DNase I sensitivity of nuclear DNA measured by flow cytometry

The DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25-30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50-55% in 30 minutes, and a limit digest of 45-50% beyond 45 minutes. Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 X 10(6) rads (137Cs-gamma-rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of 14C-TdR (thymidine)-labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15-20%). This trapped DNA was released when the digested nuclei were exposed to 0.5-1.0 M NaCl just prior to EtBr staining. Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic Models of C3h Mouse Mammary Tumor Growth: Implications Regarding Tumor Cell Loss

Three models of tumor cell loss are described. the effects of cell loss on other cellular kinetic parameters are evaluated, and experiments which may distinguish among the models are discussed. Each model is based on a different cell-loss mechanism, and equations for the cell-cycle, cell-frequency distribution, the growth of both the proliferating and non-proliferating cell population, the growth fraction (GF), and the relative rate of volumetric growth, (dV/dt)/V, are derived. The following types of data are simulated for each model: the pulse labelling index, the mitotic index, and the labeling index as a function of time after a single or a series of ³H-TdR injections. the relative volumetric growth rate has the same mathematical form for each model. the PLM curves predicted by each model for the tumor lines studied (S102F and Slow) are not appreciably different. the predicted initial labeling index and mitotic index may differ significantly among the models depending upon the tumor line. the most striking difference among the models lies in the predictions regarding the labeling index as a function of time after a single or after a series of ³H-TdR injections. These types of labeling experiments should be valuable for distinguishing the different cell-loss mechanisms in solid tumors.     

Role of ultrasonography in diagnosing early rheumatoid arthritis and remission of rheumatoid arthritis - a systematic review of the literature

Introduction

Ultrasonography (US) might have an added value to clinical examination in diagnosing early rheumatoid arthritis (RA) and assessing remission of RA. We aimed to clarify the added value of US in RA in these situations performing a systematic review.

Methods

A systematic literature search was performed for RA, US, diagnosis and remission. Methodological quality was assessed; the wide variability in the design of studies prohibited pooling of results.

Results

Six papers on the added value of US diagnosing early RA were found, in which at least bilateral metacarpophalangeal (MCP), wrists and metatarsophalangeal (MTP) joints were scanned. Compared to clinical examination, US was superior with regard to detecting synovitis and predicting progression to persistent arthritis or RA. Eleven papers on assessing remission were identified, in which at least the wrist and the MCP joints of the dominant hand were scanned. Often US detected inflammation in patients clinically in remission, irrespective of the remission criteria used. Power Doppler signs of synovitis predicted X-ray progression and future flare in patients clinically in remission.

Conclusions

US appears to have added value to clinical examination for diagnosing of RA when scanning at least MCP, wrist and MTP joints, and, when evaluating remission of RA, scanning at least wrist and MCP joints of the dominant hand. For both purposes primarily power Doppler US might be used since its results are less equivocal than those of greyscale US.     

Delaying S-phase progression rescues cells from heat-induced S-phase hypertoxicity

The mechanism by which a cell protects itself from the lethal effects of heat shock and other stress-inducing agents is the subject of much research. We have investigated the relationship between heat-induced damage to DNA replication machinery and the lethal effects of heat shock, in S-phase cells, which are more sensitive to heat shock than either G1 or G2. We found that maintaining cells in aphidicolin, which prevents the passage of cells through S-phase, can rescue S-phase HeLa cells from the lethal effects of heat shock. When S-phase, HeLa cells were held for 5-6 h in 3 microM aphidicolin the measured clonogenic survival was similar to that for exponentially growing cells. It is known, that heat shock induces denaturation or unfolding of proteins, rendering them less soluble and more likely to co-isolate with the nuclear matrix. Here, we show that enhanced binding of proteins involved in DNA replication (PCNA, RPA, and cyclin A), with the nuclear matrix, correlates with lethality of S-phase cells following heat shock under four different experimental conditions. Specifically, the amounts of RPA, PCNA, and cyclin A associated with the nuclear matrix when cells resumed progression through S-phase correlated with cell killing. Heat-induced enhanced binding of nuclear proteins involved with other aspects of DNA metabolism, (Mrell, PDI), do not show this correlation. These results support the hypothesis that heat-induced changes in the binding of proteins associated with DNA replication factories are the potentially lethal lesions, which become fixed to lethal lesions by S-phase progression but are repairable if S-phase progression is delayed.     

