Protein kinase Cepsilon is oncogenic in colon epithelial cells by interaction with the ras signal transduction pathway

We have shown previously that overexpression of the epsilon isoform of protein kinase C (PKCepsilon) in rat colonic epithelial cells causes malignant transformation, possibly by interacting with the ras signal transduction pathway (Oncogene 12: 847, 1996). We have now performed experiments to examine certain early steps in the ras signaling pathway. A marked increase of Raf-1 phosphorylation was detected in tumorigenic ras-transformed D/ras as well as in D/epsilon cells (overexpressing PKCepsilon), compared to the nontumorigenic D/WT parental line. Moreover, in the PKCepsilon-transformed D/epsilon cell line, stable transfection with a dominant-negative raf-1 (DNraf) sequence caused complete regression of the neoplastic phenotype. These results suggested that PKCepsilon-induced transformation was associated with increased Raf-1 activation, and that DNraf could block the oncogenic effect of PKCepsilon. Furthermore, transfection of D/WT cells with dominant-negative ras induced arrest of cell growth, and subsequent transfection with PKCepsilon cDNA enhanced cell proliferation and induced neoplastic transformation. These results suggest that ras acts upstream of PKCepsilon, and that overexpression of PKCepsilon circumvents the block in cell proliferation caused by dominant-negative ras. We conclude that PKCepsilon exerts its oncogenic activity in rat colonic cells by affecting the ras signaling cascade at the level of Raf-1 activation.     

Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome C and activation of caspase-3

Bcr-Abl expression in leukemic cells is known to exert a potent effect against apoptosis due to antileukemic drugs, but its mechanism has not been elucidated. Recent reports have indicated that a variety of apoptotic stimuli cause the preapoptotic mitochondrial release of cytochrome c (cyt c) into cytosol, which mediates the cleavage and activity of caspase-3 involved in the execution of apoptosis. Whether Bcr-Abl exerts its antiapoptotic effect upstream to the cleavage and activation of caspase-3 or acts downstream by blocking the ensuing degradation of substrates resulting in apoptosis, has been the focus of the present studies. In these, we used (1) the human acute myelogenous leukemia (AML) HL-60 cells that are stably transfected with the bcr-abl gene (HL-60/Bcr-Abl) and express p185 Bcr-Abl; and (2) the chronic myelogenous leukemia (CML)-blast crisis K562 cells, which have endogenous expression of p210 Bcr-Abl. Exposure of the control AML HL-60 cells to high-dose Ara-C (HIDAC), etoposide, or sphingoid bases (including C2 ceramide, sphingosine, or sphinganine) caused the accumulation of cyt c in the cytosol, loss of mitochondrial membrane potential (MMP), and increase in the reactive oxygen species (ROS). These preapoptotic events were associated with the cleavage and activity of caspase-3, resulting in the degradation of poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) and DNA fragmentation factor (DFF), internucleosomal DNA fragmentation, and morphologic features of apoptosis. In contrast, in HL-60/Bcr-Abl and K562 cells, these apoptotic stimuli failed to cause the cytosolic accumulation of cyt c and other associated mitochondrial perturbations, as well as the failure to induce the activation of caspase-3 and apoptosis. While the control HL-60 cells showed high levels of Bcl-2 and barely detectable Bcl-xL, HL-60/Bcr-Abl cells expressed high levels of Bcl-xL and undetectable levels of Bcl-2, a pattern of expression similar to the one in K562 cells. Bax and caspase-3 expressions were not significantly different between HL-60/Bcr-Abl or K562 versus HL-60 cells. These findings indicate that Bcr-Abl expression blocks apoptosis due to diverse apoptotic stimuli upstream by preventing the cytosolic accumulation of cyt c and other preapoptotic mitochondrial perturbations, thereby inhibiting the activation of caspase-3 and execution of apoptosis.     

ATP inhibition and rectification of a Ca2+-activated anion channel in sarcoplasmic reticulum of skeletal muscle

We describe ATP-dependent inhibition of the 75-105-pS (in 250 mM Cl-) anion channel (SCl) from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle. In addition to activation by Ca2+ and voltage, inhibition by ATP provides a further mechanism for regulating SCl channel activity in vivo. Inhibition by the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate (AMP-PNP) ruled out a phosphorylation mechanism. Cytoplasmic ATP (approximately 1 mM) inhibited only when Cl- flowed from cytoplasm to lumen, regardless of membrane voltage. Flux in the opposite direction was not inhibited by 9 mM ATP. Thus ATP causes true, current rectification in SCl channels. Inhibition by cytoplasmic ATP was also voltage dependent, having a K(I) of 0.4-1 mM at -40 mV (Hill coefficient approximately 2), which increased at more negative potentials. Luminal ATP inhibited with a K(I) of approximately 2 mM at +40 mV, and showed no block at negative voltages. Hidden Markov model analysis revealed that ATP inhibition 1) reduced mean open times without altering the maximum channel amplitude, 2) was mediated by a novel, single, voltage-independent closed state (approximately 1 ms), and 3) was much less potent on lower conductance substates than the higher conductance states. Therefore, the SCl channel is unlikely to pass Cl- from cytoplasm to SR lumen in vivo, and balance electrogenic Ca2+ uptake as previously suggested. Possible roles for the SCl channel in the transport of other anions are discussed.     

The impact of genetic markers on selection

Genetic marker technologies, such as marker-assisted selection, parentage identification, and gene introgression can be applied to livestock selection programs. Highly saturated genetic maps are now available for cattle, swine, and sheep to provide the genetic framework for developing MAS programs. These programs rely on three phases for commercialization of the technology: the detection phase, in which quantitative trait loci are located and their effects on the phenotype measured; the evaluation phase, in which the markers are evaluated in commercial populations; and the implementation phase, in which markers are combined with phenotypic and pedigree information in genetic evaluation for predicting the genetic merit of individuals within the population. Predicting the economic impact of genetic technologies is a complex process that requires quantitative prediction and economic analysis. Evaluating the impact of these benefits across an industry can be achieved through a process in which gains from implementation of a genetic technology are assessed at the individual, enterprise, and industry levels. A pattern of annual benefits and costs can be predicted using gene flows that can be evaluated by conventional economic analysis.     

Current methods for assessing mucociliary transport disorders in the diagnosis of chronic rhinosinusitis]

This paper summarises the findings of a systematic review of research on preschool vision screening. The review concludes that the provision of such screening programmes should be reconsidered, if not abandoned.     

Concomitant treatment with mild hyperthermia, cisplatin and low dose-rate irradiation in human ovarian cancer cells sensitive and resistant to cisplatin

Hyperthermia has been shown to be an effective radiation sensitizer. Cisplatin has also been shown to cause radiosensitization. In part, the sensitization is through the inhibition of repair of radiation damage. In this study we have set out to combine low dose-rate irradiation (during which extensive repair occurs) with both cisplatin and hyperthermia to maximize the radiation sensitizing effect. Two human ovarian carcinoma cell lines, one parental (A2780S) and the other a cisplatin resistant derivative (A2780CP) cell line were used in these experiments. Long duration hyperthermia at 40 degrees C was combined with low concentrations of cisplatin (0.5-3 microg/ml) and low dose-rate irradiation (LDRI). The responses to the individual treatments showed that there was cross resistance in the two cell lines for cisplatin and radiation, but for hyperthermia the opposite effect was found. When all treatments were given concurrently the response was greater than the calculated response of all three individual treatments, indicating a synergistic interaction. The effect was greater in the cisplatin resistant cell line. The combination of mild hyperthermia, low dose cisplatin and LDRI are a good combination for potential clinical application. In addition, this could be a good approach to deal with cisplatin resistance.     

Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis

Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential.     

Gunshot injuries in infants and children in KwaZulu-Natal--an emerging epidemic?

AIM: To report the quantitation of the lipid composition of a corneal button from a Japanese woman in her 60s with clinically and histopathologically proved Schnyder's corneal dystrophy. METHODS: Total lipids extracted from the corneal button of the patient were analysed by the method of thin layer chromatography flame ionisation detection. Two different solvent systems were used for neutral lipid analysis and phospholipid analysis. Results were compared with three age matched corneal buttons obtained from cadaveric eyes. RESULTS: The lipids that accumulated in the cornea in Schnyder's dystrophy consisted mainly of unesterified cholesterol and phospholipids. The analysis of phospholipids showed sphingomyelin to be the predominant phospholipid in the patient's cornea. CONCLUSION: Findings suggest that this disorder involves a disturbance of the metabolism of cholesterol and/or sphingomyelin metabolism that is limited to the cornea.     

Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

The enzyme nitric oxide synthase catalyzes the oxidation of the amino acid L-arginine to L-citrulline and nitric oxide in an NADPH-dependent reaction. Nitric oxide plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and is a key component of the cytostatic/cytotoxic function of the immune system. Characterization of nitric oxide synthase substrates and cofactors has outlined the broad details of the overall reaction and suggested possibilities for chemical steps in the reaction; however, the molecular details of the reaction mechanism are still poorly understood. Recent evidence suggests a role for the reduced bound pterin in the first step of the reaction--the hydroxylation of L-arginine.     

On the influence of Si−Ge additions on the aging response of Al−Cu

Al−Cu−Si−Ge alloys display a unique combination of ultrarapid aging response, high peak hardness, and extended-aging microstructural stability. The purpose of this work is to explain these properties in terms of the role that the Si−Ge additions have on modifying the conventional Al−Cu aging sequence. In both AlCu and AlCuSiGe, the room-temperature microstructure consists of both Guinier-Preston (GP) zones and ϕ″ precipitates. Upon aging at 190°C, Al−Cu displays the well-known precipitation sequence: the slow dissolution of GP zones and ϕ″ and the gradual formation of ϕ′. In the quaternary alloy, Si−Ge particles quickly nucleate and grow during elevated-temperature aging (they are detected after as little as 30 minutes at 190 °C). The Si−Ge particles then act as nucleation sites for ϕ′ precipitates, resulting in a peak-aged microstructure consisting of a dense distribution of ϕ′ attached to Si−Ge. DAVID MITLIN, Postdoctoral Fellow, formerly with the Materials Science Department, University of California-Berkeley and the Lawrence Berkeley National Laboratory.