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.     

Activity of cathepsin B,D and L in rat cerebrum after cimetidine and famotidine administration

Cathepsins are lysosomal enzymes that are used a sensitive markers in various toxicological investigations. The purpose of this study was to evaluate and compare the influence of cimetidine and famotidine on the cerebral cortex, particularly on the activity of cortical cathepsin B, D and L in the frontal lobe of rat brain. The drugs were administered intraperitoneally, twice a day, for six weeks to male Wistar rats in two doses. The initial dose was 2.85 mg/kg for cimetidine and 0.285 mg/kg for famotidine. The second dose was 10 times higher. Control animals were injected with 0.9% NaCl. Half of the animals from each of the drug-treated and control groups were sacrificed on the 42nd day of the experiment. The remaining animals were raised for another 6 weeks without any xenobiotics, and sacrificed on the 84th day. The frontal lobe of the right cerebral hemisphere was taken for biochemical investigation. The activities of free and bound fractions of cathepsin B, D and L were evaluated spectrophotometrically in cortical homogenates. The activity of bound fraction of cathepsin D and L decreased significantly in animals exposed to the higher dose of cimetidine and sacrificed on the 42nd day. Also significant elevation of the free fraction of cathepsin L was noted in the same group of rats. Cathepsin activities were normalized during the next six weeks. No behavioural changes were noted among the observed animals. Unlike cimetidine, famotidine did not change profiles of the cerebral cathepsins.     

Melatonin concentrations in patients with large goiter before and after surgery

OBJECTIVES: Surgical removal of a very large goiter may traumatize adjacent anatomical structures. The manipulations that involve superior cervical ganglia may alter melatonin secretion. To test this hypothesis we decided to study diurnal serum melatonin profiles in patients with a very large goiter before and after the surgery. MATERIAL AND METHODS:The study was performed on 10 women (mean age-46.5+/-1.6 years; mean+/-SEM; range 39-54 years) with very large non-toxic nodular goiter (mean thyroid volume-125.8+/-25.9 cm (3); mean+/-SEM; range 82.6-326.7 cm(3)). Diurnal serum melatonin profiles were estimated two days before the operation and 10 days after the surgery. Blood samples were collected at 08:00, 12:00, 16:00, 20:00, 22:00, 24:00, 02:00, 04:00, 06:00 and 08:00 h. Melatonin concentration was measured using RIA kit. RESULTS: Nocturnal serum melatonin concentrations (at 24, 02, and 04 hours) were significantly higher after the surgery than before the operation. CONCLUSIONS: Very large goiter may compress the superior cervical ganglia altering indirectly the melatonin synthesis. It cannot be excluded, however, that the presence of the large goiter in some other way affects melatonin secretion.     

A matrix-located processing peptidase of plant mitochondria

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc₁ complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the F_Ad subunit of the ATP synthetase and the tobacco F₁ subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bc₁ complex of the respiratory chain.     

The decline of antibiotic era--new approaches for antibacterial drug discovery

Infectious diseases still remain the main cause of human premature deaths; especially in developing countries. The emergence and spread of pathogenic bacteria resistant to many antibiotics (multidrug-resistant strains) have created the need for the development of novel therapeutic agents. Only two new classes of antibiotics of novel mechanisms of action (linezolid and daptomycin) have been introduced into the market during the last three decades. The recent progress in molecular biology and bacterial genome analysis has had an enormous impact on antibacterial drug research. This review presents new achievements in searching a new bacterial essential genes, a potential targets for antibacterial drugs. Application of metagenomics strategy is also shown. Some recent technologies aimed at development of anti-pathogenic drugs such as inhibitors of quorum sensing process or histidine kinases are also discussed. Extensive research efforts have provided many details concerning structure of bacterial proteins playing an important role in pathogenesis such as adherence proteins or toxins, what allowed searching for antitoxin drugs or drugs interfering with bacterial adhesion. As an example, the review focuses on anthrax therapies under development. Additionally, the article presents the progress in phage therapy; using bacteriophages or their products such as lysins in antibacterial therapy.     

A novel Amoeba proteus 120 kDa actin-binding protein with only 1 filamin repeat and a coiled-coil region.

