LPG Gas-Sensing System With SnO2 Thin-Film Transducer and 0.7-mu m CMOS Signal Conditioning ASIC

The design and implementation of a complete gas sensor system for liquified petroleum gas (LPG) gas sensing are presented. The system consists of a SnO2 transducer, a lowcost heater, an application specific integrated circuit (ASIC) with front-end interface circuitry, and a microcontroller interface for data logging. The ASIC includes a relaxation-oscillator-based heater driver circuit that is capable of controlling the sensor operating temperature from 100degC to 425degC. The sensor readout circuit in the ASIC, which is based on the resistance to time conversion technique, has been designed to measure the gas sensor response over three orders of resistance change during its interaction with gases.

Studies on Rat Ovarian Receptors for Lutropin (Luteinizing Hormone): Applicability of radioimmunoassay to measure lutropin bound to receptors.

Measurement of receptor-bound unlabelled physiologically active lutropin (luteinizing hormone, LH) was possible by a modified radioimmunoassay. The conventional radioimmunoassayconducted at 4°C was inadequate, whereas the modified assay performed at 37'C could measure receptor-bound lutropin. The radioimmunoassay at 37'C takes only 36h for completion compared with 5-7 days at 4°C. The sensitivity and range of dose-response curves are, however, unaltered. The validity of the technique was established by a number of criteria.

Vibration-rotation pattern in acetylene. II. Introduction of Coriolis coupling in the global model and analysis of emission spectra of hot acetylene around 3 mu m

A high temperature source has been developed and coupled to a high resolution Fourier transform spectrometer to record emission spectra of acetylene around 3 mu m up to 1455 K under Doppler limited resolution (0.015 cm(-1)). The nu(3)-ground state (GS) and nu(2)+nu(4)+nu(5)(Sigma(+)(u) and Delta(u))-GS bands and 76 related hot bands, counting e and f parities separately, are assigned using semiautomatic methods based on a global model to reproduce all related vibration-rotation states. Significantly higher J-values than previously reported are observed for 40 known substates while 37 new e or f vibrational substates, up to about 6000 cm(-1), are identified and characterized by vibration-rotation parameters. The 3 811 new or improved data resulting from the analysis are merged into the database presented by Robert et al. [Mol. Phys. 106, 2581 (2008)], now including 15 562 lines accessing vibrational states up to 8600 cm(-1). A global model, updated as compared to the one in the previous paper, allows all lines in the database to be simultaneously fitted, successfully. The updates are discussed taking into account, in particular, the systematic inclusion of Coriolis interaction.

Genetics of T cell co-stimulatory receptors -CD28, CTLA4, ICOS and PDCD1 in immunity and transplantation

Co-stimulatory signals are essential for the activation of naïve T cells and productive immune response. Naïve T cells receive first, antigen-specific signal through T cell receptor. Co-stimulatory receptors provide the second signal which can be either activating or inhibitory. The balance between signals determines the outcome of an immune response. CD28 is crucial for T cell activation; whereas cytotoxic T lymphocyte associated antigen 4 (CTLA4) mediates critical inhibitory signal. Inducible co-stimulator (ICOS) augments cytokine expression and plays role in immunoglobulin class switching. Programmed cell death 1 (PDCD1) acts as negative regulator of T cell proliferation and cytokine responses. The co-stimulatory receptor pathways are potentially involved in self-tolerance and thus, they provide a promising therapeutic strategy for autoimmune diseases and transplantation. The genes encoding CD28, CTLA4 and ICOS are located adjacently in the chromosome region 2q33. The PDCD1 gene maps further, to the region 2q37. CTLA4 and PDCD1 are associated with the risk of a few autoimmune diseases. There is strong linkage disequilibrium (LD) on the 2q33 region; the whole gene of CD28 exists in its own LD block but CTLA4 and the 5' part of ICOS are within a same LD block. The 3' part of ICOS and PDCD1 are in their own separate LD blocks. Extended haplotypes covering the 2q33 region can be identified. This study focuses on immune related conditions like coeliac disease (CD) which is a chronic inflammatory disease with autoimmune features. Immunoglobulin A deficiency (IgAD) belongs to the group of primary antibody deficiencies characterised by reduced levels of immunoglobulins. IgAD co-occurs often with coeliac disease. Renal transplantation is needed in the end stage kidney diseases. Transplantation causes strong immune response which is tried to suppress with drugs. All these conditions are multifactorial with complex genetic background and multiple environmental factors affecting the outcome. We have screened ICOS for polymorphisms by sequencing the exon regions. We detected 11 new variants and determined their frequencies in Finnish population. We have measured linkage disequilibrium on the 2q33 region in Finnish as well as other European populations and observed conserved haplotypes. We analysed genetic association and linkage of the co-stimulatory receptor gene region aiming to study if it is a common risk locus for immune diseases. The 2q33 region was replicated to be linked to coeliac disease in Finnish population and CTLA4-ICOS haplotypes were found to be associated with CD and IgAD being the first non-HLA risk locus common for CD and immunodeficiencies. We also showed association between ICOS and the outcome of kidney transplantation. Our results suggest new evidence for CTLA4-ICOS gene region to be involved in susceptibility of coeliac disease. The earlier published contradictory association results can be explained by involvement of both CTLA4 and ICOS in disease susceptibility. The pattern of variants acting together rather than a single polymorphism may confer the disease risk. These genes may predispose also to immunodeficiencies as well as decreased graft survival and delayed graft function. Consequently, the present study indicates that like the well established HLA locus, the co-stimulatory receptor genes predispose to variety of immune disorders.

