Overexpression of proline oxidase induces proline-dependent and mitochondria-mediated apoptosis

The neuropathogenesis of influenza-associated encephalopathy in children and Reye's syndrome remains unclear. A surveillance effort conducted during 2000-2003 in South-West Japan reveals that almost all fatal and handicapped influenza-associated encephalopathy patients exhibit a disorder of mitochondrial β-oxidation with elevated serum acylcarnitine ratios (C_16:0+C_18:1)/C₂. Here we show invasion by a non-neurotropic epidemic influenza A H3N2 virus in cerebral capillaries with progressive brain edema after intranasal infection of mice having impaired mitochondrial β-oxidation congenitally or posteriorly in the newborn/ suckling periods. Mice genetically lacking of carnitine transporter OCTN2, resulting in carnitine deficiency and impaired β-oxidation, exhibited significant higher virus-genome numbers in the brain, accumulation of virus antigen exclusively in the cerebral capillaries and increased brain vascular permeability compared to in wild type mice. Mini-plasmin, which proteolytically potentiates influenza virus multiplication in vivo and destroys the blood-brain barrier, accumulated with virus antigen in the brain capillaries of OCTN2-deficient mice but only a little in wild-type mice. These results suggest that the impaired mitochondrial β-oxidation changes the susceptibility to a non-neurotropic influenza A virus as to multiplication in the brain capillaries and to cause brain edema. These pathological findings in the brain of mice having impaired mitochondrial β-oxidation after influenza virus infection may have implications for human influenza-associated encephalopathy.     

Microbial production and application of sophorolipids

Sophorolipids are surface-active compounds synthesized by a selected number of yeast species. They have been known for over 40 years, but because of growing environmental awareness, they recently regained attention as biosurfactants due to their biodegradability, low ecotoxicity, and production based on renewable resources. In this paper, an overview is given of the producing yeast strains and various aspects of fermentative sophorolipid production. Also, the biochemical pathways and regulatory mechanisms involved in sophorolipid biosynthesis are outlined. To conclude, a summary is given on possible applications of sophorolipids, either as native or modified molecules.     

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2 to facilitate biochemical studies using thiol-specific modifying reagents. Of 10 endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site saturation mutagenesis scheme based on the Stratagene Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in Saccharomyces cerevisiae cells auxotrophic for purines, several highly functional noncysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position.     

Isolation of avian paramyxovirus 1 from a patient with a lethal case of pneumonia

An unknown virus was isolated from a lung biopsy sample and multiple other samples from a patient who developed a lethal case of pneumonia following a peripheral blood stem cell transplant. A random PCR-based molecular screening method was used to identify the infectious agent as avian paramyxovirus 1 (APMV-1; a group encompassing Newcastle disease virus), which is a highly contagious poultry pathogen that has only rarely been found in human infections. Immunohistochemical analysis confirmed the presence of APMV-1 antigen in sloughed alveolar cells in lung tissue from autopsy. Sequence from the human isolate showed that it was most closely related to virulent pigeon strains of APMV-1. This is the most completely documented case of a systemic human infection caused by APMV-1 and is the first report of an association between this virus and a fatal disease in a human.     

Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.     

The Batten disease Palmitoyl Protein Thioesterase 1 gene regulates neural specification and axon connectivity during Drosophila embryonic development

Palmitoyl Protein Thioesterase 1 (PPT1) is an essential lysosomal protein in the mammalian nervous system whereby defects result in a fatal pediatric disease called Infantile Neuronal Ceroids Lipofuscinosis (INCL). Flies bearing mutations in the Drosophila ortholog Ppt1 exhibit phenotypes similar to the human disease: accumulation of autofluorescence deposits and shortened adult lifespan. Since INCL patients die as young children, early developmental neural defects due to the loss of PPT1 are postulated but have yet to be elucidated. Here we show that Drosophila Ppt1 is required during embryonic neural development. Ppt1 embryos display numerous neural defects ranging from abnormal cell fate specification in a number of identified precursor lineages in the CNS, missing and disorganized neurons, faulty motoneuronal axon trajectory, and discontinuous, misaligned, and incorrect midline crossings of the longitudinal axon bundles of the ventral nerve cord. Defects in the PNS include a decreased number of sensory neurons, disorganized chordotonal neural clusters, and abnormally shaped neurons with aberrant dendritic projections. These results indicate that Ppt1 is essential for proper neuronal cell fates and organization; and to establish the local environment for proper axon guidance and fasciculation. Ppt1 function is well conserved from humans to flies; thus the INCL pathologies may be due, in part, to the accumulation of various embryonic neural defects similar to that of Drosophila. These findings may be relevant for understanding the developmental origin of neural deficiencies in INCL.