Site-directed mutagenesis of a conserved hexapeptide in the paramyxovirus hemagglutinin-neuraminidase glycoprotein: effects on antigenic structure and function.

The sequence NRKSCS constitutes the longest linear stretch in the amino acid sequence of the hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxoviruses that is completely conserved among all viruses in the group. We have used site-directed mutagenesis and expression of the mutated HN protein of one member of the group, Newcastle disease virus, to explore the role of this highly conserved sequence in the structure and function of the protein. Any substitution introduced for each of four residues in the sequence, N-234, R-235, K-236, or S-237, results in a drastic decrease in neuraminidase activity relative to that of the wild-type protein. Only substitutions for the terminal serine residue in the sequence had comparatively little effect on this activity. These findings are consistent with prior computer-based predictions of protein secondary structure which had suggested that this domain corresponds to one in the beta-sheet propeller structure of the neuraminidase protein of influenza virus closest to the center of the sialic acid binding site and forms part of the enzyme active site. Four of the substitutions, N-234-->Y and K-236-->E, -->Q, and -->S, apparently cause a local alteration in the antigenic structure of the protein. This is evidenced by (i) the diminished recognition of the protein only by monoclonal antibodies thought to bind at the neuraminidase active site, among an extensive panel of conformation-specific antibodies, and (ii) the slower rate of migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for all except the K-236-->Q mutation. One of the mutations, K-236-->S, completely abolishes the ability of the protein to promote cellular fusion when coexpressed with the fusion protein. The latter cannot be explained by a decrease in the relative hemadsorption activity of the protein and suggests that the globular head of the protein may contribute to this process beyond providing receptor recognition.     

Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.     

Expression of human chorionic gonadotropin uniformly labeled with NMR isotpes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures

Summary Most secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure requires confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein back bone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with ¹³C and ¹⁵N isotopes. We report the first such uniform labeling of a glycoprotein using a universal ¹³C-and ¹⁵N-labeling medium to express ¹³C, ¹⁵N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The ¹³C, ¹⁵N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.To whom correspondence should be addressed.     

Activation of smooth muscle in the airway wall, force production, and airway narrowing.

Airway narrowing depends on smooth muscle force production and muscle shortening, but the structural and geometric properties exhibited by individual generations of the bronchial tree largely determine the extent and characteristics of airway narrowing. Properties of major importance include the nature and integrity of the epithelium, the structural and mechanical properties of the airway wall, as well as airway diameter. The influence of these properties on airway narrowing measured as flow or flow resistance in large and small diameter segments of airways from pig lung is described using a novel preparation, the perfused bronchial segment.     

Use of microsphere-antibody conjugates in microparticle-enhanced nephelometric immunoassay.

A new microparticle-enhanced nephelometric immunoassay has been recently described as a sensitive, accurate, and easy-to-perform competitive immunoassay for various analytes. As initially described, this test is based on the nephelometric quantification of the inhibition, by the antigen to be assayed, of immunoagglutination of microparticle-antigen conjugates. Its applicability as a competitive immunoassay is thus limited by the necessary availability of pure antigens to prepare microparticle-antigen conjugates. In this paper, we report an adaptation of this initial test, where microparticles are coated by monoclonal antibodies, eliminating the need for purified antigens. The new configurations of particle agglutination-based immunoassays described include use of these microparticle-antibody conjugates with microparticle-antigen conjugates, free antigen, and anti-mouse immunoglobulins antiserum. The feasibility of such configurations is studied with human chorionic gonadotropin hormone, human thyroid stimulating hormone, and human myoglobin as antigens. Capture of the analyte by microparticle-antibody conjugates is evidenced by inhibition of their agglutination with microparticle-antigen conjugates and by agglutination in a sandwich assay with a complementary monoclonal antibody or polyclonal antiserum. The use of a second xenogenic antibody enhances the agglutination process and increases the assay sensitivity. Microparticle-antibody conjugates may extend the applications of microparticle-enhanced nephelometric immunoassays to unavailable analytes.     

Etiology of a Root Rot Disease Complex of Alstroemeria in Alberta, Canada

Fusarium oxysporum, Pythiu-m ultimum, and Rhizoctonia solani were isolated from the basal stems of diseased alstroemeria showing symptoms of dark brown stripes along leaf margins, leaf chlorosis, plant wilting, browning or rotting of basal stem, rhizome, and storage and fibrous roots. The pathogen isolated most frequently was Fusarium spp. (40.5 % of plants examined). Pythium spp. and R. solani were isolated less frequently (5.5 % and 6.8 % of plants examined, respectively). F. oxysporum caused the highest mortality in alstroemeria when rhizomes were grown in unsterilized soil-less mix medium. This is the first report in North America of a root-rot disease complex affecting alstroemeria.     

