Structural and functional characterization of Group B Streptococcus pilus 2b

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Characterization of mitochondrial genomes in bivalve species with doubly uniparental inheritance of mitochondria

Many bivalve species possess two distinct mtDNA lineages, called F and M, respectively inherited maternally and paternally: this system is called doubly uniparental inheritance (DUI). The main experimental project of my PhD was the quantification of the two mtDNAs during the development of the DUI species Ruditapes philippinarum, from early embryos to sub-adults, using Real-Time qPCR. I identified the time interval in which M mtDNA is lost from female individuals, while it is retained in males (which are heteroplasmic through all of their life cycle). The results also suggested absence of mtDNA replication during early embryogenesis, a process constituting a bottleneck that highly reduces the copy number of mtDNA molecules in cells of developing larvae. In males this bottleneck may produce cells homoplasmic for M mtDNA, and could be considered as a first step of the segregation of M in the male germ line. Another finding was the characterization, in young clams approaching the first reproductive season, of a significant boost in copy number of F mtDNA in females and of M in males. Given the age of animals in which this mtDNA-specific growth was observed, the finding could probably be the outcome of the first round of gonads and gametes production. Other lines of research included the characterization of the unassigned regions in mt genomes of DUI bivalves. These regions can harbor signals involved in the control of replication and/or transcription of the mtDNA molecule, as well as additional open reading frames (ORFs) not related to oxidative phosphorylation. These features in DUI species could be associated to the maintenance of separate inheritance routes for the two mtDNAs. Additional ORFs are also found in other animal mt genomes: I summarized the presence of gene duplications as a co-author in a review focusing on animal mt genomes with unusual gene content.

Functional characterization of the placenta specific protein PLAC1 and its use for cancer immunotherapy

Summary Antibody-based cancer therapies have been successfully introduced into the clinic and have emerged as the most promising therapeutics in oncology. The limiting factor regarding the development of therapeutical antibody vaccines is the identification of tumor-associated antigens. PLAC1, the placenta-specific protein 1, was categorized for the first time by the group of Prof. Sahin as such a tumor-specific antigen. Within this work PLAC1 was characterized using a variety of biochemical methods. The protein expression profile, the cellular localization, the conformational state and especially the interacting partners of PLAC1 and its functionality in cancer were analyzed. Analysis of the protein expression profile of PLAC1 in normal human tissue confirms the published RT-PCR data. Except for placenta no PLAC1 expression was detectable in any other normal human tissue. Beyond, an increased PLAC1 expression was detected in several cancer cell lines derived of trophoblastic, breast and pancreatic lineage emphasizing its properties as tumor-specific antigen. rnThe cellular localization of PLAC1 revealed that PLAC1 contains a functional signal peptide which conducts the propeptide to the endoplasmic reticulum (ER) and results in the secretion of PLAC1 by the secretory pathway. Although PLAC1 did not exhibit a distinct transmembrane domain, no unbound protein was detectable in the cell culture supernatant of overexpressing cells. But by selective isolation of different cellular compartments PLAC1 was clearly enriched within the membrane fraction. Using size exclusion chromatography PLAC1 was characterized as a highly aggregating protein that forms a network of high molecular multimers, consisting of a mixture of non-covalent as well as covalent interactions. Those interactions were formed by PLAC1 with itself and probably other cellular components and proteins. Consequently, PLAC1 localize outside the cell, where it is associated to the membrane forming a stable extracellular coat-like structure.rnThe first mechanistic hint how PLAC1 promote cancer cell proliferation was achieved identifying the fibroblast growth factor FGF7 as a specific interacting partner of PLAC1. Moreover, it was clearly shown that PLAC1 as well as FGF7 bind to heparin, a glycosaminoglycan of the ECM that is also involved in FGF-signaling. The participation of PLAC1 within this pathway was approved after co-localizing PLAC1, FGF7 and the FGF7 specific receptor (FGFR2IIIb) and identifying the formation of a trimeric complex (PLAC1, FGF7 and the specific receptor FGFR2IIIb). Especially this trimeric complex revealed the role of PLAC1. Binding of PLAC1 together with FGF7 leads to the activation of the intracellular tyrosine kinase of the FGFR2IIIb-receptor and mediate the direct phosphorylation of the AKT-kinase. In the absence of PLAC1, no FGF7 mediated phosphorylation of AKT was observed. Consequently the function of PLAC1 was clarified: PLAC1 acts as a co-factor by stimulating proliferation by of the FGF7-FGFR2 signaling pathway.rnAll together, these novel biochemical findings underline that the placenta specific protein PLAC1 could be a new target for cancer immunotherapy, especially considering its potential applicability for antibody therapy in tumor patients.

