Pneumocystis stimulates MCP-1 production by alveolar epithelial cells through a JNK-dependent mechanism

Pneumocystis carinii is an opportunistic fungal pathogen that causes pneumonia (PCP) in immunocompromised individuals. Recent studies have demonstrated that the host's immune response is clearly responsible for the majority of the pathophysiological changes associated with PCP. P. carinii interacts closely with alveolar epithelial cells (AECs); however, the nature and pathological consequences of the epithelial response remain poorly defined. Monocyte chemotactic protein-1 (MCP-1) is involved in lung inflammation, immunity, and epithelial repair and is upregulated during PCP. To determine whether AECs are an important source of MCP-1 in the P. carinii-infected lung, in vivo and in vitro studies were performed. In situ hybridization showed that MCP-1 mRNA was localized to cells with morphological characteristics of AECs in the lungs of infected mice. In vitro studies demonstrated that P. carinii stimulated a time- and dose-dependent MCP-1 response in primary murine type II cells that was preceded by JNK activation. Pharmacological inhibition of JNK nearly abolished P. carinii-stimulated MCP-1 production, while ERK, p38 MAPK, and TNF receptor signaling were not required. Furthermore, delivery of a JNK inhibitory peptide specifically to pulmonary epithelial cells using a recombinant adenovirus vector blocked the early lung MCP-1 response following intratracheal instillation of infectious P. carinii. JNK inhibition did not affect P. carinii-stimulated production of macrophage inflammatory protein-2 in vitro or in vivo, indicating that multiple signaling pathways are activated in P. carinii-stimulated AECs. These data demonstrate that AECs respond to P. carinii in a proinflammatory manner that may contribute to the generation of immune-mediated lung injury.     

Ignoring linkage disequilibrium among tightly linked markers induces false-positive evidence of linkage for affected sib pair analysis

Most multipoint linkage programs assume linkage equilibrium among the markers being studied. The assumption is appropriate for the study of sparsely spaced markers with intermarker distances exceeding a few centimorgans, because linkage equilibrium is expected over these intervals for almost all populations. However, with recent advancements in high-throughput genotyping technology, much denser markers are available, and linkage disequilibrium (LD) may exist among the markers. Applying linkage analyses that assume linkage equilibrium to dense markers may lead to bias. Here, we demonstrated that, when some or all of the parental genotypes are missing, assuming linkage equilibrium among tightly linked markers where strong LD exists can cause apparent oversharing of multipoint identity by descent (IBD) between sib pairs and false-positive evidence for multipoint model-free linkage analysis of affected sib pair data. LD can also mimic linkage between a disease locus and multiple tightly linked markers, thus causing false-positive evidence of linkage using parametric models, particularly when heterogeneity LOD score approaches are applied. Bias can be eliminated by inclusion of parental genotype data and can be reduced when additional unaffected siblings are included in the analysis.     

Influence of malaria parasite (P. vivax) on erythrocyte aggregation: a study based on dynamic imaging and analysis

Malaria due to P. vivax (PV) is prevalent in many countries. The present work is aimed to determine the cell-cell interaction through formation of aggregates under dynamic conditions. Blood samples are obtained from patients (n=11) suffering from PV malaria, and the normal subjects (n=10) in test tubes containing citrate phosphate dextrose (10:1.4), as an anticoagulant. The signature analysis of infected erythrocytes shows significant alterations in their shape and membrane. For aggregation analysis, erythrocyte suspension in plasma at hematocrit 5%, was placed in a glass chamber and mounted vertically on the stage of the video-microscope system. The aggregate images thus acquired show erythrocytes adhering with each other to form mash-like structures. With increase in parasitaemia, the erythrocytes show hyper-aggregation compared to that of normal cells. By processing of the sequence of recorded images during sedimentation, the various aggregation parameters are obtained. These parameters show that the formed aggregates are compact which produce distinct changes in sedimentation pattern with significantly higher sedimentation velocity compared to that in healthy blood samples. These changes in malaria could partly be responsible for alteration in blood flow through microcirculatory system.     

Proline-Rich Peptide from the Coral Pathogen Vibrio shiloi That Inhibits Photosynthesis of Zooxanthellae

The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29°C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH₄Cl, pure natural or synthetic toxin P (10 μM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH₄Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH₄Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH₃ into the cell. It is known that uptake of NH₃ into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.     

