Attila Balogh

Effects of sex hormones on ECG parameters and expression of cardiac ion channels in dogs

Aim:  The aim of the study was to examine the effects of testosterone and oestrogen on the ECG parameters and expression of cardiac ion channels in male and female dogs, and to compare the dofetilide‐induced lengthening of QTc interval in control, castrated and hormone‐treated animals.

Reference genes for quantitative real time PCR in UVB irradiated keratinocytes.

Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a sensitive and highly reproducible method often used for determining mRNA levels. To enable proper comparison of gene expression genes expressed at stabile levels within the cells in the studied experimental system need to be identified and used as reference. Ultraviolet B (UVB) radiation is an exogenous carcinogenic stimulus in keratinocytes, and UVB elicited changes have extensively been studied by qRT-PCR, yet a comparison of commonly used reference genes in UVB treatment is lacking. To find the best genes for compensating slight inter-sample variations in keratinocytes in UVB experiments and to understand the potential effects of improper reference gene (RG) selection we have analyzed the mRNA expression of 10 housekeeping genes in neonatal human epidermal keratinocytes (NHEK) after UVB treatment. The biological effect of the used UVB light source was validated by trypane blue exclusion, MTT and comet assays. 20-40mJ/cm(2) dose was chosen for the experiments. The stability of the 10 RGs was assessed by the GeNorm and Normfinder software tools. Regardless of their slightly different algorithm the programs found succinate dehydrogenase complex subunit A (SDHA) to be the best individual RG and SDHA and phosphoglycerate kinase-1 (PGK1) as the most suitable combination. Analysis of the expression of tumor necrosis factor alpha (TNFalpha) and vascular endothelial growth factor (VEGF) found that while the perception of changes in TNF-alpha, a gene undergoing marked upregulation after UVB irradiation is independent of the used RG, changes seen in the more modestly upregulated VEGF are greatly effected by reference gene selection. These findings highlight the importance of reference gene selection in UVB irradiation experiments, and provide evidence that using SDHA or the combination of SDHA and PGK1 as standards could be a reliable method for normalizing qRT-PCR results in keratinocytes after UVB treatment.

Altered calcium handling following the recombinant overexpression of protein kinase C isoforms in HaCaT cells

Abstract:  Both changes in intracellular calcium concentration ([Ca2+]i) and activation of certain protein kinase C (PKC) isoforms play a crucial role in keratinocyte functions. To better understand the interaction between these two signalling pathways we investigated the resting [Ca2+]i and the extracellular ATP‐induced changes in [Ca2+]i on HaCaT cell clones overexpressing either the classical α or the β PKC isoform. These PKC isoenzymes were previously shown to decrease (α) or increase (β) cell proliferation and augment (α) or suppress (β) cell differentiation. Keratinocyte clones with decreased proliferation rate were found to have unaltered resting [Ca2+]i, but responded with greater calcium transients to the application of 180 μm of ATP. In contrast, clones with increased proliferation rate had elevated resting [Ca2+]i and suppressed calcium responses to ATP. Calcium transients on PKCβ clones displayed a faster falling phase. Each clone had a distinct purinergic receptor expression pattern, some of which paralleled the altered proliferation rate and calcium handling. Keratinocytes overexpressing PKCβ revealed decreased P2X1 and increased P2Y1 receptor expression as compared with the control or PKCα clones. The expression level of P2X7 was significantly increased in keratinocytes overexpressing PKCα. On the other hand neither the P2X2 nor the P2Y2 expression was altered significantly in the cell types investigated. These data indicate that a modified proliferation and differentiation pattern is associated with altered calcium handling in keratinocytes. The observations also suggest that different PKC isoenzymes have different effects on the phosphatidyl–inositol signalling pathway.

UV-B induced alteration in purinergic receptors and signaling on HaCaT keratinocytes.