In vitro and in vivo characterization of novel 18F-labeled bombesin analogues for targeting GRPR-positive tumors

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with (18)F, and characterized in vitro and in vivo as potential PET imaging agents. Binding studies displayed nanomolar binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates 6b (K(i) = 0.7 nM) and 7b (K(i) = 0.1 nM) were chosen for further in vivo evaluation. Both tracers revealed specific uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [(18)F]6b, compound [(18)F]7b was characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget ratios. In vivo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [(18)F]7b is a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.

Characterization of a novel five-transmembrane domain cholecystokinin-2 receptor splice variant identified in human tumors

The cholecystokinin-2 receptor (CCK2R), is expressed in cancers where it contributes to tumor progression. The CCK2R is over-expressed in a sub-set of tumors, allowing its use in tumor targeting with a radiolabel ligand. Since discrepancies between mRNA levels and CCK2R binding sites were noticed, we searched for abnormally spliced variants in tumors from various origins having been previously reported to frequently express cholecystokinin receptors, such as medullary thyroid carcinomas, gastrointestinal stromal tumors, leiomyomas and leiomyosarcomas, and gastroenteropancreatic tumors. A variant of the CCK2R coding for a putative five-transmembrane domains receptor has been cloned. This variant represented as much as 6% of CCK2R levels. Ectopic expression in COS-7 cells revealed that this variant lacks biological activity due to its sequestration in endoplasmic reticulum. When co-expressed with the CCK2R, this variant diminished membrane density of the CCK2R and CCK2R-mediated activity (phospholipase-C and ERK activation). In conclusion, a novel splice variant acting as a dominant negative on membrane density of the CCK2R may be of importance for the pathophysiology of certain tumors and for their in vivo CCK2R-targeting.

The Effects of Altered Prenatal Melatonin Signaling on Adult Behavior and Hippocampal Gene Expression of the Male Rat: A Circadioneuroendocrine-Axis Hypothesis of Psychopathology

Disturbances in melatonin - the neurohormone that signals environmental darkness as part of the circadian circuit of mammals - have been implicated in various psychopathologies in humans. At present, experimental evidence linking prenatal melatonin signaling to adult physiology, behavior, and gene expression is lacking. We hypothesized that administration of melatonin (5 mg/kg) or the melatonin receptor antagonist luzindole (5 mg/kg) to rats in utero would permanently alter the circadian circuit to produce differential growth, adult behavior, and hippocampal gene expressionin the male rat. Prenatal treatment was found to increase growth in melatonin-treated animals. In addition, subjects exposed to melatonin prenatally displayed increased rearing in the open field test and an increased right turn preference in the elevated plusmaze. Rats administered luzindole prenatally, however, displayed greater freezing and grooming behavior in the open field test and improved learning in the Morris water maze. Analysis of relative adult hippocampal gene expression with RT-PCR revealed increasedexpression of brain-derived neurotrophic factor (BDNF) with a trend toward increased expression of melatonin 1A (MEL1A) receptors in melatonin-exposed animals whereas overall prenatal treatment had a significant effect on microtubule-associated protein 2(MAP2) expression. Our data support the conclusion that the manipulation of maternal melatonin levels alters brain development and leads to physiological and behavioral abnormalities in adult offspring. We designate the term circadioneuroendocrine (CNE)axis and propose the CNE-axis hypothesis of psychopathology.

