Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.

Influence of the spxB gene on competence in Streptococcus pneumoniae

In Streptococcus pneumoniae expression of pyruvate oxidase (SpxB) peaks during the early growth phase, coincident with the time of natural competence. This study investigated whether SpxB influences parameters of competence, such as spontaneous transformation frequency, expression of competence genes, and DNA release. Knockout of the spxB gene in strain D39 abolished spontaneous transformation (compared to a frequency of 6.3 x 10(-6) in the parent strain [P < 0.01]). It also reduced expression levels of comC and recA as well as DNA release from bacterial cells significantly during the early growth phase, coincident with the time of spontaneous competence in the parent strain. In the spxB mutant, supplementation with competence-stimulating peptide 1 (CSP-1) restored transformation (rate, 1.8 x 10(-2)). This speaks against the role of SpxB as a necessary source of energy for competence. Neither supplementation with CSP-1 nor supplementation with the SpxB products H2O2 and acetate altered DNA release. Supplementation of the parent strain with catalase did not reduce DNA release significantly. In conclusion, the pneumococcal spxB gene influences competence; however, the mechanism remains elusive.

A homologue of aliB is found in the capsule region of nonencapsulated Streptococcus pneumoniae

The epidemiology, phylogeny, and biology of nonencapsulated Streptococcus pneumoniae are largely unknown. Increased colonization capacity and transformability are, however, intriguing features of these pneumococci and play an important role. Twenty-seven nonencapsulated pneumococci were identified in a nationwide collection of 1,980 nasopharyngeal samples and 215 blood samples obtained between 1998 and 2002. On the basis of multilocus sequence typing and capsule region analysis we divided the nonencapsulated pneumococci into two groups. Group I was closely related to encapsulated strains. Group II had a clonal population structure, including two geographically widespread clones able to cause epidemic conjunctivitis and invasive diseases. Group II strains also carried a 1,959-bp homologue of aliB (aliB-like ORF 2) in the capsule region, which was highly homologous to a sequence in the capsule region of Streptococcus mitis. In addition, strains of the two major clones in group II had an additional sequence, aliB-like ORF 1 (1,968 to 2,004 bp), upstream of aliB-like ORF 2. Expression of aliB-like ORF 1 was detected by reverse transcription-PCR, and the corresponding RNA was visualized by Northern blotting. A gene fragment homologous to capN of serotypes 33 and 37 suggests that group II strains were derived from encapsulated pneumococci some time ago. Therefore, loss of capsule expression in vivo was found to be associated with the importation of one or two aliB homologues in some nonencapsulated pneumococci.

Evidence that the novel receptor FGFRL1 signals indirectly via FGFR1

Fibroblast growth factor (FGF) receptor-like protein 1 (FGFRL1) is a recently discovered member of the FGF receptor (FGFR) family. Similar to the classical FGFRs, it contains three extracellular immunoglobulin-like domains and interacts with FGF ligands. However, in contrast to the classical receptors, it does not contain any intracellular tyrosine kinase domain and consequently cannot signal by transphosphorylation. In mouse kidneys, FgfrL1 is expressed primarily at embryonic stages E14-E15 in regions where nascent nephrons develop. In this study, we used whole-mount in situ hybridization to show the spatial pattern of five different Fgfrs in the developing mouse kidney. We compared the expression pattern of FgfrL1 with that of other Fgfrs. The expression pattern of FgfrL1 closely resembled that of Fgfr1, but clearly differed from that of Fgfr2‑Fgfr4. It is therefore conceivable that FgfrL1 signals indirectly via Fgfr1. The mechanisms by which FgfrL1 affects the activity of Fgfr1 remain to be elucidated.