A whole-genome scan for obstructive sleep apnea and obesity

Obstructive sleep apnea (OSA) is a common, chronic, complex disease associated with serious cardiovascular and neuropsychological sequelae and with substantial social and economic costs. Along with male gender, obesity is the most characteristic feature of OSA in adults. To identify susceptibility loci for OSA, we undertook a 9-cM genome scan in 66 white pedigrees (n=349 subjects) ascertained on the basis of either an affected individual with laboratory-confirmed OSA or a proband who was a neighborhood control individual. Multipoint variance-component linkage analysis was performed for the OSA-associated quantitative phenotypes apnea-hypopnea index (AHI) and body mass index (BMI). Candidate regions on chromosomes 1p (LOD score 1.39), 2p (LOD score 1.64), 12p (LOD score 1.43), and 19p (LOD score 1.40) gave the most evidence for linkage to AHI. BMI was also linked to multiple regions, most significantly to markers on chromosomes 2p (LOD score 3.08), 7p (LOD score 2.53), and 12p (LOD score 3.41). Extended modeling indicated that the evidence for linkage to AHI was effectively removed after adjustment for BMI, with the exception of the candidate regions on chromosomes 2p (adjusted LOD score 1.33) and 19p (adjusted LOD score 1.45). After adjustment for AHI, the primary linkages to BMI remained suggestive but were roughly halved. Our results suggest that there are both shared and unshared genetic factors underlying susceptibility to OSA and obesity and that the interrelationship of OSA and obesity in white individuals may be partially explained by a common causal pathway involving one or more genes regulating both AHI and BMI levels.     

Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia

Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA•TTC)_n sequences (9–105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8–107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)_n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.     

Genetic admixture of European FRDA genes is the cause of Friedreich ataxia in the Mexican population

Friedreich ataxia accounts for approximately 75% of European recessive ataxia patients. Approximately 98% of pathogenic chromosomes have large expansions of a GAA triplet repeat in the FRDA gene (E alleles), and strong linkage disequilibrium among polymorphisms spanning the FRDA locus indicates a common origin for all European E alleles. In contrast, we found that only 14 of 151 (9.3%) Mexican Mestizo patients with recessive ataxia were homozygous for E alleles. Analysis of polymorphisms spanning the FRDA locus revealed that all Mestizo E alleles had the common European haplotype, indicating that they share a single origin. Genetic admixture levels were determined, which revealed that the relative contributions to the Mestizo FRDA gene pool by Native American and European genes were 76-87% and 13-24%, respectively, commensurate with the observed low prevalence of Friedreich ataxia in Mestizos. This indicates that Friedreich ataxia in Mexican Mestizos is due to genetic admixture of European mutant FRDA genes in the Native American gene pool that existed prior to contact with Europeans.     

Human embryonic kidney cells (HEK-293 cells): characterization and dose-response relationship for modulated release of nerve growth factor for nerve regeneration

The development of engineered constructs to bridge nerve gaps may hold the key to improved functional outcomes in the repair of injured peripheral nerves. These constructs must be rendered bioactive by providing the growth factors required for successful peripheral nerve regeneration. Previous studies demonstrated that harvested human and rat dermal fibroblasts could be genetically engineered to release nerve growth factor (NGF) both in vitro and in vivo. The use of fibroblasts, however, has the potential to cause scarring, and the expression of NGF from those cells was transient. To overcome these potential difficulties, human embryonic kidney cells were modified for use with the ecdysone-inducible mammalian expression system. These cells (hNGF-EcR-293) have been engineered and regulated to secrete human NGF in response to the ecdysone analogue ponasterone A. HEK-293 cells were transfected with human NGF cDNA with the ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, Calif.). Stable clones were then selected. Ponasterone A, an analogue of ecdysone, was used as the inducing agent. The secretion of NGF into the medium was analyzed with two different methods. After 24 hours of exposure to the inducing agent, cell medium was transferred to PC-12 cells seeded in 12-well plates, for determination of whether the secreted NGF was bioactive. Medium from untreated or ponasterone A-treated hNGF-EcR-293 cells was deemed bioactive on the basis of its ability to induce PC-12 cell differentiation. The concentrations of secreted NGF were also quantified with an enzyme-linked immunosorbent assay, in triplicate. NGF production was measured in successive samples of the same medium during a 9-day period, with maximal release of 9.05 +/- 2.6 ng/ml at day 9. Maximal NGF production was 8.46 +/- 2.1 pg/10(3) cells at day 9. These levels were statistically significantly different from levels in noninduced samples (p 相似文献