Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop formation

Bis (cysteinyl) octapeptides related to the active sites of the oxidoreductases protein disulfide isomerase (PDI), thioredoxin reductase (trr), glutaredoxin (grx), and thioredoxin (trx) were analyzed for their propensity to form the intramolecular 14-membered disulfide ring in oxidation experiments. The rank order of percentage of cyclic monomer formed in aqueous buffer (pH 7.0) at 10^?3 M concentration was found to be very similar, but opposite to that of the K_ox and, correspondingly, of the redox potentials of the native enzymes. Attempts to induce intrinsic conformational preferences of the peptides by addition of trifluoroethanol led to enhancements of β-turn structures as reflected by the CD and Fourier transform ir spectra. The induced secondary structure, instead of aligning the tendencies of the excised fragments for loop formation with those of the intact proteins, was found to suppress the differences by significantly increasing the preference for cyclic monomers (≈ 90%). Similarly, operating under denaturing conditions, i.e., in 6M guanidinium hydrochloride, only for the trx peptide was the statistical product distribution obtained. For the remaining peptides, again a strong increase of cyclic monomer contents was observed that could not be correlated with dissolution of β-sheet type aggregates. The CD spectra are more consistent with the presence of ordered structure to some extent, possibly resulting from an hydrophobic collapse of the sparingly soluble peptides. The results of the oxidation experiments further support previous findings from thiol disulfide interchange equilibria, which clearly revealed a decisive role of the characteristic thioredoxin structural motif in dictating the redox properties of the enzymes. Point mutations in the active sites of the oxidoreductases allowed us to affect their redox potentials strongly, but apparently only in the constraint form of the three-dimensional structure as similar exchanges in the excised fragments did not produce the expected effect. This observation contrasts with numerous reports that the conformation of short disulfide loops is mainly dictated by the amino acid sequence. © 1994 John Wiley & Sons, Inc. © 1994 John Wiley & Sons, Inc.     

Polyester Wadding for Specimen Orientation During Embedding in Methacrylates

Polyester fibers are not dissolved by either glycol methacrylate or methyl methacrylate. Commercial polyester wadding is consequently an advantageous material to use in getting precise orientation of tissue specimens during embedding in methacrylate.     

Neuropeptides Control the Dynamic Behavior of Airway Mucosal Dendritic Cells

The airway mucosal epithelium is permanently exposed to airborne particles. A network of immune cells patrols at this interface to the environment. The interplay of immune cells is orchestrated by different mediators. In the current study we investigated the impact of neuronal signals on key functions of dendritic cells (DC). Using two-photon microscopic time-lapse analysis of living lung sections from CD11c-EYFP transgenic mice we studied the influence of neuropeptides on airway DC motility. Additionally, using a confocal microscopic approach, the phagocytotic capacity of CD11c⁺ cells after neuropeptide stimulation was determined. Electrical field stimulation (EFS) leads to an unspecific release of neuropeptides from nerves. After EFS and treatment with the neuropeptides vasoactive intestinal peptide (VIP) or calcitonin gene-related peptide (CGRP), airway DC in living lung slices showed an altered motility. Furthermore, the EFS-mediated effect could partially be blocked by pre-treatment with the receptor antagonist CGRP_8–37. Additionally, the phagocytotic capacity of bone marrow-derived and whole lung CD11c⁺ cells could be inhibited by neuropeptides CGRP, VIP, and Substance P. We then cross-linked these data with the in vivo situation by analyzing DC motility in two different OVA asthma models. Both in the acute and prolonged OVA asthma model altered neuropeptide amounts and DC motility in the airways could be measured. In summary, our data suggest that neuropeptides modulate key features motility and phagocytosis of mouse airway DC. Therefore altered neuropeptide levels in airways during allergic inflammation have impact on regulation of airway immune mechanisms and therefore might contribute to the pathophysiology of asthma.     

Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.     

Transformation of distinct mycobacterial species by shuttle vectors derived from theMycobacterium fortuitum pAL5000 plasmid

Mycobacterium tuberculosis H37Ra,M. smegmatisATCC 607,M. smegmatis MC²155,M. aurum A +,M. aurum A11, and one representative strain ofM. flavescens were transformed by electroporation with plasmid pMY10 and cosmid pDC100. Plasmid pMY 10 contained the origin of replication of pAL5000, the origin of replication of pBR322, a kanamycin resistance gene, and the origin of transfer of the Inc plasmid RK2; the cosmid pDC100 contained the pHC79 SS cosmid, the origin of replication of pAL5000, and a kanamycin resistance gene. The efficiency of transformation varied with the recipient cells used and was in decreasing order: 7×10⁵ forM. smegmatis MC²155, 6×10³ forM. tuberculosis H37Ra, 10³ forM. aurum, 50 forM. smegmatis ATCC 607, and 5 forM. flavescens. A rapid protocol for plasmid extraction from mycobacteria was developed.The satisfactory transformation of the nonvirulentM. tuberculosis strain H37Ra was of interest for future studies on cloning of virulence genes, while the satisfactory transformation ofM. aurum was of interest for future studies on the genetics of drug resistance because these bacteria are sensitive to drugs specifically used in the treatment of tuberculosis and leprosy. However, neither vector was stably maintained inM. smegmatis, indicating that further investigations are still necessary to resolve this difficulty.     