Measurements of alkali-labile DNA damage and protein-DNA crosslinks after 2450 MHz microwave and low-dose gamma irradiation in vitro

In vitro experiments were performed to determine whether 2450 MHz microwave radiation induces alkali-labile DNA damage and/or DNA-protein or DNA-DNA crosslinks in C3H 10T(1/2) cells. After a 2-h exposure to either 2450 MHz continuous-wave (CW) microwaves at an SAR of 1.9 W/kg or 1 mM cisplatinum (CDDP, a positive control for DNA crosslinks), C3H 10T(1/2) cells were irradiated with 4 Gy of gamma rays ((137)Cs). Immediately after gamma irradiation, the single-cell gel electrophoresis assay was performed to detect DNA damage. For each exposure condition, one set of samples was treated with proteinase K (1 mg/ml) to remove any possible DNA-protein crosslinks. To measure DNA-protein crosslinks independent of DNA-DNA crosslinks, we quantified the proteins that were recovered with DNA after microwave exposure, using CDDP and gamma irradiation, positive controls for DNA-protein crosslinks. Ionizing radiation (4 Gy) induced significant DNA damage. However, no DNA damage could be detected after exposure to 2450 MHz CW microwaves alone. The crosslinking agent CDDP significantly reduced both the comet length and the normalized comet moment in C3H 10T(1/2) cells irradiated with 4 Gy gamma rays. In contrast, 2450 MHz microwaves did not impede the DNA migration induced by gamma rays. When control cells were treated with proteinase K, both parameters increased in the absence of any DNA damage. However, no additional effect of proteinase K was seen in samples exposed to 2450 MHz microwaves or in samples treated with the combination of microwaves and radiation. On the other hand, proteinase K treatment was ineffective in restoring any migration of the DNA in cells pretreated with CDDP and irradiated with gamma rays. When DNA-protein crosslinks were specifically measured, we found no evidence for the induction of DNA-protein crosslinks or changes in amount of the protein associated with DNA by 2450 MHz CW microwave exposure. Thus 2-h exposures to 1.9 W/ kg of 2450 MHz CW microwaves did not induce measurable alkali-labile DNA damage or DNA-DNA or DNA-protein crosslinks.     

Evaluation of parameters of oxidative stress after in vitro exposure to FMCW- and CDMA-modulated radiofrequency radiation fields

The goal of this study was to determine whether radiofrequency (RF) radiation is capable of inducing oxidative stress or affecting the response to oxidative stress in cultured mammalian cells. The two types of RF radiation investigated were frequency-modulated continuous-wave with a carrier frequency of 835.62 MHz (FMCW) and code division multiple access centered on 847.74 MHz (CDMA). To evaluate the effect of RF radiation on oxidative stress, J774.16 mouse macrophage cells were stimulated with gamma-interferon (IFN) and bacterial lipopolysaccharide (LPS) prior to exposure. Cell cultures were exposed for 20-22 h to a specific absorption rate of 0.8 W/kg at a temperature of 37.0 +/- 0.3 degrees C. Oxidative stress was evaluated by measuring oxidant levels, antioxidant levels, oxidative damage and nitric oxide production. Oxidation of thiols was measured by monitoring the accumulation of glutathione disulfide (GSSG). Cellular antioxidant defenses were evaluated by measuring superoxide dismutase activity (CuZnSOD and MnSOD) as well as catalase and glutathione peroxidase activity. The trypan blue dye exclusion assay was used to measure any changes in viability. The results of these studies indicated that FMCW- and CDMA-modulated RF radiation did not alter parameters indicative of oxidative stress in J774.16 cells. FMCW- and CDMA-modulated fields did not alter the level of intracellular oxidants, accumulation of GSSG or induction of antioxidant defenses in IFN/LPS-stimulated cells. Consistent with the lack of an effect on oxidative stress parameters, no change in toxicity was observed in J774.16 cells after either optimal (with or without inhibitors of nitric oxide synthase) or suboptimal stimulation.     

