Defective P2Y purinergic receptor function: A possible novel mechanism for impaired glucose transport

Extracellular ATP is an ubiquitous mediator that regulates several cellular functions via specific P2 plasma membrane receptors (P2Rs), for which a role in modulating intracellular glucose metabolism has been recently suggested. We have investigated glucose uptake in response to P2Rs stimulation in fibroblasts from type 2 diabetic (T2D) patients and control subjects. P2Rs expression was evaluated by RT-PCR; intracellular calcium release by fluorometry; glucose transporter (GLUT1) translocation by immunoblotting and chemiluminescence; glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose (2-DOG) and ATP by luminometry. Cells from T2D patients, in contrast to those from healthy controls, showed no increase in glucose uptake after ATP stimulation; extracellular ATP caused, however, a similar GLUT1 recruitment to the plasma membrane in both groups. P2Rs expression did not differ between fibroblasts from diabetic and healthy subjects, but while plasma membrane depolarization, a P2X-mediated response was similar in both groups, no evident intracellular calcium increase was detectable in the cells from the former group. The calcium response in fibroblasts from diabetics was restored by co-incubation with apyrase or hexokinase, suggesting that P2YRs in those cells were normally expressed but chronically desensitised. In support to this finding, fibroblasts from T2D subjects secreted a two-fold larger amount of ATP compared to controls. Pre-treatment with apyrase or hexokinase also restored ATP stimulated glucose uptake in fibroblasts from diabetic subjects. These results suggest that extracellular ATP plays a role in the modulation of glucose transport via GLUT1, and that the P2Y-dependent GLUT1 activation is deficient in fibroblasts from T2D individuals. Our observations may point to additional therapeutic targets for improving glucose utilization in diabetes.     

Preservation of rat skeletal muscle energy metabolism by illumination

Skeletal muscle viability is crucially dependent on the tissue levels of its high energy phosphates. In this study we investigated the effect of the preservation medium Perfadex and illumination with Singlet Oxygen Energy (SOE). Singlet oxygen can be produced photochemically by energy transfer from an excited photosensitizer. The energy emitted from singlet oxygen upon relaxation to its triplet state is captured as photons at 634 nm and is here referred to as SOE. Rat hind limb rectus femoris muscles were preserved for five hours at 22 degrees C in Perfadex, saline, SOE illuminated Perfadex or SOE illuminated saline. Extracts of the muscles were analysed by 31P NMR. Data were analysed using two-way analysis of variance and are given as mean values micromol/g dry weight) +/- SEM. The ATP concentration was higher (p = 0.006) in saline groups (4.52) compared with Perfadex groups (2.82). There was no statistically significant difference in PCr between the saline groups (1.25) and Perfadex groups (0.82). However, there were higher (p = 0.003) ATP in the SOE illuminated groups (4.61) compared with the non-illuminated groups (2.73). The PCr was also higher (p < 0.0001) in the SOE illuminated groups (1.89) compared with the non-illuminated groups (0.18). In conclusion, Perfadex in this experimental model was incapable of preserving the high energy phosphates in skeletal muscle during 5 hours of ischemia. Illumination with SOE at 634 nm improved the preservation potential, in terms of a positive effect on the energy status of the muscle cell.     

Angiogenesis and nerve regeneration in a model of human skin equivalent transplant

The angiogenesis and reinnervation were studied in a porcine model of human skin equivalent (SE) graft and the relationship between the two processes was investigated. Confocal laser scanning microscopy was used to monitor, during the healing process, the pattern of vascularization and reinnervation at different time points. The SE was obtained by co-culturing fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan biopolymer and grafted on dorsal wounds generated by full-thickness resection in 25/30 Kg Large white pigs. Frozen sections were obtained from biopsies performed in autograft and xenograft, then were immunolabeled by using the endothelial marker lectin Lactifolia and with the neuronal marker gene product PGP9.5. Cajal staining was also used to visualize the nerve fibers. The results show that the vascularization precedes the innervation process. These data are consistent with the view that the development of nervous tissue is driven by nutritional and trophic factors provided by the vascular system. The arborization of the two systems observed during the third week from the graft might play a key role in maintaining the healing process and the graft survival.     

Siderophores and hemolysins in iron aquisition by Acinetobacter genus

In 60 strains of Acinetobacter genus isolated from clinical material belonging to species A. baumannii, A. haemolyticus, A. junii and A. lwoffii a hydroxamate and phenol-catechol class siderophores was identified by chemical and biological testes. A correlation between siderophores production and growth intensity, species affiliation and origin of strains was found.     

Cytotoxicity of DMSO for MRC5, Chang liver and CV1 cells evaluated in vitro by LK, MTT and NR assays

Evaluation of chemicals cytotoxicity plays fundamental role in many in vitro investigations. The way of assessment of cytotoxicity depend on aim of study, characteristic of used cells and mode of action of investigated chemicals. The principal aspect of these investigations is validation of used method. In this paper validation of three different cytotoxicity assays is presented: total cell number measurement (LK), microplate assay measured mitochondrial dehydrogenase activity (MTT) and colorimetric assay measured ability of live cell to uptake neutral red (NR). The investigation was performed on different cells (MRC5, CV1 i Chang Liver) with DMSO as reference agent.     