Although there are a number of recognized risk factors resulting in cutaneous malignancies, very little is known about the exact mechanism. In keratinocytes different purinergic receptors have been implicated to play essential roles in deciding the fate of the cells through regulating proliferation and differentiation. While P2Y receptors seem to control the former, P2X receptors, among which the P2X(7) receptor is associated with the induction of apoptosis, are likely to be responsible for the latter. Forty mJ/cm(2) UV-B irradiation decreased the number of viable cells as assessed using MTT assay. This irradiation decreased the amount of both P2X(1) and P2Y(2) receptors and essentially destroyed the P2X(7) receptors in surviving cells. Morphology of ATP-induced Ca(2+) transients were altered in irradiated cells compared to control. The amplitude and the rate of rise of the transients were decreased and the return to resting [Ca(2+)](i) prolonged. This observation is consistent with the finding that in control cells mostly ionotropic, while in irradiated cells mostly metabotropic receptors were underlying the response to ATP. These alterations in the expression pattern of purinergic receptors and in the Ca(2+) transients could explain the observed decreased tendency for ATP-induced apoptosis and possibly contribute to the malignant transformation of keratinocytes.

Whole genome transcriptional profiling identifies novel differentiation regulated genes in keratinocytes

Please cite this paper as: Whole genome transcriptional profiling identifies novel differentiation regulated genes in keratinocytes. Experimental Dermatology 2010; 19: 100–107.

Keratinocyte ATP binding cassette transporter expression is regulated by ultraviolet light

Many ATP binding cassette (ABC) transporters are important regulators of lipid homeostasis and have been implicated in keratinocyte lipid transport. Ultraviolet (UV) light exposure is a known epidermal stressor, which amongst other effects causes lipid alterations and defective lamellar body biogenesis. To elucidate the background of these lipid changes we studied the effect of UVB light on ABC transporter expression. The effect of UVB treatment on the levels of 47 known human ABC transporter mRNAs was analyzed in normal human epidermal keratinocytes. Immunoblots and promoter assays were carried out for ABCA1 and ABCG1. The mRNA levels of cholesterol transport regulators ABCA1 and ABCG1 were markedly downregulated by UVB, parallel to the lamellar ichthyosis related glucosylceramide transporter ABCA12 and the suspected sphingosine-1-phosphate and cholesterol sulfate transporter ABCC1. The long but not the short alternative splice variant of the ABCF2 was found to be markedly upregulated rapidly after UVB irradiation. Immunoblot confirmed ABCA1 and ABCG1 protein downregulation, and luciferase assays showed suppression of their promoters by UVB. These proteins mostly transport lipids, which account for the integrity of the epidermal barrier; therefore our findings on the UVB regulation of ABC transporters may explain the appearance of barrier dysfunction after UVB exposure.

Transtracheal electrical stimulation of the spinal cord for intraoperative monitoring of the motor pathway

Because of the suppressant effects of anesthetic drugs and muscle relaxants on motor responses elicited by either magnetic or electrical transcranial stimulation, intraoperative monitoring of the motor system, and especially monitoring of lower limb function, presents many difficulties.The upper part of the spinal cord was stimulated in 14 anesthetized and relaxed dogs with a cathode attached to the intratracheal tube and an anode fixed above the upper cervical spinous processes. Action potentials evoked by single and serial stimuli were recorded from the exposed right femoral nerve and quadriceps muscle Averaging was necessary for serial stimulations.Reproducible early and late responses to both single and serial stimulations were recorded during regular anesthesia. The origin of the different responses is discussed.Transtracheal stimulation of the spinal cord is easy to perform and the responses recorded from the peripheral nerve or limb muscle are well reproducible in regular anesthesia. The method seems to be appropriate for intraoperative monitoring of the thoracolumbar spine.

Atomization of cadmium compounds under reactive and inert high-temperature environment with stationary sample introduction.

Atomization of cadmium compounds (acetate, chloride, nitrate, perchlorate, sulfate, formate, propionate) was studied using flame atomic absorption spectrometry. Our goal was to study processes influencing atomization separately, the focus was on the contribution of thermal properties of substances to atomization. For this purpose new techniques and equipment have been developed, such as a special separated three-slot burner, quartz flame furnace, and an electrically heated thermospectrometer. According to quartz flame furnace and thermospectrometric measurements, cadmium salts do not atomize below 600 °C in an inert atmosphere. We found that in the thermospectrometer the atomization of cadmium compounds follows at least two different reaction courses. At lower temperatures (650–700 °C) a slower mechanism is dominant at higher regions of the furnace, while at 800 °C a faster mechanism demanding less residence time in the furnace becomes dominant. Under inert atmosphere the degree of atomization strongly depends on the thermal properties of substances.