Identification of PhIL1, a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide

The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[(125)I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [(125)I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.     

Hydrophobic inactivation of influenza viruses confers preservation of viral structure with enhanced immunogenicity

The use of inactivated influenza virus for the development of vaccines with broad heterosubtypic protection requires selective inactivation techniques that eliminate viral infectivity while preserving structural integrity. Here we tested if a hydrophobic inactivation approach reported for retroviruses could be applied to the influenza virus. By this approach, the transmembrane domains of viral envelope proteins are selectively targeted by the hydrophobic photoactivatable compound 1,5-iodonaphthyl-azide (INA). This probe partitions into the lipid bilayer of the viral envelope and upon far UV irradiation reacts selectively with membrane-embedded domains of proteins and lipids while the protein domains that localize outside the bilayer remain unaffected. INA treatment of influenza virus blocked infection in a dose-dependent manner without disrupting the virion or affecting neuraminidase activity. Moreover, the virus maintained the full activity in inducing pH-dependent lipid mixing, but pH-dependent redistribution of viral envelope proteins into the target cell membrane was completely blocked. These results indicate that INA selectively blocks fusion of the virus with the target cell membrane at the pore formation and expansion step. Using a murine model of influenza virus infection, INA-inactivated influenza virus induced potent anti-influenza virus serum antibody and T-cell responses, similar to live virus immunization, and protected against heterosubtypic challenge. INA treatment of influenza A virus produced a virus that is noninfectious, intact, and fully maintains the functional activity associated with the ectodomains of its two major envelope proteins, neuraminidase and hemagglutinin. When used as a vaccine given intranasally (i.n.), INA-inactivated influenza virus induced immune responses similar to live virus infection.     

Reduction of Hydrogen Peroxide Accumulation and Toxicity by a Catalase from Mycoplasma iowae

Mycoplasma iowae is a well-established avian pathogen that can infect and damage many sites throughout the body. One potential mediator of cellular damage by mycoplasmas is the production of H₂O₂ via a glycerol catabolic pathway whose genes are widespread amongst many mycoplasma species. Previous sequencing of M. iowae serovar I strain 695 revealed the presence of not only genes for H₂O₂ production through glycerol catabolism but also the first documented mycoplasma gene for catalase, which degrades H₂O₂. To test the activity of M. iowae catalase in degrading H₂O₂, we studied catalase activity and H₂O₂ accumulation by both M. iowae serovar K strain DK-CPA, whose genome we sequenced, and strains of the H₂O₂-producing species Mycoplasma gallisepticum engineered to produce M. iowae catalase by transformation with the M. iowae putative catalase gene, katE. H₂O₂-mediated virulence by M. iowae serovar K and catalase-producing M. gallisepticum transformants were also analyzed using a Caenorhabditis elegans toxicity assay, which has never previously been used in conjunction with mycoplasmas. We found that M. iowae katE encodes an active catalase that, when expressed in M. gallisepticum, reduces both the amount of H₂O₂ produced and the amount of damage to C. elegans in the presence of glycerol. Therefore, the correlation between the presence of glycerol catabolism genes and the use of H₂O₂ as a virulence factor by mycoplasmas might not be absolute.     

A mouse model for human hearing loss DFNB30 due to loss of function of myosin IIIA

The motor protein myosin IIIA is critical for maintenance of normal hearing. Homozygosity and compound heterozygosity for loss-of-function mutations in MYO3A, which encodes myosin IIIA, are responsible for inherited human progressive hearing loss DFNB30. To further evaluate this hearing loss, we constructed a mouse model, Myo3a ^KI/KI , that harbors the mutation equivalent to the nonsense allele responsible for the most severe human phenotype. Myo3a ^KI/KI mice were compared to their wild-type littermates. Myosin IIIA, with a unique N-terminal kinase domain and a C-terminal actin-binding domain, localizes to the tips of stereocilia in wild-type mice but is absent in the mutant. The phenotype of the Myo3a ^KI/KI mouse parallels the phenotype of human DFNB30. Hearing loss, as measured by auditory brainstem response, is reduced and progresses significantly with age. Vestibular function is normal. Outer hair cells of Myo3a ^KI/KI mice degenerate with age in a pattern consistent with their progressive hearing loss.     

Pseudozyma aphidis activates reactive oxygen species production,programmed cell death and morphological alterations in the necrotrophic fungus Botrytis cinerea

Many types of yeast have been studied in the last few years as potential biocontrol agents against different phytopathogenic fungi. Their ability to control plant diseases is mainly through combined modes of action. Among them, antibiosis, competition for nutrients and niches, induction of systemic resistance in plants and mycoparasitism have been the most studied. In previous work, we have established that the epiphytic yeast Pseudozyma aphidis inhibits Botrytis cinerea through induced resistance and antibiosis. Here, we demonstrate that P. aphidis adheres to B. cinerea hyphae and competes with them for nutrients. We further show that the secreted antifungal compounds activate the production of reactive oxygen species and programmed cell death in B. cinerea mycelium. Finally, P. aphidis and its secreted compounds negatively affect B. cinerea hyphae, leading to morphological alterations, including hyphal curliness, vacuolization and branching, which presumably affects the colonization ability and infectivity of B. cinerea. This study demonstrates additional modes of action for P. aphidis and its antifungal compounds against the plant pathogen B. cinerea.     

The Protein Quality Control Machinery Regulates Its Misassembled Proteasome Subunits

Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with various aggregation diseases. In eukaryotes, the ubiquitin proteasome system (UPS) plays a vital role in protein quality control (PQC), by selectively targeting misfolded proteins for degradation. While the assembly of the proteasome can be naturally impaired by many factors, the regulatory pathways that mediate the sorting and elimination of misassembled proteasomal subunits are poorly understood. Here, we reveal how the dysfunctional proteasome is controlled by the PQC machinery. We found that among the multilayered quality control mechanisms, UPS mediated degradation of its own misassembled subunits is the favored pathway. We also demonstrated that the Hsp42 chaperone mediates an alternative pathway, the accumulation of these subunits in cytoprotective compartments. Thus, we show that proteasome homeostasis is controlled through probing the level of proteasome assembly, and the interplay between UPS mediated degradation or their sorting into distinct cellular compartments.     

Tear production in three captive wild herbivores in Israel

The Schirmer tear test (STT) I was performed to evaluate tear production in 12 captive Nubian ibex (Capra ibex nubiana), 10 captive Burchell's zebras (Equus burchelli) and five Arabian oryx (Oryx leucoryx) at the Tel-Aviv Ramat-Gan Zoological Center (Israel). Mean (+/- standard deviation) STT values were 13.2 +/- 5.1 mm/min in the ibex, 23.4 +/- 3.4 mm/min in the zebra and 12.7 +/- 4.8 mm/min in the oryx. There were no significant effects of gender, age, weight, or side of the eye. There were no significant differences in STT values between ibex and oryx, but tear production in both species was significantly lower than in zebras. Knowledge of normal tear production values is important for the differential diagnosis of conjunctivitis and keratitis in these species.