Deficient membrane integration of the novel p.N14D-GJB2 mutant associated with non-syndromic hearing impairment.

Mutations in GJB2, the gene encoding for the Gap Junction protein Connexin 26 (Cx26), have been established as the major cause of hereditary, non-syndromic hearing impairment (HI). We report here the identification of a novel point mutation in GJB2, c.40A>G [p.N14D], detected in compound heterozygosity with the c.35delG mutation in two brothers with moderate non-syndromic sensorineural HI. The mother who carried one wildtype and a p.N14D allele displayed normal hearing. The mutation leads to substitution of the neutral amino acid asparagine (N) by the negatively charged aspartic acid (D) at amino acid number 14, a position that is conserved among Cx26 of different organisms and among many other connexin isoforms. To investigate the impact of this mutation on protein function, Cx26 activity was measured by depolarization activated hemichannel conductance in non-coupled Xenopus laevis oocytes. Oocytes injected with the p.N14D mutant cRNA showed strongly reduced currents compared to wildtype. Coinjection of wildtype and mutant cRNA at equimolar levels restored the conductive properties supporting the recessive character of this mutation. Total Cx26 protein expression and cell surface abundance examined by western blotting and by quantitative immunoassays revealed that the hemichannel was properly synthesized but not integrated into the plasma membrane. In this study we have shown that the GJB2 mutation p.N14D is associated with recessively inherited HI and exhibits a defective phenotype due to diminished expression at the cell surface.

Isolation of Tn916-like conjugal elements from swine lot effluent.

Isolates of Enterococcus faecalis obtained from a swine farrowing house outflow were examined for genetic elements similar to Tn916. Of the enterococci isolated, 71% were resistant to tetracycline. Among the tetracycline-resistant enterococci isolated from the outflow samples, approximately 34% were able to transfer the tetracycline resistance phenotype to Bacillus thuringiensis in cross-genus matings. The frequencies of transfer for 10 random isolates were comparable to those for transfer of Tn916 from E. faecalis to B. thuringiensis. In addition, these elements were shown to mobilize plasmid pC194 between Bacillus species, as did Tn916. Southern blot and polymerase chain reaction (PCR) analysis showed these elements share extensive structural homology with Tn916. The selected conjugal elements were capable of transfer to a Bacillus recipient in a soil environment. When the swine waste was introduced into the soil, the tetracycline resistant fecal enterococci levels rose from essentially undetectable levels to approximately 4 x 10(4) and remained at this level for 4 weeks. After six months, including one winter, levels had decreased to 5 x 10(3).

Heterogeneity in the attachment and uptake mechanisms of the Legionnaires' disease bacterium, Legionella pneumophila, by protozoan hosts.

Invasion and intracellular replication of Legionella pneumophila within protozoa in the environment plays a major role in the transmission of Legionnaires' disease. Intracellular replication of L. pneumophila within protozoa occurs in a rough endoplasmic reticulum (RER)-surrounded phagosome (Y. Abu Kwaik, Appl. Environ. Microbiol. 62:2022-2028, 1996). Since the subsequent fate of many intracellular pathogens is determined by the route of entry, we compared the mechanisms of attachment and subsequent uptake of L. pneumophila by the two protozoa Hartmannella vermiformis and Acanthamoeba polyphaga. Our data provide biochemical and genetic evidence that the mechanisms of attachment and subsequent uptake of L. pneumophila by the two protozoan hosts are, in part, different. First, uptake of L. pneumophila by H. vermiformis is completely blocked by the monovalent sugars galactose and N-acetyl-D-galactosamine, but these sugars partially blocked A. polyphaga. Second, attachment of L. pneumophila to H. vermiformis is associated with a time-dependent and reversible tyrosine dephosphorylation of multiple host proteins. In contrast, only a slight dephosphorylation of a 170-kDa protein of A. polyphaga is detected upon infection. Third, synthesis of H. vermiformis proteins but not of A. polyphaga proteins is required for uptake of L. pneumophila. Fourth, we have identified L. pneumophila mutants that are severely defective in attachment to A. polyphaga but which exhibit minor reductions in attachment to H. vermiformis and, thus, provide a genetic basis for the difference in mechanisms of attachment to both protozoa. The data indicate a remarkable adaptation of L. pneumophila to attach and invade different protozoan hosts by different mechanisms, yet invasion is followed by a remarkably similar intracellular replication within a RER-surrounded phagosome and subsequent killing of the host cell.