Molecular and biochemical characterisation of a novel mutation in POLG associated with Alpers syndrome

Background DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes. Methods mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE. Results We characterise a novel splice site mutation in POLG found in trans with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities. Conclusions mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore, POLG analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.

Immobilization and characterization of recombinant esterase enzyme on chitosan nanoparticles

Immobilization of biologically important molecules on myriad nano-sized materials has attracted great attention. Through this study, thermophilic esterase enzyme was obtained using recombinant DNA technology and purified applying one-step His-Select HF nickel affinity gel. The synthesis of chitosan was achieved from chitin by deacetylation process and degree of deacetylation was calculated as 89% by elemental analysis. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. The physicochemical properties of the chitosan and chitosan nanoparticles were determined by several methods including SEM (Scanning Electron Microscopy), FT-IR (Fourier Transform Infrared Spectroscopy) and DLS (Dynamic Light Scattering). The morphology of chitosan nanoparticles was spherical and the nanospheres’ average diameter was 75.3 nm. The purified recombinant esterase was immobilized efficiently by physical adsorption onto chitosan nanoparticles and effects of various immobilization conditions were investigated in details to develope highly cost-effective esterase as a biocatalyst to be utilized in biotechnological purposes. The optimal conditions of immobilization were determined as follows; 1.0 mg/mL of recombinant esterase was immobilized on 1.5 mg chitosan nanoparticles for 30 min at 60°C, pH 7.0 under 100 rpm stirring speed. Under optimized conditions, immobilized recombinant esterase activity yield was 88.5%. The physicochemical characterization of enzyme immobilized chitosan nanoparticles was analyzed by SEM, FT-IR and AFM (Atomic Force Microscopy).

Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei

ABSTRACT: Clostridium chauvoei is the causative agent of blackleg, a wide spread serious infection of cattle and sheep with high mortality. In this study we have analyzed the sialidase activity of the NanA protein of C. chauvoei and cloned the sialidase gene nanA. Sialidase is encoded as a precursor protein of 722 amino acids with a 26 amino acid signal peptide. The mature sialidase has a calculated molecular mass of 81 kDa and contains the carbohydrate binding module 32 (CBM32, or F5/8 type C domain), the sialic acid binding module CBM40 and the enzymatically active sialidase domain found in all pro- and eukaryotic sialidases. Sialidase activity does not require the CBM32 domain. The NanA protein is secreted by C. chauvoei as a dimer. The nanA gene was found to be conserved and sialidase activity was found in C. chauvoei strains isolated over a period of 50 years from various geographical locations. Antiserum directed against a recombinant 40 kDa peptide containing CBM40 and part of the enzymatically active domain of NanA neutralized the secreted sialidase activity of all C. chauvoei strains tested.

Molecular cloning and characterization of equine thymic stromal lymphopoietin

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that plays a central role in T helper 2 (Th2) cell differentiation and allergic inflammation. It is predominantly expressed by epithelial cells, and its expression is increased in patients with atopic dermatitis and asthma. Mice overexpressing TSLP in the skin develop allergic dermatitis and mice overexpressing TSLP in lungs develop asthma-like disease. However, it is not known whether TSLP plays an important role in equine allergies. Therefore, we cloned and sequenced the complete translated region of equine TSLP gene and measured its expression in various tissues. The equine TSLP gene is organized in 4 exons and encodes a protein of 143 amino acids, which has 62% amino acid identity with human TSLP.

