Biochemical and immunohistochemical study on physiological activity and distribution of hepatic cathepsin D

Cathepsin D (EC 3.4.23.5) is a lysosomal endopeptidase physiologically present at very low concentration in different tissues. The aim of the study was to estimate the physiological activity and distribution of cathepsin D in the liver. Four groups of ten-week-old male Wistar rats were raised without xenobiotics and sacrificed on day 4, 42, 47 and 84 of the experiment, and their livers were taken for immunohistochemical and biochemical investigation. Immunostaining for cathepsin D was evaluated by light microscope. Activity of the free and bound fractions of hepatic cathepsin D was measured spectrophotometrically. Immunohistochemical staining for cathepsin D was positive in Browicz-Kupffer cells in some but not in all rat liver specimens of each experimental group. The staining pattern was cytoplasmic and granular. Occasionally the positive stained endothelial cells were also found. No activity of cathepsin D in hepatocytes was detected. The positive immunostaining was found in livers with high enzyme activity in the biochemical investigation. No significant differences in activity of the free and bound fractions of cathepsin D among the different age groups were noted. However, the higher, age-dependent activity (p>0.05) of the free fraction was observed in the youngest and the two-middle groups of rats that were sacrificed on day 42 and 47 than in the oldest one. The bound fraction did not reveal such changes. It could be concluded that there were no differences in the activity of hepatic free and bound fractions of cathepsin D in male Wistar rats of various reproductive age. The rat Browicz-Kupffer cells revealed the highest activity of cathepsin D.     

Adhesion of blood platelets to collagen and fibrinogen after treatment with cisplatin and its complex with glutathione

Cisplatin (cis-diamminedichloroplatinum II, CDDP) is one of the most widely used chemotherapy drugs. Unfortunately, it induces serious side effects such as haematological toxicity. The aim of the present study was to evaluate the effect of CDDP on the first step in blood platelet activation-platelet adhesion, induced by thrombin or adenosine diphosphate (ADP), to collagen and fibrinogen. The action of cisplatin was compared with the action of cisplatin glutathione complex (GS-Pt) on platelet adhesion and on free radical generation measured by chemiluminescence. Pretreatment of blood platelets with cisplatin (0.1-20 microM) caused a dose- and time-dependent reduction of platelet adhesion to collagen and fibrinogen (p <0.05). The GS-Pt complex (20 microM, 30 min) had a stronger inhibitory effect on this process. Moreover, the complex (R2 = 0.992; p <0.05) also stimulated the chemiluminescence of blood platelets to a greater extent than CDDP alone (R2 = 0.999; p <0.01). The results suggest that inhibition of platelet adhesion in the presence of cisplatin and its complex with glutathione correlates with the generation of reactive oxygen species in these cells.     

Runoff water quality from intensive and extensive vegetated roofs

Vegetated roofs are becoming a trend in urban design, among others as a tool for city greening, mitigating urban heat island effect, and lowering urban storm runoff. Additionally, pollutant removal within vegetated roofs is often expected; however, it is commonly not a design feature. This study investigated influence on runoff water quality from two full-scale vegetated roofs (an intensive from Japan and an extensive from Sweden). Results show that both extensive and intensive vegetated roofs are a sink of nitrate nitrogen and ammonium nitrogen with similar performance. The intensive vegetated roof is also a sink of total nitrogen in contrast to the extensive roof. Phosphorus release is observed from the extensive vegetated roof but not from the intensive vegetated roof; release of dissolved organic carbon and potassium is observed from both roofs. The vegetated roofs, if not retaining the metal pollutants, were generally not a significant source. The increase of average pH during rainwater passage through the intensive vegetated roof indicated rapid neutralization of the acid depositions.     

Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly

Background

Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient’s prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules ‘(i.e. nucleotides, aminoacids, fatty acids). In cancer, Malic enzyme (ME) and ATP-citrate lyase (ACLY) are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biological and prognostic significance in non-small cell lung cancer (NSCLC).

