Three paradigms of airway smooth muscle hyperresponsiveness in young guinea pigs

Evidence for contributions of airway smooth muscle (ASM) to the hyperresponsiveness of newborn and juvenile airways continues to accumulate. In our laboratory, 3 novel paradigms of hyperresponsiveness of newborn and young ASM have recently emerged using a guinea pig model of maturation in 3 age groups: 1 week (newborn), 3 weeks (juvenile), and 2-3 months (adult). The first paradigm includes evidence for a natural decline after newborn and juvenile life of the velocity of ASM shortening associated with a decrease in regulatory myosin light chain phosphorylation and a parallel decline in the content of myosin light chain kinase. Associated with the decrease in ASM shortening with age is an increase in the internal resistance to shortening. Dynamic stiffness is shown to relate inversely to the expression of myosin light chain kinase. This suggests that developmental changes in shortening relate inversely to the stiffness of the ASM early in shortening, suggesting a dynamic role for the cytoskeleton in facilitating and opposing ASM shortening. This relationship can be approximated as (dP/dt)max approximately (dP/dL)passive x (dL/dt)max (the maximal rate of increase of active stress generation approximately to the passive stiffness x the maximal shortening velocity). The second paradigm demonstrates that newborn ASM, unlike that in adults, does not relax during prolonged electric field stimulation. The impaired relaxation is related to changes in prostanoid synthesis and acetylcholinesterase function. The third paradigm demonstrates that, whereas oscillatory strain serves to transiently relax adult ASM, in newborns it induces (after the initial relaxation) a sustained potentiation of active stress. This is related to developmental changes in the prostanoid release. Together, these paradigms demonstrate that ASM contributes by multiple mechanisms to the natural hyperresponsiveness of newborn and juvenile airways. Future studies will elaborate the mechanisms and extend these paradigms to ASM hyperresponsiveness following sensitization in early life.     

Functional and structural aspects of poplar cytosolic and plastidial type a methionine sulfoxide reductases

The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys(46) is the catalytic cysteine, and the two C-terminal cysteines, Cys(196) and Cys(202), are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys(202), but not Cys(196), is the first recycling cysteine that forms a disulfide bond with the catalytic Cys(46). Then Cys(202) forms a disulfide bond with the second recycling cysteine Cys(196) that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys(202) is located closer to Cys(46) compared with Cys(196) and is included in a (202)CYG(204) signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine.     

Conformation-sensitive antibodies against alzheimer amyloid-beta by immunization with a thioredoxin-constrained B-cell epitope peptide

Immunotherapy against the amyloid-beta (Abeta) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Abeta, and yet be capable of eliciting antibodies that recognize Abeta fibrils and neurotoxic Abeta oligomers but not the physiological monomeric species of Abeta. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Abeta1-15 (Abeta15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Abeta15)n polypeptides bearing one, four, or eight copies of Abeta15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Abeta15)4 antibody, in particular, recognized Abeta42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Abeta. We have also demonstrated that anti-Trx(Abeta15)4, which binds to human AD plaques, markedly reduces Abeta pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Abeta fragment repeat and identify Trx(Abeta15)4 as a promising new tool for AD immunotherapy.     

Differences in variation of human immunodeficiency virus type 1 sequences from Henan and Shanghai regions of China

Illegally paid blood donation was a risk factor for HIV acquisition exclusively in Henan and Hubei Provinces of China, and not in Shanghai. Nucleotide sequences in the gag and env genes of HIV-1 were compared between isolates from Henan and Shanghai regions of China to test whether an expected higher degree of a common source of infections from this unique blood donation transmission risk would be evident as decreased variation among Henan isolates in an exploratory cross-sectional analysis. Among 38 isolates studied, 23 of 23 (100%) from Henan and 8 of 15 (54%) from Shanghai were subtype B. In addition, fewer sequence differences were found in gp41 of subtype B isolates from Henan than from Shanghai isolates. Further studies with additional controls are therefore warranted to confirm the role of the degree of a common source of infections in differences in HIV variation across populations. Fundation items: The Vanderbilt-Meharry Center for AIDS Research (P30 AI 54999); R.T.D (R01 AI 29193); Start Fund of Ministry of Education of China (for Hong-zhou LU, 2004BA719A10).     

