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.     

Quantitative trait loci for plant height in Maresi × CamB barley population and their associations with yield-related traits under different water regimes

High-yielding capacity of the modern barley varieties is mostly dependent on the sources of semi-dwarfness associated with the sdw1/denso locus. The objective of the study was to identify quantitative trait loci (QTLs) associated with the plant height and yield potential of barley recombinant inbred lines (RILs) grown under various soil moisture regimes. The plant material was developed from a hybrid between the Maresi (European cv.) and CamB (Syrian cv.). A total of 103 QTLs affecting analysed traits were detected and 36 of them showed stable effects over environments. In total, ten QTLs were found to be significant only under water shortage conditions. Nine QTLs affecting the length of main stem were detected on 2H-6H chromosomes. In four of the detected QTLs, alleles contributed by Maresi had negative effects on that trait, the most significant being the QLSt-3H.1-1 in the 3H.1 linkage group. The close linkage between QTLs identified around the sdw1/denso locus, with positive alleles contributed by Maresi, indicates that the semi-dwarf cv. Maresi could serve as a donor of favourable traits resulting in grain yield improvement, also under water scarcity. Molecular analyses revealed that the Syrian cv. also contributed alleles which increased the yield potential. Available barley resources of genomic annotations were employed to the biological interpretation of detected QTLs. This approach revealed 26 over-represented Gene Ontology terms. In the projected support intervals of QGWS_l-5H.3-2 and QLSt-5H.3 on the chromosome 5H, four genes annotated to ‘response to stress’ were found. It suggests that these QTL-regions may be involved in a response of plant to a wide range of environmental disturbances.     

Antimicrobial and antioxidative potential of free and immobilised cellobiose dehydrogenase isolated from wood degrading fungi

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC₅₀ (10.04 ± 0.75 $\text{μg}$/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 $\text{μg}$/ml). The EC₅₀value reached 12.6 ± 1.51 $\text{μg}$/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.     

Fibrous Aggregates of Short Peptides Containing Two Distinct Aromatic Amino Acid Residues

The aim of the study was the assessment of the ability of short peptides to form aggregates under physiological conditions. The dipeptides studied were derived from different aromatic amino acids (heteroaromatic peptides). Tripeptides were obtained from two distinct aromatic amino acids and cysteine or methionine residue in the C‐terminal, N‐terminal, or central position. The ability of the peptides to form fibrous aggregates under physiological conditions was evaluated using three independent methods: the Congo Red assay, the Thioflavin T assay, and microscopic examinations using normal and polarized light. Materials potentially useful for regenerative medicine were selected based on their cytotoxicity to the endothelial cell line EA.hy 926 and physicochemical properties of films formed by peptides. The required parameters of biocompatibility were fulfilled by H?PheCysTrp?OH, H?PheCysTyr?OH, H?PheTyrMet?OH, and H?TrpTyr?OH.     

Transmembrane localization of cis-isomers of zeaxanthin in the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the 9-cis and 13-cis isomers of zeaxanthin on the molecular organization and dynamics of dimyristoylphosphatidylcholine (DMPC) membranes were investigated using conventional and saturation recovery EPR observations of the 1-palmitoyl-2-(14-doxylstearoyl)phosphatidylcholine (14-PC) spin label. The results were compared with the effects caused by the all-trans isomer of zeaxanthin. Effects on membrane fluidity, order, hydrophobicity, and the oxygen transport parameter were monitored at the center of the fluid phase DMPC membrane. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane center, studied by saturation-recovery EPR, decreased by 47% and 27% by including 10 mol% 13-cis and 9-cis zeaxanthin, respectively; whereas, incorporation of all-trans zeaxanthin decreased this parameter by only 11%. At a zeaxanthin-to-DMPC mole ratio of 1:9, all investigated isomers decreased the membrane fluidity and increased the alkyl chain order in the membrane center. They also increased the hydrophobicity of the membrane interior. The effects of these isomers of zeaxanthin on the membrane properties mentioned above increase as: all-trans < 9-cis ≤ 13-cis. Obtained results suggest that the investigated cis-isomers of zeaxanthin, similar to the all-trans isomer, are located in the membrane interior, adopting transmembrane orientation with the polar terminal hydroxyl groups located in the opposite leaflets of the bilayer. However, the existence of the second pool of cis-zeaxanthin molecules located in the one leaflet and anchored by the terminal hydroxyl groups in the same polar headgroup region cannot be completely ruled out.     

