Assessment of the genetic diversity of wild hops (Humulus lupulus L.) in Europe using chemical and molecular analyses

The goal of this study was to characterize European wild hops (Humulus lupulus L.) by chemical and molecular genetic analyses in comparison to cultivated hops and North American wild hops. The contents of alpha and beta and acids varied from 0.45% to 5.55% and from 1.22% to 5.73% in European wild hops, respectively. Low bitter acid contents, alpha/beta acid ratios of lower than 1.0 and cohumulone content not exceeding 30% were typical as well as for traditional European cultivars. The lower myrcene content, the presence of farnesene and high selinene content were typical for European wild hops. We evaluated molecular genetic diversity in European wild hops by microsatellite and gene-specific markers and found that this variability did not correlate with the chemical characteristics. Our phylogenetic analysis confirmed overlapping variability and close genetic relationships in Europe, the separation of wild hops from the Caucasus region and the high diversity of North American wild hops.     

ADAM10 is expressed in human podocytes and found in urinary vesicles of patients with glomerular kidney diseases

Background

Influenza viruses are a major cause of morbidity and mortality around the world. More recently, a swine-origin influenza A (H1N1) virus that is spreading via human-to-human transmission has become a serious public concern. Although vaccination is the primary strategy for preventing infections, influenza antiviral drugs play an important role in a comprehensive approach to controlling illness and transmission. In addition, a search for influenza-inhibiting drugs is particularly important in the face of high rate of emergence of influenza strains resistant to several existing influenza antivirals.

Methods

We searched for novel anti-influenza inhibitors using a cell-based neutralization (inhibition of virus-induced cytopathic effect) assay. After screening 20,800 randomly selected compounds from a library from ChemDiv, Inc., we found that BPR1P0034 has sub-micromolar antiviral activity. The compound was resynthesized in five steps by conventional chemical techniques. Lead optimization and a structure-activity analysis were used to improve potency. Time-of-addition assay was performed to target an event in the virus life cycle.

Results

The 50% effective inhibitory concentration (IC₅₀) of BPR1P0034 was 0.42 ± 0.11 μM, when measured with a plaque reduction assay. Viral protein and RNA synthesis of A/WSN/33 (H1N1) was inhibited by BPR1P0034 and the virus-induced cytopathic effects were thus significantly reduced. BPR1P0034 exhibited broad inhibition spectrum for influenza viruses but showed no antiviral effect for enteroviruses and echovirus 9. In a time-of-addition assay, in which the compound was added at different stages along the viral replication cycle (such as at adsorption or after adsorption), its antiviral activity was more efficient in cells treated with the test compound between 0 and 2 h, right after viral infection, implying that an early step of viral replication might be the target of the compound. These results suggest that BPR1P0034 targets the virus during viral uncoating or viral RNA importation into the nucleus.

Conclusions

To the best of our knowledge, BPR1P0034 is the first pyrazole-based anti-influenza compound ever identified and characterized from high throughput screening to show potent (sub-μM) antiviral activity. We conclude that BPR1P0034 has potential antiviral activity, which offers an opportunity for the development of a new anti-influenza virus agent.     

Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH₂ (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys–His-Ile-Leu-Lys-NH₂ (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic α-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, α-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.     

Characterization of endocytic compartments after holo-high density lipoprotein particle uptake in HepG2 cells

Holo-high density lipoprotein (HDL) particle uptake, besides selective lipid uptake, constitutes an alternative pathway to regulate cellular cholesterol homeostasis. In the current study, the cellular path of holo-HDL particles was investigated in human liver carcinoma cells (HepG2) using combined light and electron microscopical methods. The apolipoprotein moiety of HDL was visualized with different markers: horseradish peroxidase, colloidal gold and the fluorochrome Alexa⁵⁶⁸, used in fluorescence microscopy and after photooxidation correlatively at the ultrastructural level. Time course experiments showed a rapid uptake of holo-HDL particles, an accumulation in endosomal compartments, with a plateau after 1–2 h of continuous uptake, and a clearance 1–2 h upon replacement by unlabeled HDL. Correlative microscopy, using HDL-Alexa⁵⁶⁸-driven diaminobenzidine (DAB) photooxidation, identified the fluorescent organelles as DAB-positive multivesicular bodies (MVBs) in the electron microscope; their luminal contents but not the internal vesicles were stained. Labeled MVBs increased in numbers and changed shapes along with the duration of uptake, from polymorphic organelles with multiple surface domains and differently shaped protrusions dominating at early times of uptake to compact bodies with mainly tubular appendices and densely packed vesicles after later times. Differently shaped and labeled surface domains and appendices, as revealed by three dimensional reconstructions, as well as images of homotypic fusions indicate the dynamics of the HDL-positive MVBs. Double staining visualized by confocal microscopy, along with the electron microscopic data, shows that holo-HDL particles after temporal storage in MVBs are only to a minor degree transported to lysosomes, which suggests that different mechanisms are involved in cellular HDL clearance, including resecretion.     