Hydrodynamic Characterization of Bile Salt Host-Guest Complexes by Pulsed Field Gradient NMR Spectroscopy

The work described herein is aimed at understanding primary and secondary aggregation of bile salt micelles and how micelles can perform chiral recognition of binapthyl analytes. Previous work with cholate and deoxycholate using micellar electrokinetic chromatography (MEKC) and nuclear magnetic resonance (NMR) has provided insightinto cholate and deoxycholate micelle formation, especially with respect to the critical micelle concentration (CMC). Chiral separations of the model analyte, 1,1â??-binaphthyl-2,2â??-diyl hydrogen phosphate (BNDHP), via cholate (C) and deoxycholate (DC) mediated MEKC separataions previously have shown the DC CMC to be 7-10 mM andthe cholate CMC at 14 mM at ph 12. A second model analyte,1,1â??-binaphthol (BN), was also previously investigated to probe micellar structure, but the MEKC data for this analyte implied a higher CMC, which may be interpreted as secondary aggregation. Thiswork extends the investigation of bile salts to include pulsed field gradient spin echo (PFGSE) NMR experiments being used to gain information about the size and degree of polydispersity of cholate and deoxycholate micelles. Concentrations of cholate below 10mM show a large variation in effective radius likely due to the existence of transient preliminary aggregates. The onset of the primary micelle shows a dramatic increase in effective radius of the micelle in cholate and deoxycholate. In the region of expectedsecondary aggregation a gradual increase of effective radius was observed with cholate; deoxycholate showed a persistent aggregate size in the secondary micelle region that is modulated by the presence of an analyte molecule. Effective radii of cholate anddeoxycholate (individually) were compared with and without R- and S-BNDHP in order to observe the effective radius difference of micelles with and without analyte present. The presence of S-BNDHP consistently resulted in a larger effective aggregate radius incholate and deoxycholate, confirming previous data of the S-BNDHP interacting more with the micelle than R-BNDHP. In total, various NMR techniques, like diffusion NMR can be used to gain a greater understanding of the bile salt micellization process and chiral resolution.

Multiscale Characterization of Acrylic Bone Cement Modified with Functionalized Mesoporous Silica Nanoparticles

Acrylic bone cement is widely used to anchor orthopedic implants to bone and mechanical failure of the cement mantle surrounding an implant can contribute to aseptic loosening. In an effort to enhance the mechanical properties of bone cement, a variety of nanoparticles and fibers can be incorporated into the cement matrix. Mesoporous silica nanoparticles (MSNs) are a class of particles that display high potential for use as reinforcement within bone cement. Therefore, the purpose of this study was to quantify the impact of modifying an acrylic cement with various low-loadings of mesoporous silica. Three types of MSNs (one plain variety and two modified with functional groups) at two loading ratios (0.1 and 0.2 wt/wt) were incorporated into a commercially available bone cement. The mechanical properties were characterized using four-point bending, microindentation and nanoindentation (static, stress relaxation, and creep) while material properties were assessed through dynamic mechanical analysis, differential scanning calorimetry, thermogravimetric analysis, FTIR spectroscopy, and scanning electron microscopy. Four-point flexural testing and nanoindentation revealed minimal impact on the properties of the cements, except for several changes in the nano-level static mechanical properties. Conversely, microindentation testing demonstrated that the addition of MSNs significantly increased the microhardness. The stress relaxation and creep properties of the cements measured with nanoindentation displayed no effect resulting from the addition of MSNs. The measured material properties were consistent among all cements. Analysis of scanning electron micrographs images revealed that surface functionalization enhanced particle dispersion within the cement matrix and resulted in fewer particle agglomerates. These results suggest that the loading ratios of mesoporous silica used in this study were not an effective reinforcement material. Future work should be conducted to determine the impact of higher MSN loading ratios and alternative functional groups. (C) 2014 Elsevier Ltd. All rights reserved.

