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.     

Detection of colorectal adenoma and cancer based on transthyretin and C3a-desArg serum levels

Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop. To discover sensitive and specific biomarkers for improvement of pre‐clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI‐TOF‐MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data. Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p<0.001) were identified as transthyretin (pre‐albumin) and C3a‐desArg by MS/MS and were further validated by antibody‐based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut‐off of <0.225 g/L for transthyretin and >1974 ng/mL for C3a‐desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.     

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.     

Flow induced particle migration in fresh concrete: Theoretical frame,numerical simulations and experimental results on model fluids

In this paper, we describe and compare the various physical phenomena which potentially lead to flow induced particle migration in concrete. We show that, in the case of industrial casting of concrete, gravity induced particle migration dominates all other potential sources of heterogeneities induced by flow. We then show, from comparisons between experiments using model materials, dimensional analysis and numerical simulations, that, from a quantitative point of view, the viscous drag force, which prevents particles from migrating during a casting process, shall neither be computed from the apparent viscosity nor from the plastic viscosity of the suspending phase but from its tangential viscosity. Finally, the transfer of this type of numerical prediction tool to real concrete is discussed.     

Comparison of methods for enumeration of yeasts and molds in shredded low-moisture, part-skim mozzarella cheese

Two studies were conducted to compare established and new methods for enumerating yeasts and molds in shredded low-moisture, part-skim mozzarella cheese stored under refrigeration and temperature-abuse conditions. Yeast and mold counts covered a range of 6 log10 units. In study 1, the potato dextrose agar plus chlortetracycline (PDA) pour plate, dichloran rose bengal chloramphenicol (DRBC) spread plate, Petrifilm, and Iso-Grid hydrophobic grid-membrane filtration methods were used to analyze samples after < or = 1 day of unopened storage at 7 degrees C and after opening, resealing, and 2 days of storage at 25 degrees C. The results of all methods were highly correlated (r2 > or = 0.96). In study 2, the PDA, DRBC, and Iso-Grid methods were compared with the Simplate 2-day method in an analysis of 42 samples stored for various times at 8, 11, 15, and/or 22 degrees C. The results of all methods except the Simplate method were again highly correlated (r2 > or = 0.94), although yeasts and molds were not always detected by all methods. Compared with the PDA, DRBC, and Iso-Grid methods, the Simplate method most often (10 of 42 samples, 23.8%) failed to detect yeasts and molds when at least one other method did, and the results were less highly correlated with those of other methods (r2 = 0.88 to 0.90). Our results suggest that the PDA, DRBC, Petrifilm, and Iso-Grid methods are equivalent for enumerating yeasts and molds in shredded low-moisture, part-skim mozzarella cheese samples.     

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.