Mouse knockout of the cholesterogenic cytochrome P450 lanosterol 14alpha-demethylase (Cyp51) resembles Antley-Bixler syndrome

Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51(-/-) mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.     

Kinetics of Torpedo californica acetylcholinesterase inhibition by bisnorcymserine and crystal structure of the complex with its leaving group

Natural and synthetic carbamates act as pseudo-irreversible inhibitors of AChE (acetylcholinesterase) as well as BChE (butyrylcholinesterase), two enzymes involved in neuronal function as well as in the development and progression of AD (Alzheimer's disease). The AChE mode of action is characterized by a rapid carbamoylation of the active-site Ser(200) with release of a leaving group followed by a slow regeneration of enzyme action due to subsequent decarbamoylation. The experimental AD therapeutic bisnorcymserine, a synthetic carbamate, shows an interesting activity and selectivity for BChE, and its clinical development is currently being pursued. We undertook detailed kinetic studies on the activity of the carbamate bisnorcymserine with Tc (Torpedo californica) AChE and, on the basis of the results, crystallized the complex between TcAChE and bisnorcymserine. The X-ray crystal structure showed only the leaving group, bisnoreseroline, trapped at the bottom of the aromatic enzyme gorge. Specifically, bisnoreseroline interacts in a non-covalent way with Ser(200) and His(440), disrupting the existing interactions within the catalytic triad, and it stacks with Trp(84) at the bottom of the gorge, giving rise to an unprecedented hydrogen-bonding contact. These interactions point to a dominant reversible inhibition mechanism attributable to the leaving group, bisnoreseroline, as revealed by kinetic analysis.     

Hysteretic Behavior of Imidacloprid Sorption-Desorption in Soils of Croatian Coastal Regions

Sorption and desorption are important processes that influence the transport, transformation, and bioavailability of imidacloprid in the soils. Equilibrium batch experiments were carried out using six coastal Croatian soils. The equilibrium sorption and desorption experimental data showed the best fit to the Freundlich equation. Sorption parameters predicted with the Freundlich model, K_F ^sor and 1/n ranged from 2.92 to 5.74 (mg/kg)/(mg/L)^1/n, and 0.888 to 0.919, respectively. The sorption of imidacloprid was found to be sensitive to organic carbon (OC) content. The highest sorption was observed in Krk soil (OC 4.74%) and the lowest in Zadar soil (OC 1.06%). Fitted desorption parameter values, K_F ^des , were consistently higher than those associated with sorption. The opposite trend was observed for the exponential parameter 1/n. Results also suggested that imidacloprid sorption-desorption by soil is concentration-dependent, i.e. at lower imidacloprid concentrations a greater sorption percentage and lower desorption percentage occurred. Desorption studies revealed that there was a hysteresis effect in all the tested soils. Hysteresis coefficient values (H) varied from 0.656 to 0.859. The study results emphasize that the controlled application of imidacloprid is obligatory, especially in soils with a low organic carbon content, in order to minimize a risk of environmental and groundwater pollution.     

Influence of stressful fermentation conditions on neutral lipids of a Saccharomyces cerevisiae brewing strain

The neutral lipid fraction of the aerobically grown starter yeast culture of a Saccharomyces cerevisiae brewing strain, and three-first recycled yeast generations exposed to multiple stress factors during beer fermentation was studied. No pronounced changes in the cellular neutral lipid content between the non-stressed starter and stressed recycled cells were found. However, it was found that recycled yeast generations modulate their neutral lipid composition during fermentation. The ergosterol content was increased at the expense of steryl esters (SEs) and squalene, which resulted in a higher ergosterol/SEs molar ratio and a slightly higher ergosterol/squalene molar ratio. In addition, the proportion of unsaturated fatty acids, mainly palmitoleic acid increased in the neutral lipid fraction of the stressed recycled yeast generations. These results suggest that some specific neutral lipid species and fatty acids stored in the neutral lipid fraction are involved in the adaptive response of the brewer’s yeast to stressful fermentation conditions. The striking finding was a high squalene content in the neutral lipid fraction of both the starter yeast culture and recycled yeast generations (22.4 vs. 19–20%, respectively), implying a possible biotechnological exploitation of this biologically active molecule from the yeast biomass.     

Histological differences in healing following experimental transmural infarction in rats

Mechanisms of cardiac regeneration following transmural myocardial infarction were analysed in rat hearts using immunohistochemistry for a-SMA, caspase-3, Ki-67 and nestin markers. Seven weeks after experimental myocardial infarction, two different types of healing processes were revealed in rats with and without aneurysmatic bulging of the left ventricular wall. Besides thinning of the ventricular wall, three zones characterized both types of scars: the scar zone (divided into central and peripheral parts), the peri-infarct zone and the border zone. The main difference between the types of scars was the presence of a central necrotic zone inside the aneurysmatic wall, while connective tissue with myofibroblasts characterized the same zone in non-bulging wall. Apoptotic caspase-3 positive cells were found in the granulation tissue of the border zone in aneurysmatic scar, while in non-bulging scar they characterized all three zones. Proliferating Ki-67 positive cells displayed reverse expression pattern compared to apoptotic cells. Quantification of a-SMA positive cells revealed 60% a-SMA positive cells inside the central part of the aneurysmatic scar zone and 39% in invaginating areas, versus 19% in non-invaginating areas of the peripheral zone, but only 30% in the peripheral part of the non-bulging scar zone. Nestin positive cells were found in both types of scars, but with different distribution. These results suggest that even seven weeks after myocardial infarction, the healing processes in non-bulging scars are in chronic phase, while aneurysmatic scars are still in subacute phase. Histological differences in scar healing might be important for functional properties of the heart wall and for heart recovery prognosis.     