Mass‐trapping trials for the control of pine processionary moth in a pine woodland recreational area

The pine processionary moth, Thaumetopoea pityocampa, causes serious defoliation to Cedrus, Pinus and Pseudotsuga trees, as well as health problems in humans, pets and farm animals due to their urticating hairs. Environmentally friendly strategies for the management of T. pityocampa include: removal of egg batches, removal of nests, trapping of migrant larvae, spraying microbial or Insect Growth Regulator (IGR) insecticides and biocontrol, as well as pheromone‐based adult trapping and mating‐disruption. In the present paper, results on innovative technology for the control of T. pityocampa infestation using pheromone mass‐trapping are reported. Two 1‐ha plots were identified in the study area (central‐south Italy), a pine woodland recreational site growing Pinus halepensis. In the experimental plot (MT‐plot), 10 G‐traps (funnel trap type) baited with (Z)‐13‐hexadecen‐11‐ynyl acetate sex pheromone component were placed for mass‐trapping of adults; the other plot was used as a control‐plot (C‐plot). The T. pityocampa population was monitored using the two central traps in the MT‐plot and two traps positioned in the C‐plot. In addition, the winter nests made by T. pityocampa larvae overwintering on pine trees were counted. After 2 years of mass‐trapping, the number of adults trapped by the monitoring pheromone traps decreased in the MT‐plot, but not in the C‐plot, whereas the number of nests decreased in both plots. Statistical results highlighted significant differences in trap catches between the two plots but not between years. In the case of nests, differences among plots were not significant before the mass‐trapping, but significant after 1‐year treatment. According to our results, the mass‐trapping technique is able to reduce T. pityocampa infestations. This pheromone method can be applied in combination with other control systems in the context of integrated pest management in recreational areas.     

Activation of the phosphatidylinositol 3-kinase-Akt/protein kinase B signaling pathway in arachidonic acid-stimulated human myeloid and endothelial cells: involvement of the ErbB receptor family

Although arachidonic acid has been demonstrated to stimulate a wide variety of cellular functions, the responsible mechanisms remain poorly defined. We now report that arachidonic acid stimulated the activity of class Ia phosphatidylinositol 3-kinase (PI3K) in human umbilical vein endothelial cells, HL60 cells, and human neutrophils. Pretreatment of endothelial cells with AG-1478, an inhibitor of the ErbB receptor family, resulted in the suppression of PI3K activation by arachidonic acid. The fatty acid enhanced the tyrosine phosphorylation of ErbB4 but not of ErbB2 or ErbB3. The ability of arachidonic acid to stimulate PI3K activity in neutrophils was suppressed by indomethacin and nordihydroguaiaretic acid, inhibitors of the cyclooxygenases and lipoxygenases, respectively, but not by 17-octadecynoic acid, an inhibitor of omega-hydroxylation of arachidonic acid by cytochrome P450 monooxygenases. Consistent with this, the activity of PI3K in neutrophils was stimulated by 5-hydroxyeicosatetraenoic acid. Arachidonic acid also transiently stimulated the phosphorylation of Akt on Thr-308 and Ser-473. Although PI3K was not required for the activation of the mitogen-activated protein kinases, ERK1, ERK2, and p38, in arachidonic acid-stimulated neutrophils, the fatty acid acted via PI3K to stimulate the respiratory burst. These results not only define a novel mechanism through which some of the actions of arachidonic acid are mediated but also demonstrate that, in addition to ErbB1 (epidermal growth factor receptor), ErbB4 can also be transactivated by a non-epidermal growth factor-like ligand.     

In vivo tyrosine phosphorylation sites of activated ephrin-B1 and ephB2 from neural tissue

EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, we have identified three tyrosines of ephrin-B1 as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ. Furthermore, many of the tyrosines of EphB2 previously identified as phosphorylation sites in 293 cells (Kalo, M. S., and Pasquale, E. B. (1999) Biochemistry 38, 14396-14408) are also phosphorylated in retinal tissue. Our data underline the complexity of ephrin-Eph bidirectional signaling by implicating many tyrosine phosphorylation sites of the ligand-receptor complex.