There is intense interest in induction and characterization of strain-transcending neutralizing Ab against antigenically variable human pathogens. We have recently identified the human malaria ...parasite Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) as a target of broadly neutralizing Abs, but there is little information regarding the functional mechanism(s) of Ab-mediated neutralization. In this study, we report that vaccine-induced polyclonal anti-PfRH5 Abs inhibit the tight attachment of merozoites to erythrocytes and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 mAbs, we provide evidence of the following: 1) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; 2) neutralizing mAbs bind spatially related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; 3) a brief exposure window of PfRH5 is likely to necessitate rapid binding of Ab to neutralize parasites; and 4) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly neutralizing anti-malaria Abs and further encourage anti-PfRH5-based malaria prevention efforts.
IL-25 is implicated in the pathogenesis of viral asthma exacerbations. However, the effect of IL-25 on antiviral immunity has yet to be elucidated. We observed abundant expression and colocalization ...of IL-25 and IL-25 receptor at the apical surface of uninfected airway epithelial cells and rhinovirus infection increased IL-25 expression. Analysis of immune transcriptome of rhinovirus-infected differentiated asthmatic bronchial epithelial cells (BECs) treated with an anti-IL-25 monoclonal antibody (LNR125) revealed a re-calibrated response defined by increased type I/III IFN and reduced expression of type-2 immune genes CCL26, IL1RL1 and IL-25 receptor. LNR125 treatment also increased type I/III IFN expression by coronavirus infected BECs. Exogenous IL-25 treatment increased viral load with suppressed innate immunity. In vivo LNR125 treatment reduced IL-25/type 2 cytokine expression and increased IFN-β expression and reduced lung viral load. We define a new immune-regulatory role for IL-25 that directly inhibits virus induced airway epithelial cell innate anti-viral immunity.
Oncogenic osteomalacia (OO), a tumor-associated phosphate-wasting syndrome, provides an opportunity to identify regulators of renal phosphate homeostasis. We established cultures from OO-associated ...tumors. Conditioned medium from these cultures inhibited phosphate uptake in renal tubular epithelial cells. We then compared RNA from tumor-derived cultures expressing inhibitory activity with RNA from tumor-derived cultures in which inhibitory activity was not evident and identified candidate mRNAs specifically expressed by cultures inhibiting renal phosphate transport. Testing of identified candidates revealed that one protein, fibroblast growth factor 7 (FGF7), was a potent and direct inhibitor of phosphate uptake in vitro. A neutralizing monoclonal antibody to FGF7 reversed FGF7-dependent phosphate transport inhibition and inhibitory activity in conditioned medium from tumor cell cultures. Immunoassay revealed abundant FGF7 in inhibitory conditioned medium and minimal amounts in nonconditioned medium or conditioned medium with no phosphate transport inhibitory activity. Furthermore, only small amounts of FGF23 were present in inhibitory conditioned medium, comparable to concentrations found in conditioned medium with no phosphate transport inhibitory activity. Thus, FGF7 was specifically identified when selecting for in vitro phosphate transport inhibitory activity of tumor-derived cultures and was confirmed as a potent inhibitor of phosphate transport. Finally, FGF7 message was confirmed in PCR products of mRNA extracted from fragments of each tumor. Members of the FGF family (other than FGF23) are expressed by OO-associated tumors and may play a role in mediating this syndrome.
Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family ...currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-α, whereas PDGF-B activates both PDGFR-α and PDGFR-β. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-β but not PDGFR-α. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-α activation may result from PDGFR-α/β heterodimerization.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Autosomal recessive pseudohypoaldosteronism type I is a rare life-threatening disease characterized by severe neonatal salt wasting, hyperkalaemia, metabolic acidosis, and unresponsiveness to ...mineralocorticoid hormones. Investigation of affected offspring of consanguineous union reveals mutations in either the alpha or beta subunits of the amiloride-sensitive epithelial sodium channel in five kindreds. These mutations are homozygous in affected subjects, co-segregate with the disease, and introduce frameshift, premature termination or missense mutations that result in loss of channel activity. These findings demonstrate the molecular basis and explain the pathophysiology of this disease.
