Familial Hemophagocytic Lymphohistiocytosis Type 5 (FHL-5) Is Caused by Mutations in Munc18-2 and Impaired Binding to Syntaxin 11

Rapid intracellular transport and secretion of cytotoxic granules through the immunological synapse requires a balanced interaction of several proteins. Disturbance of this highly regulated process underlies familial hemophagocytic lymphohistiocytosis (FHL), a genetically heterogeneous autosomal-recessive disorder characterized by a severe hyperinflammatory phenotype. Here, we have assigned FHL-5 to a 1 Mb region on chromosome 19p by using high-resolution SNP genotyping in eight unrelated FHL patients from consanguineous families. Subsequently, we found nine different mutations, either truncating or missense, in STXBP2 in twelve patients from Turkey, Saudi Arabia, and Central Europe. STXBP2 encodes syntaxin binding protein 2 (Munc18-2), involved in the regulation of vesicle transport to the plasma membrane. We have identified syntaxin 11, a SNARE protein mutated in FHL-4, as an interaction partner of STXBP2. This interaction is eliminated by the missense mutations found in our FHL-5 patients, which leads to a decreased stability of both proteins, as shown in patient lymphocytes. Activity of natural killer and cytotoxic T cells was markedly reduced or absent, as determined by CD107 degranulation. Our findings thus identify a key role for STXBP2 in lytic granule exocytosis.     

Synthesis and antibacterial activity of new N-linked 5-triazolylmethyl oxazolidinones

A new series of N-linked 5-triazolylmethyl oxazolidinones with varying substitution at the piperazine nitrogen 4-position were synthesized and tested against a panel of Gram-positive and Gram-negative bacteria including clinical isolates. Most of the compounds showed excellent antibacterial activity against susceptible and resistant Gram-positive organisms. One of the compounds showed enhanced antibacterial activity against Moraxella catarrhalis.     

Influence of maternal age, gestational age and fetal gender on expression of immune mediators in amniotic fluid

ABSTRACT: BACKGROUND: Variations in cytokine and immune mediator expression patterns in amniotic fluid due to gestational age, maternal age and fetal gender were investigated. METHODS: Amniotic fluid samples were obtained from 192 women, 82 with a mid-trimester amniocentesis (median gestational age 17 weeks) and 110 with a caesarean section not in labor (median gestational age 39 weeks). Amniotic fluid was screened by commercial ELISAs for the TH1/TH2/TH17 cytokines and immune mediators IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, TNF alpha, GROalpha, MIP1alpha, MIP1beta, histone, and IP10. Analysis was by Bonferroni correction for multiple comparisons. RESULTS: None of the 15 examined cytokines revealed any differences in expression patterns regarding fetal gender and age of the mothers. Significant differences were found in IL-4, IL-10, IL-12 TNF- alpha and MIP1-beta with respect to gestational age. CONCLUSIONS: Cytokines utilized as biomarkers in the diagnosis of intrauterine infections are not influenced in their expression pattern by fetal gender or maternal age, but may vary with respect to gestational age.     

Translation initiation and the fate of bacterial mRNAs

Studies in pro- and eukaryotes have revealed that translation can determine the stability of a given messenger RNA. In bacteria, intrinsic mRNA signals can confer efficient ribosome binding, whereas translational feedback inhibition or environmental cues can interfere with this process. Such regulatory mechanisms are often controlled by RNA-binding proteins, small noncoding RNAs and structural rearrangements within the 5' untranslated region. Here, we review molecular events occurring in the 5' untranslated region of primarily Escherichia coli mRNAs with regard to their effects on mRNA stability.     

The interaction domain of the redox protein adrenodoxin is mandatory for binding of the electron acceptor CYP11A1, but is not required for binding of the electron donor adrenodoxin reductase

Adrenodoxin (Adx) is a [2Fe-2S] ferredoxin involved in electron transfer reactions in the steroid hormone biosynthesis of mammals. In this study, we deleted the sequence coding for the complete interaction domain in the Adx cDNA. The expressed recombinant protein consists of the amino acids 1-60, followed by the residues 89-128, and represents only the core domain of Adx (Adx-cd) but still incorporates the [2Fe-2S] cluster. Adx-cd accepts electrons from its natural redox partner, adrenodoxin reductase (AdR), and forms an individual complex with this NADPH-dependent flavoprotein. In contrast, formation of a complex with the natural electron acceptor, CYP11A1, as well as electron transfer to this steroid hydroxylase is prevented. By an electrostatic and van der Waals energy minimization procedure, complexes between AdR and Adx-cd have been proposed which have binding areas different from the native complex. Electron transport remains possible, despite longer electron transfer pathways.     

