Identification of genes responsive to gamma radiation in rat hepatocytes and rat liver by cDNA array gene expression analysis

The mechanisms underlying hepatocellular damage after irradiation are obscure. We identified genes induced by radiation in isolated rat hepatocytes in vitro by cDNA array gene expression analysis and then screened in vivo experiments with those same genes using real-time PCR and Western blotting. Hepatocytes were irradiated and cDNA array analyses were performed 6 h after irradiation. The mRNA of differentially expressed genes was quantitatively analyzed by real-time PCR. cDNA array analyses showed an up-regulation of 10 genes in hepatocytes 6 h after irradiation; this was confirmed by real-time PCR. In vivo, rat livers were irradiated selectively. Treated and sham-irradiated controls were killed humanely 1, 3, 6, 12, 24 and 48 h after irradiation. Liver RNA was analyzed by real-time PCR; expression of in vivo altered genes was also analyzed at the protein level by Western blotting. Up-regulation was confirmed for three of the in vitro altered genes (multidrug resistance protein, proteasome component C3, eukaryotic translation initiation factor 2). Histologically, livers from irradiated animals were characterized by steatosis of hepatocytes. Thus we identified genes that may be involved in liver steatosis after irradiation. The methods shown in this work should help to further clarify the consequences of radiation exposure in the liver.     

ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca(2+)-ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin

Sarcoplasmic reticulum Ca(2+)-ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca(2+). Unphosphorylated enzyme was rapidly degraded. In contrast, ADP-insensitive phosphoenzyme formed with P(i) and phosphorylated state analogues produced by the binding of F(-) or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg(505)). The results indicate that the phosphoenzyme and its analogues have a very compact form in the cytoplasmic region, being consistent with large domain motions (gathering of three cytoplasmic domains). Results further show that the structure of the enzyme with bound decavanadate is very similar to ADP-insensitive phosphoenzyme. Thapsigargin did not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues.     

Structural analysis of dental fear in children with and without dental trauma experience

The aim of the study was to evaluate dental fear in children with and without dental injuries in a randomly selected children in Croatia (59 girls and 88 boys). Children were divided into three groups depending on dental trauma experience. They were also divided into two age groups: 5-8 and 9-12 years. Only dental trauma to the permanent teeth was included in the study. The CFSS-DS, CDAS and CMFQ were used for evaluation of dental anxiety and the ISP Hollingshead Index of Social Position was calculated for evaluation of social status. The mean values of CDAS, CFSS-DS and CMFQ tests revealed that the anxiety level decreases with increasing experience of dental injury. The analysis of variance performed for CDAS showed a significant difference between children with and without dental trauma (p = 0,010). Regarding the groups, the analysis of variance for CMFQ (p = 0,021) and CFSS-DS (p = 0,001) showed a significant difference, as well as regarding age (CMFQ; p = 0,001 and CFSS-DS; p = 0,016). Cronbach's alpha coefficients revealed the highest reliability for CFSS-DS (alpha = 0,910). Pearson's correlation coefficients revealed significant correlations between the anxiety scores, age and ISP values for children without dental trauma, and between the anxiety scores and age for children with repeated dental trauma. The results of the ISP Hollingshead Index exhibited the highest frequency in children with dental injuries who belonged to the families with poor social background (ISP = 44-60). No significant difference was obtained between children with and without dental injuries depending on either gender or the ISP value.     

Hemolymph ion composition and volume changes in the supralittoral isopod Ligia pallasii Brandt, during molt