Hijacking of Host Cell Signaling by Theileria

The apicomplexan parasites Theileria annulata and T. parva possess the ability to transform the infected host cell and induce uncontrolled proliferation. Residing free in the cytosol of its host leukocyte, the schizont is in a perfect position to manipulate host cell signaling pathways involved in regulating apoptosis, proliferation, and cell motility. While extensive Theileria-induced changes in host cell protein phosphorylation patterns have been reported, no Theileria-encoded kinases or phosphatases have been demonstrated - or are even predicted - to be associated with the schizont surface or secreted into the host cell. Instead, it seems that Theileria has evolved the capacity to modulate kinases of the host cell. In certain cases this involves “hijacking” pivotal kinases, such as the IκB kinase complex or the mitotic kinase polo-like kinase 1, recruiting them to the schizont surface. In this chapter the current understanding of Theileria-induced changes in host cell kinase activation is reviewed, and an attempt is made to link these events to phenotypic changes that occur in the cell in response to Theileria infection.

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

Suitability of Common Collagen Scaffolds for Anterior Cruciate Ligament Repair

Introduction: Anterior cruciate ligament (ACL) injuries are very common; in Germany incidence of ACL ruptures is estimated at 32 per 100 000 in the general population and in the sports community this rate more than doubles. Current gold standard for anterior cruciate lig- ament repair is reconstruction using an autograft [1]. However, this approach has shown some limitations. A new method has been her- alded by the Knee Team at the Bern University Hospital (Inselspital) and the Sonnenhof clinic called Dynamic Intraligamentary Stabilization (DIS), which keeps ACL remnants in place in order to promote biologi- cal healing and makes use of a dynamic screw system [2]. The aim of this study was to investigate the cytocompatibility of collagen patches in combination with DIS to support regeneration of the ACL. The spe- cific hypothesis we tested was whether MSCs would differentiate towards TCs in co-culture. Materials and methods: Primary Tenocytes (TCs) and human bone marrow derived mesenchymal stem cells (MSCs) were harvested from ACL removed during knee prothesis or from bone marrow aspirations (Ethical Permit 187/10). Cells were seeded on two types of three dimensional carriers currently approved for cartilage repair, Novocart (NC, B. Brown) and Chondro-Gide (CG, Geistlich). These scaffolds comprise collagen structures with interconnecting pores originally developed for seeding of chondrocytes in the case of CG. ~40k cells were seeded on punched zylindrical cores of 8 mm in Ø and cultured on CG or NC patches for up to 7 days. The cells were either cultured as TC only, MSC only or co-cultured in a 1:1 mix on the scaffolds and on both sides of culture inserts (PET, high density pore Ø 0.4 mm, BD, Fal- con) with cell-cell contact. We monitored DNA content, GAG and HOP-content, tracked the cells using DIL and DIO fluorescent dyes (Molecular Probes, Life technologies) and confocal laser scanning and SEM microscopy as well as RT-PCR of tenocyte specific markers (i.e. col 1 and 3, TNC, TNMD, SCXA&B, and markers of dedifferentiation ACAN, col2, MMP3, MMP13). Finally, H&E stain was interpreted on cryosections and SEM images of cells on the scaffold were taken. Results: ThecLSMimagesshowedcellproliferationoverthe7dayson both matrices, however, on CG there were much fewer MSCs attached than on NC. SEM images showed a roundish chondrocyte-like pheno- type of cells on CG whereas on NC the phenotype was more teno- cyte-like (Fig. 1). Gene expression of both, MSC and TC seem to confirm a more favorable environment in 3D for both patches rather than monolayer control.

Ceramide kinase contributes to proliferation but not to prostaglandin E2 formation in renal mesangial cells and fibroblasts

Background/Aims: Ceramide kinase (CerK) catalyzes the generation of the sphingolipid ceramide-1-phosphate (C1P) which regulates various cellular functions including cell growth and death, and inflammation. Here, we used a novel catalytic inhibitor of CerK, NVP-231, and CerK knockout cells to investigate the contribution of CerK to proliferation and inflammation in renal mesangial cells and fibroblasts. Methods: Cells were treated with NVP-231 and [3H]-thymidine incorporation into DNA, [3H]-arachidonic acid release, prostaglandin E2 (PGE2) synthesis, cell cycle distribution, and apoptosis were determined. Results: Treatment of rat mesangial cells and mouse renal fibroblasts with NVP-231 decreased DNA synthesis, but not of agonist-stimulated arachidonic acid release or PGE2 synthesis. Similarly, proliferation but not arachidonic acid release or PGE2 synthesis was reduced in CERK knockout renal fibroblasts. The anti-proliferative effect of NVP-231 on mesangial cells was due to M phase arrest as determined using the mitosis markers phospho-histone H3, cdc2 and polo-like kinase-1, and induction of apoptosis. Moreover, loss of CerK sensitized cells towards stress-induced apoptosis. Conclusions: Our data demonstrate that CerK induces proliferation but not PGE2 formation of renal mesangial cells and fibroblasts, and suggest that targeted CerK inhibition has potential for treating mesangioproliferative kidney diseases.