In situ activation of syngeneic tumour-specific cytotoxic T lymphocytes: Intra-pinna immunization followed by restimulation in the peritoneal cavity

Summary Tumour-specific cytotoxic T lymphocytes (CTL) are usually obtained after immunization in vivo and restimulation of immune cells in vitro. We here describe the generation of syngeneic tumour-specific CTL within no more than 9 days by priming and restimulation in vivo. This is achieved only if the correct sites are used both for primary immunization (ear pinna) and for restimulation (peritoneal cavity). The kinetics of immune T cell induction and of the secondary response in vivo will be reported. While a secondary CTL response could be generated in the peritoneal cavity, this was not possible in the spleen, no matter which routes of antigen restimulation were used. Upon transfer of immune spleen cells into the peritoneal cavity but not into the spleen, a secondary response could be generated upon in situ restimulation, indicating the importance of the correct microenvironment for this type of response. The peritoneal effector cells were true T cells and recognized a tumour-associated antigen in association with the K^d major histocompatibility (MHC class I) antigen. Finally the activated tumour-specific peritoneal exudate cells were able to transfer protective immunity without exogenous interleukin-2 into normal syngeneic mice.     

Sequence Capture and Next Generation Resequencing of the MHC Region Highlights Potential Transplantation Determinants in HLA Identical Haematopoietic Stem Cell Transplantation

How cells coordinate the immune system activities is important for potentially life-saving organ or stem cell transplantations. Polymorphic immunoregulatory genes, many of them located in the human major histocompatibility complex, impact the process and assure the proper execution of tolerance-versus-activity mechanisms. In haematopoietic stem cell transplantation, on the basis of fully human leukocyte antigen (HLA)-matched donor–recipient pairs, adverse effects like graft versus leukaemia and graft versus host are observed and difficult to handle. So far, high-resolution HLA typing was performed with Sanger sequencing, but for methodological reasons information on additional immunocompetent major histocompatibility complex loci has not been revealed. Now, we have used microarray sequence capture and targeted enrichment combined with next generation pyrosequencing for 3.5 million base pair human major histocompatibility complex resequencing in a clinical transplant setting and describe 3025 variant single nucleotide polymorphisms, insertions and deletions among recipient and donor in a single sequencing experiment. Taken together, the presented data show that sequence capture and massively parallel pyrosequencing can be used as a new tool for risk assessment in the setting of allogeneic stem cell transplantation.     

Developmental regulation of presence of binding sites for neoglycoproteins and endogenous lectins in various embryonic stages of human lung,liver and heart

Protein-carbohydrate interactions are supposed to play key roles in the mechanisms of cell adhesion, biosignalling and intracellular routing, warranting the analysis of the developmental course of expression of epitopes of this system. Thus, a panel of carrier-immobilized carbohydrate ligands was used as probes, namely lactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, fucose and maltose. Additionally, an antibody to an endogenous -galactoside-binding lectin (anti-galectin-1), the biotinylated lectin and two further human lectins, namely the macrophage migration inhibitory factor-binding sarcolectin and serum amyloid P component (SAP) that displays selectivity for sulphated sugars and mannose-6-phosphate, were included. They enabled us to assess the extent of the presence of respective binding sites in fixed sections from human lungs (pulmonary epithelial cells), livers (hepatocytes) and hearts (myocard cells) of 10–50 weeks gestation. Invariably, specific binding was detected in the three organ types, at least in certain stages. In most of the cases, the intensity of staining exhibited developmental regulation. The apparent patterns reveal similarities between the different cell types, as seen with immobilizedN-acetylglucosamine as well as with labelled galectin-1 and sarcolectin. However, drastic differences among such patterns with nearly opposite developmental courses do also occur, as detected for carrier-attached mannose and maltose residues. These results point to a potential importance for the detected glycohistochemical features in human development and substantiate the possibility of differential regulation of the presence of binding sites for distinct sugars within a certain organ and between the individual cell types of the monitored organs.