Characterization of the molecular components and function of BARE-1, Hin-Mu and Mu transposition machineries

Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important.

GDNF family receptors in peripheral target innervation and hormone production

Glial cell line-derived neurotrophic factor (GDNF) family ligands: GDNF, neurturin, persephin and artemin, signal through a receptor tyrosine kinase Ret by binding first to a co-receptor (GFRα1-4) that is attached to the plasma membrane. The GDNF family factors can support the survival of various peripheral and central neuronal populations and have important functions also outside the nervous system, especially in kidney development. Activating mutations in the RET gene cause tumours in neuroendocrine cells, whereas inactivating mutations in RET are found in patients with Hirschsprung s disease (HSCR) characterized by loss of ganglionic cells along the intestine. The aim of this study was to examine the in vivo functions of neurturin receptor GFRα2 and persephin receptor GFRα4 using knockout (KO) mice. Mice lacking GFRα2 grow poorly after weaning and have deficits in parasympathetic and enteric innervation. This study shows that impaired secretion of the salivary glands and exocrine pancreas contribute to growth retardation in GFRα2-KO mice. These mice have a reduced number of intrapancreatic neurons and decreased cholinergic innervation of the exocrine pancreas as well as reduced excitatory fibres in the myenteric plexus of the small intestine. This study also demonstrates that GFRα2-mediated Ret signalling is required for target innervation and maintenance of soma size of sympathetic cholinergic neurons and sensory nociceptive IB4-binding neurons. Furthermore, lack of GFRα2 in mice results in deficient perception of temperatures above and below thermoneutrality and in attenuated inflammatory pain response. GFRα4 is co-expressed with Ret predominantly in calcitonin-producing thyroid C-cells in the mouse. In this study GFRα4-deficient mice were generated. The mice show no gross developmental deficits and have a normal number of C-cells. However, young but not adult mice lacking GFRα4 have a lower production of calcitonin in thyroid tissue and consequently, an increased bone formation rate. Thus, GFRα4/Ret signalling may regulate calcitonin production. In conclusion, this study reveals that GFRα2/Ret signalling is crucial for the development and function of specific components of the peripheral nervous system and that GFRα4-mediated Ret signalling is required for controlling transmitter synthesis in thyroid C-cells.

Mu in vitro Transposition Technology in Functional Genetics and Genomics: Applications on Mouse and Bacteriophages

