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1.
Valiunas V Mui R McLachlan E Valdimarsson G Brink PR White TW 《American journal of physiology. Cell physiology》2004,287(6):C1596-C1604
A subset of connexins can form unopposed hemichannels in expression systems, providing an opportunity for comparison of hemichannel gating properties with those of intact gap junction channels. Zebrafish connexin35 (Cx35) is a member of the Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that Cx35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole cell, cell-attached, and excised patch configurations, we obtained multichannel and single-channel current recordings attributable to the Cx35 hemichannels (Ihc) that were activated and increased by stepwise depolarization of membrane potential (Vm) and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense Cx38. However, water-injected oocytes that were not treated with antisense showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance (hc: 250320 pS). The hc of Cx35 hemichannels exhibited a pronounced Vm dependence; i.e., hc increased/decreased with relative hyperpolarization/depolarization (hc was 72 pS at Vm = 100 mV and 35 pS at Vm = 100 mV). Extrapolation to Vm = 0 mV predicted a hc of 48 pS, suggesting a unitary conductance of intact Cx35 gap junction channels of 24 pS. Channel gating was also Vm dependent: open time declined with negative Vm and increased with positive Vm. The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo. connexin; gating; retina 相似文献
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Cheng S Shakespeare T Mui R White TW Valdimarsson G 《The Journal of biological chemistry》2004,279(35):36993-37003
Gap junctions are composed of connexin (Cx) proteins and mediate intercellular communication required for many developmental and physiological processes. Here we describe the isolation and characterization of Cx48.5, a zebrafish connexin with the highest sequence identity to mammalian Cx46. Expression analysis showed that Cx48.5 is expressed in the adult and embryonic lens and heart, adult testis, and transiently in the embryonic otic vesicles. Injection of Cx48.5 cRNA into Xenopus oocytes elicited intercellular electrical coupling with voltage sensitivity similar to mammalian Cx46. In single oocytes, Cx48.5 also induced large outward currents on depolarization, consistent with gap-junctional hemichannels. Disruption of Cx48.5 expression in embryos with antisense morpholino oligos (morpholinos) revealed that Cx48.5 has an essential role in the maintenance of lens homeostasis. The morpholino-treated embryos also developed small lenses and eyes as well as severe cardiovascular abnormalities. 相似文献
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Adjemian JZ Adjemian MK Foley P Chomel BB Kasten RW Foley JE 《Journal of wildlife diseases》2008,44(3):737-742
This study aimed to describe the occurrence of Yersinia pestis, Rickettsia rickettsii, Anaplasma phagocytophilum, and ectoparasites in a wild rodent community in the eastern Sierra Nevada. From May to September 2006, rodents were live-trapped, examined for ectoparasites, and blood was collected. All rodents were serologically tested for antibodies to Y. pestis, R. rickettsii, and A. phagocytophilum; in addition, blood samples and ectoparasites were tested by PCR to detect the presence of these zoonotic agents. Overall, 89 rodents, 46 fleas, and four ticks were collected. Antibody prevalence rates observed for rodents were 14% for R. rickettsii or antigenically related spotted-fever group rickettsiae, and 8% for A. phagocytophilum. No samples were positive for antibodies to Y. pestis. Positive PCR results included one yellow-pine chipmunk for Y. pestis (CT=32.8), one golden-mantled ground squirrel for R. rickettsii (CT=33), and one flea found to be co-infected with both R. rickettsii (CT=17) and A. phagocytophilum (CT=36). The results of this study provide evidence of multiple zoonoses overlapping within a single, located rodent community. 相似文献
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Adam Vigil Rocio Ortega Aarti Jain Rie Nakajima-Sasaki Xiaolin Tan Bruno B. Chomel Rickie W. Kasten Jane E. Koehler Philip L. Felgner 《PloS one》2010,5(7)
Background
Bartonella henselae is the zoonotic agent of cat scratch disease and causes potentially fatal infections in immunocompromised patients. Understanding the complex interactions between the host''s immune system and bacterial pathogens is central to the field of infectious diseases and to the development of effective diagnostics and vaccines.Methodology
