Aquaporins（AQPs） are molecular water channels that play important physiological roles in fluid trans-porting organs. The expression and function of AQPs in the immune system are largely unknown. CD 11 （a-d）/CD18 integrins are adhesion molecules expressed on leukocytes, which play a critical role in leukocyte adhesion, migration and host defense. In the present study, we discovered the expression of aquaporin-3（AQP3） on spleen CD1 lb positive cells, and the content of CDllb positive splenocytes in aquaporin 3-null mice is significantly decreased. Further analysis suggested remarkably decreased monocyte/macrophage subpopulation and significantly decreased granulocyte subpopulation. It is the first report suggesting an important role of AQP in the development and maturation of imrnunocytes.
mCLCA3 is a member of calcium activated chloride channel（CACC） family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung. To study the protein structure and expression of mCLCA3 in asthmatic mouse lung, an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E. coli, purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated. Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen, Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3. The results demonstrate that the 90 kDa N-terminal peptide, neither the flail-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.
Dictamine is a furoquinoline alkaloid isolated from Dictamus dasycarpus Turcz.In the present study,we found that dictamine is able to stimulate the chloride transport activity of wild-type and ΔF508 mutant CFTR.The activity is cAMP-dependent and can be completely reversed by specific CFTR inhibitor CFTRinh-172.In addition,dictamine can further increase the chloride transport activity when CFTR is maximally activated by the combination of cAMP stimulators forskolin（FSK）and IBMX,suggesting direct interaction of dictamine with CFTR.Dictamine may be useful for probing CFTR channel gating mechanisms and used as a lead compound to develop the pharmacological therapy of CFTR-related diseases such as idiopathic chronic pancreatitis and keratoconjunctivitis sicca and cystic fibrosis.
In the present study, we identified the natural compound curcumin to be an effective G551D-CFTR activator by cell-based fluorescent assay and electrophysiological measurement. We demonstrated that curcumin can restore the impaired chloride conductance of G551D mutant CFTR. The activity is rapid, reversible, and cAMP-dependent. Our study identified a new natural lead compound for the pharmacological therapy of cystic fibrosis caused by G551D mutation of CFTR.
The asparagine-proline-alanine sequences (NPA motifs) are highly conserved in aquaporin water channel family. Crystallographic studies of AQP1 structure demonstrated that the two NPA motifs are in the narrow central constriction of the channel, serving to bind water molecules for selective and effi-cient water passage. To investigate the importance of the two NPA motifs in the structure, function and biogenesis of aquaporin water channels, we generated AQP1 mutations with NPA1 deletion, NPA2 de-letion and NPA1,2 double deletion. The coding sequences of the three mutated cDNAs were subcloned into the mammalian expression vector pcDNA3.1 to form expression plasmids. We established stably transfected CHO cell lines expressing these AQP1 mutants. Immunofluorescence indicated that all the three mutated AQP1 proteins are expressed normally on the plasma membrane of stably transfected CHO cells, suggesting that deletion of NPA motifs does not influence the expression and intracellular processing of AQP1. Functional analysis demonstrated that NPA1 or NPA2 deletion reduced AQP1 water permeability by 49.6% and 46.7%, respectively, while NPA1,2 double deletion had little effect on AQP1 water permeability. These results provide evidence that NPA motifs are important for water per-meation but not essential for the expression, intracellular processing and the basic structure of AQP1 water channel.
We reported the expression and function of aquaglyceroporin AQP3 in mouse peripheral nervous system.AQP3 mRNA was identified in freshly isolated mouse sciatic nerve by RT-PCR.Immunofluorescence using AQP3 antibody localized the protein expression in the myelin sheathe of sciatic nerve fibers.Although primary morpholo-gical evaluation of sciatic nerves from AQP3-knockout and wildtype mice revealed no significant difference,biochemical analysis demonstrated remarkably decreased glycerol and ATP contents in freshly isolated AQP3-knockout sciatic nerve.The study indicates an important role of facilitated glycerol transport mechanism in peripheral nerve energy metabolism.
Magnolin is a herbal compound from Magnolia biondii Pamp. It possesses numerous biological activities. Cystic fibrosis transmembrane conductance regulator（CFTR） is an epithelial chloride channel that plays a key role in the fluid secretion of various exocrine organs. In the present study, the activation of CFTR-mediated chloride transport by magnolin is indentified and characterized. In CFTR stably transfected FRT cells, magnolin increases CFTR CI- currents in a concentration-dependent manner. The activation of magnolin on CFTR is rapid, reversible, and cAMP-dependent. Magnolin does not elevate cellular cAMP level, indicating that it activates CFTR by direct binding and interaction with CFTR protein. Magnolin selectively activates wildtype CFTR rather than mutant CFTR. Magnolin may present a novel class of therapeutic lead compound tbr the treatment of diseases associated with reduced CFTR function such as keratoconjunctivitis sicca, idiopathic chronic pancreatiti, and chronic constipation.
JIN Ling-lingLIU XinSUN YanLIN SenZHOU NaXU Li-naYU BoHOU Shu-guangYANG Hong
An overt phenotype of aquaporin-1 knockout（AQP1 ko） mice is growth retardation, suggesting possible defects in bone development and metabolism. In the present study, we analyzed the bone mineral density（ BMD）, bone calcium and phosphorus contents, and bone metabolism in an AQP1 ko mouse model. The BMD of femurs in AQP1 ko mice was significantly lower than that of litter-matched wildtype mice as measured by dual energy X-ray absorptiometry. Consistently, the contents of bone total calcium and phosphorus were also significantly lower in AQP1 ko mice. The reduced BMD caused by AQP1 deficiency mainly affect male mice. Bone metabolic activity, as indicated by 99m^Tc-MDP absorption measurements, was remarkably reduced in AQP1 ko mice. These results provide the first evidence that AQP1 play an important role in bone structure and metabolism.
The cystic fibrosis transmembrane conductance regulator （CFFR） is a cAMP-activated chloride channel expressed in intestinal exoerine glands, which plays a key role in intestinal fluid secretion. A natural anthraquinone ac tivator of CFTR Cl^- channel, rhein, was identified by screening 217 single compounds from Chinese herbs via a cellbased halide-sensitive fluorescent assay. Rhein activates CFTR Cl^- transportation in a dose-dependent manner in the presence of cAMP with a physiological concentration. This study provides a novel molecular pharmacological mechanism for the laxative drugs in Treditional Chinese Medicine such as aloe, cascara and senna.
SHI Lin-linXU Li-naHOU Shu-guangLIN SenYANG HongMA Tong-hui
Molecular cloning of the porcine leukemia inhibitor factor（pLIF） has not been reported. A full-length eDNA encoding pLIF was cloned, expressed and characterized. The full-length porcine LIF cDNA encodes a 202 amino acid protein that has an 84% sequence identity to mouse LIF and 86% sequence identity to human LIF. The deduced amino acid sequence of a pLIF protein contains six conserved consensus N-linked glycosylation sites and six cysteine groups to form potential disulfide bonds. The pLIF was expressed in E coli, as a mature form, and in CHO cells as a secreted form. Both the forms of the recombinant pLIFs can maintain murine embryonic stem cells in an undifferentiated state in a culture. The recombinant pLiFs will be useful in establishing a long-term culture of stable pluripotent porcine embryonic stem cells for further manipulation.
LI Ming-tangJIANG YongWANG Shu-meiLI Yong-mingWANG FangHOU XiaYANG HongMA Tong-hui
