsulata N22

Two models of the action of uncoupler molecules in inhibiting photophosphorylation in bacterial chromatophores are considered: either uncoupler molecules shuttle rapidly between energy-coupling sites, or uncoupler molecules that are bound to particular sites in the chromatophores for a time that is comparable with the turnover time of the photophosphorylation apparatus may uncouple by a co-operative "substoichiometric' mechanism. It is found that the titre of uncoupler necessary to cause complete uncoupling is lowered if the rate of photophosphorylation is initially decreased by partially restricting electron flow with an appropriate titre of antimycin A. This result indicates that uncoupler molecules shuttle rapidly between energy coupling in which the energized intermediate between electron transport and phosphorylation is delocalized over the entire chromatophore membrane and those in which it is not. If the rate of photophosphorylation is partially restricted with the covalent H+-translocating ATP synthase inhibitor dicyclohexylcarbodi-imide, the titre of uncoupler necessary to effect complete inhibition of photophosphorylation is also decreased relative to that in which the covalent H+-ATP synthase inhibitor is absent. This important result appears to be inconsistent with models of electron-transport phosphorylation in which the "energized state' of the chromatophore membrane that is set up by electron transport and utilized in photophosphorylation is delocalized over the entire chromatophore membrane.     

Drinking patterns as predictors of alcohol withdrawal reactions in DBA/2J mice

Individually housed DBA/2J mice were fed a liquid diet in which ethanol supplied 33% of the calories. The level of physical dependence that developed was estimated by scoring convulsions, elicited by handling the mice, after discontinuing the alcohol diet. The severity of the withdrawal reaction increased progressively with duration (5-12 days) of alcohol administration. A 2-day period on the diet produced no withdrawal reaction. Pretreatment of the mice with alcohol in their drinking water slightly increased the subsequent intake of the liquid diet. "Effective" alcohol intake was defined as uninterrupted alcohol consumption above 10 g/kg/day. Withdrawal scores correlated better with effective intake than with total intake under a variety of conditions. We interpret this to mean that brief interruptions in drinking (1 day) may allow the accrued physical dependence to disappear. On the basis of their effective alcohol intake, mice could be assigned to nondependent, moderately dependent or severely dependent groups for further study of the nature of physical dependence.     

The effect of converting from pravastatin to simvastatin on the pharmacodynamics of warfarin

Glycosylated amino acids and glycosylated human serum albumin reduce nitrite to nitric oxide under anaerobic conditions. The amount of nitric oxide produced was recorded by generation of nitrosoHb from deoxyHb. Without preincubation after the addition of sodium nitrite, glucose or a mixture of glucose with amino acid or serum albumin did not cause spectrophotometrically detectible transformation of deoxyHb into nitrosoHb. The generation of NO increased with an increase in content of colored "final" products of amino acid and serum albumin glycosylation in the incubation mixture. The incubation of blood plasma of patients with diabetes mellitus with nitrite also resulted in the increased production of NO as compared to blood plasma of healthy subjects. During the incubation of healthy subjects' blood plasma with nitrite a small amount of NO was produced. The removal of low-molecular-weight compounds was accompanied by a significantly decreased generation of NO by blood plasma.     

Isolation and characterization of the Saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase

Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a Mg2+-independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. A dpp1Delta mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1Delta mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg2+-independent PA phosphatase activity in S. cerevisiae.     

Extraction, quantification, and biological availability of fumonisin B1 incorporated into the Oregon test diet and fed to rainbow trout

The purpose of this study was (i) to determine whether pure fumonisin B1 could be incorporated into, recovered, and detected by high-pressure liquid chromatographic analysis from the semipurified Oregon test diet (OTD) used in rainbow trout feeding studies, and (ii) to determine if the incorporated fumonisin B1 was biologically available using the change in free sphingoid bases in liver, kidney, and serum as a mechanism-based biomarker. The results indicate that fumonisin is not easily quantified in the OTD. Recoveries ranged from 12 to 81% of the calculated concentrations based on the fumonisin B1 added to the OTD. However, the fumonisin B1 in the OTD was readily absorbed and biologically active as evidenced by marked increases in free sphinganine in liver, kidney, and serum. The magnitude of the increase in free sphinganine at 100 ppm in the OTD was comparable to that known to be associated with liver toxicity in rats, pigs, and ponies.     

