Shark rectal gland vasoactive intestinal peptide receptor: cloning, functional expression, and regulation of CFTR chloride channels

Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 +/- 66 to 2,604 +/- 286 microeq x h(-1) x g(-1); the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-R1. It maintains a long, extracellular NH2 terminus with seven cysteine residues, and has three N-glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 +/- 2 to 127 +/- 34 microS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R.     

Enhanced accumulation of the isoprostane, 8-epi-PGF2 alpha,in human aortic and pulmonary valves of patients with coronary heart disease

The aim of the present study was to investigate whether the isoprostane 8-epi-PGF2 alpha differently accumulates in semilunar valves of patients suffering from coronary heart disease (CHD, n = 19) as compared to valves from healthy heart donors (controls, n = 6). Sections from isolated aortic and pulmonary valves were analyzed by semiquantitative immunohistochemistry. The 8-epi-PGF2 alpha-content was determined by using a specific radioimmunoassay. The accumulation of 8-epi-PGF2 alpha in both valves was higher in CHD-patients in comparison to controls (Aortic valves: 36.49 +/- 11.26% vs. 15.78 +/- 3.04%; pulmonary valves: 46.79 +/- 9.80% vs. 14.99 +/- 3.57%). The results from the radioimmunoassay revealed comparable findings in both groups (CHD vs. controls: 395.95 +/- 86.09 vs. 139.50 +/- 47.46 pg/mg protein in the aortic valves and 430.47 +/- 76.30 vs. 147.33 +/- 53.84 pg/mg protein in pulmonary valves). Pulmonary valves seem to be more susceptible to oxidative stress than aortic valves as evidenced by a higher accumulation of 8-epi-PGF2 alpha in CHD patients. Considering the data presented in this study, we suggest that 8-epi-PGF2 alpha is a valuable indicator of oxidative injury in human semilunar valves.     

Cloning of regulatory sequences mediating guard-cell-specific gene expression

This report describes the use of promoter trap lines from the model plant Arabidopsis thaliana to clone regulatory sequences that mediate guard-cell-specific reporter gene expression. Stomatal guard cells represent a highly differentiated cell type within the epidermis of green tissues of higher plants. They control the stomatal aperture in response to different endogenous and environmental signals in order to optimize carbon fixation while minimizing water loss. We screened available promoter trap lines for guard-cell-specific activation of a beta-glucuronidase (uidA) reporter gene in order to obtain marker lines for guard-cell development and to gain access to regulatory pathways leading to gene expression which is restricted to this cell type. From two lines identified we successfully cloned upstream regulatory sequences. For one line, guard-cell-specific promoter activity was confirmed by re-introducing the uidA gene, fused to the newly identified regulatory sequences, into the Arabidopsis nuclear genome. However, DNA sequences downstream of the uidA/T-DNA insertion sites in the original promoter trap lines revealed no obvious coding regions in the corresponding orientation, indicating that we have probably identified 'cryptic' promoters, being active in guard cells.     

The K+ channel SKT1 is co-expressed with KST1 in potato guard cells--both channels can co-assemble via their conserved KT domains

An appreciable number of potassium channels mediating K+ uptake have been identified in higher plants. Promoter-beta-glucuronidase reporter gene studies were used here to demonstrate that SKT1, encoding a potato K+ inwardly rectifying channel, is expressed in guard cells in addition to KST1 previously reported. However, whereas KST1 was found to be expressed in essentially all mature guard cells, SKT1 expression was almost exclusively restricted to guard cells of the abaxial leaf epidermis. This suggests that different types of K+ channel subunits contribute to channel formation in potato guard cells and therefore differential regulation of stomatal movements in the two leaf surfaces. The overlapping expression pattern of SKT1 and KST1 in abaxial guard cells indicates that K+in channels of different sub-families contribute to ionic currents in this cell type, thus explaining the different properties of channels expressed solely in heterologous systems and those endogenous to guard cells. Interaction studies had previously suggested that plant K+ inward rectifiers form clusters via their conserved C-terminal domain, KT/HA. K+ channels co-expressed in one cell type may therefore form heteromers, which increase functional variability of K+ currents, a phenomenon well described for animal voltage-gated K+ channels. Co-expression of KST1 and SKT1 in Xenopus oocytes resulted in currents with an intermediate sensitivity towards Cs+, suggesting the presence of heteromers, and a sensitivity towards external Ca2+, which reflected the property of the endogenous K+in current in guard cells. Modulation of KST1 currents in oocytes by co-expressing KST1 with a SKT1 pore-mutant, which by itself was not able to confer activating K+ currents, demonstrated the possibility that KST1 and SKT1 co-assemble to hetero-oligomers. Furthermore, various C-terminal deletions of the mutated SKT1 channel restored KST1 currents, showing that the C-terminal KT motif is essential for heteromeric channel formation.     

Recent advances in the diagnosis and treatment of hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease of severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and macrophages secreting high amounts of inflammatory cytokines. It is a frequent manifestation in patients with predisposing genetic defects, but can occur secondary to various infectious, malignant, and autoimmune triggers in patients without a known genetic predisposition. Clinical hallmarks are prolonged fever, cytopenias, hepatosplenomegaly, and neurological symptoms, but atypical variants presenting with signs of chronic immunodeficiency are increasingly recognized. Impaired secretion of perforin is a key feature in several genetic forms of the disease, but not required for disease pathogenesis. Despite progress in diagnostics and therapy, mortality of patients with severe HLH is still above 40%. Reference treatment is an etoposide-based protocol, but new approaches are currently explored. Key for a favorable prognosis is the rapid identification of an underlying genetic cause, which has been facilitated by recent immunological and genetic advances. In patients with predisposing genetic disease, hematopoietic stem cell transplantation is performed increasingly with reduced intensity conditioning regimes. Current research aims at a better understanding of disease pathogenesis and evaluation of more targeted approaches to therapy, including anti-cytokine antibodies and gene therapy.