The PDZ Protein Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) Regulates Planar Cell Polarity and Motile Cilia Organization

Directional flow of the cerebrospinal fluid requires coordinated movement of the motile cilia of the ependymal epithelium that lines the cerebral ventricles. Here we report that mice lacking the Na⁺/H⁺ Exchanger Regulatory Factor 1 (NHERF1/Slc9a3r1, also known as EBP50) develop profound communicating hydrocephalus associated with fewer and disorganized ependymal cilia. Knockdown of NHERF1/slc9a3r1 in zebrafish embryos also causes severe hydrocephalus of the hindbrain and impaired ciliogenesis in the otic vesicle. Ultrastructural analysis did not reveal defects in the shape or organization of individual cilia. Similar phenotypes have been described in animals with deficiencies in Wnt signaling and the Planar Cell Polarity (PCP) pathway. We show that NHERF1 binds the PCP core genes Frizzled (Fzd) and Vangl. We further show that NHERF1 assembles a ternary complex with Fzd4 and Vangl2 and promotes translocation of Vangl2 to the plasma membrane, in particular to the apical surface of ependymal cells. Taken together, these results strongly support an important role for NHERF1 in the regulation of PCP signaling and the development of functional motile cilia.     

A Clinical,Neuropathological and Genetic Study of Homozygous A467T POLG-Related Mitochondrial Disease

Mutations in the nuclear gene POLG (encoding the catalytic subunit of DNA polymerase gamma) are an important cause of mitochondrial disease. The most common POLG mutation, A467T, appears to exhibit considerable phenotypic heterogeneity. The mechanism by which this single genetic defect results in such clinical diversity remains unclear. In this study we evaluate the clinical, neuropathological and mitochondrial genetic features of four unrelated patients with homozygous A467T mutations. One patient presented with the severe and lethal Alpers-Huttenlocher syndrome, which was confirmed on neuropathology, and was found to have a depletion of mitochondrial DNA (mtDNA). Of the remaining three patients, one presented with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), one with a phenotype in the Myoclonic Epilepsy, Myopathy and Sensory Ataxia (MEMSA) spectrum and one with Sensory Ataxic Neuropathy, Dysarthria and Ophthalmoplegia (SANDO). All three had secondary accumulation of multiple mtDNA deletions. Complete sequence analysis of muscle mtDNA using the MitoChip resequencing chip in all four cases demonstrated significant variation in mtDNA, including a pathogenic MT-ND5 mutation in one patient. These data highlight the variable and overlapping clinical and neuropathological phenotypes and downstream molecular defects caused by the A467T mutation, which may result from factors such as the mtDNA genetic background, nuclear genetic modifiers and environmental stressors.     

Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 10³ PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses.     

Effects of fire on abundance of Eragrostis intermedia in a semi-arid grassland in southeastern Arizona

Abstract. Eragrostis intermedia (Plains lovegrass) is a midheight perennial bunchgrass native to semi-arid grasslands of the southwestern USA, that becomes an abundant and dominant component of these grasslands in areas long protected from livestock grazing. Substantial mortality of plains lovegrass occurred on a large livestock exclosure in southeastern Arizona, after a period of declining precipitation, but only in areas that had not burned in the previous three years. Lovegrass abundance subsequently increased on both undisturbed and burned sites, but remained substantially higher on the burned area. Long-term abundance of plains lovegrass may depend on episodic fire, particularly during periods of reduced precipitation.     

Analysis of the biomacromolecular architecture of eukaryotic and prokaryotic serine proteases

Summary We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered on the serine proteases and their natural inhibitors and substrates. It is essential that these approaches emphasize the comparison of these macromolecules at the separate levels of secondary, tertiary and quaternary structure. We assume in our analysis that in functionally related macromolecules (i.e., a family of evolutionarily related enzymes), regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of observed specificity. It is the intent of our research to encapsulate such knowledge in a form which is capable of observing patterns which may serve as generalizable rules for macrostructural analysis (Liebman, M.N. 1986. Enzyme 36: 150–163), and to serve as the essential tools for the rational design of modified serine proteases and/or their natural inhibitors by the methods available through genetic engineering.     

Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: Assessment of cross-reactivity with Pseudomonas aeruginosa

Abstract Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa . The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa . The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. This study suggests that there is a specific anti- B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa . Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS.     

Organisation and expression of a wound/ripening-related small multigene family from tomato

cDNA clones derived from a ripe tomato fruit cDNA library were used to investigate changes in the abundance of specific mRNAs in ripening fruit and wounded leaves. mRNAs related to one cDNA clone (pTOM 13) were expressed in both situations. This clone was used to identify homologous sequences in a tomato genomic library. Three groups of related clones that hybridised to the pTOM 13 cDNA insert were identified and subcloned into plasmid vectors. Genomic Southern analysis of tomato DNA using gene-specific DNA fragments isolated from the subcloned DNAs indicated that all pTOM 13 closely related genes had been isolated. RNA dot blot analysis with these DNA fragments as probes indicated differential expression of this small multigene family in leaves and fruit.     

Lipopolysaccharide heterogeneity in strains ofSerratia marcescens agglutinated by O14 antiserum

Lipopolysaccharides (LPS) from 71 strains ofSerratia marcescens that were agglutinated by O14 antiserum were examined by SDS-PAGE. Four major profiles were found, designated LPS1 to LPS4. These groups accounted for 51, 7, 5, and 3 strains respectively. Five strains were unclassified. Immunoblotting showed that O14 antibodies bound only to LPS1 and not to LPS2, 3, or 4. LPS1 also bound antibodies in O1, O4, O12, and O23 antisera. LPS2 reacted specifically with O8 antiserum, LPS3 with O6, and LPS4 with O2, O3, O6, O12, and O21 antisera. These reactions were not found in agglutination tests with boiled, whole-cell antigens. However, tests with autoclaved antigens (45 min at 121°C) corroborated the immunoblotting classifications; LPS1 strains belonged to serotype O14, LPS2 to serotype O8, LPS3 to serotype O6, and LPS4 to serotype O21. We conclude that there is a heat-stable antigen on many clinical strains ofS. marcescens that masks the expression of O-specific LPS antigens and which binds with nonspecific antibody in serum O14. We propose that O-antigens should be prepared from autoclaved cultures and that the H-reference strain O14H9 CDC 1783-57 (LPS2) should be reclassified as serotype O8.     

Sterilizing Effects of High-Intensity Airborne Sonic and Ultrasonic Waves

The lethal effects of high-intensity airborne sonic (9.9 kc/sec) and ultrasonic waves (30.4 kc/sec) on spores of Bacillus subtilis var. niger ATCC 9372 were determined. The spores, which were deposited on filter-paper strips, were exposed to sound waves for periods varying from 1 to 8 hr, at a temperature of 40 C and a relative humidity of 40%. Significant reductions in the viable counts of spores exposed to airborne sonic or ultrasonic irradiations were obtained. The antibacterial activity of airborne sound waves varied with the sound intensity level, the period of irradiation, and the distance of the sample from the sound source. At similar intensity levels, the amplitude of motion of the sound waves appeared to be a factor in acoustic sterilization.