We analyzed ion composition and volume of the hemolymph of Ligia pallasii in four different stages of the molt cycle using capillary electrophoresis and ³H-inulin. The main ions in the hemolymph were Na⁺, K⁺, Mg²⁺, Ca²⁺, and Cl⁻. The Ca²⁺concentration increased significantly during the molt by 47% from intermolt to intramolt and by 37% from intermolt to postmolt, probably due to resorption of Ca²⁺ from the cuticle and sternal CaCO₃ deposits. The K⁺ concentration increased significantly by 20% during molt. The hemolymph volume normalized to the dry mass of the animals decreased by 36% from intermolt to late premolt. This was due to a reduction in the hemolymph volume and to an increase in dry mass of the animals during premolt. A sudden increase in the hemolymph volume occurring between late premolt and intramolt served to expand the cuticle. Since the Na⁺, K⁺, Mg²⁺, and Cl⁻ concentrations did not change significantly from late premolt to intramolt, the increase in hemolymph volume suggests an uptake of seawater rather than freshwater. Accepted: 7 March 2000     

Changes of hepatic lactoferrin gene expression in two mouse models of the acute phase reaction

Lactoferrin (Ltf), an iron binding glycoprotein, is a pleiotropic molecule whose serum concentration increases under acute phase conditions. The physiological roles of this protein have been well elucidated, but the source and serum regulation of Ltf gene expression have not been investigated in detail as part of the acute phase reaction (APR). In the current work, the changes in hepatic Ltf-gene-expression during turpentine oil- (TO-) or LPS-induced APR were investigated. Ltf was upregulated at both the mRNA and protein levels in the liver of TO- and LPS-treated wild type (WT) mice. The pattern of induction however was different in both animal models indicating distinctive signalling patterns resulting in an acute phase reaction. Cytokines are the core regulators of APR. Among the major cytokines, IL-6 is an important signalling molecule, which also regulates iron homeostasis in response to an inflammatory situation. In this study, the administration of IL-6 induced Ltf gene expression in the liver of WT mice, in murine hepatocytes and in hepa 1-6 cells. Ltf-gene-expression was upregulated also in the liver of TO- and LPS-treated IL-6 knockout (KO) mice. The increase in serum Ltf after LPS injection was greater than after TO-injection both in WT and IL-6-KO mice. To evaluate the contribution of other acute phase cytokines in the regulation of Ltf-gene-expression in the liver, both in vitro and in vivo studies with IL-1β, TNF-α, or IFN-γ were performed. The results demonstrate that TNF-α and IFN-γ also upregulated Ltf-gene-expression, while IL-1β has no role in the regulation of Ltf-gene-expression.     

EEG beta and gamma powers when comparing certain psychophysiological states with normal and weakened electromyogram of facial muscles

With the advancement of contemporary techniques for studies of high-frequency electroencephalograms (EEGs), possible contamination of the EEG with the electromyogram (EMG) of pericranial muscles has raised more and more concern. The aim of the present study was to demonstrate if certain EEG correlates of mental activities can be revealed in a high-frequency scalp EEG in spite of EMG contamination. Nineteen healthy women who performed similar test tasks before and after cosmetic injections of Dysport in various facial regions for reduction of the activity of facial muscles took part in the study. Inductions of emotional states with different valences, memory storing, and extraction of verbal information were used in the test tasks. The default state of rest was examined as well. During performance of the tasks, parallel registrations of the EEG from the scalp surface (19 channels) and EMG from several facial muscles (6 channels) were carried out. Changes in the spectral power in β₂ and low γ frequency bands (18–40 Hz) in EEG- and EMG-derivations after Dysport injections were analyzed. Changes in the spectral power in the same bands in pairwise comparisons for the test tasks before and after Dysport injections were also analyzed separately. It was demonstrated that Dysport injections lead to reduction of the EMG power in areas of the injections and to reduction of EEG power in the frontal and temporal derivations. However, the EEG-correlates revealed when comparing different test tasks remained qualitatively invariable as for after and before Disport injections. These facts confirm that EMG makes a noticeable contribution to the electric activity registered from the scalp in the frequency ranges greater than 18 Hz. At the same time, one can see that at least in certain experimental situations the influence of EMG does not make impossible identification of EEG-correlates of mental activity with EEG registration from the head surface at least in the β₂ and low γ frequency bands (18–40 Hz).     