Identification of a Gal/GalNAc lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the Legionnaires' disease bacterium.

The Legionnaire's disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen which invades and replicates within two evolutionarily distant hosts, free-living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaire's disease. Although attachment and invasion of human macrophages by L. pneumophila is mediated in part by the complement receptors CR1 and CR3, the protozoan receptor involved in bacterial attachment and invasion has not been identified. To define the molecular events involved in invasion of protozoa by L. pneumophila, we examined the role of protein tyrosine phosphorylation of the protozoan host Hartmannella vermiformis upon attachment and invasion by L. pneumophila. Bacterial attachment and invasion were associated with a time-dependent tyrosine dephosphorylation of multiple host cell proteins. This host cell response was highly specific for live L. pneumophila, required contact with viable bacteria, and was completely reversible following washing off the bacteria from the host cell surface. Tyrosine dephosphorylation of host proteins was blocked by a tyrosine phosphatase inhibitor but not by tyrosine kinase inhibitors. One of the tyrosine dephosphorylated proteins was identified as the 170-kD galactose/N-acetylgalactosamine-inhibitable lectin (Gal/GalNAc) using immunoprecipitation and immunoblotting by antibodies generated against the Gal/GalNAc lectin of the protozoan Entamoeba histolytica. This Gal/GalNAc-inhibitable lectin has been shown previously to mediate adherence of E. histolytica to mammalian epithelial cells. Uptake of L. pneumophila by H. vermiformis was specifically inhibited by two monovalent sugars, Gal and GalNAc, and by mABs generated against the 170-kD lectin of E. histolytica. Interestingly, inhibition of invasion by Gal and GalNAc was associated with inhibition of bacterial-induced tyrosine dephosphorylation of H. vermiformis proteins. High stringency DNA hybridization confirmed the presence of the 170-kD lectin gene in H. vermiformis. We conclude that attachment of L. pneumophila to the H. vermiformis 170-kD lectin is required for invasion and is associated with tyrosine dephosphorylation of the Gal lectin and other host proteins. This is the first demonstration of a potential receptor used by L. pneumophila to invade protozoa.

Monitoring Wetland Changes with Remote Sensing: An East African Example

Environmental managers need current, accurate information upon which to base decisions. Viable information, especially in developing countries, is often unavailable. Satellite remote sensing is an appropriate and effective data source for mapping the surface of the earth, including a variety of environmental features. Remote-sensing-derived information is enhanced by being one component within a geographic information system (GIS). These techniques were employed to study an expanding delta in East Africa.The Omo River flows from the Ethiopian Highlands into the northern end of Lake Turkana, creating a large delta extending between Ethiopia and Kenya. This isolated and unique wetland feature has expanded by over 500 sq km in the last 15 years as measured by space-borne remote sensing techniques and corroborated by low-altitude aircraft reconnaissance flights.The growth of the delta appears to be a function of both increased sedimentation and decreased lake levels and river flows. Within the delta there has been a selective decline in wildlife and an increase in human activity, both pastoral and agricultural. The uniqueness of this isolated delta suggests that consideration be given to its possible protection and management.

Cholera toxin inhibits T cell receptor signaling by covalent modification of the CD3-zeta subunit.

In the Jurkat T cell line, triggering of the TCR leads to activation of phospholipase C, resulting in an increase in inositol trisphosphate (IP3) release followed by a rise in intracellular Ca2+. This signaling pathway is interrupted by cholera toxin (CTX) treatment. To possibly explain this inhibition, we demonstrate that CTX can affect the TCR/CD3 complex itself by causing a covalent modification of the CD3-zeta subunit. After exposure of Jurkat cells to CTX, CD3-zeta increases its apparent m.w. and becomes more acidic in isoelectric focusing. The time course of the modification correlates well with the reduction in IP3 generation and Ca2+ release after CTX treatment, suggesting that the modification of zeta might be the cause of the impaired TCR/CD3 signaling. As is true for the CTX-mediated decrease in TCR signaling, the change in CD3-zeta was cAMP-independent and cannot be evoked by the enzymatically inactive CTX-B subunit alone.