Material and Methods

ME and ACLY expression was analyzed in 258 NSCLC in correlation with clinico-pathological parameters including patient’s survival.

Results

Though, overall expression of both enzymes correlated positively, ACLY was associated with local tumor stage, whereas ME correlated with occurrence of mediastinal lymph node metastases. Young patients overexpressing ACLY and/or ME had a significantly longer overall survival. This proved to be an independent prognostic factor. This contrasts older NSCLC patients, in whom overexpression of ACLY and/or ME appears to predict the opposite.

Conclusion

In NSCLC, ME and ACLY show different enzyme expressions relating to local and mediastinal spread. Most important, we detected an inverse prognostic impact of ACLY and/or ME overexpression in young and elderly patients. It can therefore be expected, that treatment of NSCLC especially, if targeting metabolic pathways, requires different strategies in different age groups.     

Genetic Structure of Daphnia galeata Populations in Eastern China

This study presents the first examination of the genetic structure of Daphnia longispina complex populations in Eastern China. Only one species, D. galeata, was present across the eight investigated lakes; as identified by taxon assignment using allelic variation at 15 microsatellite loci. Three genetically differentiated D. galeata subgroups emerged independent of the type of statistical analysis applied. Thus, Bayesian clustering, discriminant analysis based on results from factorial correspondence analysis, and UPGMA clustering consistently showed that populations from two neighbouring lakes were genetically separated from a mixture of genotypes found in other lakes, which formed another two subgroups. Clonal diversity was high in all D. galeata populations, and most samples showed no deviation from Hardy-Weinberg equilibrium, indicating that clonal selection had little effect on the genetic diversity. Overall, populations did not cluster by geographical origin. Further studies will show if the observed pattern can be explained by natural colonization processes or by recent anthropogenic impact on predominantly artificial lakes.     

Analysis of the Behavior of Mitochondria in the Ovaries of the Earthworm Dendrobaena veneta Rosa 1839

We examined six types of cells that form the ovary of the earthworm Dendrobena veneta ogonia, prooocytes, vitellogenic oocytes, trophocytes, fully grown postvitellogenic oocytes and somatic cells of the gonad. The quantitative stereological method revealed a much higher “volume density” of mitochondria in all of the types of germ-line cells except for the somatic cells. Fluorescent vital stain JC-1, however, showed a much higher oxidative activity of mitochondria in the somatic cells than in the germ-line cells. The distribution of active and inactive mitochondria within the studied cells was assessed using the computer program ImageJ. The analysis showed a higher luminosity of inactive mitochondria in all of the types of germ-line cells and a higher luminosity of active mitochondria in somatic cells. The OXPHOS activity was found in somatic cells mitochondria and in the peripheral mitochondria of the vitellogenic oocytes. The detection of reactive oxygen species (ROS) revealed a differentiated distribution of ROS in the different cell types. The amount of ROS substances was lower in somatic cells than in younger germ-line cells. The ROS level was also low in the cytoplasm of fully grown postwitellogenic oocytes. The distribution of the MnSOD enzyme that protects mitochondria against destructive role of ROS substances was high in the oogonia and in prooocytes and it was very high in vitellogenic and postvitellogenic oocytes. However, a much lower level of this protective enzyme was observed in the trophocytes and the lowest level was found in the cytoplasm of somatic cells. The lower mitochondrial activity and higher level of MnSOD activity in germ-line cells when compared to somatic cells testifies to the necessity of the organisms to protect the mitochondria of oocytes against the destructive role of the ROS that are produced during oxidative phosphorylation. The protection of the mitochondria in oocytes is essential for the transfer of healthy organelles to the next generation.     