Comparative study of the topographic localization of NADPH diaphorase positive cells in the rabbit and pheasant thymuses

Nicotinamide adenine dinucleotide hydrogen phosphatediaphorase (NADPHd) histochemistry was used as a marker for nitric oxide synthase (NOS). In the rabbit thymus, NADPHd staining was observed between the capsule and corticomedullary junction in radially oriented blood vessels in the cortex. The outer surface of the thymic lobule and interlobular septa showed adipocytes clumped together. There was a high density of NADPHd positive cells in the medulla, without a sharp boundary in corticomedullary space. In addition to radially oriented blood vessels in the cortex, they were also found as solitary profiles with well stained walls in the medulla. Neuronal plexuses were localized in perivascular topography. In the pheasant thymus, NADPHd positive cells were present as clusters which were distributed in the medulla and the corticomedullar area. NADPHd positive nerve fibres were localized in perivascular topography.     

Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum

Sarcoplasmic reticulum isolated from rabbit skeletal muscle was labeled with a limited (0.625 nmol/mg sarcoplasmic reticulum protein) amount of the fluorescent thiol reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The fluorescence intensity of the membrane-attached DACM decreased concurrently with (Ca2+ and caffeine)-induced Ca2+ release, depolarization-induced Ca2+ release and Ca2+-dependent dependent passive efflux of Ca2+. The decreased DACM fluorescence level initiated by a Ca2+ jump was subsequently reversed under passive efflux conditions when there was no ATP-dependent Ca2+ uptake, suggesting spontaneous closing of the channels. Therefore, the higher fluorescence level corresponds to a larger population of closed channels, whereas the lower level represents a larger population of opened channels. Under conditions when the Ca2+ release-coupled fluorescence change was maximal, a stoichiometric incorporation of DACM took place only into a 32-kDa protein. Furthermore, reconstituted vesicles, in which purified DACM-labeled 32-kDa protein was incorporated into unlabeled sarcoplasmic reticulum vesicles, were capable of both (Ca2+ and caffeine)-induced Ca2+ release and the release-coupled DACM fluorescence change. These results suggest that the 32-kDa protein is a constituent of the Ca2+ release channel or a protein which is in close contact with the channel.     

Exposure of Chlorpromazine to 266 nm Laser Beam Generates New Species with Antibacterial Properties: Contributions to Development of a New Process for Drug Discovery

Introduction

Phenothiazines when exposed to white light or to UV radiation undergo a variety of reactions that result in degradation of parental compound and formation of new species. This process is slow and may be sped up with exposure to high energy light such as that produced by a laser.

Methods

Varying concentrations of Chlorpromazine Hydrochloride (CPZ) (2–20 mg/mL in distilled water) were exposed to 266 nm laser beam (time intervals: 1–24 hrs). At distinct intervals the irradiation products were evaluated by spectrophotometry between 200–1500 nm, Thin Layer Chromatography, High Pressure Liquid Chromatography (HPLC) - Diode Array Detection, HPLC tandem mass spectrometry, and for activity against the CPZ sensitive test organism Staphylococcus aureus ATCC 25923.

Results

CPZ exposure to 266 nm laser beam of given energy levels yielded species, whose number increased with duration of exposure. Although the major species produced were Promazine (PZ), hydroxypromazine or PZ sulfoxide, and CPZ sulfoxide, over 200 compounds were generated with exposure of 20 mg/mL of CPZ for 24 hrs. Evaluation of the irradiation products indicated that the bioactivity against the test organism increased despite the total disappearance of CPZ, that is due, most probably, to one or more new species that remain yet unidentified.

Conclusions

Exposure of CPZ to a high energy (6.5 mJ) 266 nm laser beam yields rapidly a large number of new and stable species. For biological grade phenothiazines (in other words knowing the impurities in the samples: solvent and solute) this process may be reproducible because one can control within reasonably low experimental errors: the concentration of the parent compound, the laser beam wavelength and average energy, as well as the duration of the exposure time. Because the process is “clean” and rapid, it may offer advantages over the pyrogenically based methods for the production of derivatives.