Interference of plant extracts,phytoestrogens and antioxidants with the MTT tetrazolium assay

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely used screening method to measure cell viability and proliferation. When testing the effects of kaempferol on breast cancer cell number (crystal violet staining) and viability (MTT tetrazolium assay) conflicting results were obtained. Cell number decreased but MTT formazan formation increased, suggesting a direct interaction of kaempferol with the MTT tetrazolium reduction. Direct reductive potential was observed in a cell-free system for the presumptive phytoestrogens kaempferol and resveratrol, and extracts of Hypericum perforatum L. and Cimicifuga racemosa L. All agents led to instantaneous dark blue formazan formation in the absence of cells. Additionally, antioxidants such as ascorbic acid, vitamin E and N-acetylcysteine interfered with the MTT tetrazolium assay. When MCF7 and HS578 cells treated with kaempferol were washed before addition of MTT tetrazolium, the direct reduction of dye was reduced significantly. These results indicate that the MTT tetrazolium assay may lead to false positive results when testing natural compounds with intrinsic reductive potential.     

Construction noise: exposure,effects, and the potential for remediation; a review and analysis

More than one-half million construction workers are exposed to potentially hazardous levels of noise, yet federal and state Occupational Safety and Health Administration (OSHA) programs provide little incentive to protect them against noise-induced hearing loss. Construction noise regulations lack the specificity of general industry noise regulations. In addition, problems that characterize the construction industry, such as worker mobility and the large proportion of small businesses, make implementing hearing conservation measures more difficult. The apparent severity of exposure depends greatly on the measurement method, with the 3-dB exchange rate almost always showing higher average exposure levels than the 5-dB (OSHA) rule. Construction workers demonstrate hearing threshold levels that generally conform to those expected in manufacturing. The prevalence of hearing protection device (HPD) use among U.S. construction workers is very poor, partly because of perceived difficulties in hearing and understanding speech communication and warning signals. In addition, masking by noise of necessary communication and warning signals is of particular concern in construction, where recent research demonstrated the association between fatalities and the failure to hear reverse alarms. Judicial use of HPDs is of the utmost importance, along with avoiding overattenuation, selecting HPDs with uniform attenuation, and using noise-attenuating communication systems when possible. A successful hearing conservation program in British Columbia can serve as a model for the United States, with a long-standing positive safety culture, a high percentage of HPD use, improvement in average hearing threshold levels over the last decade, and a centralized record-keeping procedure, which helps solve the problem of worker mobility. However, controlling construction noise at the source is the most reliable way to protect worker hearing. U.S. manufacturers and contractors should benefit from the activities of the European Community, where noise control and product labeling in construction has been carried out for more than 20 years.     

Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module

Mutations in the myotubularin (MTM)-related protein 2 (MTMR2) gene are responsible for the severe autosomal recessive neuropathy Charcot-Marie-Tooth disease type 4B1. MTMR2 belongs to the MTM family of dual-specific phosphatases that use phosphatidylinositol (PI) 3,5-bisphosphate [PI(3,5)P2] and PI 3-phosphate [PI(3)P] as their substrate. Because these substrates are localized in the membrane bilayer, membrane targeting of Mtmr2 is an important regulatory mechanism. In hypoosmotically stressed COS cells with increased levels of PI(3,5)P2, Mtmr2 is bound to the membrane of vacuoles formed under these conditions. Using several mutant forms of Mtmr2, we identified two domains that are necessary for membrane association: (i) A pleckstrin homology-GRAM domain; and (ii) a coiled-coil module. Protein-lipid overlay assays show that the pleckstrin homology-GRAM domain binds to PI(3,5)P2 and PI(5)P, a substrate and a product of the Mtmr2 enzyme, respectively. We also demonstrate that Mtmr2 forms a dimer and that the C-terminal coiled-coil is responsible for homodimerization, in addition to membrane association. Our data indicate that phosphoinositide-protein interactions, as well as protein-protein interactions, are necessary for the correct regulation of MTMR2.     