Transposons are mobile elements of genetic material that are able to move in the genomes of their host organisms using a special form of recombination called transposition. Bacteriophage Mu was the first transposon for which a cell-free in vitro transposition reaction was developed. Subsequently, the reaction has been refined and the minimal Mu in vitro reaction is useful in the generation of comprehensive libraries of mutant DNA molecules that can be used in a variety of applications. To date, the functional genetics applications of Mu in vitro technology have been subjected to either plasmids or genomic regions and entire genomes of viruses cloned on specific vectors. This study expands the use of Mu in vitro transposition in functional genetics and genomics by describing novel methods applicable to the targeted transgenesis of mouse and the whole-genome analysis of bacteriophages. The methods described here are rapid, efficient, and easily applicable to a wide variety of organisms, demonstrating the potential of the Mu transposition technology in the functional analysis of genes and genomes. First, an easy-to-use, rapid strategy to generate construct for the targeted mutagenesis of mouse genes was developed. To test the strategy, a gene encoding a neuronal K+/Cl- cotransporter was mutagenised. After a highly efficient transpositional mutagenesis, the gene fragments mutagenised were cloned into a vector backbone and transferred into bacterial cells. These constructs were screened with PCR using an effective 3D matrix system. In addition to traditional knock-out constructs, the method developed yields hypomorphic alleles that lead into reduced expression of the target gene in transgenic mice and have since been used in a follow-up study. Moreover, a scheme is devised to rapidly produce conditional alleles from the constructs produced. Next, an efficient strategy for the whole-genome analysis of bacteriophages was developed based on the transpositional mutagenesis of uncloned, infective virus genomes and their subsequent transfer into susceptible host cells. Mutant viruses able to produce viable progeny were collected and their transposon integration sites determined to map genomic regions nonessential to the viral life cycle. This method, applied here to three very different bacteriophages, PRD1, ΦYeO3 12, and PM2, does not require the target genome to be cloned and is directly applicable to all DNA and RNA viruses that have infective genomes. The method developed yielded valuable novel information on the three bacteriophages studied and whole-genome data can be complemented with concomitant studies on individual genes. Moreover, end-modified transposons constructed for this study can be used to manipulate genomes devoid of suitable restriction sites.

Mu in vitro transposition applications in protein engineering

Transposons, mobile genetic elements that are ubiquitous in all living organisms have been used as tools in molecular biology for decades. They have the ability to move into discrete DNA locations with no apparent homology to the target site. The utility of transposons as molecular tools is based on their ability to integrate into various DNA sequences efficiently, producing extensive mutant clone libraries that can be used in various molecular biology applications. Bacteriophage Mu is one of the most useful transposons due to its well-characterized and simple in vitro transposition reaction. This study establishes the properties of the Mu in vitro transposition system as a versatile multipurpose tool in molecular biology. In addition, this study describes Mu-based applications for engineering proteins by random insertional transposon mutagenesis in order to study structure-function relationships in proteins. We initially characterized the properties of the minimal Mu in vitro transposition system. We showed that the Mu transposition system works efficiently and accurately and produces insertions into a wide spectrum of target sites in different DNA molecules. Then, we developed a pentapeptide insertion mutagenesis strategy for inserting random five amino acid cassettes into proteins. These protein variants can be used especially for screening important sites for protein-protein interactions. Also, the system may produce temperature-sensitive variants of the protein of interest. Furthermore, we developed an efficient screening system for high-resolution mapping of protein-protein interfaces with the pentapeptide insertion mutagenesis. This was accomplished by combining the mutagenesis with subsequent yeast two-hybrid screening and PCR-based genetic footprinting. This combination allows the analysis of the whole mutant library en masse, without the need for producing or isolating separate mutant clones, and the protein-protein interfaces can be determined at amino acid accuracy. The system was validated by analysing the interacting region of JFC1 with Rab8A, and we show that the interaction is mediated via the JFC1 Slp homology domain. In addition, we developed a procedure for the production of nested sets of N- and C-terminal deletion variants of proteins with the Mu system. These variants are useful in many functional studies of proteins, especially in mapping regions involved in protein-protein interactions. This methodology was validated by analysing the region in yeast Mso1 involved in an interaction with Sec1. The results of this study show that the Mu in vitro transposition system is versatile for various applicational purposes and can efficiently be adapted to random protein engineering applications for functional studies of proteins.

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.