Cell cycle-dependent phosphorylation of Theileria annulata schizont surface proteins.

The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr), serine (p-Ser) and threonine-proline (p-Thr-Pro) epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state.

Is "milk crust" a transient form of golden retriever ichthyosis?

BACKGROUND A recessive inherited form of lamellar ichthyosis is well recognized in golden retrievers. In this breed, young puppies demonstrate a self-limiting scaling disorder which is commonly recognized by breeders, who use the term "milk crust" to describe this syndrome. HYPOTHESIS/OBJECTIVES To determine whether "milk crust" is a new keratinization disorder or a self-limiting form of golden retriever ichthyosis. ANIMALS A total of 179 golden retriever dogs (21 dams and 158 puppies) were examined. METHODS Dermatological examination and assessment of the patatin-like phospholipase-1 (PNPLA1) genotype by PCR testing of buccal mucosal swabs. Skin biopsies from one affected puppy were evaluated for histopathological abnormalities. RESULTS Forty-five of 158 (28%) puppies exhibited scaling at 8 weeks of age; 113 of 158 (72%) were dermatologically normal. Of 144 analysed samples, 40 of 144 (28%) puppies demonstrated a homozygous mutation of the PNPLA1 genotype [of which, 36 of 40 (90%) had signs of scaling], 77 of 144 (53%) demonstrated a heterozygous mutation and 27 of 144 (19%) were a normal wild-type. In six of 17 (35%) dams, a homozygous mutation of the PNPLA1 genotype was found, eight of 17 (47%) demonstrated a heterozygous mutation and three of 17 (18%) were normal wild-type. Dams with a homozygous mutation were clinically unaffected. A 1 year follow-up revealed that 23 of 28 (82%) puppies affected with this syndrome failed to develop typical signs of ichthyosis. In five of 28 (18%) dogs there was persistence of mild scaling. CONCLUSIONS AND CLINICAL IMPORTANCE We hypothesize that the clinical syndrome termed "milk crust" could represent a transient form of golden retriever ichthyosis. Remission is not fully linked to PNPLA1 genotype, suggesting that unknown factors may contribute to the clinical disease.

Intervertebral Disc Repair using Fleece-Membrane Composite Silk Material and Genipin-enhanced Fibrin Hydrogel

Introduction: Treating low back pain (LBP) has become an increasing challenge, as it is one of the main factors causing pain and is accompanied by high costs for the individual and the society. LBP can be caused by trauma of the intervertebral disc (IVD) or IVD degeneration. In the case of disc herniation the inner gelatinous part of the IVD, called nucleus pulposus, is pressed through the fibrous, annulus fibrosus that forms the outer part of the IVD. Today’s gold standard for treatment is extensive surgery as removal of the IVD and fusion of the vertebrae. In order to find a more gentle way to treat LBP and restore the native IVD we use a novel silk fleece-membrane composite from genetically modified silk worms whose silk contains a growth factor (GDF-6) that is associated with pushing stem cells towards a disc like phenotype (1). By combining it with a genipin-enhanced fibrin hydrogel we tested its suitability in organ culture on prior injured bovine IVD in our custom built two-degree of freedom bioreactor to mimic natural loading conditions. Material & Methods: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue followed by cutting out the IVDs as previously described (2). Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described (2). On the next day injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35-55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d (2). After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy- proline) were determined. Finally, real-time qPCR of major IVD marker and inflammation genes was performed to judge integrity of IVDs. Results: The fibrin hydrogel is able to keep the silk seal in place throughout the 14 days of in organ culture under all conditions. Additionally, cell activity showed optimistic results and we could not confirm negative effects of the repaired discs regarding overexpression of inflammation markers. Conclusions: The genipin-enhanced fibrin hydrogel in combination with the silk fleece- membrane composite seems to be a promising approach for IVD repair. Currently we assess the capability of GDF-6 incorporated in our silk composites on human mesenchymal stem cells and later on in organ culture. References 1. Clarke LE, McConnell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA. Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition and micromechanical properties of nucleus pulposus constructs. Arthritis Res Ther 2014, Mar 12;16(2):R67. 2. Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp 2012, Feb 2;60(60):e3490. Acknowledgements. This work is funded by the Gebert Rüf Foundation, project number GRS-028/13.