We report the development of a microarray comprised of proteins expressed from 96% (1433/1493) of the predicted ORFs encoded by the genome of the zoonotic pathogen Bartonella henselae. The array was probed with a collection of 62 uninfected, 62 infected, and 8 “specific-pathogen free” naïve cat sera, to profile the antibody repertoire elicited during natural Bartonella henselae infection.Conclusions
We found that 7.3% of the B. henselae proteins on the microarray were seroreactive and that seroreactivity was not evenly distributed between predicted protein function or subcellular localization. Membrane proteins were significantly most likely to be seroreactive, although only 23% of the membrane proteins were reactive. Conversely, we found that proteins involved in amino acid transport and metabolism were significantly underrepresented and did not contain any seroreactive antigens. Of all seroreactive antigens, 52 were differentially reactive with sera from infected cats, and 53 were equally reactive with sera from infected and uninfected cats. Thirteen of the seroreactive antigens were found to be differentially seroreactive between B. henselae type I and type II. Based on these results, we developed a classifier algorithm that was capable of accurately discerning 93% of the infected animals using the microarray platform. The seroreactivity and diagnostic potential of these antigens was then validated on an immunostrip platform, which correctly identified 98% of the infected cats. Our protein microarray platform provides a high-throughput, comprehensive analysis of the feline humoral immune response to natural infection with the alpha-proteobacterium B. henselae at an antigen-specific, sera-specific, and genome-wide level. Furthermore, these results provide novel insight and utility in diagnostics, vaccine development, and understanding of host-pathogen interaction. 相似文献7.
A cDNA encoding metallothionein (MT) was isolated from a library constructed with poly A(+) RNA purified from 48 h etiolated cotton (Gossypium hirsutum L.) cotyledons. This cDNA encodes a deduced protein with 63 residues and a molecular weight of 6.3 kDa. The protein has 10 cysteines of which 4 are within the CXXCXCXXXXXC amino-terminus motif and six are within the CXCXXXCXCXXCXC carboxyl-terminus motif characteristic of the type III MT (MT3). The cotton MT3 protein sequence is 76.2, 69.8, 66.7, 60.3 and 33.5% identical to MT3 from Carica papaya, Rubus idaeus, Ribes nigrum, Citrus unshiu, and Gossypium hirsutum type I MT, respectively. A fusion protein was constructed by producing PCR primers for the 5' and 3' ends of the cotton MT3 cDNA and ligating the PCR product inframe at the 3' end of a bacterial glutathione S-transferase (GST) gene in the pGEX3 vector. The 5' PCR primer incorporated a segment of the cotton MT3 noncoding region, resulting in an addition of 9 residues to the MT3 (after Factor Xa digestion site) which increased the size of the expressed protein to 72 residues and 7.6 kDa. Expression of the 7.6 kDa protein in bacteria was confirmed by SDS-PAGE. Induction and accumulation of the GST-MT3 protein began inhibiting bacterial growth after 1 h. Addition of Cu (1 muM to 1 mM), 1 mM cysteine, or 1 mM cystine to the media did not rescue growth. Additionally, this protein was evaluated for its ability to bind Cd, Cu, Ni and Zn in the bacterial expression system. We found that cotton MT3 preferentially binds Cu. 相似文献
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Eria A. Rebollar Myra C. Hughey Reid N. Harris Rickie J. Domangue Daniel Medina Roberto Ibá?ez Lisa K. Belden 《PloS one》2014,9(4)