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

177Lu-AMBA: Synthesis and characterization of a selective 177Lu-labeled GRP-R agonist for systemic radiotherapy of prostate cancer

Gastrin-releasing peptide receptors (GRP-R) are upregulated in many cancers, including prostate, breast, and lung. We describe a new radiolabeled bombesin (BBN) analog for imaging and systemic radiotherapy that has improved pharmacokinetics (PK) and better retention of radioactivity in the tumor. METHODS: DO3A-CH2CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH2 (AMBA) was synthesized and radiolabeled. The human prostate cancer cell line PC-3 was used to determine the binding (Kd), retention, and efflux of 177Lu-AMBA. Receptor specificity was determined by in vitro autoradiography in human tissues. PK and radiotherapy studies were performed in PC-3 tumor-bearing male nude mice. RESULTS: 177Lu-AMBA has a high affinity for the GRP-R (Kd, 1.02 nmol/L), with a maximum binding capacity (Bmax) of 414 fmol/10(6) cells (2.5 x 10(5) GRP-R/cell). Internalization was similar for 177Lu-AMBA (76.8%), 177Lu-BBN8 (72.9%), and 125I-[Tyr4]-BBN (74.9%). Efflux was markedly lower for 177Lu-AMBA (2.9%) compared with 177Lu-BBN8 (15.9%) and 125I-[Tyr4]-BBN (46.1%). By receptor autoradiography, Lu-AMBA binds specifically to GRP-R (0.8 nmol/L) and to the neuromedin B receptor (NMB-R) (0.9 nmol/L), with no affinity for the bb3 receptor (>1,000 nmol/L). 177Lu-AMBA was renally excreted (55 %ID 1 h [percentage injected dose at 1 h]); tumor uptake at 1 and 24 h was 6.35 %ID/g and 3.39 %ID/g, respectively. One or 2 doses of 177Lu-AMBA (27.75 MBq/dose) significantly prolonged the life span of PC-3 tumor-bearing mice (P < 0.001 and P < 0.0001, respectively) and decreased PC-3 tumor growth rate over controls. When compared using World Health Organization criteria, mice receiving 2 doses versus 1 dose of 177Lu-AMBA demonstrated a shift away from stable/progressive disease toward complete/partial response; by RECIST (Response Evaluation Criteria in Solid Tumors), median survival increased by 36% and time to progression/progression-free survival increased by 65%. CONCLUSION: 177Lu-AMBA binds with nanomolar affinity to GRP-R and NMB-R, has low retention of radioactivity in kidney, demonstrates a very favorable risk-benefit profile, and is in phase I clinical trials.

Morphological and immunocytochemical characterization of feline odontoclastic resorptive lesions

Objective: Feline odontoclastic resorptive lesions (FORL) are common in domestic cats. The disease is painful, and leads to root resorption and replacement by bone, and tooth loss. The cause of this non-curable disease has not been established. The present study focused on the periodontal ligament (PL) in clinically and histologically healthy teeth and in teeth exhibiting various degrees of FORL. Methods: A total of 176 tissue blocks from 29 teeth was available for light and transmission electron microscopy. An antibody against osteopontin (OPN) was used for high-resolution immunocytochemistry, since this protein is involved in bone remodeling, cell adhesion, and inflammation. Results: A partial low cell number and an occasional translucent perivascular zone characterized the PL in healthy looking teeth. In teeth exhibiting small or initial FORL, the perivascular hyalinization was more pronounced, cell number was reduced, and widened channels connected the PL with the alveolar bone. In teeth exhibiting severe forms of FORL, the PL tissue was drastically affected. PL regions with massive tissue destruction were characterized by a lack of both cells and extracellular matrix, while other regions were replaced by bone marrow stroma. OPN labeling was restricted to the PL-facing matrix portions of bone and cementum. Conclusion: PL alterations seem to be a common feature in feline teeth. PL degeneration may initially be associated with vasculitis and appeared to increase with the severity of FORL. Since the PL did not recover and bone marrow stroma occupied the degenerated PL, ankylosis and replacement resorption are regarded as unavoidable sequelae.