A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histi-dine tag and an enterokinase cleavage site exhibits     

Characterization of the state of differentiation of six newly established human non-small-cell lung cancer cell lines

Six new non-small-cell lung cancer (NSCLC) cell lines were established directly from human tissue or indirectly via nude mouse xenografts in serum-supplemented media with success rates of 8% and 13%, respectively. They comprised one adenocarcinoma (ADLC-5M2), two squamous cell carcinomas (EPLC-32M1, EPLC-65H), two large cell carcinomas (LCLC-97TM1, LCLC-103H), and one malignant biphasic mesothelioma (MSTO-211H). All cell lines grew adherent to culture vessels with population doubling times (PDT) of 16-40 h, formed colonies in soft agarose with efficiencies of 0.1%-5.1%, and all grew in athymic nude mice. Xenograft histologies appeared as follows: (a) undifferentiated carcinomas with feeble resemblance to the original tumors in the case of adenocarcinomas and squamous cell carcinomas; (b) large cell carcinoma with high resemblance to the original tumor; (c) an undifferentiated tumor with predominance of large epithelial cells and few fibrous cells in the case of mesothelioma. Human chorionic gonadotropin (HCG) was found by radioimmunoassay and high-affinity binding sites for epidermal growth factor (EGF) by radio-receptor assay in 4/4 cell lines. A very low activity of L-DOPA decarboxylase (DDC) was detectable only in the adenocarcinoma cell line. All cell lines overexpressed the c-myc protooncogene, and no gene rearrangement or amplification was observed. Chromosome analysis revealed modal chromosome numbers of 70-73 in ADLC-5M2, EPLC-32M1, EPLC-65H, and MSTO-211H. Cell lines derived from large cell carcinoma had modal values of 65 and 170 and a wider chromosome distribution than all other cell lines. A NSCLC specific chromosomal aberration has been undetectable until now. These cell lines may aid in elucidating the biology of NSCLC and its interrelationship to other lung tumors.     

Subtypes of the plasmid-encoded serine protease EspP in Shiga toxin-producing Escherichia coli: distribution, secretion, and proteolytic activity

We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espPalpha, espPbeta, espPgamma, and espPdelta), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPbeta, espPgamma, and espPdelta genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espPalpha, espPbeta, espPgamma, and espPdelta in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspPalpha (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspPgamma cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPbeta and EspPdelta either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.     

Binding of N-terminal fragments of anthrax edema factor (EF(N)) and lethal factor (LF(N)) to the protective antigen pore

Anthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA(63), forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA(63)-channels. Here, in experiments with artificial lipid bilayer membranes, EF(N) and LF(N) show block of PA(63)-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EF(N), increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA(63)-channel.     

CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern of Gram-negative bacterial infections, and includes smooth (S-LPS) and rough (R-LPS) chemotypes. Upon activation by LPS through CD14, TLR4/MD-2 heterodimers sequentially induce two waves of intracellular signaling for macrophage activation: the MyD88-dependent pathway from the plasma membrane and, following internalization, the TRIF-dependent pathway from endosomes. We sought to better define the role of scavenger receptors CD36 and CD204/SR-A as accessory LPS receptors that can contribute to pro-inflammatory and microbicidal activation of macrophages. We have found that CD36 differently regulates activation of mouse macrophages by S-LPS versus R-LPS. The ability of CD36 to substitute for CD14 in loading R-LPS, but not S-LPS onto TLR4/MD-2 allows CD14-independent macrophage responses to R-LPS. Conversely, S-LPS, but not R-LPS effectively stimulates CD14 binding to CD36, which favors S-LPS transfer from CD14 onto TLR4/MD-2 under conditions of low CD14 occupancy with S-LPS in serum-free medium. In contrast, in the presence of serum, CD36 reduces S-LPS binding to TLR4/MD-2 and the subsequent MyD88-dependent signaling, by mediating internalization of S-LPS/CD14 complexes. Additionally, CD36 positively regulates activation of TRIF-dependent signaling by both S-LPS and R-LPS, by promoting TLR4/MD-2 endocytosis. In contrast, we have found that SR-A does not function as a S-LPS receptor. Thus, by co-operating with CD14 in both R- and S-LPS loading onto TLR4/MD-2, CD36 can enhance the sensitivity of tissue-resident macrophages in detecting infections by Gram-negative bacteria. However, in later phases, following influx of serum to the infection site, the CD36-mediated negative regulation of MyD88-dependent branch of S-LPS-induced TLR4 signaling might constitute a mechanism to prevent an excessive inflammatory response, while preserving the adjuvant effect of S-LPS for adaptive immunity.     