The Geodia cydonium galectin exhibits prototype and chimera-type characteristics and a unique sequence polymorphism within its carbohydrate recognition domain

The ancestral galectin from the sponge Geodia cydonium (GCG) is classified on a structural basis to the prototype subfamily, whereas its carbohydrate-binding specificity is related to that of the mammalian chimera-type galectin-3. This dual coordination reveals GCG as a potential precursor of the later evolved galectin subfamilies, which is reflected in the primary structure of the protein. This study provides evidence that GCG is the LECT1 gene product, while neither a previously described LECT2 gene nor a functional LECT2 gene product was found in the specimen under investigation. The electrophoretically separated protein isomers with apparent molecular masses of 13, 15, and 16 kDa correspond to variants of the LECT1 protein-exhibiting peptide sequence polymorphisms that concern critical positions of the carbohydrate recognition domain (13 kDa: Leu51, Asn55, His130, Gly137; 15 kDa: Ser51, Asn55, Asn130, Gly137; 16 kDa: Ser51, Tyr55, Asn130, Glu137). Four residues, highly conserved in the galectin family, are substituted. None of the residues claimed to be involved in interactions with GalNAcalpha1-3 moieties at an extended binding subsite of galectin-3 was identified in the corresponding positions of GCG. Apparently, the substitutions do not confer distinct binding characteristics to the GCG variants as evidenced by binding studies with a recombinantly expressed 15-kDa isoform. The natural isoforms as well as the recombinant 15-kDa isoform oligomerize by the formation of non-covalent heteromeric or homomeric complexes. A phosphorylation of the galectin was confirmed neither by mass spectrometry nor by alkaline phosphatase treatment combined with isoelectric focusing.     

Virus–host cell interactions in vaccine production cell lines infected with different human influenza A virus variants: A proteomic approach

A proteomic approach was used to investigate the dynamic cellular host cell response induced by influenza virus infection in two different vaccine production cell lines, MDCK and Vero. For identification of proteins possibly involved in global host cell response mechanisms and virus–host cell interactions in these producer cell lines, quantitative 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were performed. In particular, host cell proteome alterations caused by infection with influenza virus variants showing differences in replication characteristics in MDCK cells were compared. Moreover, the host cell response to virus infection in Vero cells with respect to their deficiency in interferon (IFN) production and the need for virus adaptation to optimize productivity of this cell line were analyzed. Several proteins with differential abundance profiles were identified and Western blot analysis was performed for further confirmation of selected proteins. IFN-induced signal transduction, cytoskeleton remodeling, vesicle transport and proteolysis were the principal pathways that changed in infected MDCK cells. In Vero cells alterations of cell interactions, heat shock and oxidative stress response were detected. The findings will improve understanding of host cell response mechanisms during influenza vaccine production and viral strategies to evade these responses and to replicate efficiently in different cell lines.     

An approach to quality management in structural biology: biophysical selection of proteins for successful crystallization

Aggregation, incorrect folding and low stability are common obstacles for protein structure determination, and are often discovered at a very late state of protein production. In many cases, however, the reasons for failure to obtain diffracting crystals remain entirely unknown. We report on the contribution of systematic biophysical characterization to the success in structural determination of human proteins of unknown fold. Routine analysis using dynamic light scattering (DLS), differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) was employed to evaluate fold and stability of 263 purified protein samples (98 different human proteins). We found that FTIR-monitored temperature scanning may be used to detect incorrect folding and discovered a positive correlation between unfolding enthalpy measured with DSC and the size of small, globular proteins that may be used to estimate the quality of protein preparations. Furthermore, our work establishes that the risk of aggregation during concentration of proteins may be reduced through DLS monitoring. In summary, our study demonstrates that biophysical characterization provides an ideal tool to facilitate quality management for structural biology and many other areas of biological research.     

Selectins Mediate Small Cell Lung Cancer Systemic Metastasis

Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC) patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181). However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.     

A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes

For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.