Troglitazone antagonizes metabolic effects of glucocorticoids in humans: effects on glucose tolerance,insulin sensitivity,suppression of free fatty acids,and leptin

Glucocorticoids induce insulin resistance in humans, whereas thiazolidinediones enhance insulin sensitivity. Although the effects of glucocorticoids and thiazolidinediones have been assessed in isolation, interaction between these drugs, which both act as ligands for nuclear receptors, has been less well studied. Therefore, we examined the metabolic effects of dexamethasone and troglitazone, alone and in combination, for the first time in humans. A total of 10 healthy individuals with normal glucose tolerance (age 40 +/- 11 years, BMI 31 +/- 6.1 kg/m(2)) were sequentially studied at baseline, after 4 days of dexamethasone (4 mg/day), after 4-6 weeks on troglitazone alone (400 mg/day), and again after 4 days of dexamethasone added to troglitazone. Key metabolic variables included glucose tolerance assessed by blood glucose and insulin responses to an oral glucose tolerance test (OGTT), insulin sensitivity evaluated via hyperinsulinemic-euglycemic clamp, free fatty acids (FFAs) and FFA suppressibility by insulin during the clamp study, and fasting serum leptin. Dexamethasone drastically impaired glucose tolerance, with fasting and 2-h OGTT insulin values increasing by 2.3-fold (P < 0.001) and 4.4-fold (P < 0.001) over baseline values, respectively. The glucocorticoid also induced a profound state of insulin resistance, with a 34% reduction in maximal glucose disposal rates (GDRs; P < 0.001). Troglitazone alone increased GDRs by 20% over baseline (P = 0.007) and completely prevented the deleterious effects of dexamethasone on glucose tolerance and insulin sensitivity, as illustrated by a return of OGTT glucose and insulin values and maximal GDR to near-baseline levels. Insulin-mediated FFA suppressibility (FFA decline at 30 min during clamp/FFA at time 0) was also markedly reduced by dexamethasone (P = 0.002). Troglitazone had no effect per se, but it was able to normalize FFA suppressibility in subjects coadministered dexamethasone. Futhermore, the magnitudes of response of FFA suppressibility and GDR to dexamethasone were proportionate. The same was true for the reversal of dexamethasone-induced insulin resistance by troglitazone, but not in response to troglitazone alone. Leptin levels were increased 2.2-fold above baseline by dexamethasone. Again, troglitazone had no effect per se but blocked the dexamethasone-induced increase in leptin. Subjects experienced a 1.7-kg weight gain while taking troglitazone but no other untoward effects. We conclude that in healthy humans, thiazolidinediones antagonize the action of dexamethasone with respect to multiple metabolic effects. Specifically, troglitazone reverses both glucocorticoid-induced insulin resistance and impairment of glucose tolerance, prevents dexamethasone from impairing the antilipolytic action of insulin, and blocks the increase in leptin levels induced by dexamethasone. Even though changes in FFA suppressibility were correlated with dexamethasone-induced insulin resistance and its reversal by troglitazone, a cause-and-effect relationship cannot be established. However, the data suggest that glucocorticoids and thiazolidinediones exert fundamentally antagonistic effects on human metabolism in both adipose and muscle tissues. By preventing or reversing insulin resistance, troglitazone may prove to be a valuable therapeutic agent in the difficult clinical task of controlling diabetes in patients receiving glucocorticoids.     

Hepatitis C virus infection in young, low-income women: the role of sexually transmitted infection as a potential cofactor for HCV infection

OBJECTIVES: We evaluated risk for hepatitis C virus (HCV) infection in women residing in low-income neighborhoods of northern California. METHODS: A population-based sample of 1707 women, aged 18 to 29, were surveyed and screened for sexually transmitted infections and HCV. RESULTS: Women infected with HCV (2.5%) were more likely to have a history of injection and noninjection drug use, to exchange sex for money or drugs, and to have sexually transmitted infections. HCV was independently associated with history of injection drug use, herpes simplex virus type 2 (HSV-2) infection, and heroin and cocaine use. CONCLUSIONS: Injection drug use is the highest risk exposure for HCV, but HSV-2 and noninjection drug use contribute significantly to increased risk. HCV prevention programs in impoverished areas should integrate drug treatment and sexually transmitted infection control.     