A Small Motor Cortex Lesion Abolished Ocular Dominance Plasticity in the Adult Mouse Primary Visual Cortex and Impaired Experience-Dependent Visual Improvements

It was previously shown that a small lesion in the primary somatosensory cortex (S1) prevented both cortical plasticity and sensory learning in the adult mouse visual system: While 3-month-old control mice continued to show ocular dominance (OD) plasticity in their primary visual cortex (V1) after monocular deprivation (MD), age-matched mice with a small photothrombotically induced (PT) stroke lesion in S1, positioned at least 1 mm anterior to the anterior border of V1, no longer expressed OD-plasticity. In addition, in the S1-lesioned mice, neither the experience-dependent increase of the spatial frequency threshold (“visual acuity”) nor of the contrast threshold (“contrast sensitivity”) of the optomotor reflex through the open eye was present. To assess whether these plasticity impairments can also occur if a lesion is placed more distant from V1, we tested the effect of a PT-lesion in the secondary motor cortex (M2). We observed that mice with a small M2-lesion restricted to the superficial cortical layers no longer expressed an OD-shift towards the open eye after 7 days of MD in V1 of the lesioned hemisphere. Consistent with previous findings about the consequences of an S1-lesion, OD-plasticity in V1 of the nonlesioned hemisphere of the M2-lesioned mice was still present. In addition, the experience-dependent improvements of both visual acuity and contrast sensitivity of the open eye were severely reduced. In contrast, sham-lesioned mice displayed both an OD-shift and improvements of visual capabilities of their open eye. To summarize, our data indicate that even a very small lesion restricted to the superficial cortical layers and more than 3mm anterior to the anterior border of V1 compromised V1-plasticity and impaired learning-induced visual improvements in adult mice. Thus both plasticity phenomena cannot only depend on modality-specific and local nerve cell networks but are clearly influenced by long-range interactions even from distant brain regions.     

The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.     

The conformational properties of dehydrobutyrine and dehydrovaline: theoretical and solid‐state conformational studies

Dehydrobutyrine is the most naturally occurring dehydroamino acid. It is also the simplest dehydroamino acid having the geometrical isomers E/Z. To investigate its conformational properties, a theoretical analysis was performed on N‐acetyl‐α,β‐dehydrobutyrine N′‐methylamides, Ac‐(E)‐ΔAbu‐NHMe and Ac‐(Z)‐ΔAbu‐NHMe, as well as the dehydrovaline derivative Ac‐ΔVal‐NHMe. The ?, ψ potential energy surfaces and the localised conformers were calculated at the B3LYP/6‐311 + + G(d,p) level of theory both in vacuo and with inclusion of the solvent (chloroform, water) effect (SCRF method). The X‐ray crystal structures of Ac‐(Z)‐ΔAbu‐NHMe and Ac‐ΔVal‐NHMe were determined at 85 and 100 K, respectively. The solid‐state conformational preferences for the studied residues have been analysed and compared with the other related structures. Despite the limitations imposed by the C^α = C^β double bond on the topography of the side chains, the main chains of the studied dehydroamino acids are more flexible than in standard alanine. The studied dehydroamino acids differ in their conformational preferences, which depend on the polarity of the environment. This might be a reason why the nature quite precisely differentiates between ΔVal and each of the ΔAbu isomers, and why, particularly so with the latter, they are used as a conformational tool to influence the biological action of usually small, cyclic dehydropeptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Cadmium and copper toxicity to tench <Emphasis Type="Italic">Tinca</Emphasis><Emphasis Type="Italic">tinca</Emphasis> (L.) larvae after a short-term exposure

At the onset of swimbladder inflation, Tinca tinca larvae were exposed for 24 h to cadmium or copper at 0.0 (control concentration), 0.1, 0.2 and 0.3 mg dm⁻³ at 22°C. From then larvae were reared at 25°C for 9 days in un-supplemented water. Both metals resulted in a significantly reduced growth, survival, and retarded swimbladder inflation in a dose-response manner. The highest Cd and Cu concentration delayed the onset of exogenous feeding (live artemia nauplii) for 2 or 1 days, respectively, comparing to the control concentration. Our results demonstrate a highly toxic effect of Cd and Cu in the studied period of larval ontogeny, when fish seem especially sensitive. Although, at low concentrations and long exposure period, Cu is considered more toxic to fish than Cd, our study revealed the reverse effect for first-feeding larvae of both metals at high concentrations and short exposure.