A novel 120 kDa actin-binding protein (ApABP-F1) was found in Amoeba proteus. It was distributed throughout the cytoplasm, mainly in the subplasma membrane and perinuclear-nuclear areas, enriched in actin. The full-length cDNA of ApABP consisted of 2672 nucleotides with an open reading frame of 878 amino acids, giving a ~95 kDa protein with a theoretical pI value of 5.11. It had a novel domain organization pattern: the N terminus (residues 1-104) contained 1 calponin-homology (CH) domain, followed by only 1 region that was homologous to the filamin repeat (FR, residues 209-324), and a central region (residues 344-577) exhibiting a very high probability of coiled-coil formation, probably engaged in the observed protein dimerization. A phylogenetic tree constructed for CH domains from 25 various proteins revealed that the CH domain of ApABP was most related to that of the hypothetical mouse KIAA0903-like protein, whereas not much relationship to either filamins or the gelation factor (ABP-120) of Dictyostelium discoideum and Entamoeba histolytica was found.     

Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia

Emerging fungal and oomycete pathogens are increasingly threatening animals and plants globally. Amongst oomycetes, Saprolegnia species adversely affect wild and cultivated populations of amphibians and fish, leading to substantial reductions in biodiversity and food productivity. With the ban of several chemical control measures, new sustainable methods are needed to mitigate Saprolegnia infections in aquaculture. Here, PhyloChip-based community analyses showed that the Pseudomonadales, particularly Pseudomonas species, represent one of the largest bacterial orders associated with salmon eggs from a commercial hatchery. Among the Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs. Subsequent in vivo activity bioassays showed that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by Saprolegnia diclina. Live colony mass spectrometry showed that strain H6 produces a viscosin-like lipopeptide surfactant. This biosurfactant inhibited growth of Saprolegnia in vitro, but no significant protection of salmon eggs against Saprolegniosis was observed. These results indicate that live inocula of aquatic Pseudomonas strains, instead of their bioactive compound, can provide new (micro)biological and sustainable means to mitigate oomycete diseases in aquaculture.     

The circadian clock starts ticking at a developmentally early stage

Although overt diurnal rhythms of behavior do not begin until well after birth, molecular studies suggest that the circadian clock may begin much earlier at a cellular level: mouse embryonic fibroblasts, for example, already possess robust clocks. By multiple criteria, we found no circadian clock present in mouse embryonic stem cells. Nevertheless, upon their differentiation into neurons, circadian gene expression was observed. In the first steps along the pathway from ES cells to neurons, a neural precursor cell (NPC) line already showed robust circadian oscillations. Therefore, at a cellular level, the circadian clock likely begins at the very earliest stages of mammalian development.     

In vitro antiproliferative and antifungal activity of essential oils from Erigeron acris L. and Erigeron annuus (L.) Pers

Antiproliferative and antifungal activities of essential oils from Erigeron acris root and herb and from Erigeron annuus herb were investigated. The cell viability assay was performed in cultured fibroblasts, cancer cell lines (MCF-7 and MDA-MBA-231), and endometrial adenocarcinoma (Ishikawa) cells as well as colon adenocarcinoma (DLD-1) cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The essential oil from E. acris root showed the highest antiproliferative activity in the MCF-7 cell line with an IC50 value of 14.5 microg/mL. No effect of the essential oil on normal cells at that concentration was found. Antifungal activity against various strains of five Candida species, i.e. C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. parapsilosis, was tested by the microdilution method. It was found that all examined oils can be useful as antifungal agents against the above-mentioned species, but the essential oil of E. acris herb was the most active. Their minimum inhibitory concentrations (MIC) ranged from 30 to 0.4 microL/mL. The data presented suggest that essential oils from E. acris and E. annuus possess antifungal activity against Candida spp. and antiproliferative activity against breast cancer MCF-7 cells.     

Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+‐associated opening of plasma membrane anion channels

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe‐associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN‐SENSING 2 (FLS2) and EF‐Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode‐impaled mesophyll cells and root hairs of Col‐0 Arabidopsis plants revealed rapid, dose‐dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion‐selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species‐independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2‐associated receptor‐like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1‐dependent, calcium‐associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.