Habitat connectivity, more than species’ biology, influences genetic differentiation in a habitat specialist, the short-eared rock-wallaby (Petrogale brachyotis)

It is difficult to assess the relative influence of anthropogenic processes (e.g., habitat fragmentation) versus species’ biology on the level of genetic differentiation among populations when species are restricted in their distribution to fragmented habitats. This issue is particularly problematic for Australian rock-wallabies (Petrogale sp.), where most previous studies have examined threatened species in anthropogenically fragmented habitats. The short-eared rock-wallaby (Petrogale brachyotis) provides an opportunity to assess natural population structure and gene flow in relatively continuous habitat across north-western Australia. This region has reported widespread declines in small-to-medium sized mammals, making data regarding the influence of habitat connectivity on genetic diversity important for broad-scale management. Using non-invasive and standard methods, 12 microsatellite loci and mitochondrial DNA were compared to examine patterns of population structure and dispersal among populations of P. brachyotis in the Kimberley, Western Australia. Low genetic differentiation was detected between populations separated by up to 67?km. The inferred genetic connectivity of these populations suggests that in suitable habitat P. brachyotis can potentially disperse far greater distances than previously reported for rock-wallabies in more fragmented habitat. Like other Petrogale species male-biased dispersal was detected. These findings suggest that a complete understanding of population biology may not be achieved solely by the study of fragmented populations in disturbed environments and that management strategies may need to draw on studies of populations (or related species) in undisturbed areas of contiguous habitat.     

Chondrocyte-specific phenotype confers susceptibility of rat chondrocytes to lysis by NK cells

Normal chondrocytes are targets for natural killer (NK) cells. Since the mechanism of this phenomenon remains unknown, the present study was aimed at testing whether it is associated with chondrocyte-specific phenotype defined as ability of cartilage cells to produce sulfated glycosaminoglycans (GAG) and express collagen II and aggrecan mRNA. Lysis of rat epiphyseal chondrocytes by syngeneic spleen mononuclear cells (SMCs) was evaluated by ⁵¹Cr-release assay. Loss of chondrocyte phenotype following long-term culture resulted in their decreased susceptibility to lysis. Similar effect was also observed after suppression of chondrocyte phenotype by TNF. On the other hand, stimulation of cartilage-specific matrix component synthesis by IGF-1 resulted in increased chondrocyte killing and exogenous chondroitin sulfate A stimulated NK cell-mediated cytotoxicity against chondrocytes and human K562 cells. This suggests that chondrocyte susceptibility to lysis by NK cells depends on chondrocyte-specific phenotype, especially sulfated GAG production.     

Neuronal non-CG methylation is an essential target for MeCP2 function

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Basal dinosauriform and theropod dinosaurs from the mid–late Norian (Late Triassic) of Poland: implications for Triassic dinosaur evolution and distribution

The rise of dinosaurs during the Triassic is a widely studied evolutionary radiation, but there are still many unanswered questions about early dinosaur evolution and biogeography that are hampered by an unevenly sampled Late Triassic fossil record. Although very common in western North America and parts of South America, dinosaur (and more basal dinosauriform) remains are relatively rare in the Upper Triassic deposits of Europe, making any new discoveries critically important. One of the most diverse dinosauriform assemblages from Europe comes from the Por?ba site in Poland, a recently described locality with exposures of the Zb?szynek Beds, which have a palynomorph assemblage characteristic for the mid–late Norian in the biostratigraphic schemes of the Germanic Basin. Using a synapomorphy‐based approach, we evaluate several isolated dinosauriform specimens from Por?ba. This assemblage includes a silesaurid, a herrerasaurid and remains of another type of theropod (potentially a neotheropod). The Por?ba herrerasaurid is the first record of this rare group of primitive dinosaurs from Europe and one of the youngest records worldwide, whereas the silesaurid is the youngest record of a silesaurid from Europe. These findings indicate that silesaurids persisted alongside true dinosaurs into the mid–late Norian of Europe and that silesaurid–herrerasaurid–neotheropod assemblages (which are also known from the Norian of North America, at low latitudes) were more widespread geographically and latitudinally than previously thought. Silesaurid–herrerasaurid–neotheropod assemblages may have been a common ecological structuring of dinosaurs during their early evolution, and their widespread distribution may indicate weak palaeolatitudinal controls on early dinosaur biogeography during the latest Triassic.     

Morphological,genetic and molecular characteristics of barley root hair mutants

Root hairs are tubular outgrowths of specialized epidermal cells called trichoblasts. They affect anchoring plants in soil, the uptake of water and nutrients and are the sites of the interaction between plants and microorganisms. Nineteen root hair mutants of barley representing different stages of root hair development were subjected to detailed morphological and genetic analyses. Each mutant was monogenic and recessive. An allelism test revealed that nine loci were responsible for the mutated root hair phenotypes in the collection and 1–4 mutated allelic forms were identified at each locus. Genetic relationships between the genes responsible for different stages of root hair formation were established. The linkage groups of four loci rhl1, rhp1, rhi1 and rhs1, which had previously been mapped on chromosomes 7H, 1H, 6H and 5H, respectively, were enriched with new markers that flank the genes at a distance of 0.16 cM to 4.6 cM. The chromosomal position of three new genes – two that are responsible for the development of short root hairs (rhs2 and rhs3) and the gene that controls an irregular root hair pattern (rhi2) – were mapped on chromosomes 6H, 2H and 1H, respectively. A comparative analysis of the agrobotanical parameters between some mutants and their respective parental lines showed that mutations in genes responsible for root hair development had no effect on the agrobotanical performance of plants that were grown under controlled conditions. The presented mutant collection is a valuable tool for further identification of genes controlling root hair development in barley.