Saccharomyces kluyveri as a model organism to study pyrimidine degradation

The yeast Saccharomyces kluyveri (Lachancea kluyveri), a far relative of Saccharomyces cerevisiae, is not a widely studied organism in the laboratory. However, significant contributions to the understanding of nucleic acid precursors degradation in eukaryotes have been made using this model organism. Here we review eukaryotic pyrimidine degradation with emphasis on the contributions made with S. kluyveri and how this increases our understanding of human disease. Additionally, we discuss the possibilities and limitations of this nonconventional yeast as a laboratory organism.     

Comparison of essential and non-essential element distribution in leaves of the Cd/Zn hyperaccumulator Thlaspi praecox as revealed by micro-PIXE

A detailed localization of elements in leaf tissues of the field-collected Cd/Zn hyperaccumulator Thlaspi praecox (Brassicaceae) growing at a highly metal-polluted site was determined by micro-proton-induced X-ray emission (micro-PIXE) in order to reveal and compare nutrient and non-essential element accumulation patterns in the case of multiple metal accumulation within particular leaf tissues, including the detailed distribution between apoplast and symplast regions. On the larger scans, the highest concentrations of metals were observed in the epidermis, S and Ca in the palisade mesophyll, Cl in the spongy mesophyll and vascular bundles, and P and K in the vascular bundles. On the more detailed scans, the highest Cd, Pb, Cl and K concentrations were observed in vascular bundle collenchyma. The relative element distribution (%) was calculated based on concentrations of elements in particular leaf tissues and their relative weight portions, indicating that most of the accumulated Zn was located in epidermises, while the majority of Cd and Pb was distributed within the mesophyll. Detailed scans of epidermal/mesophyll tissues revealed that Zn was mainly accumulated and detoxified in the symplast of large vacuolated epidermal cells, Cd in the mesophyll symplast, and Pb in the mesophyll symplast and apoplast.     

Anti-inflammatory effects of exercise training in the early period after myocardial infarction

The aim of this investigation was to determine the effect of exercise training on the levels of plasma cytokines and acute phase reactants in the early post acute myocardial infarction (AMI) period. Sixty patients were enrolled into this three-week cardiac rehabilitation study. The mean time from AMI was 7.08 +/- 1.60 days, and the patient mean age was 60 +/- 10 years. Subjects were randomly assigned to one of the two groups: the control group treated with standard measures, and the group with additional regular moderate-intensity exercise training. Physical activity was based on the ergospirometry test results. Apart from clinical follow-up and routine laboratory analysis we determined the levels of plasma cytokines: tumor necrosis factor (TNF-alpha), soluble TNF-alpha receptor 1 (TNF-alphaSR1), interleukin (IL)-8, IL-10, and acute phase reactants: high sensitivity C-reactive protein (hsCRP) and fibrinogen. The obtained results confirmed the hypothesis that the early post AMI period is an inflammatory state the intensity of which gradually decreases with standard treatment during the first month after AMI, while including patients into early exercise training improves their inflammatory profile by decreasing the level of acute phase reactant and TNF-alphaSR1.     

Age, sexual and seasonal differences of haematological values and antibody status to Chlamydophila sp. in feral and racing pigeons (Columba livia forma domestica) from an urban environment (Zagreb, Croatia)

The aim of this study was to determine the basic haematological parameters in feral and racing pigeons and to compare these parameters according to age, sex and season in healthy feral pigeons as well as between Chlamydophila-serologically positive and negative feral pigeons. Red blood cells (RBC), packed cell volume (PCV), haemoglobin (Hb), mean cell volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood count (WBC), thrombocyte count and differential WBC, were determined in 366 pigeons (Columba livia forma domestica) captured in the City of Zagreb between 1999 and 2002. Of these, 232 feral (179 adult and 53 juvenile, 104 male and 75 female) and 57 racing pigeons (25 male and 32 female) were clinically healthy and bacteriologically and serologically negative, but 77 birds had antibody titres against Chlamydophila sp. Significantly lower values of RBC, PCV, Hb, MCH, WBC and thrombocyte (P<0.05) were observed in young compared to adult pigeons, while the differences in MCV and MCH were not significant between age classes. In differential WBC of young pigeons, a significantly higher percentage of heterophils, eosinophils, basophils and monocytes and a significantly smaller percentage of lymphocytes (P<0.01) was found than in adult pigeons. Significant sex-related differences were seen only in MCV values and in the percentage of lymphocytes (higher in females) and neutrophils (higher in males). PCV, Hb, MCV and MCH increased, while WBC decreased during wintertime (P<0.05). In differential WBC, percentage of heterophils was low in summer and autumn. At the same time, a higher percentage of basophils was found. Low numbers of monocytes were found in summer and low values of eosinophils in winter. In racing pigeons, values of eosinophils and basophils were significantly lower than in feral pigeons. Pigeons which had antibodies against Chlamydophila sp. possessed a higher percentage of monocytes and less lymphocytes than sero-negative animals, while WBC was significant lower than in sero-negative feral pigeons.