Anaerobic bacteria in bronchoalveolar lavage fluid (BAL) after thoracic surgery

Purpose of this study was to find out what kind of anaerobic bacteria were in lower respiratory tract and how often they were present there considering patients after thoracic surgery. Also, what is susceptibility of bacteria to antibiotics. Research covered 30 patients after operation. Material for research was bronchoalveolar lavage (BAL) taken during bronchoscopy. Collected sample was cultivated in anaerobic and aerobic conditions. Anaerobic bacteria were found in 28 samples (93%). Totally there were 100 anaerobic bacteria strains. The most common Gram-negative rods were from genus Prevotella (24 strains, 24%) and Bacteroides (15 strains, 15%). Gram-negative bacteria except Bacteroides characterised biggest susceptibility to imipenem, piperacillin/tazobactam, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin, clindamycin and metronidazol. Bacteroides were susceptible to imipenem, piperacillin/tazobactam and metronidazol. Among Gram-positive anaerobic bacteria mostly were isolated from cocci Peptostreptococcus (18 strains, 18%) and were susceptible to all antibiotics. Gram-positive rods were in most cases represented by Actinomyces (12 strains 12%) and were highly susceptible to all antibacterial means except metronidazol (100% is resistant).     

Biological activities of organic compounds adsorbed onto ambient air particles: comparison between the cities of Teplice and Prague during the summer and winter seasons 2000-2001

The capital of the Czech Republic, Prague, appears today to be one of the most polluted residential areas in the country, whereas air pollution in the Northern Bohemia region (the former "Black Triangle Region") has substantially decreased during the last decade, especially with respect to the gaseous pollutant SO(2). This study evaluated the biological activities of complex mixtures of organic compounds adsorbed onto ambient air particles (PM10) collected during the summer and winter seasons of 2000-2001 at three monitoring sites--Teplice (TP), Prague-Smíchov (PRG-SM) (city centre) and Prague-Libus (PRG-LB) (suburban area). The following short-term in vitro assays with strikingly different endpoints were used: a bacterial mutagenicity test using the Salmonella typhimurium tester strain TA98 and YG1041, an acellular assay (CT DNA) combined with 32P-postlabelling to evaluate DNA adduct-forming potency and the chick embryotoxicity screening test (CHEST). The results of the mutagenicity test with the YG1041 strain, the acellular genotoxicity (DNA adducts) and the embryotoxicity tests responded to the amount of eight carcinogenic polycyclic aromatic hydrocarbons (PAHs) analysed in the EOM (dichloromethane extractable organic matter) samples tested. Nevertheless, the biological effects of the EOM did not differ between locations. The highest biological activity of the ambient air in terms of organic compounds associated with particles (per unit volume of air) was seen in the Prague city centre during both summer and winter seasons. At this location, B[a]P concentration ranged from 0.1 to 8.9 ng/m(3) (mean 0.3 and 3.6 ng/m(3) for summer and winter seasons, respectively), 13 PAHs ranged from 11 to 343 ng/m(3) (mean 52 and 160 ng/m(3) for summer and winter seasons, respectively). Generally, using in vitro tests, higher ambient air activity was found in the winter season as compared with the summer season at all three monitoring sites (TA98 +S9, approximately 4-fold; YG1041 -S9, approximately 5-fold; YG1041 +S9, approximately 8-fold; CT DNA +S9, approximately 10-fold; CHEST, approximately 10-fold; B[a]P, carcinogenic PAHs and total PAHs analysed, more than 10-fold). The different proportions of individual PAHs found in the summer and winter samples suggested traffic as a major emission source in the summer and, additionally, residential heating in the winter season at all three monitoring sites. The DNA adduct patterns resulting from the in vitro acellular assay also demonstrated similar major emission sources at all three locations. The study shows that particle-bound carcinogenic-PAH concentrations may be taken as an index for the biologically active (mutagenic, genotoxic, embryotoxic) components in air particulate samples. Therefore, high-quality monitoring data of carcinogenic PAHs may be useful for epidemiological studies of the impact of air pollution on the health of the population and for helping decision makers to improve our environment.     

A new approach for evaluating in vivo anti-leukemic activity using the SCE assay. An application on three newly synthesised anti-tumour steroidal esters

Three newly synthesised steroidal esteric derivatives of nitrogen mustard (compounds 1-3) were comparatively studied on a molar basis regarding their ability to induce sister chromatid exchanges (SCEs) in normal human lymphocytes in vitro and therapeutic effects on leukemia P388 bearing mice. Compounds 1 and 3 are modified steroidal esters of p-methyl-m-N,N-bis(2-chloroethyl)amino benzoic acid, and compound 2 is a modified steroidal ester of chlorambucil. All compounds induced statistically significant increases in SCEs and decreases in proliferation rate indices (PRIs) of cultured human lymphocytes and significantly increased the life span of P388 bearing mice. In this study, the doses applied for therapeutic purposes upon leukemia P388 bearing mice in vivo were derived from cytogenetic observations in normal human lymphocytes in vitro. A substantially better therapeutic effect was obtained compared to the effect achieved after the use of quite higher doses related with LD(10) values. We have demonstrated that the order of anti-tumour effectiveness of the treatment schedules of the three newly synthesised compounds tested (at doses derived from cytogenetic observations) coincides with the order of the cytogenetic effects they induce. The SCE assay appears to have an application in the clinical prediction of tumour sensitivity to potential chemotherapeutics.