Localization and behaviors in null mice suggest that ASIC1 and ASIC2 modulate responses to aversive stimuli.

Acid-sensing ion channels (ASICs) generate H(+) -gated Na(+) currents that contribute to neuronal function and animal behavior. Like ASIC1, ASIC2 subunits are expressed in the brain and multimerize with ASIC1 to influence acid-evoked currents and facilitate ASIC1 localization to dendritic spines. To better understand how ASIC2 contributes to brain function, we localized the protein and tested the behavioral consequences of ASIC2 gene disruption. For comparison, we also localized ASIC1 and studied ASIC1(-/-) mice. ASIC2 was prominently expressed in areas of high synaptic density, and with a few exceptions, ASIC1 and ASIC2 localization exhibited substantial overlap. Loss of ASIC1 or ASIC2 decreased freezing behavior in contextual and auditory cue fear conditioning assays, in response to predator odor and in response to CO2 inhalation. In addition, loss of ASIC1 or ASIC2 increased activity in a forced swim assay. These data suggest that ASIC2, like ASIC1, plays a key role in determining the defensive response to aversive stimuli. They also raise the question of whether gene variations in both ASIC1 and ASIC2 might affect fear and panic in humans.

Expressing acid-sensing ion channel 3 in the brain alters acid-evoked currents and impairs fear conditioning.

Previous studies on mice with a disruption of the gene encoding acid-sensing ion channel 1a (ASIC1a) suggest that ASIC1a is required for normal fear behavior. To investigate the effects of altering the subunit composition of brain ASICs on behavior, we developed transgenic mice expressing ASIC3 via the pan-neuronal synapsin I promoter. These mice express ASIC3 in the brain, where the endogenous ASIC3 protein is not detected. We found that in ASIC3 transgenic mice, ASIC3 co-immunoprecipitated with the endogenous ASIC1a protein and distributed in the same subcellular brain fractions as ASIC1a. In addition, ASIC3 significantly increased the rate of desensitization of acid-evoked currents in cultured cortical neurons. Importantly, ASIC3 reduced Pavlovian fear conditioning to both context and auditory cues. These observations suggest that ASIC3 can heteromultimerize with ASIC1a in the brain and alter the biophysical properties of the endogenous channel complex. Moreover, these data suggest that ASIC subunit composition and channel desensitization may be critical determinants for ASIC-dependent behavior.

The piglet as a model for B cell and immune system development.

The ability to identify factors responsible for disease in all species depends on the ability to separate those factors which are environmental from those that are intrinsic. This is particularly important for studies on the development of the adaptive immune response of neonates. Studies on laboratory rodents or primates have been ambiguous because neither the effect of environmental nor maternal factors on the newborn can be controlled in mammals that: (i) transmit potential maternal immunoregulatory factors in utero and (ii) are altricial and cannot be reared after birth without their mothers. Employing the newborn piglet model can address each of these concerns. However, it comes at the price of having first to characterize the immune system of swine and its development. This review focuses on the porcine B cell system, especially on the methods used for its characterization in fetal studies and neonatal piglets. Understanding these procedures is important in the interpretation of the data obtained. Studies on neonatal piglets have (a) provided valuable information on the development of the adaptive immune system, (b) lead to important advances in evolutionary biology, (c) aided our understanding of passive immunity and (d) provided opportunities to use swine to address specific issues in veterinary and biomedical research and immunotherapy. This review summarizes the history of the development of the piglet as a model for antibody repertoire development, thus providing a framework to guide future investigators.

Endothelial barrier dysfunction caused by LPS correlates with phosphorylation of HSP27 in vivo.