Discordance of databases designed for claims payment versus clinical information systems. Implications for outcomes research

OBJECTIVE: To determine the suitability of insurance claims information for use in clinical outcomes research in ischemic heart disease. DESIGN: Concordance study of two databases. SETTING: Tertiary care referral center. PATIENTS: A total of 12,937 consecutive patients hospitalized for cardiac catheterization for suspected ischemic heart disease between July 1985 and May 1990. INTERVENTIONS: Two-by-two tables were used to compute overall and kappa measures of agreement comparing clinical versus claims data for 12 important predictors of prognosis in patients with ischemic heart disease. MEASUREMENTS: Kappa statistics (agreement adjusted for chance agreement) were used to quantify agreement rates. RESULTS: Agreement rates between the clinical and claims databases ranged from 0.83 for the diagnosis of diabetes to 0.09 for the diagnosis of unstable angina (kappa values). Claims data failed to identify more than one half of the patients with prognostically important conditions, including mitral insufficiency, congestive heart failure, peripheral vascular disease, old myocardial infarction, hyperlipidemia, cerebrovascular disease, tobacco use, angina, and unstable angina, when compared with the clinical information system. CONCLUSIONS: Our results suggest that insurance claims data lack important diagnostic and prognostic information when compared with concurrently collected clinical data in the study of ischemic heart disease. Thus, insurance claims data are not as useful as clinical data for identifying clinically relevant patient groups and for adjusting for risk in outcome studies, such as analyses of hospital mortality.     

In vivo radiofrequency thermal balloon angioplasty of porcine coronary arteries: histologic effects and safety

The purpose of this study was to assess the safety and histologic effects of radiofrequency thermal balloon angioplasty in the coronary vasculature of normal pigs. Radiofrequency thermal balloon angioplasty was performed in 30 coronary arteries of 16 nonatherosclerotic pigs. Heated inflations were performed at either 50 degrees, 60 degrees, or 70 degrees C for 30 or 60 seconds, and were compared with five nonheated inflations in five additional arteries. All balloon inflations were performed at 2 atm pressure with a balloon/vessel diameter ratio of 1.2 to 1. Heart rate, arterial pressure, and left ventricular pressure were monitored continuously for each animal. A 12-lead ECG, coronary angiography, and two-dimensional transthoracic echocardiography were performed before and 1 hour after each balloon inflation. Each animal was subsequently put to death for postmortem cardiac examination. Heated inflations were well tolerated in 28 of the 30 arteries without significant adverse effects. During one inflation, ventricular fibrillation occurred because of prolonged ischemia from an occlusive guiding catheter. In another artery, a heated inflation resulted in a dissection with a transient decrease in distal coronary flow. Histologic examination revealed a significant increase in wall thinning and elastic fiber straightening with heating at 70 degrees C for both 30 and 60 seconds, and a significant increase in intracoronary thrombus with heating at 70 degrees C for 60 seconds. Depth of periarterial myocardial heat necrosis paralleled the increase in temperature, with an average depth of 166 microns at 50 degrees C, 312 microns at 60 degrees C, and 1031 microns at 70 degrees C. In vivo, radiofrequency coronary angioplasty can be performed relatively safely without significant electrical, hemodynamic, or ischemic changes beyond those seen with conventional nonthermal angioplasty. The extent of heat-induced vessel wall thinning, elastic tissue straightening, intracoronary thrombus formation, and periarterial myocardial necrosis are all related to balloon temperature or duration of heating.     

Islet amyloid polypeptide decreases the effects of insulin-like growth factor-I on glucose transport and glycogen synthesis in skeletal muscle

Previous studies have shown that islet amyloid polypeptide (IAPP) is co-secreted with insulin from the beta-cell. IAPP reduces insulin-stimulated rates of glycogen synthesis in skeletal muscle but the mechanisms are unclear. Insulin-like growth factor I (IGF-I) is an important regulator of glucose metabolism in skeletal muscle and acts through its own receptor, which has many structural and functional similarities with the insulin receptor. Despite this, the effects of IGF-I on glucose utilization are not identical to those of insulin. The aim of the study was to determine the effects of IAPP on IGF-I-stimulated rates of glucose transport and metabolism (measured by 3-O-methyl[3H]glucose and [U-14C]glucose, respectively) in rat soleus muscle, and compare them with those simulated by insulin. IAPP (10 nM) decreased the sensitivity of 3-O-methylglucose transport, the flux of glucose to hexosemonophosphate and the sensitivity of glycogen synthesis to IGF-I. In contrast, IAPP had no effect on IGF-I-stimulated rates of lactate formation (i.e., glycolysis). IAPP decreased the sensitivity of 3-O-methylglucose transport and glycogen synthesis to insulin. It is concluded that IAPP blunts the stimulation of glucose uptake and deposition by IGF-I or insulin in skeletal muscle. These observations expand those made initially for IAPP and insulin and suggest that IAPP affects IGF-I- or insulin-stimulated glucose metabolism in muscle by a mechanism which is common for both hormones. These experiments may serve as a framework for future studies in order to clarify the mechanisms by which IAPP affects glucose metabolism in skeletal muscle.