GDNF Receptors: Veterans and Novices

Neurotrophic factors play essential role in the development and functioning of the nervous system and other organs. Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands (GFLs) are of particular interest because they promote the survival of dopaminergic neurons in vitro, in Parkinson s disease animal models and in patients. GDNF is also a potent survival factor for the central motoneurons and thus is considered as a potential lead for the treatment of amyotrophic lateral sclerosis. The survival promoting receptor complex for GFLs consists of a ligand-specific co-receptor, GFRα and a signal transducing module, receptor tyrosine kinase RET. At least GDNF and persephin, a GFL, have established functions outside central nervous system. GDNF is crucial for enteric nervous system and kidney development as well as for spermatogenesis. Persephin controls calcitonin secretion. Communication between cells often occurs in the extracellular matrix (ECM), a meshwork, which is secreted and deposited by the cells and is mainly composed of fibrillar proteins and polymerized sugars. We evaluated the relationship between GFLs and extracellular matrix components and demonstrated that three GFLs - GDNF, neurturin and artemin bind heparan sulfates with nanomolar affinities. The fourth member of the family - persephin binds these polysaccharides thousand times less tightly. GDNF, neurturin and artemin also bind with high affinity to heparan sulfate proteoglycan (HSPG) isolated from the nervous system, syndecan-3. GDNF signals through HSPGs, evoking Src family kinase activation. This signaling induces cell spreading, hippocampal neurite outgrowth in vitro and cellular migration. Specifically, GDNF signaling through syndecan-3 is important for embryonic cortical neuron migration. Syndecan-3-deficient mice, similarly to mice lacking GDNF, have less GABAergic neurons in their cortex, as compared to the wild-type mice. This fact provides indirect evidence that GDNF interaction with syndecan-3 is important for cortical brain development. Noteworthy, in non-neuronal tissues GFLs may signal via other syndecans. We also present the structural model for a GDNF co-receptor, GFRα1. The X-ray structure of the GFRα1 domain 3 was solved with 1.8 Å resolution, revealing a new protein fold. Later we also solved the structure of the truncated GFRα1 in the complex with GDNF and this model was confirmed by site-directed mutagenesis. In summary, our work contributed to the structural characterization of GFRα-based receptor complex and revealed a new receptor for GDNF, neurturin and artemin the HSPG syndecan-3. This information is critically important for the development of GFRα/RET agonists for the treatment of neurodegenerative diseases.

Neurotrophic factors and their receptors

Four GDNF ligands (GDNF, neurturin, artemin and persephin), and mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF) protect midbrain dopaminergic neurons that degenerate in Parkinson's disease. Each GDNF ligand binds a specific coreceptor GDNF family receptor α (GFRα), leading to the formation of a heterotetramer complex, which then interacts with receptor tyrosine kinase RET, the signalling receptor. The present thesis describes the structural and biochemical characterization of the GDNF2-GFRα12 complex and the MANF and CDNF proteins. Previous and current mutation data and comparison between GDNF-GFRα1 and artemin-GFRα3 binding interfaces show that N162GFRα1, I175GFRα1, V230GFRα1, Y120GDNF and L114GDNF are the specificity determinants among different ligand-coreceptor pairs. The structure suggests that sucrose octasulphate, a heparin mimic, interacts with a region R190-K202 within domain 2 of GFRα1. Mutating these residues on the GFRα1 surface, which are not in the GDNF binding region, affected RET phosphorylation, which provides a putative RET binding region in domain 2 and 3 of GFRα1. The structural comparison of the GDNF-GFRα1 and artemin-GFRα3 complexes shows a difference in bend angle between the ligand monomers. This variation in bend angle of the ligand may affect the kinetics of RET phosphorylation. To confirm that the difference is not due to crystallization artefacts, I crystallized the GDNF-GFRα1 complex without SOS in different cell dimensions. The structure of the second GDNF-GFRα1 complex is very similar to the previous one, suggesting that the difference between the artemin-GFRα3 and GDNF-GFRα1 complexes are intrinsic, not due to crystal packing. Finally, MANF and CDNF are bifunctional proteins with extracellular neurotrophic activity and ER resident cytoprotective role. The crystal structures of MANF and CDNF are presented here. Intriguingly, the structures of both the neurotrophic factors do not show structural similarity to any of previously known growth factor superfamilies; instead they are similar to saposins, the lipid-binding proteins. The N-terminal domain of MANF and CDNF contain conserved lysines and arginines on its surface, which may interact with negatively charged head groups of phospholipids, as saposins do. Thus MANF and CDNF may provide neurotrophic activities by interacting with a lipo-receptor. The structure of MANF shows a CXXC motif forming internal disulphide bridge in the natively unfolded C-terminus. This motif is common to reductases and disulphide isomerases. It is thus tempting to speculate that the CXXC motif of MANF and CDNF may be involved in oxidative protein folding, which may explain its cytoprotective role in the ER.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.