Lung edema during sepsis is triggered by formation of gaps between endothelial cells followed by macrophage infiltration. Endothelial gap formation has been proposed to involve changes in the structure of the actin filament cytoskeleton. Heat shock protein 27 (HSP27) is believed to modulate actin filament dynamics or structure, in a manner dependent on its phosphorylation status. We hypothesized that HSP27 may play a role in endothelial gap formation, by affecting actin dependent events in endothelial cells. As there has been no report concerning HSP27 in lung edema in vivo, we examined induction and phosphorylation of HSP27 in lung following LPS injection, as a model of sepsis. In lung, HSP27 mainly localized in capillary endothelial cells of the alveolus, and in smooth muscle cells of pulmonary arteries. HSP27 became significantly more phosphorylated at 3 h after LPS treatment, while the distribution of HSP27 remained unchanged. Pre-treatment with anti-TNFalpha antibody, which has been shown to reduce lung injury, blocked increases in HSP27 phosphorylation at 3 h. HSP27 phosphorylation was also increased in cultured rat pulmonary arterial endothelial cells (RPAEC) by treatment with TNFalpha, LPS, or H2O2. This phosphorylation was blocked by pre-treatment with SB203580, an inhibitor of the upstream kinase, p38 MAP kinase. Increased endothelial permeability caused by H2O2 in vitro was also blocked by SB203580. The amount of actin associated with HSP27 was reduced after treatment with LPS, or H2O2. In summary, HSP27 phosphorylation temporally correlated with LPS induced pathological endothelial cell gap formation in vivo and in a cell culture model system. This is the first report of increased HSP27 phosphorylation associated with pathological lung injury in an animal model of sepsis.

Adenovirus calcium phosphate coprecipitates enhance squamous cell carcinoma gene transfer.

OBJECTIVES/HYPOTHESIS: Adenoviral-mediated gene transfer offers a potential new treatment strategy for squamous cell cancer of the head and neck (SCCHN). Initial studies on some SCCHN cell lines have shown that these cells can be resistant to adenovirus-mediated gene transfer, requiring large amounts of vector and long infection times. The objectives of this study were to identify the barriers to gene transfer in three SCCHN lines, FaDu, SCC-9, and SCC-15, and to develop a method to circumvent the obstacles. We hypothesized that a low expression of adenovirus receptors may limit adenovirus infection and this may be overcome by using adenovirus complexed with calcium phosphate coprecipitates. METHODS: Using standard cell and molecular biology techniques, the infectability of SCCHN cells was investigated. RESULTS: Using Cy3-labeled adenovirus, we found minimal binding of adenovirus to FaDu cells and variable levels of binding among SCC-9 and SCC-15 cells. Northern blot analysis indicated that messenger RNA (mRNA) transcripts for coxsackie-adenovirus receptor, which binds adenovirus, were absent in FaDu cells but present in SCC-9 and SCC-15 cells. Integrin alphavbeta5, which binds and facilitates internalization of adenovirus, were expressed at low levels in all three cell types. We overcame these barriers by using adenovirus complexed with calcium phosphate precipitates. Total transgene expression and the number of cells expressing transgene were increased in all three cancer lines using adenovirus complexed with calcium phosphate precipitates compared with adenovirus that was not complexed. CONCLUSIONS: Data in the present study suggest that adenovirus-mediated gene transfer to SCCHN cell lines is a result of limited viral receptors. Delivering adenovirus in a calcium phosphate coprecipitate enhanced gene transfer and, perhaps, the therapeutic index.

Identification and characterization of hic-5/ARA55 as an hsp27 binding protein.

hsp27 has been reported to participate in a wide variety of activities, including resistance to thermal and metabolic stress, regulation of growth and differentiation, and acting as a molecular chaperone or a regulator of actin polymerization. We hypothesized that these diverse functions are regulated in a cell- or tissue-specific manner via interaction with various binding proteins. To investigate this hypothesis, we used hsp27 as a "bait" to screen a yeast two-hybrid cDNA library from rat kidney glomeruli and identified a novel hsp27 binding protein, hic-5 (also known as ARA55), a focal adhesion protein and steroid receptor co-activator. Biochemical interaction between hsp27 and hic-5 was confirmed by co-immunoprecipitation, and critical protein.protein interaction regions were mapped to the hic-5 LIM domains and the hsp27 C-terminal domain. Initial analysis of the functional role of hsp27.hic-5 interaction revealed that hic-5 significantly inhibited the protection against heat-induced cell death conferred by hsp27 overexpression in co-transfected 293T cells. In contrast, when a non-hsp27-interacting hic-5 truncation mutant (hic-5/DeltaLIM4) was co-expressed with hsp27, the hic-5 inhibition of hsp27 protection was absent. We conclude that hic-5 is a true hsp27 binding protein and inhibits the ability of hsp27 to provide protection against heat shock in an interaction-dependent manner.

