Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease

To understand the pathogenesis of chronic inflammatory disease, we analyzed an experimental mouse model of chronic lung disease with pathology that resembles asthma and chronic obstructive pulmonary disease (COPD) in humans. In this model, chronic lung disease develops after an infection with a common type of respiratory virus is cleared to only trace levels of noninfectious virus. Chronic inflammatory disease is generally thought to depend on an altered adaptive immune response. However, here we find that this type of disease arises independently of an adaptive immune response and is driven instead by interleukin-13 produced by macrophages that have been stimulated by CD1d-dependent T cell receptor-invariant natural killer T (NKT) cells. This innate immune axis is also activated in the lungs of humans with chronic airway disease due to asthma or COPD. These findings provide new insight into the pathogenesis of chronic inflammatory disease with the discovery that the transition from respiratory viral infection into chronic lung disease requires persistent activation of a previously undescribed NKT cell-macrophage innate immune axis.     

Determination of amprenavir, a HIV-1 protease inhibitor, in human seminal plasma using high-performance liquid chromatography-tandem mass spectrometry

A HPLC-MS-MS method to measure amprenavir in human seminal plasma has been developed and validated. The procedure uses stable, isotopically labeled 13C6-amprenavir as an internal standard and 100 microl of sample. The method is accurate (bias less than or equal to 7.2%) and precise (within- and between-day RSDs less than or equal to 4.2%) over the dynamic range of 30-4,000 ng/ml. Recently, this simple and sensitive method was used to determine amprenavir concentrations in seminal samples collected from HIV-1 positive subjects receiving amprenavir antiretroviral therapy as part of a multicenter clinical trial.     

Poor physical fitness is independently associated with mild cognitive impairment in elderly Koreans

The purpose of this study was to investigate the association between physical fitness and mild cognitive impairment (MCI) in elderly Koreans. This was a cross-sectional study that involved 134 men and 299 women aged 65 to 88 years. Six senior fitness tests were used as independent variables: 30 s chair stand for lower body strength, arm curl for upper body strength, chair-sit-and-reach for lower body flexibility, back scratch for upper body flexibility, 8-ft up-and-go for agility/dynamic balance, and 2-min walk for aerobic endurance. Global cognitive function was assessed using the Korean version of the Mini-Mental State Examination (MMSE). Potential covariates such as age, education levels, blood lipids, and insulin resistance (IR) markers were also assessed. Compared to individuals without MMSE-based MCI, individuals with MMSE-based MCI had poor physical fitness based on the senior fitness test (SFT). There were significant positive trends observed for education level (p=0.001) and MMSE score (p<0.001) across incremental levels of physical fitness in this study population. Individuals with moderate (OR=0.341, p=0.006) and high (OR=0.271, p=0.007) physical fitness based on a composite score of the SFT measures were less likely to have MMSE-based MCI than individuals with low physical fitness (referent, OR=1). The strength of the association between moderate (OR=0.377, p=0.038) or high (OR=0.282, p=0.050) physical fitness and MMSE-based MCI was somewhat attenuated but remained statistically significant even after adjustment for the measured compounding factors. We found that poor physical fitness was independently associated with MMSE-based MCI in elderly Koreans.     

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives

The 18S and 5.8S rDNA genes and the internal transcribed spacers ITS-1 and ITS-2 of ciliates living in the hindgut of frogs, millipedes, and cockroaches were analyzed in order to study the evolution of intestinal protists. All ciliates studied here belong to the genus Nycrotherus. Phylogenetic analysis revealed that these ciliates from a monophyletic group that includes the distantly related anaerobic free-living heterotrichous ciliates Metopus palaeformis and Metopus contortus. The intestinal ciliates from the different vertebrate and invertebrate hosts are clearly divergent at the level of their rDNA repeats. This argues for the antiquity of the associations and a predominantly vertical transmission. This mode of transmission seems to be controlled primarily by the behavior of the host. The different degrees of divergence between ciliates living in different strains of one and the same cockroach species most likely reflect the different geographical origins of the hosts. In addition, host switches must have occurred during the evolution of cockroaches, since identical ciliates were found only in distantly related hosts. These phenomena prevent the reconstruction of potential cospeciation events.      

Concanavalin A distorts the beta-GlcNAc-(1-->2)-Man linkage of beta- GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man- (1-- >6)]-Man upon binding

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1-->2)-alpha- Man-(1-->3)-[beta- GlcNAc-(1-->2)-alpha-Man-(1-->6)]-Man) of N-linked oligosaccharides with a Ka of 1.41 x 10(6 )M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta- GlcNAc-(1-->2)-alpha-Man- (1-->6)]-Man to 2.7A. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1-->6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1-->3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1-->2)-Man glycosidic linkage PSI torsion angle on the 1-->6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1-->6 arm GlcNAc site.      

Inhibition of topoisomerases from Pneumocystis carinii by aromatic dicationic molecules.

Pentamidine and related derivatives inhibit an ATP-dependent topoisomerase activity from Pneumocystis carinii extracts. Since it would be extremely difficult to purify ample quantities of the organisms to allow characterization of the enzyme and carry out drug binding experiments, we have begun the cloning of the topoisomerase genes with a goal towards expression of each gene in a heterologous system. Following construction of genomic libraries in the vectors lambda DASH and lambda ZAP, oligonucleotides corresponding to conserved regions of both topoisomerases I and II were used in the polymerase chain reaction (PCR) of P. carinii DNA to generate probes. Candidate clones for both genes have been identified. Partial DNA sequence of the topoisomerase II gene has been determined.     

Construction of genomic libraries of Cryptosporidium parvum and identification of antigen-encoding genes.

Genomic libraries have been constructed from bovine C. parvum DNA in the lambda ZAP and lambda DASH vectors. Based on an estimated genome size of 2 x 10(4) kilobases (kb), each recombinant library contains greater than 10 genomic equivalents. The average recombinant size for the lambda ZAP library is 2.1 kb and for the lambda DASH library is 14 kb. We have identified genes to major antigens recognized by hyperimmune bovine antiserum. These recombinants are currently being purified and characterized. Limited DNA sequence analysis of random C. parvum clones confirms suggestions that the genome is quite AT-rich. The DNA sequence of random lambda ZAP fusion proteins has identified a potential ATPase, a structural protein and a DNA-binding protein.     

MARCH6 and TRC8 facilitate the quality control of cytosolic and tail‐anchored proteins

Misfolded or damaged proteins are typically targeted for destruction by proteasome‐mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome‐mediated degradation of the soluble misfolded reporter, mCherry‐CL1, involves two ER‐resident E3 ligases, MARCH6 and TRC8. mCherry‐CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail‐anchored protein heme oxygenase‐1 (HO‐1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO‐1 following intramembrane proteolysis. Our results highlight how ER‐resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail‐anchored proteins.