Different mechanisms of decreased drug accumulation in doxorubicin and mitoxantrone resistant variants of the MCF7 human breast cancer cell line.

We selected two drug resistant variants of the MCF7 human breast cancer cell line by chronic in vitro exposure to doxorubicin (MCF7/D40 cell line) and mitoxantrone (MCF7/Mitox cell line), respectively. The cell lines are similar in growth characteristics including doubling time, DNA synthetic phase and cell size. Resistance to mitoxantrone conferred only partial resistance to doxorubicin; whereas resistance selected for doxorubicin appeared to confer complete resistance to mitoxantrone. Both agents selected for cross resistance to the Vinca alkaloids. MCF7/D40 cells display a classic-multi-drug resistance phenotype with expression of P-glycoprotein, decreased drug accumulation relative to the parental line and reversal of drug accumulation and drug resistance by verapamil. MCF7/Mitox cells likewise display resistance to multiple drugs, but in contrast to MCF7/D40 cells do not express P-glycoprotein by immunoblot or RNA blot analysis. Net drug accumulation in MCF7/Mitox cells was decreased relative to the parental cells but there was no selective modulation of drug accumulation or in vitro drug resistance by the addition of verapamil. Efflux of mitoxantrone was enhanced in both the MCF7/D40 and MCF7/Mitox cell lines relative to the MCF7/S cell line. We conclude that the two drug resistant cell lines have different mechanisms of decreased drug accumulation.     

Creating a professional practice environment on a shoestring

Did you ever think, “If we just had a little money we could…”? The current health care environment is wrought with financial stressors that can be overwhelming and take up most of our time. Such stress can limit the development of a professional practice environment if you let it. How do you not only survive but thrive in this financial climate?     

Dietary carbohydrate, a Big Mac, and insulin requirements in type I diabetes

Using the artificial beta-cell (Biostator), we determined the insulin requirements in five nonobese type I (insulin-dependent) diabetic subjects who received isocaloric 40 and 60% mixed-carbohydrate diets in a crossover randomized fashion for 4 days, each day consisting of four equal meals. This was followed on day 5 by a "Big Mac Attack" lunch consisting of a Big Mac, french fries, and milk shake. Insulin requirements to maintain normoglycemia were calculated for each 24-h period and for the 2 h after each meal. The mean 24-h insulin requirements to maintain normoglycemia was greater for the 60% carbohydrate diet than the 40% diet. Although the four meals were of equal size, in all patients the insulin required to cover breakfast greater than lunch greater than dinner greater than or equal to snack. Expressed as milliunits per kilocalorie, the amount of insulin to cover breakfast was greater for the 60% (P less than .05) than the 40% carbohydrate diet and greater for breakfast than the other meals (P less than .01). Insulin requirements for the Big Mac (43% carbohydrate) were 58% greater than for the 40% carbohydrate diet, even after correction for caloric differences. In summary, 1) increasing dietary carbohydrate from 40 to 60% results in an increased insulin requirement for meals only; 2) insulin requirements are greater in the morning than in the evening, even when meal size is constant; and 3) very large meals with high fat and carbohydrate content result in a major increase in insulin requirement. These data indicate that diet has an important impact on insulin requirements in diabetes.     

Mitochondrial Ca(2+) buffering regulates synaptic transmission between retinal amacrine cells

The diverse functions of retinal amacrine cells are reliant on the physiological properties of their synapses. Here we examine the role of mitochondria as Ca(2+) buffering organelles in synaptic transmission between GABAergic amacrine cells. We used the protonophore p-trifluoromethoxy-phenylhydrazone (FCCP) to dissipate the membrane potential across the inner mitochondrial membrane that normally sustains the activity of the mitochondrial Ca(2+) uniporter. Measurements of cytosolic Ca(2+) levels reveal that prolonged depolarization-induced Ca(2+) elevations measured at the cell body are altered by inhibition of mitochondrial Ca(2+) uptake. Furthermore, an analysis of the ratio of Ca(2+) efflux on the plasma membrane Na-Ca exchanger to influx through Ca(2+) channels during voltage steps indicates that mitochondria can also buffer Ca(2+) loads induced by relatively brief stimuli. Importantly, we also demonstrate that mitochondrial Ca(2+) uptake operates at rest to help maintain low cytosolic Ca(2+) levels. This aspect of mitochondrial Ca(2+) buffering suggests that in amacrine cells, the normal function of Ca(2+)-dependent mechanisms would be contingent upon ongoing mitochondrial Ca(2+) uptake. To test the role of mitochondrial Ca(2+) buffering at amacrine cell synapses, we record from amacrine cells receiving GABAergic synaptic input. The Ca(2+) elevations produced by inhibition of mitochondrial Ca(2+) uptake are localized and sufficient in magnitude to stimulate exocytosis, indicating that mitochondria help to maintain low levels of exocytosis at rest. However, we found that inhibition of mitochondrial Ca(2+) uptake during evoked synaptic transmission results in a reduction in the charge transferred at the synapse. Recordings from isolated amacrine cells reveal that this is most likely due to the increase in the inactivation of presynaptic Ca(2+) channels observed in the absence of mitochondrial Ca(2+) buffering. These results demonstrate that mitochondrial Ca(2+) buffering plays a critical role in the function of amacrine cell synapses.     