Wheat variety carrying 2NvS chromosomal segment provides yield advantage through lowering terminal heat–induced oxidative stress

A 2N^vS chromosomal segment carrying bread wheat variety, BARI Gom 33 (‘BG33’), showed tolerance to terminal heat stress and higher yield over a heat-tolerant non-2N^vS BARI Gom 26 (‘BG26’) and a heat-susceptible Pavon 76 (‘Pavon’). This study aimed to ascertain the potential of the 2N^vS ‘BG33’ in terminal heat-induced oxidative stress tolerance compared to non-2N^vS ‘BG26’ and heat-susceptible ‘Pavon’ under two heat regimes at the reproductive stages viz. control (optimum sowing time) and heat stress (late sowing). We found that both ‘BG26’ and ‘BG33’ showed significantly higher tolerance to oxidative stress by limiting the generation of reactive oxygen species (ROS), methylglyoxal under heat stress. During terminal heat stress, both ‘BG33’ and ‘BG26’ exhibited greater cellular homeostasis than heat-susceptible ‘Pavon’, which was maintained by the increased accumulation of osmolytes, nonenzymatic antioxidants, and enzymes associated with ROS scavenging, ascorbate–glutathione cycle, and glyoxalase system. Lesser cellular damage in ‘BG26’ and ‘BG33’ was eventually imitated in a smaller reduction in grain yield (15 and 12%, respectively) than in ‘Pavon’, which had a 33% reduction owing to heat stress. Collectively, our findings revealed that the chromosomal segment 2N^vS provides yield advantage to ‘BG33’ under terminal heat stress by lowering oxidative damage. As 2N^vS translocation contains multiple nucleotide-binding domain leucine-rich repeat containing, cytochrome P450, and other gene families associated with plant stress tolerance, further studies are warranted to dissect the underlying molecular mechanisms associated with higher heat stress tolerance of 2N^vS carrying ‘BG33’.

     

Synthetic, EPR spectroscopic, magnetic and X-ray crystallographic structural studies on copper(II) complexes of the tridentate N2S donor ligand formed from 6-methyl-2-formylpyridine and S-methyldithiocarbazate (Hmpsme)

New copper(II) complexes of general empirical formula, Cu(mpsme)X · xCH₃COCH₃ (mpsme = anionic form of the 6-methyl-2-formylpyridine Schiff base of S-methyldithiocarbazate; X = Cl, N₃, NCS, NO₃; x = 0, 0.5) have been synthesized and characterized by IR, electronic, EPR and susceptibility measurements. Room temperature μ_eff values for the complexes are in the range 1.75-2.1 μ_B typical of uncoupled or weakly coupled Cu(II) centres. The EPR spectra of the [Cu(mpsme)X] (X = Cl, N₃, NO₃, NCS) complexes reveal a tetragonally distorted coordination sphere around the mononuclear Cu(II) centre. We have exploited second derivative EPR spectra in conjunction with Fourier filtering (sine bell and Hamming functions) to extract all of the nitrogen hyperfine coupling matrices. While the X-ray crystallography of [Cu(mpsme)NCS] reveals a linear polymer in which the thiocyanate anion bridges the two copper(II) ions, the EPR spectra in solution are typical of a magnetically isolated monomeric Cu(II) centres indicating dissociation of the polymeric chain in solution. The structures of the free ligand, Hmpsme and the {[Cu(mpsme)NO₃] · 0.5CH₃COCH₃}₂ and [Cu(mpsme)NCS]_n complexes have been determined by X-ray diffraction. The {[Cu(mpsme)NO₃] 0.5CH₃COCH₃}₂ complex is a centrosymmetric dimer in which each copper atom adopts a five-coordinate distorted square-pyramidal geometry with an N₂OS₂ coordination environment, the Schiff base coordinating as a uninegatively charged tridentate ligand chelating through the pyridine and azomethine nitrogen atoms and the thiolate, an oxygen atom of a unidentate nitrato ligand and a bridging sulfur atom from the second ligand completing the coordination sphere. The [Cu(mpsme)(NCS)]_n complex has a novel staircase-like one dimensional polymeric structure in which the NCS⁻ ligands bridge two adjacent copper(II) ions asymmetrically in an end-to-end fashion providing its nitrogen atom to one copper and the sulfur atom to the other.     

Analysis of transcriptional profiles and functional clustering of global cerebellar gene expression in PCD3J mice

The Purkinje cell degeneration (PCD) mutant mouse is characterized by a degeneration of cerebellar Purkinje cells and progressive ataxia. To identify the molecular mechanisms that lead to the death of Purkinje neurons in PCD mice, we used Affymetrix microarray technology to compare cerebellar gene expression profiles in pcd3J mutant mice 14 days of age (prior to Purkinje cell loss) to unaffected littermates. Microarray analysis, Ingenuity Pathway Analysis (IPA) and expression analysis systematic explorer (EASE) software were used to identify biological and molecular pathways implicated in the progression of Purkinje cell degeneration. IPA analysis indicated that mutant pcd3J mice showed dysregulation of specific processes that may lead to Purkinje cell death, including several molecules known to control neuronal apoptosis such as Bad, CDK5 and PTEN. These findings demonstrate the usefulness of these powerful microarray analysis tools and have important implications for understanding the mechanisms of selective neuronal death and for developing therapeutic strategies to treat neurodegenerative disorders.     

Stability of dystrophin STR fragments in relation to junction helicity

Dystrophin is a rod shaped protein consisting of amino- and carboxy-terminal binding domains linked by a large central rod composed of 24 homologous copies of the STR motif and 4 non-homologous regions termed hinges. These hinges are proposed to confer local flexibility; conversely, the tacit implication is that the STR regions away from the hinges are comparatively rigid. This, and the repeating nature of this rod, has contributed to the view that the STR region of the rod is uniform and monolithic. However, we have produced various 2 STR fragments, chosen to have high and low alpha-helix content at their junctions with each other, and show that they exhibit markedly different stabilities. In contrast to a related protein, spectrin, these differences are not correlated with the calculated helicity, but appear to be an intrinsic property of the motifs themselves. A full understanding of how these properties vary along the length of the rod has implications for the engineering of these rods regions in exon skipping and minidystrophin therapies.