The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure) mediates activation of the immune response in bacterial infection of the central nervous system (CNS). The chemotactic G-protein-coupled receptor (GPCR) formyl-peptide-receptor like-1 (FPRL1) plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear.

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5'-3'- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.

Insulin-like growth factor type-1 receptor down-regulation associated with dwarfism in Holstein calves

Perturbations in endocrine functions can impact normal growth. Endocrine traits were studied in three dwarf calves exhibiting retarded but proportionate growth and four phenotypically normal half-siblings, sired by the same bull, and four unrelated control calves. Plasma 3,5,3'-triiodothyronine and thyroxine concentrations in dwarfs and half-siblings were in the physiological range and responded normally to injected thyroid-releasing hormone. Plasma glucagon concentrations were different (dwarfs, controls>half-siblings; P<0.05). Plasma growth hormone (GH), insulin-like growth factor-1 (IGF-1) and insulin concentrations in the three groups during an 8-h period were similar, but integrated GH concentrations (areas under concentration curves) were different (dwarfs>controls, P<0.02; half-siblings>controls, P=0.08). Responses of GH to xylazine and to a GH-releasing-factor analogue were similar in dwarfs and half-siblings. Relative gene expression of IGF-1, IGF-2, GH receptor (GHR), insulin receptor, IGF-1 type-1 and -2 receptors (IGF-1R, IGF-2R), and IGF binding proteins were measured in liver and anconeus muscle. GHR mRNA levels were different in liver (dwarfs1, respectively) and in muscle tissue was 2.3- and 1.8-fold higher (P=0.01 and P=0.08, respectively) than in dwarfs. Hepatic IGF-1R protein levels (Western blots) in muscle were 2.5-fold higher (P<0.05) and in liver and muscle (quantitative immunohistochemistry) were higher (P<0.02 and P<0.07, respectively) in half-siblings than in dwarfs. The reduced presence of IGF-1R may have been the underlying cause of dwarfism in studied calves.

Glucagon, catecholamine and pancreatic polypeptide secretion in type I diabetic recipients of pancreas allografts

Successful pancreas transplantation in type I diabetic patients restores normal fasting glucose levels and biphasic insulin responses to glucose. However, virtually no data from pancreas recipients are available relative to other islet hormonal responses or hormonal counterregulation of hypoglycemia. Consequently, glucose, glucagon, catecholamine, and pancreatic polypeptide responses to insulin-induced hypoglycemia and to stimulation with arginine and secretin were examined in 38 diabetic pancreas recipients, 54 type I diabetic nonrecipients, and 26 nondiabetic normal control subjects. Glucose recovery after insulin-induced hypoglycemia in pancreas recipients was significantly improved. Basal glucagon levels were significantly higher in recipients compared with nonrecipients and normal subjects. Glucagon responses to insulin-induced hypoglycemia were significantly greater in the pancreas recipients compared with nonrecipients and similar to that observed in control subjects. Glucagon responses to intravenous arginine were significantly greater in pancreas recipients than that observed in both the nonrecipients and normal subjects. No differences were observed in epinephrine responses during insulin-induced hypoglycemia. No differences in pancreatic polypeptide responses to hypoglycemia were observed when comparing the recipient and nonrecipient groups, both of which were less than that observed in the control subjects. Our data demonstrate significant improvement in glucose recovery after hypoglycemia which was associated with improved glucagon secretion in type I diabetic recipients of pancreas transplantation.