Preexposure of murine macrophages to CpG oligonucleotide results in a biphasic tumor necrosis factor alpha response to subsequent lipopolysaccharide challenge

Bacterial DNA and synthetic oligonucleotides containing CpG sequences (CpG-DNA and CpG-ODN) provoke a proinflammatory cytokine response (tumor necrosis factor alpha [TNF-alpha], interleukin-12 [IL-12], and IL-6) and increased mortality in lipopolysaccharide (LPS)-challenged mice via a TNF-alpha-mediated mechanism. It was hypothesized that preexposure of macrophages to CpG-ODN would result in an increased TNF-alpha response to subsequent LPS challenge in vitro. Using the murine macrophage cell line RAW 264.7, we demonstrated both a rapid proinflammatory cytokine response (TNF-alpha) and a delayed inhibitory cytokine response (IL-10) with CpG-ODN. Preexposure of macrophages to CpG-ODN for brief periods (1 to 3 h) augmented TNF-alpha secretion and mRNA accumulation following subsequent LPS challenge (1 microg/ml). However, prolonged preexposure to CpG-ODN (6 to 9 h) resulted in suppression of the TNF-alpha protein and mRNA response to LPS. The addition of anti-IL-10 antibody to CpG-ODN during preexposure resulted in an increase in the LPS-induced TNF-alpha response over that induced by CpG-ODN preexposure alone. Thus, while brief preexposure of macrophages to CpG-ODN augments the proinflammatory cytokine response to subsequent LPS challenge, prolonged preexposure elicits IL-10 production, which inhibits the TNF-alpha response. Although the initial proinflammatory effects of CpG-DNA are well established, the immune response to CpG-DNA may also include autocrine or paracrine feedback mechanisms, leading to a complex interaction of proinflammatory and inhibitory cytokines.     

Association of ambient air‐pollution levels with acute asthma exacerbation among children in Singapore

BACKGROUND: Air-pollution levels have been shown to be associated with increased morbidity of respiratory diseases. METHODS: Data for ambient air-pollutant levels, meteorologic factors, and hospitalization or emergency room (ER) visits for acute asthma in Singapore children over a 5-year period (1990-4) were obtained and analyzed for associations by time-series methods. RESULTS: Throughout this period, the annual mean and 24-h mean levels for sulfur dioxide (SO2), nitrogen dioxide (NO2), and total suspended particles (TSP) and maximum 1-h daily average for ozone were generally within the air-quality guidelines established by the World Health Organization (WHO). However, positive correlation between levels of each of these pollutants and daily ER visits for asthma was observed in children aged 3-12 years, but not among adolescents and young adults (13-21 years old). The association with SO2 and TSP persisted after standardization for meteorologic and temporal variables. An adjusted increase in 2.9 ER visits for every 20 microg/m3 increase in atmospheric SO2 levels, lagged by 1 day, was observed on days when levels were above 68 microg/m3. With TSP, an adjusted increase of 5.80 ER visits for every 20 microg/m3 increase in its daily atmospheric levels, lagged by 1 day, was observed on days with levels above 73 microg/m3. Similar results were also obtained after controlling for autocorrelation by time-series analysis. CONCLUSIONS: These associations were observed even though the overall levels of all pollutants were generally within the air-quality guidelines established by the WHO. These findings suggest that asthmatic children are susceptible to increased levels of air pollutants, particularly SO2 and TSP, although the ambient levels are generally within "acceptable" ranges.     

Effect of estradiol and progesterone on daily rhythm in food intake and feeding patterns in Fischer rats

The product of meal number x meal size, over time, is food intake. Because estrogens modulate feeding activity via their action on the hypothalamus, and because there is a diurnal rhythm in the expression of cytoplasmic estrogen receptors and in estrogen binding activity, the present study examined the effects of ovariectomy and later hormone therapy on acute changes in body weight, and on the meal number-to-meal size relationship as reflected by food intake in the dark/light feeding patterns, in adult female rats in the intact state and after ovariectomy. Twelve female Fischer rats were randomized into ovariectomy and sham operation groups. A rat eater meter measured the feeding indexes for 15 days before and 25 days after ovariectomy, and later for 35 days with hormone therapy. We report: (a) mean body weight gain was linear before and up to ovariectomy, while exponential after ovariectomy; (b) increase in daily food consumption is mainly via an increase in food intake during the light phase; (c) light phase meal number remains unchanged, meal size significantly increases, with the resultant increase in overall food intake; (d) during the dark phase, meal size also significantly increases, but is accompanied by a proportional decrease in meal number, resulting in unchanged dark-phase food intake; and (e) estrogen restoration with either estradiol valerate or estradiol-progesterone combination, reversed the above changes. Data show that in the female Fischer 344 rat: (a) changes in daily rhythm in food intake are brought about by differential effects of the hormones on both meal size and meal number in both the total daily levels as well as in the dark-to-light distribution; (b) estadiol appears to have a tonic inhibitory effect on the light phase meal size and a phasic effect on the dark phase meal size and number, but no significant effect on the light-phase meal number; and (c) in the Fischer rats, progesterone augments estradiol's effect on these indicies.