ENaC subunits are molecular components of the arterial baroreceptor complex.

Mechanosensation is essential to the perception of our environment. It is required for hearing, touch, balance, proprioception, and blood pressure homeostasis. Yet little is known about the identity of ion-channel complexes that transduce mechanical stimuli into neuronal responses. Genetic studies in Caenorhabditis elegans suggest that members of the DEG/ENaC family may be mechanosensors. Therefore we tested the hypothesis that mammalian epithelial Na(+)-channel (ENaC) subunits contribute to the mechanosensor in baroreceptor neurons. The data presented here show that ENaC transcripts and proteins are expressed in mechanosensory neurons and at the putative sites of mechanotransduction in baroreceptor sensory-nerve terminals. Additionally, known ENaC inhibitors, amiloride and benzamil, disrupt mechanotransduction in arterial baroreceptor neurons. These data are consistent with the hypothesis that DEG/ENaC proteins are components of mechanosensitive ion-channel complexes.

KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response.

Keratinocyte growth factor (KGF) regulates several functions in adult and developing lung epithelia; it causes proliferation, stimulates secretion of fluid and electrolytes, enhances repair, and may minimize injury. To gain insight into the molecular processes influenced by KGF, we applied KGF to primary cultures of well-differentiated human airway epithelia and used microarray hybridization to assess the abundance of gene transcripts. Of 7,069 genes tested, KGF changed expression levels of 910. Earlier studies showed that KGF causes epithelial proliferation, and as expected, treatment altered expression of numerous genes involved in cell proliferation. We found that KGF stimulated transepithelial Cl(-) transport, but the number of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) transcripts fell. Although transcripts for ClC-1 and ClC-7 Cl(-) channels increased, KGF failed to augment transepithelial Cl(-) transport in CF epithelia, suggesting that KGF-stimulated Cl(-) transport in differentiated airway epithelia depends on the CFTR Cl(-) channel. Interestingly, KGF decreased transcripts for many interferon (IFN)-induced genes. IFN causes trafficking of Stat dimers to the nucleus, where they activate transcription of IFN-induced genes. We found that KGF prevented the IFN-stimulated trafficking of Stat1 from the cytosol to the nucleus, suggesting a molecular mechanism for KGF-mediated suppression of the IFN-signaling pathway. These results suggest that in addition to stimulating proliferation and repair of damaged airway epithelia, KGF stimulates Cl(-) transport and may dampen the response of epithelial cells to inflammatory mediators.

Apical localization of the coxsackie-adenovirus receptor by glycosyl-phosphatidylinositol modification is sufficient for adenovirus-mediated gene transfer through the apical surface of human airway epithelia.

In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl(-) transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis.

Condom use during a community intervention trial in Kenya.

We conducted a cluster-randomized community intervention trial at Kenyan agricultural sites to measure the impact of female condom introduction on sexually transmitted infection (STI) prevalence. We present male and female condom use data here. Six Intervention sites received a community risk-reduction campaign and distribution of female condoms and male condoms, while 6 Control sites received the same campaign with male condoms only. Male and female condom distribution increased throughout follow-up. Self-reported male condom use increased substantially during follow-up to over 60% of the participants. The proportion of consistent male condom users at Control sites was higher than at Intervention sites, 23% vs 14% at 6 months and 24% vs 22% at 12 months. At Intervention sites, 11% and 7% of women used the female condoms all the time at 6 and 12 months, respectively, while the percentage of female condom non-users grew. Male and female condom use was hindered by male partner objections; suspicion of the study and the devices among residents; and bias against condoms by clinic service providers. A large proportion of coital acts remained unprotected during the trial. Our female condom intervention did not reduce STI prevalence, compared with male condom promotion only.

Female condom introduction and sexually transmitted infection prevalence: results of a community intervention trial in Kenya.