Comparison of fatty acid contents and composition in major lipid classes of larvae and adults of mosquitoes (Diptera: Culicidae) from a steppe region

Emerging aquatic insects, including mosquitoes, are known to transfer to terrestrial ecosystems specific essential biochemicals, such as polyunsaturated fatty acids (PUFA). We studied fatty acid (FA) composition and contents of dominant mosquito populations (Diptera: Culicidae), that is, Anopheles messeae, Ochlerotatus caspius, Oc. flavescens, Oc. euedes, Oc. subdiversus, Oc. cataphylla, and Aedes cinereus, inhabited a steppe wetland of a temperate climate zone to fill up the gap in their lipid knowledge. The polar lipid and triacylglycerol fractions of larvae and adults were compared. In most studied mosquito species, we first found and identified a number of short‐chain PUFA, for example, prominent 14:2n‐6 and 14:3n‐3, which were not earlier documented in living organisms. These PUFA, although occurred in low levels in adult mosquitoes, can be potentially used as markers of mosquito biomass in terrestrial food webs. We hypothesize that these acids might be synthesized (or retroconverted) by the mosquitoes. Using FA trophic markers accumulated in triacylglycerols, trophic relations of the mosquitoes were accessed. The larval diet comprised green algae, cryptophytes, and dinoflagellates and provided the mosquitoes with essential n‐3 PUFA, linolenic, and eicosapentaenoic acids. As a result, both larvae and adults of the studied mosquitoes had comparatively high content of the essential PUFA. Comparison of FA proportions in polar lipids versus storage lipids shown that during mosquito metamorphosis transfer of essential eicosapentaenoic and arachidonic acids from the reserve in storage lipids of larvae to functional polar lipids in adults occurred.     

Understanding the effect of landscape fragmentation and vegetation productivity on elephant habitat utilization in Amboseli ecosystem,Kenya

Understanding factors affecting the distribution of the African elephant is important for its conservation in increasingly human‐dominated savannah landscapes. However, understanding how landscape fragmentation and vegetation productivity affect elephant habitat utilization remains poorly understood. In this study, we tested whether landscape fragmentation and vegetation productivity explain elephant habitat utilization in the Amboseli ecosystem in Kenya. We used GPS (Global Positioning System) telemetry data from five elephants to quantify elephant habitat utilization. Habitat utilization was determined by calculating the time elephants spent within a unit area. We then used generalized additive models (GAMs) to model the relationship between time density and landscape fragmentation, as well as vegetation productivity. Results show that landscape fragmentation and vegetation productivity significantly (P < 0.05) explain elephant habitat utilization. A significant (P < 0.05) unimodal relationship between vegetation productivity and habitat utilization was observed. Results suggest that elephants spend much of their time in less fragmented landscapes of intermediate productivity.     

Is the combination of topsoil replacement and inoculation with plant material an effective tool for the restoration of threatened sandy grassland?

Question: Is it possible to restore dry calcareous inland sand ecosystems with their characteristic plant community structure within a 4‐yr period by means of combined abiotic–biotic techniques (topsoil replacement, inoculation with raked/mown plant material from target areas)? Location: Upper Rhine valley, Germany. Methods: Two 4‐year experiments were carried out on former arable land, each in the proximity of a reference area bearing a similar complex of threatened sandy grasslands (experiment 1: fine‐scale; experiment 2: landscape scale). In both experiments we used nutrient‐poor deep‐sand substrate (abiotic approach), raked/mown inoculation material from target areas and grazing as management tool (biotic and management approach). The vegetation of the restoration and donor areas was sampled once a year and analysed by non‐metric multidimensional scaling (NMDS) ordination and target‐species ratios. Mixed linear models were calculated to determine effects of grazing (experiment 1) and year (both experiments). Results: NMDS revealed a continuous development of the restored sites towards the corresponding donor sites. Similarly, target‐species ratios of the restored sites tended towards the ratios of the donor sites. To date, grazing effects have mainly been structural: reduction of a carpet‐forming pleurocarpous moss species and of litter. In addition, cover of target species in relation to total plant cover was significantly enhanced by grazing in the last two study years. Conclusions: The combination of nutrient‐poor substrate, inoculation with raked/mown plant material and grazing proved to be a very effective restoration method for dry base‐rich sand ecosystems. After 4 yr the restored plant communities serve as well‐developed parts of a habitat network.     

Pilot study: potential transcription markers for adult attention-deficit hyperactivity disorder in whole blood

Attention-deficit hyperactivity disorder (ADHD) is a common behavioural disorder that affects not only children and adolescents but also adults; however, diagnosis of adult ADHD is difficult because patients seem to have reduced externalized behaviour. ADHD is a multifactorial disorder in which many genes, all with small effects, are thought to cause the disorder in the presence of unfavourable environmental conditions. Therefore, in this pilot study, we explored the expression profile of a list of previously established candidate genes in peripheral blood samples from adult ADHD subjects (n = 108) and compared these results with those of healthy controls (n = 35). We demonstrate that combining the gene expression levels of dopamine transporter (SLC6A3), dopamine D5 receptor, tryptophan hydroxylase-1, and SNAP25 as predictors in a regression model resulted in sensitivity and specificity of over 80 % (ROC: max R(2) = 0.587, AUC = 0.917, P < 0.001, 95 % CI: 0.900-0.985). In conclusion, the combination of these four genes could represent a potential method for estimating risk and could be of diagnostic value for ADHD. Nevertheless, further investigation in a larger independent population including different subtypes of ADHD (inattentive, hyperactive, or combined type) patients is required to obtain more specific sets of biomarkers for each subtype as well as to differentiate between child, adolescent, and adulthood forms.     

Associations of HLA DR and DQ molecules with Lyme borreliosis in Latvian patients

ABSTRACT: BACKGROUND: Many autoimmune diseases are associated with variants of HLA genes such as those encoding the MHC complex. This correlation is not absolute, but may help in understanding of the molecular mechanism of disease. The purpose of this study was to determine HLA-DR,-DQ alleles in Latvian patients with Lyme borreliosis and control (healthy) persons. Case patients and control subjects were similar in age, gender and ethnic heritage and differed only as regards the presence of Borrelia burgdorferi infection. The study included 20 patients with clinical stage - erythema migrans and 25 control (healthy) persons. HLA genotyping was performed by PCR with sequence-specific primers. RESULTS: The results show difference in HLA-DRB1 alleles distribution between patients and control subjects. The frequencies of HLA-DRB1 *04 (OR 8.65; p<0.022) and HLA-DRB1 *17 (03) (OR 7.00; p<0.048) were increased in the Lyme disease patients. And the frequency of allele DRB1*13 (OR 0.13; p<0.033) was lower in Borreliosis patients and higher in control group. But, significant differences in frequencies of HLA-DQ alleles we did not detect. CONCLUSIONS: HLA predisposition to Lyme borreliosis appears not to be limited to HLA molecules, but some HLA-DR alleles also have a significant influence, and, may have implications in our understanding of pathogenesis of this disease. In particular, HLA-DRB1*04 and DRB1 *17 (03) may contribute to the Lyme borreliosis development in Latvian population KEYWORDS: Lyme borreliosis, HLA alleles, PCR.