The activity of the epithelial sodium channel (ENaC) in the distal nephron is regulated by an antidiuretic hormone, aldosterone, and insulin, but the molecular mechanisms that mediate these hormonal ...effects are mostly unknown. We have investigated whether aldosterone, insulin, or activation of protein kinases has an effect on the phosphorylation of the channel. Experiments were performed in an epithelial cell line generated by stable cotransfection of the three subunits (α ,β , and γ ) of ENaC. We found that β and γ , but not the α subunit, are phosphorylated in the basal state. Aldosterone, insulin, and protein kinases A and C increased phosphorylation of the β and γ subunits in their carboxyl termini, but none of these agents induced de novo phosphorylation of α subunits. Serines and threonines but not tyrosines were found to be phosphorylated. The results suggest that aldosterone, insulin, and protein kinases A and C modulate the activity of ENaC by phosphorylation of the carboxyl termini of the β and γ subunits.
We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput ...reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We have studied the functional consequences of a mutation in the epithelial Na+channel that causes a heritable form of salt-sensitive hypertension, Liddle disease. This mutation, identified in the ...original kindred described by Liddle, introduces a premature stop codon in the channel β subunit, resulting in a deletion of almost all of the C terminus of the encoded protein. Coexpression of the mutant β subunit with wild-type α and γ subunits in Xenopus laevis oocytes resulted in an ≈3-fold increase in the macroscopic amiloride-sensitive Na+current (INa) compared with the wild-type channel. This change in INareflected an increase in the overall channel activity characterized by a higher number of active channels in membrane patches. The truncation mutation in the β subunit of epithelial Na+channel did not alter the biophysical and pharmacological properties of the channel-including unitary conductance, ion selectivity, or sensitivity to amiloride block. These results provide direct physiological evidence that Liddle disease is related to constitutive channel hyperactivity in the cell membrane. Deletions of the C-terminal end of the β and γ subunits of rat epithelial Na+channel were functionally equivalent in increasing INa, suggesting that the cytoplasmic domain of the γ subunit might be another molecular target for mutations responsible for salt-sensitive forms of hypertension.
Activity of the epithelial sodium channel (ENaC) is a key determinant of sodium homeostasis and blood pressure. Liddle's syndrome, an inherited form of hypertension, is caused by mutations that ...delete or alter PY domains in the carboxyl termini of β or γ ENaC subunits, leading to increased channel activity. In this study we investigated the mechanism of this effect by analysis of wild-type and mutant ENaC activity in Xenopus oocytes. By inhibiting insertion of new channels into the plasma membrane with brefeldin A, we demonstrate that the half-life of the activity of channels containing Liddle's mutations is markedly prolonged compared with wild-type channels (t½ of 30 h in mutant versus 3.6 in wild-type, p < 0.001). We investigated the involvement of clathrin-coated pit-mediated endocytosis by co-expressing a dominant-negative dynamin mutant with wild-type ENaC in oocytes. Expression of this specific inhibitor of endocytosis leads to a large increase in the activity of wild-type channels, demonstrating that normal turnover of this channel is through the clathrin-coated pit pathway. In contrast, co-expression of Liddle's mutations and dynamin mutants leads to no further increase in channel activity, consistent with one of the effects of Liddle's mutations being the loss of endocytosis of these channels. These findings demonstrate the normal mechanism of turnover of ENaC from the cell surface and demonstrate a mechanism that can account for the increased number of channels in the plasma membrane seen in Liddle's syndrome.
We describe here the GeneCalling method for the discovery of differentially expressed genes, both known and novel, from any species and with useful sequence information to determine the potential ...function of novel genes captured. The method relies on transcript visualization coupled to a database query to rapidly and quantitatively identify differentially expressed transcripts. The method has been applied to a wide variety of disease models in a wide variety of species, addressing problems as diverse as identifying novel human cancer gene targets, understanding how drugs and diet affect animal models of disease, and understanding the basis of trait differences in related strains of corn.
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