OBJECTIVE: To measure the impact on sexually transmitted infection (STI) prevalence of a female condom introduction and risk-reduction program at Kenyan agricultural sites. DESIGN: We conducted a cluster-randomized trial to determine whether a replicable, community-level intervention would reduce STI prevalence. METHODS: Six matched pairs of tea, coffee and flower plantations were identified. The six intervention sites received an information/motivation program with free distribution of female and male condoms, and six control sites received only male condoms and related information. Participants were tested for cervical gonorrhea and chlamydia by ligase chain reaction on urine specimens, and vaginal trichomoniasis by culture, at baseline, 6 and 12 months. RESULTS: Participants at intervention (n = 969) and control sites (n = 960) were similar; baseline STI prevalence was 23.9%. Consistent male condom use was more than 20% at 12 months. Consistent female condom use was reported by 11 and 7% of intervention site women at 6 and 12 months. Unadjusted STI prevalence was 16.5 and 17.4% at 6 months, and 18.3 and 18.5% at 12 months, at the intervention and control sites, respectively. Logistic regression models confirmed the null effect of the female condom intervention. CONCLUSIONS: Female condom introduction did not enhance STI prevention at these sites. It is unclear which aspects of the intervention -- STI education, condom promotion, case management -- were associated with decreased STI prevalence from baseline to follow-up.

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature.

Several DEG/ENaC cation channel subunits are expressed in the tongue and in cutaneous sensory neurons, where they are postulated to function as receptors for salt and sour taste and for touch. Because these tissues are exposed to large temperature variations, we examined how temperature affects DEG/ENaC channel function. We found that cold temperature markedly increased the constitutively active Na(+) currents generated by epithelial Na(+) channels (ENaC). Half-maximal stimulation occurred at 25 degrees C. Cold temperature did not induce current from other DEG/ENaC family members (BNC1, ASIC, and DRASIC). However, when these channels were activated by acid, cold temperature potentiated the currents by slowing the rate of desensitization. Potentiation was abolished by a "Deg" mutation that alters channel gating. Temperature changes in the physiologic range had prominent effects on current in cells heterologously expressing acid-gated DEG/ENaC channels, as well as in dorsal root ganglion sensory neurons. The finding that cold temperature modulates DEG/ENaC channel function may provide a molecular explanation for the widely recognized ability of temperature to modify taste sensation and mechanosensation.

HSP22, a new member of the small heat shock protein superfamily, interacts with mimic of phosphorylated HSP27 ((3D)HSP27).

Most of the members of the superfamily of mammalian small heat shock or stress proteins are abundant in muscles where they play a role in muscle function and maintenance of muscle integrity. One member of this protein superfamily, human HSP27, is rapidly phosphorylated on three serine residues (Ser(15), Ser(78), and Ser(82)) during cellular response to a number of extracellular factors. To understand better the role of HSP27, we performed a yeast two-hybrid screen of a human heart cDNA library for HSP27-interacting proteins. By using the triple aspartate mutant, a mimic of phosphorylated HSP27, as "bait" construct, a protein with a molecular mass of 21.6 kDa was identified as an HSP27-binding protein. Sequence analysis revealed that this new protein shares an overall sequence identity of 33% with human HSP27. This protein also contains the alpha-crystallin domain in its C-terminal half, a hallmark of the superfamily of small stress proteins. Thus, the new protein itself is a member of this protein superfamily, and consequently we designated it HSP22. According to the two-hybrid data, HSP22 interacts preferentially with the triple aspartate form of HSP27 as compared with wild-type HSP27. HSP22 is expressed predominantly in muscles. In vitro, HSP22 is phosphorylated by protein kinase C (at residues Ser(14) and Thr(63)) and by p44 mitogen-activated protein kinase (at residues Ser(27) and Thr(87)) but not by MAPKAPK-2.

Evidence of diffusion from a targeted HIV/AIDS intervention in the Dominican Republic.