Identification of the regulatory region of the peripheral myelin protein 22 (PMP22) gene that directs temporal and spatial expression in development and regeneration of peripheral nerves

Minor changes in PMP22 gene dosage have profound effects on the development and maintenance of peripheral nerves. This is evident from the genetic disease mechanisms in Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) as well as transgenic animals with altered PMP22 gene dosage. Thus, regulation of PMP22 is a crucial aspect in understanding the function of this protein in health and disease. In this study, we have generated transgenic mice containing 10 kb of the 5'-flanking region of the PMP22 gene, including the two previously identified alternative promoters, fused to a lacZ reporter gene. We show that this part of the PMP22 gene contains the necessary information to mirror the endogenous expression pattern in peripheral nerves during development and regeneration and in mouse models of demyelination due to genetic lesions. Transgene expression is strongly regulated during myelination, demyelination, and remyelination in Schwann cells, demonstrating the crucial influence of neuron-Schwann cell interactions in the regulation of PMP22. In addition, the region of the PMP22 gene present on this transgene confers also neuronal expression in sensory and motor neurons. These results provide the crucial basis for further dissection of the elements that direct the temporal and spatial regulation of the PMP22 gene and to elucidate the molecular basis of the master program regulating peripheral nerve myelination.     

Dose reduction in CT examination of children by an attenuation-based on-line modulation of tube current (CARE Dose)

In a controlled patient study we investigated the potential of attenuation-based on-line modulation of the tube current to reduce milliampere values (mAs) in CT examinations of children without loss of image quality. mAs can be reduced for non-circular patient cross sections without an increase in noise if tube current is reduced at those angular positions where the patient diameter and, consequently, attenuation are small. We investigated a technical approach with an attenuation-based on-line control for the tube current realised as a work-in-progress implementation. The CT projection data are analysed in real time to determine optimal mAs values for each projection angle. We evaluated mAs reduction for 100 spiral CT examinations with attenuation-based on-line modulation of the tube current in a group of children. Two radiologists evaluated image quality by visual interpretation in consensus. We compared the mAs values read from the CT scanner with preset mAs of a standard protocol. Four different scan regions were examined in spiral technique (neck, thorax, abdomen, thorax and abdomen). We found the mAs product to be reduced typically by 10-60% depending on patient geometry and anatomical regions. The mean reduction was 22.3% (neck 20%, thorax 23%, abdomen 23%, thorax and abdomen 22%). In general, no deterioration of image quality was observed. There was no correlation between the age and the mean mAs reduction in the different anatomical regions. By classifying the children respectively to their weight, there is a positive trend between increasing weight and mAs reduction. We conclude that mAs in spiral CT examinations of children can be reduced substantially by attenuation-based on-line modulation of the tube current without deterioration of image quality. Attenuation-based on-line modulation of tube current is efficient and practical for reducing dose exposure to children.     

Noninvasive visualization of coronary arteries using contrast-enhanced multidetector CT: influence of heart rate on image quality and stenosis detection

OBJECTIVE: Although multidetector CT (MDCT) with retrospectively ECG-gated image reconstruction has been shown to permit noninvasive visualization of the coronary arteries, the 125-250 msec required for image acquisition frequently causes motion artifacts. We investigated the influence of a patient's heart rate on the presence of motion artifacts and on accuracy of stenosis detection on contrast-enhanced MDCT. MATERIALS AND METHODS: In 100 patients, MDCT was performed, and ECG-gated cross-sectional images were retrospectively reconstructed. From the 10 data sets obtained for each patient (reconstructed at 0-90% of the cardiac cycle in increments of 10%), we chose the best data set for every coronary artery. The images of the arteries were evaluated for occurrence of artifacts and the presence of high-grade stenosis (diameter reduction exceeding 70%) or occlusions. MDCT results were compared with coronary angiographic findings. RESULTS: Of the 400 coronary arteries, 115 (29%) could not be evaluated because of motion artifacts (n = 84) or other reasons (n = 31). Overall, 51 (49%) of 104 stenoses were revealed on MDCT. For detecting stenosis in those arteries that we could evaluate, MDCT had a sensitivity of 91% (51 of 56 stenoses detected) and a specificity of 89%. As the heart rate increased, the number of arteries that could be evaluated decreased, and overall sensitivity for stenosis detection decreased from 62% (heart rate < or = 70 beats per minute) to 33% (heart rate > 70 beats per minute). CONCLUSION: MDCT can reveal coronary stenoses, but the usefulness of MDCT as an aid in accurately evaluating stenoses decreases as a patient's heart rate increases.