Virgin forests in Romania and Bulgaria: results of two national inventory projects and their implications for protection

Despite extensive forest destruction in the Middle Ages and later intensive commercial forest management, remnants of virgin forests remained spared in some Central, Eastern and South-Eastern European countries. These virgin forests are the last examples of original forests in this part of Europe. That is why their protection becomes an important issue of current European forestry and nature protection policy. But the knowledge about the location and the area of virgin forests in these countries is incomplete up till now. This article has the prime goal to present a conceptual framework what virgin forests might be (“A conceptual framework for defining of virgin forests” section). Based on this framework, a working methodology has been tested in Bulgaria and Romania (“Results of the two national projects in Romania and in Bulgaria” section and further). For this reason two projects have been carried out by the Royal Dutch Society of Nature Conservation (KNNV) in close co-operation with the Forestry Institutes in Romania and in Bulgaria. The results of these projects are described in general terms and further analysis in the future is necessary to describe specific features like forest structure and spatial heterogeneity of these forests. Based on the results of the inventory, principles of sustainable protection and management of the mapped virgin forests were defined and described in the research reports. The usefulness of the inventory became evident already during the EU pre-accession period of both countries while preparing the NATURA 2000 network. The remaining virgin forests of temperate Europe are an inexhaustible source of ecological information about biodiversity, structure, natural processes and overall functioning of undisturbed forest ecosystems. Their research will reveal information which can be used for ecological restoration of man-made forests which are degraded through intensive forestry practices over the last centuries. The last virgin forests of temperate Europe represent an irreplaceable part of the natural capital of Europe and are worth to be protected by law. Their last remnants in South-Eastern and Eastern Europe are endangered by commercial activities. A full inventory of remaining virgin forests in all countries of temperate Europe is a matter of highest urgency. A representative selection of virgin forest sites should be declared by UNESCO as World Heritage Sites.     

Discovery of dihydrothieno- and dihydrofuropyrimidines as potent pan Akt inhibitors

Herein we report the discovery and synthesis of a novel series of dihydrothieno- and dihydrofuropyrimidines (2 and 3) as potent pan Akt inhibitors. Utilizing previous SAR and analysis of the amino acid sequences in the binding site we have designed inhibitors displaying increased PKA and general kinase selectivity with improved tolerability compared to the progenitor pyrrolopyrimidine (1). A representative dihydrothieno compound (34) was advanced into a PC3-NCI prostate mouse tumor model in which it demonstrated a dose-dependent reduction in tumor growth and stasis when dosed orally daily at 200 mg/kg.     

Cyanide inhibition and pyruvate-induced recovery of cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

The mechanism of cyanide’s inhibitory effect on the mitochondrial cytochrome c oxidase (COX) as well as the conditions for its recovery have not yet been fully explained. We investigated three parameters of COX function, namely electron transport (oxygen consumption), proton transport (mitochondrial membrane potential Δψ _m) and the enzyme affinity to oxygen (p ₅₀ value) with regard to the inhibition by KCN and its reversal by pyruvate. 250 μM KCN completely inhibited both the electron and proton transport function of COX. The inhibition was reversible as demonstrated by washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60–80% of the original values. When using low KCN concentrations of up to 5 μM, we observed a profound, 30-fold decrease of COX affinity for oxygen. Again, this decrease was completely reversed by washing mitochondria while pyruvate induced only a partial, yet significant recovery of oxygen affinity. Our results demonstrate that the inhibition of COX by cyanide is reversible and that the potential of pyruvate as a cyanide poisoning antidote is limited. Importantly, we also showed that the COX affinity for oxygen is the most sensitive indicator of cyanide toxic effects.     

Role of FtsH2 in the repair of Photosystem II in mutants of the cyanobacterium Synechocystis PCC 6803 with impaired assembly or stability of the CaMn4 cluster

The FtsH2 protease, encoded by the slr0228 gene, plays a key role in the selective degradation of photodamaged D1 protein during the repair of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. To test whether additional proteases might be involved in D1 degradation during high rates of photodamage, we have studied the synthesis and degradation of the D1 protein in ΔPsbO and ΔPsbV mutants, in which the CaMn₄ cluster catalyzing oxygen evolution is less stable, and in the D1 processing mutants, D1-S345P and ΔCtpA, which are unable to assemble a functional cluster. All four mutants exhibited a dramatically increased rate of D1 degradation in high light compared to the wild-type. Additional inactivation of the ftsH2 gene slowed the rate of D1 degradation dramatically and increased the level of PSII complexes. We conclude that FtsH2 plays a major role in the degradation of both precursor and mature forms of D1 following donor-side photoinhibition. However, this conclusion concerned only D1 assembled into larger complexes containing at least D2 and CP47. In the ΔpsbEFLJ deletion mutant blocked at an early stage in PSII assembly, unassembled D1 protein was efficiently degraded in the absence of FtsH2 pointing to the involvement of other protease(s). Significantly, the ΔPsbO mutant displayed unusually low levels of cellular chlorophyll at extremely low-light intensities. The possibilities that PSII repair may limit the availability of chlorophyll for the biogenesis of other chlorophyll-binding proteins and that PsbO might have a regulatory role in PSII repair are discussed.     

Gait changes precede overt arthritis and strongly correlate with symptoms and histopathological events in pristane-induced arthritis

Introduction  

Pristane-induced arthritis (PIA) in the rat has been described as an animal model of inflammatory arthritis which exhibits features similar to rheumatoid arthritis in humans, such as a chronic, destructive, and symmetrical involvement of peripheral joints. However, so far little is known about the earliest inflammatory events and their influence on locomotor behaviour during the course of PIA. To investigate this issue a detailed analysis of the pathologic changes occurring during the prodromal and early stages of PIA was performed.