The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is one of the main causes of amphibian population declines and extinctions all over the world. In the Neotropics, this fungal disease has caused catastrophic declines in the highlands as it has spread throughout Central America down to Panamá. In this study, we determined the prevalence and intensity of Bd infection in three species of frogs in one highland and four lowland tropical forests, including two lowland regions in eastern Panamá in which the pathogen had not been detected previously. Bd was present in all the sites sampled with a prevalence ranging from 15–34%, similar to other Neotropical lowland sites. The intensity of Bd infection on individual frogs was low, ranging from average values of 0.11–24 zoospore equivalents per site. Our work indicates that Bd is present in anuran communities in lowland Panamá, including the Darién province, and that the intensity of the infection may vary among species from different habitats and with different life histories. The population-level consequences of Bd infection in amphibian communities from the lowlands remain to be determined. Detailed studies of amphibian species from the lowlands will be essential to determine the reason why these species are persisting despite the presence of the pathogen. 相似文献
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Becker MH Harris RN Minbiole KP Schwantes CR Rollins-Smith LA Reinert LK Brucker RM Domangue RJ Gratwicke B 《EcoHealth》2011,8(4):501-506
Populations of native Panamanian golden frogs (Atelopus zeteki) have collapsed due to a recent chytridiomycosis epidemic. Reintroduction efforts from captive assurance colonies are unlikely to be successful without the development of methods to control chytridiomycosis in the wild. In an effort to develop a protective treatment regimen, we treated golden frogs with Janthinobacterium lividum, a skin bacterium that has been used to experimentally prevent chytridiomycosis in North American amphibians. Although J. lividum appeared to colonize A. zeteki skin temporarily, it did not prevent or delay mortality in A. zeteki exposed to Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis. After introduction of J. lividum, average bacterial cell counts reached a peak of 1.7 × 10(6) cells per frog ~2 weeks after treatment but declined steadily after that. When J. lividum numbers declined to ~2.8 × 10(5) cells per frog, B. dendrobatidis infection intensity increased to greater than 13,000 zoospore equivalents per frog. At this point, frogs began to die of chytridiomycosis. Future research will concentrate on isolating and testing antifungal bacterial species from Panama that may be more compatible with Atelopus skin. 相似文献
10.
Polysaccharide and glycoprotein distribution in the epidermis of cotton ovules during early fiber initiation and growth 总被引:1,自引:0,他引:1
The cotton fiber is a model system to study cell wall biosynthesis because the fiber cell elongates (∼3 cm in ∼20 days) without
mitosis. In this study, developing cotton ovules, examined from 1 day before anthesis (DBA) to 2 days post-anthesis (DPA),
that would be difficult to investigate via classical carbohydrate biochemistry were probed using a battery of antibodies that
recognize a large number of different wall components. In addition, ovules from these same stages were investigated in three
fiberless lines. Most antibodies reacted with at least some component of the ovule, and several of the antibodies reacted
specifically with the epidermal layer of cells that may give clues as to the nature of the development of the fibers and the
neighboring, nonfiber atrichoblasts. Arabinogalactan proteins (AGPs) labeled the epidermal layers more strongly than other
ovular tissue, even at 1 DBA. One of the AGP antibodies, CCRC-M7, which recognizes a 1➔6 galactan epitope of AGPs, is lost
from the fiber cells by 2 DPA, although labeling in the atrichoblasts remained strong. In contrast, LM5 that recognizes a
1➔4 galactan RGI side chain is unreactive with sections until the fibers are produced and only the fibers are reactive. Dramatic
changes also occur in the homogalacturonans (HGs). JIM5, which recognizes highly de-esterified HGs, only weakly labels epidermal
cells of 1 DBA and 0 DPA ovules, but labeling increases in fibers cells, where a pectinaceous sheath is produced around the
fiber cell and stronger reaction in the internal and external walls of the atrichoblast. In contrast, JIM7-reactive, highly
esterifed HGs are present at high levels in the epidermal cells throughout development. Fiberless lines displayed similar
patterns of labeling to the fibered lines, except that all of the cells had the labeling pattern of atrichoblasts. That is,
CCRC-M7 labeled all cells of the fiberless lines, and LM5 labeled no cells at 2 DPA. These data indicate that a number of
polysaccharides are unique in quantity or presence in the epidermal cell layers, and some of these might be critical participants
in the early stages of initiation and elongation of cotton fibers. 相似文献