Synthesis of enzymatically resistant nociceptin-related peptides containing a carbamic acid residue.

Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue [...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change.     

Detection of Staphylococcus aureus nasal carriage in healthy young adults from a Hungarian University

Asymptomatic carriage of Staphylococcus aureus in healthy individuals has a high prevalence, especially in children and young adults. Nasal colonisation is a well-known risk factor for subsequent severe infection, or can be the source of transmission of this bacterium to other susceptible persons. In this study, we have surveyed the nasal carriage rate of students of the Semmelweis University, by screening 300 volunteers. We have determined the antibiotic sensitivity of the isolates by Etest, and their genetic relatedness by pulsed-fieled gel electrophoresis. The nasal carriage rate of S. aureus was found to be 29.3%, and that of MRSA only 0.67% (2/300). The isolates were generally sensitive to antibiotics, except for macrolides. We could observe a noticeably great genetic diversity, even among strains deriving from students of the same university group.     

A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro

Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.     

Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance

Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.     

Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells

Presenilin (PSEN) deficiency is accompanied by accumulation of endosomes and autophagosomes, likely caused by impaired endo-lysosomal fusion. Recently, Lee et al. (2010. Cell. doi: http://dx.doi.org/10.1016/j.cell.2010.05.008) attributed this phenomenon to PSEN1 enabling the transport of mature V0a1 subunits of the vacuolar ATPase (V-ATPase) to lysosomes. In their view, PSEN1 mediates the N-glycosylation of V0a1 in the endoplasmic reticulum (ER); consequently, PSEN deficiency prevents V0a1 glycosylation, compromising the delivery of unglycosylated V0a1 to lysosomes, ultimately impairing V-ATPase function and lysosomal acidification. We show here that N-glycosylation is not a prerequisite for proper targeting and function of this V-ATPase subunit both in vitro and in vivo in Drosophila melanogaster. We conclude that endo-lysosomal dysfunction in PSEN(-/-) cells is not a consequence of failed N-glycosylation of V0a1, or compromised lysosomal acidification. Instead, lysosomal calcium storage/release is significantly altered in PSEN(-/-) cells and neurons, thus providing an alternative hypothesis that accounts for the impaired lysosomal fusion capacity and accumulation of endomembranes that accompanies PSEN deficiency.     

Comparative study on energy partitioning in photosystem II of two <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> mutants with reduced non-photochemical quenching capacity

Lhcb1-2 and PsbS proteins of photosystem II (PSII) have important roles in photoprotective thermal energy dissipation of the absorbed excess light energy. The light responses of chlorophyll fluorescence parameters were analyzed to examine how the absence of Lhcb1-2 or PsbS proteins can modify the energy allocation patterns of absorbed light energy in PSII using an antisense construct of lhcb2 and a psbS deletion (npq4-1) mutant of Arabidopsis thaliana. Both mutants exhibit reduced Stern–Volmer non-photochemical chlorophyll fluorescence quenching (NPQ). Here, we have adopted an approach, presented by Hendrickson et al. (Photosynth Res 82:73–81, 2004), to gain a better insight into the mechanism of the NPQ in these mutants. We have found no significant differences in the quantum yields of photochemical energy conversion (Φ_PSII) between the mutants and the wild type. Nevertheless, as it was expected, the fraction of the energy, which is dissipated as heat via regulated pathways in PSII (Φ_NPQ) for both mutants, were reduced as compared to the wild type. In a complementary way, the extent of non-regulated non-photochemical energy loss in PSII (Φ_NO) for both mutants was significantly higher than that in the wild type. This reflects, together with the lower Φ_NPQ (or NPQ) values, suboptimal capacity of photoprotective reactions at higher light intensities.     

Ceratomyxa aegyptiaca n. sp. (Myxozoa: Myxosporea) from the gall-bladder of Solea aegyptiaca Chabanaud (Pleuronectiformes: Soleidae) in a Tunisian coastal lagoon

A new marine myxosporean species, Ceratomyxa aegyptiaca n. sp. is described from the gall-bladder of Solea aegyptiaca Chabanaud collected from the Ghar El Melh Lagoon in northeastern Tunisia. Mature spores are elongate and crescent-shaped, measuring 8-11?μm in length and 48-58?μm in width. The polar capsules are spherical, 3.2-4?μm in diameter and equal in size. Trophozoites are polysporous and float free in the bile or are attached on the epithelium of the gall-bladder. Morphological data and molecular analysis based on 18S rDNA sequences are provided. The 18S rDNA of C. aegyptiaca is readily distinguishable from that of other myxozoan species, as the genetically most similar myxozoan parasite, C. seriolae Yokoyama & Fukuda, 2001 (AB530265) collected from Seriola quinqueradiata Temminck & Schlegel in Japanese waters, shares with it only 67.5% identical nucleotides over a 1,680-bp long fragment of 18S rDNA.     

Essential oil composition and preliminary molecular study of four Hungarian Thymus species

Chemical and genetic differences of twenty taxa belonging to four Thymus species were studied in order to determine whether molecular characters and essential oil components could be used as taxonomic markers and to examine the correlation between them. Plant samples, representing different taxa and geographic regions, were collected from experimentally grown populations. Essential oil samples were analysed by GC/MS and cluster analysis of volatile composition resulted in segregation of thymol chemotypes from sesquiterpenic ones. Thymol was characteristic for all the populations of Thymus glabrescens and T. pannonicus as well as for certain taxa belonging to T. praecox and T. pulegioides. Sesquiterpenes occurred in only two taxa of T. glabrescens, in each sample of T. praecox and in three taxa of T. pulegioides. Plant samples were analysed by random amplified polymorphic DNA (RAPD). The obtained dendrogram revealed high gene diversity. The 13 primers resulted 114 polymorphic RAPD bands, and the average percentage of polymorphism was 80.8%. The RAPD dendogram showed separation neither at interspecific nor at interpopulational levels. Therefore, further specific molecular studies involving more taxa are suggested. Partial correlation have been found between molecular and chemical assessments.