Interaction with GM130 during HERG ion channel trafficking. Disruption by type 2 congenital long QT syndrome mutations. Human Ether-à-go-go-Related Gene

Many mutations in the Human Ether-à-go-go-Related Gene (HERG) cause type 2 congenital long QT syndrome (LQT2) by disrupting trafficking of the HERG-encoded potassium channel. Beyond observations that some mutations trap channels in the endoplasmic reticulum, little is known about how trafficking fails. Even less is known about what checkpoints are encountered in normal trafficking. To identify protein partners encountered as HERG channels are transported among subcellular compartments, we screened a human heart library with the C terminus of HERG using yeast two-hybrid technology. Among the proteins isolated was GM130, a Golgi-associated protein involved in vesicular transport. The interaction mapped to two non-contiguous regions of HERG and to a region just upstream of the GRASP-65 interaction domain of GM130. GM130 did not interact with the N or C terminus of either KvLQT1 or Shaker channels. LQT2-causing mutations in the HERG C terminus selectively disrupted interactions with GM130 but not Tara, another HERG-interacting protein. Native GM130 and stably expressed HERG were co-immunoprecipitated from HEK-293 cells using GM130 antibodies. In rat cardiac myocytes and HEK-293 cells, confocal immunocytochemistry showed co-localization of GM130 and HERG to the Golgi apparatus. Overexpression of GM130 suppressed HERG current amplitude in Xenopus oocytes, as if by providing an excess of substrate at the Golgi checkpoint. These findings indicate that GM130 plays a previously undefined role in cargo transport. We propose that the cytoplasmic C terminus of HERG participates in the tethering or possibly targeting of HERG-containing vesicles within the Golgi via its interaction with GM130.     

The effect of chronic exposure to 835.62 MHz FDMA or 847.74 MHz CDMA radiofrequency radiation on the incidence of spontaneous tumors in rats

This study was designed to determine whether chronic exposure to radiofrequency (RF) radiation from cellular phones increased the incidence of spontaneous tumors in F344 rats. Eighty male and 80 female rats were randomly placed in each of three irradiation groups. The sham group received no irradiation; the Frequency Division Multiple Access (FDMA) group was exposed to 835.62 MHz FDMA RF radiation; and the Code Division Multiple Access (CDMA) group was exposed to 847.74 MHz CDMA RF radiation. Rats were irradiated 4 h per day, 5 days per week over 2 years. The nominal time-averaged specific absorption rate (SAR) in the brain for the irradiated animals was 0.85 +/- 0.34 W/kg (mean +/- SD) per time-averaged watt of antenna power. Antennas were driven with a time-averaged power of 1.50 +/- 0.25 W (range). That is, the nominal time-averaged brain SAR was 1.3 +/- 0.5 W/kg (mean +/- SD). This number was an average from several measurement locations inside the brain, and it takes into account changes in animal weight and head position during irradiation. All major organs were evaluated grossly and histologically. The number of tumors, tumor types and incidence of hyperplasia for each organ were recorded. There were no significant differences among final body weights or survival days for either males or females in any group. No significant differences were found between treated and sham-exposed animals for any tumor in any organ. We conclude that chronic exposure to 835.62 MHz FDMA or 847.74 MHz CDMA RF radiation had no significant effect on the incidence of spontaneous tumors in F344 rats.