The diffusion potential of a targeted HIV/AIDS intervention that enlisted peer educators to disseminate 'safer sex' messages and condoms among female commercial sex workers and their clients was evaluated in the Dominican Republic. Levels of interurban interaction potential were ascertained that linked the targeted city of La Romana with the proximate cities of San Pedro de Macoris and Guaymate. Weekly service statistics generated over an 8-month period were analysed to establish activity areas for the peer educators. Data were entered and analysed using a geographic information system and interurban linkages were established. Project outcomes were examined via a series of three cross-sectional Knowledge, Attitudes and Practices (KAP) surveys conducted among convenience samples of commercial sex workers at the start of the intervention and at 4 and 8 months. The results attest to a high degree of interconnectivity between the targeted and proximate cities, and a pattern of interurban mobility that links commercial sex workers, clients and establishments in all three cities. The examination of project outcomes revealed statistically significant changes in condom use in the targeted city of La Romana among commercial sex workers, as well as among their counterparts interviewed in the proximate cities of San Pedro de Macoris and Guaymate. These data suggest a diffusion effect. It is concluded that a targeted intervention may influence proximate cities within a relatively compressed period of time. The findings suggest the importance of considering geographic diffusion principles, such as urban hierarchies, regional nodes and transportation linkages, when designing HIV/AIDS prevention efforts. It also has important implications in the selection of control sites when conducting experimental studies of HIV/AIDS interventions.

Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer.

Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. Whereas AAV serotype 2-mediated gene transfer to muscle has partially replaced factor IX deficiency in hemophilia patients, its ability to mediate gene transfer to the lungs for cystic fibrosis is hindered by lack of apical receptors. However, AAV serotype 5 infects human airway epithelia from the lumenal surface. We found that in contrast to AAV2, the apical membrane of airway epithelia contains abundant high affinity receptors for AAV5. Binding and gene transfer with AAV5 was abolished by genetic or enzymatic removal of sialic acid from the cell surface. Furthermore, binding and gene transfer to airway epithelia was competed by lectins that specifically bind 2,3-linked sialic acid. These observations suggest that 2,3-linked sialic acid is either a receptor for AAV5 or it is a necessary component of a receptor complex. Further elucidation of the receptor for this virus should enhance understanding of parvovirus biology and expand the therapeutic targets for AAV vectors.

A conditional probability analysis of cystic fibrosis transmembrane conductance regulator gating indicates that ATP has multiple effects during the gating cycle.

ATP-binding cassette (ABC) transporters bind and hydrolyze ATP. In the cystic fibrosis transmembrane conductance regulator Cl(-) channel, this interaction with ATP generates a gating cycle between a closed (C) and two open (O1 and O2) conformations. To understand better how ATP controls channel activity, we examined gating transitions from the C to the O1 and O2 states and from these open states to the C conformation. We made three main observations. First, we found that the channel can open into either the O1 or O2 state, that the frequency of transitions to both states was increased by ATP concentration, and that ATP increased the relative proportion of openings into O1 vs. O2. These results indicate that ATP can interact with the closed state to open the channel in at least two ways, which may involve binding to nucleotide-binding domains (NBDs) NBD1 and NBD2. Second, ATP prolonged the burst duration and altered the way in which the channel closed. These data suggest that ATP also interacts with the open channel. Third, the channel showed runs of specific types of open-closed transitions. This finding suggests a mechanism with more than one cycle of gating transitions. These data suggest models to explain how ATP influences conformational transitions in cystic fibrosis transmembrane conductance regulator and perhaps other ABC transporters.

Antimicrobial peptides and proteins in the innate defense of the airway surface.

Recent studies have advanced our understanding of innate immune mechanisms that protect the airways and maintain a sterile lung. Multiple antimicrobial peptides and proteins have been identified in airway secretions and their roles are beginning to be established in animal models. Moreover, evidence for coupling between the innate and adaptive immune systems is beginning to emerge. The understanding of the innate airway defense system offers the opportunity for the development of novel therapeutic approaches.

Cystic fibrosis transmembrane conductance regulator Cl- channels with R domain deletions and translocations show phosphorylation-dependent and -independent activity.

Phosphorylation of the R domain regulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may prevent constitutive activity, i.e. opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this study we asked whether the two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducing constitutive activity. However, when residues 760-783 were deleted, channels opened without phosphorylation. Translocation of the R domain to the C terminus did not prevent constitutive activity. These results suggest that different parts of the phosphorylated R domain can stimulate activity and that their location within the protein is not critical. In contrast, prevention of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than one phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel.