Streptomycetes possess peptidyl-prolyl cis-trans isomerases that strongly resemble cyclophilins from eukaryotic organisms

A functionally active 17.5 kDa peptidyl-prolyl cis-trans isomerase was purified to homogeneity from Streptomyces chrysomallus, a Gram-positive filamentous bacterium. Characterization of the enzyme revealed inhibition and binding characteristics, against the immunsuppressive drug cyclosporin A, which were similar to cyclophilins from eukaryotes such as mammals, plants, fungi and yeasts, but different from those of cyclophilins from enterobacteria such as Escherichia coli. The amino acid sequence of the S. chrysomallus cyclophilin, as deduced from the gene sequence, revealed a striking degree of amino acid sequence identity with the corresponding 17 kDa proteins of humans (66%), Neurospora (70%) and yeast (69%). Comparison with cyclophilin sequences from the Gram-negative enterobacteria revealed much less homology (25% identity with E. coli b, 23% identity with E. coli a). Cyclophilin was detected in each of the four other Streptomyces species tested. The cyclophilins from the various streptomycetes differed in size, varying between 17 and 20.5 kDa. The cyclophilins were abundant in the Streptomyces cells, and present throughout growth.     

Host-cell cyclophilin is important for the intracellular replication of Leishmania major

The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA-binding proteins (cyclophilins) in the host–parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N-terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host-cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host-cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.     

Determining the appropriate pretreatment procedures and the utility of liver tissue for bulk stable isotope (δ13C and δ15N) studies in sharks

Stable-isotope analysis (SIA) provides a valuable tool to address complex questions pertaining to elasmobranch ecology. Liver, a metabolically active, high turnover tissue (~166 days for 95% turnover), has the potential to reveal novel insights into recent feeding/movement behaviours of this diverse group. To date, limited work has used this tissue, but ecological application of SIA in liver requires consideration of tissue preparation techniques given the potential for high concentrations of urea and lipid that could bias δ¹³C and δ¹⁵N values (i.e., result in artificially lower δ¹³C and δ¹⁵N values). Here we investigated the effectiveness of (a) deionized water washing (WW) for urea removal from liver tissue and (b) chloroform-methanol for extraction of lipids from this lipid rich tissue. We then (a) established C:N thresholds for deriving ecologically relevant liver isotopic values given complications of removing all lipid and (b) undertook a preliminary comparison of δ¹³C values between tissue pairs (muscle and liver) to test if observed isotopic differences correlated with known movement behaviour. Tests were conducted on four large shark species: the dusky (DUS, Carcharhinus obscurus), sand tiger (RAG, Carcharias taurus), scalloped hammerhead (SCA, Sphyrna lewini) and white shark (GRE, Carcharodon carcharias). There was no significant difference in δ¹⁵N values between lipid-extracted (LE) liver and lipid-extracted/water washed (WW) treatments, however, WW resulted in significant increases in %N, δ¹³C and %C. Following lipid extraction (repeated three times), some samples were still biased by lipids. Our species-specific “C:N thresholds” provide a method to derive ecologically viable isotope data given the complexities of this lipid rich tissue (C:N thresholds of 4.0, 3.6, 4.7 and 3.9 for DUS, RAG, SCA and GRE liver_LEWW tissue, respectively). The preliminary comparison of C:N threshold corrected liver and muscle δ¹³C values corresponded with movement/habitat behaviours for each shark; minor differences in δ¹³C values were observed for known regional movements of DUS and RAG (δ¹³C_Diffs = 0.24 ± 0.99‰ and 0.57 ± 0.38‰, respectively), while SCA and GRE showed greater differences (1.24 ± 0.63‰ and 1.08 ± 0.71‰, respectively) correlated to large-scale movements between temperate/tropical and pelagic/coastal environments. These data provide an approach for the successful application of liver δ¹³C and δ¹⁵N values to examine elasmobranch ecology.     

Antibody-dependent cell lysis by NK cells is preserved after sarcoma-induced inhibition of NK cell cytotoxicity

Osteosarcoma and Ewing’s sarcoma tumor cells are susceptible to IL15-induced or antibody-mediated cytolytic activity of NK cells in short-term cytotoxicity assays. When encountering the tumor environment in vivo, NK cells may be in contact with tumor cells for a prolonged time period. We explored whether a prolonged interaction with sarcoma cells can modulate the activation and cytotoxic activity of NK cells. The 40 h coculture of NK cells with sarcoma cells reversibly interfered with the IL15-induced expression of NKG2D, DNAM-1 and NKp30 and inhibited the cytolytic activity of NK cells. The inhibitory effects on receptor expression required physical contact between NK cells and sarcoma cells and were independent of TGF-β. Five days pre-incubation of NK cells with IL15 prevented the down-regulation of NKG2D and cytolytic activity in subsequent cocultures with sarcoma cells. NK cell FcγRIIIa/CD16 receptor expression and antibody-mediated cytotoxicity were not affected after the coculture. Inhibition of NK cell cytotoxicity was directly linked to the down-regulation of the respective NK cell-activating receptors. Our data demonstrate that the inhibitory effects of sarcoma cells on the cytolytic activity of NK cells do not affect the antibody-dependent cytotoxicity and can be prevented by pre-activation of NK cells with IL15. Thus, the combination of cytokine-activated NK cells and monoclonal antibody therapy may be required to improve tumor targeting and NK cell functionality in the tumor environment.     

X-ray sterilization of biopharmaceutical manufacturing equipment—Extractables profile of a film material and copolyester Tritan™ compared to gamma irradiation

The biopharmaceutical industry gains enormous flexibility in production processes by using sterilized preassembled single-use devices. Gamma irradiation is an established sterilization technology that may be restricted in the future by the availability of ⁶⁰Co as irradiation source and irradiation capacities. X-ray technology is considered an alternative type of radiation for sterilizing SU equipment. In the context of extractables and leachables—one concern connected with the use of single-use process equipment—the effect of X-ray irradiation on the extractables profile of the materials needs to be compared to established gamma irradiation to qualify this alternative technology. An approach is presented to obtain robust and comprehensive extractables data for materials used in SU devices after sterilization either using X-ray or gamma irradiation. A careful selection of the test items and the test design allows a one-to-one comparison of data obtained from a combination of orthogonal analytical techniques. The extractables of a modern SU film material and the copolyester Tritan™ are evaluated. The data presented allow a risk evaluation on the safety of this new sterilization modality for biopharmaceutical applications. It is demonstrated that the extractables profile of a polymer is not affected by the type of irradiation used for sterilization.     

NMR solution structure and dynamics of the peptidyl-prolyl cis-trans isomerase domain of the trigger factor from Mycoplasma genitalium compared to FK506-binding protein

We have solved the solution structure of the peptidyl-prolyl cis-trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy. Our results lead to a well-defined structure with a backbone rmsd of 0.23 A. As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold. Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar. Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the beta sheet. Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor. Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central beta sheet. Our data indicate, however, that this strand may be partially structured as "nascent" beta strand. This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases.