A 500-kb region on chromosome 16p13.1 contains the pseudoxanthoma elasticum locus: high-resolution mapping and genomic structure.

We have recently mapped the genetic defect underlying pseudoxanthoma elasticum (PXE), an inherited disorder characterized by progressive calcification of elastic fibers in skin, eye, and cardiovascular system, to chromosome 16p 13.1. Here we report further data on the fine-mapping and genomic structure of this locus. Haplotype analysis of informative PXE families narrowed the locus to an interval of less than 500 kb located between markers D16B9621 and D16S764. Three overlapping YAC clones were found to cover this region through YAC-STS content mapping. An overlapping BAC contig was then constructed to cover this interval and the surrounding region. About 80% of this chromosomal region has been fully sequenced using the BAC shotgun technique. Gene content and sequence analysis predicted four genes (MRP1, MRP6, PM5, and a novel transcript) and two pseudogenes (ARA and PKDI) within this interval. By screening a somatic cell hybrid panel we were able to precision-map the breakpoint of Cy185 and the starting point of a chromosomal duplication within 20 kb of BAC A962B4. The present data further refine the localization of PXE, provide additional physical cloning resources, and will aid in the eventual identification of the genetic defect causing PXE.

Characterization of four mammalian numb protein isoforms. Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain.

Numb is a membrane-associated, phosphotyrosine binding (PTB) domain-containing protein that functions as an intrinsic determinant of cell fate during Drosophila development. We have identified four isoforms of mammalian Numb with predicted molecular masses of 65, 66, 71, and 72 kDa that are generated by alternative splicing of the Numb mRNA. The different isoforms result from the presence of two sequence inserts within the PTB domain and the central region of the protein. The endogenous expression pattern of these isoforms, examined using specific antisera, varied in different tissues and cell lines. In addition, differentiation of P19 cells with retinoic acid leads to the specific loss of expression of the 71- and 72-kDa Numb proteins, suggesting that the expression of certain forms of Numb protein is regulated in a cell type-specific manner. Expression of Numb proteins fused to green fluorescent protein revealed that the form of the PTB domain with the alternatively spliced insert constitutively associated with the plasma membrane in polarized Madin-Darby canine kidney cells. In contrast, the isoform without the insert was cytoplasmic, suggesting that different PTB domain isoforms may regulate the subcellular localization of Numb proteins. The membrane localization may be due, in part, to differential affinity for acidic phospholipids. The distinct expression and localization patterns of the different mammalian Numb isoforms suggest that they have distinct functional properties.

A 12-Mb complete coverage BAC contig map in human chromosome 16p13.1-p11.2.

We have constructed a complete coverage BAC contig map that spans a 12-Mb genomic segment in the human chromosome 16p13.1-p11.2 region. The map consists of 68 previously mapped STSs and 289 BAC clones, 51 of which-corresponding to a total of 7.721 Mb of genomic DNA-have been sequenced, and provides a high resolution physical map of the region. Contigs were initially built based mainly on the analysis of STS contents and restriction fingerprint patterns of the clones. To close the gaps, probes derived from BAC clone ends were used to screen deeper BAC libraries. Clone end sequence data obtained from chromosome 16-specific BACs, as well as from public databases, were used for the identification of BACs that overlap with fully sequenced BACs by means of sequence match. This approach allowed precise alignment of clone overlaps in addition to restriction fingerprint comparison. A freehand contig drawing software tool was developed and used to manage the map data graphically and generate a real scale physical map. The map we present here is approximately 3.5 x deep and provides a minimal tiling path that covers the region in an array of contigous, overlapping BACs.

The mammalian numb phosphotyrosine-binding domain. Characterization of binding specificity and identification of a novel PDZ domain-containing numb binding protein, LNX.

Numb is a phosphotyrosine-binding (PTB) domain-containing protein implicated in the control of cell fate decisions during development. A modified two-hybrid screen in yeast was used to identify Numb PTB domain-interacting proteins important for Numb function. Here we report the identification of a novel protein, LNX, which interacts specifically with the Numb PTB domain. Two differentially expressed LNX messages encode overlapping proteins with predicted molecular masses of 80 kDa (LNX) and 70 kDa (LNX-b). LNX and LNX-b contain unique amino-terminal sequences and share four PDZ domains. The unique amino-terminal region of LNX includes a RING finger domain. The Numb PTB domain binding region of LNX was mapped to the sequence motif LDNPAY, found in both protein isoforms. Mutational analysis of LNX and peptide competition experiments showed that phosphorylation of the tyrosine residue within this motif was not required for binding to the Numb PTB domain. Finally, we also provide evidence that tyrosine phosphorylation of the LDNPAY sequence motif in LNX could generate a binding site for the phosphorylation-dependent binding of other PTB domain-containing proteins such as SHC. We speculate that LNX may be important for clustering PTB-containing proteins with functionally related transmembrane proteins in specific membrane compartments.

Experimental determination of a geometric form factor in a lidar equation for an inhomogeneous atmosphere.

We experimentally determine a geometric form factor for an inhomogeneous atmosphere by using the polynomial regression method in a lidar equation.

Deconvolution of long-pulse lidar signals with matrix formulation.

A deconvolution technique for deriving more resolved signals from lidar signals with typical CO(2) laser pulses is proposed, utilizing special matrices constructed from the temporal profile of laser pulses. It is shown that near-range signals can be corrected and small-scale variations of backscattered signals can be retrieved with this technique. Deconvolution errors as a result of noise in lidar data and in the laser pulse profile are also investigated numerically by computer simulation.

WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in sodium transport in kidney and other epithelia, and in regulating blood pressure. The channel is composed of three subunits (alphabetagamma) each containing two proline-rich sequences (P1 and P2) at its C-terminus. The P2 regions in human beta and gammaENaC, identical to the rat betagammarENaC, were recently shown to be deleted in patients with Liddle's syndrome (a hereditary form of hypertension), leading to hyperactivation of the channel. Using a yeast two-hybrid screen, we have now identified the rat homologue of Nedd4 (rNedd4) as the binding partner for the P2 regions of beta and gammarENaC. rNedd4 contains a Ca2+ lipid binding (CaLB or C2) domain, three WW domains and a ubiquitin ligase (Hect) domain. Our yeast two-hybrid and in vitro binding studies revealed that the rNedd4-WW domains mediate this association by binding to the P2 regions, which include the PY motifs (XPPXY) of either betarENaC (PPPNY) or gammarENaC (PPPRY). SH3 domains were unable to bind these sequences. Moreover, mutations to Ala of Pro616 or Tyr618 within the betarENaC P2 sequence (to PPANY or PPPNA, respectively), recently described in Liddle's patients, led to abrogation of rNedd4-WW binding. Nedd4-WW domains also bound to the proline-rich C-terminus (containing the sequence PPPAY) of alpharENaC, and endogenous Nedd4 co-immunoprecipitated with alpharENaC expressed in MDCK cells. These results demonstrate that the WW domains of rNedd4 bind to the PY motifs deleted from beta or gammaENaC in Liddle's syndrome patients, and suggest that Nedd4 may be a regulator (suppressor) of the epithelial Na+ channel.

Estrogen-dependent expression of the cystic fibrosis transmembrane regulator gene in a novel uterine epithelial cell line.

We have demonstrated previously the modulation of CFTR expression by estrogen in vivo in the rat uterine epithelium. The purpose of this study was to establish a suitable in vitro system to investigate the regulation of CFTR by steroid hormones. Primary cultures of rat uterine epithelial cells, which showed high levels of CFTR expression in vitro, were infected with an adeno/SV40 virus. One clone, UIT 1.16, which retained the morphology of the primary epithelial cells yet proliferated beyond the life span of the primary culture, was isolated and characterized. Successful immortalization of UIT 1.16 cells was verified by the presence of a band corresponding to the SV40 large T-antigen in western blots, as well as by their ability to proliferate continuously. Transmission electron microscopy studies revealed that these cells maintained the characteristics of a polarized epithelium with well-established membrane domains and specialized intercellular junctions. A high transepithelial electrical resistance was also observed when cells were assayed in modified Ussing chambers. When the basolateral cellular membrane of cells grown in vitrogen-coated filters was permeabilized with nystatin, a forskolin-stimulated Cl- permeability was observed in the apical membrane, similar to that present in other CFTR-expressing epithelial cells. UIT 1.16 cells showed high levels of CFTR expression on northern blots. The expression of CFTR was dependent on the presence of estrogen in the culture medium, since almost undetectable levels of CFTR mRNA were observed when the cells were cultured in medium containing serum depleted of steroid hormones. However, addition of estrogen to this medium prevented the disappearance of CFTR mRNA, confirming estrogen-regulated expression of CFTR in the UIT 1.16 cell line. The newly developed UIT 1.16 cell line provides a valuable model to analyze the regulation of CFTR expression by steroid hormones. Moreover, the cell line could also be used to investigate the role of CFTR in the uterus during the normal female cycle as well as for the study of other uterine epithelial functions and the agents that regulate them.

Plasma membrane recycling in CFTR-expressing CHO cells.

The hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) participates in plasma membrane recycling was tested experimentally. Using CHO cells, we determined the effects of CFTR expression and of elevated intracellular cAMP on exocytosis, measured as the incorporation into the plasma membrane of endosomes pre-labelled with biotinylated wheat-germ agglutinin (WGA). CFTR expression was without effect on the rate of exocytosis. Furthermore, cAMP did not affect endosomal recycling to the plasma membrane in either CFTR-expressing or control cells. These findings suggest that CFTR is not involved in regulating plasma membrane recycling in all cells.

Optical imaging of Cl- permeabilities in normal and CFTR-expressing mouse L cells.

Single cell optical imaging techniques were used to compare Cl- conductances in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing and control mouse L cell fibroblasts. Elevation of intracellular cAMP levels in control cells was without effect on plasma membrane Cl- permeability, whereas cells engineered to stably express CFTR displayed a 20-fold enhancement of plasma membrane Cl- permeability in response to cAMP. Control L cells displayed Ca(2+)-, as well as swelling-activated Cl- permeabilities, which were small compared with cAMP-stimulated permeability in CFTR-expressing cells. CFTR-expressing cells also displayed a similar swelling-activated Cl- permeability. Whereas 50% of the CFTR-expressing cells possessed a small Ca(2+)-activated Cl- permeability similar to control cells, the other cells displayed an enhanced response which was never observed in control cells. Intracellular cAMP determinations suggested that this latter result might be explained by a Ca(2+)-induced rise of cAMP. The cAMP-activated and Ca(2+)-activated Cl- conductances had different anion selectivities, as measured by light scattering of suspended cells. Activation of protein kinase C was without effect on Cl- permeability in CFTR-expressing cells, nor did it modify cAMP-activation of Cl- permeability. Thus, expression of human CFTR in L cells does not confer cAMP-sensitivity to pre-existing, endogenous Ca(2+)- or swelling-activated Cl- channels, but rather confers a novel Cl- conductance which is regulated by cAMP. Osmotic cell swelling and PKC activation are without specific effect in CFTR-expressing L cells. However, elevated [Ca2+]i may play a role in activating a Cl- conductance specifically associated with CFTR.

Activation of Cl- currents by intracellular chloride in fibroblasts stably expressing the human cystic fibrosis transmembrane conductance regulator.

The Cl- conductance of a mouse fibroblast cell line (LTK- cells) that was stably transfected with the human CFTR (cystic fibrosis transmembrane conductance regulator) complementary DNA was studied. Single Cl- channel activity was observed only after treatment of the cells with forskolin, the single-channel conductance being 6.2 +/- 0.2 pS with a linear current-voltage relationship. In CFTR+ cells, the whole-cell current at +90 mV increased from 7.3 +/- 2.7 pA/pF (n = 12) to 46.1 +/- 11.2 pA/pF (n = 5) after addition of dibutyryl-cyclic AMP (10(-4) M) to the bath. Increasing the intracellular Cl- concentration to 150 mM activated linear Cl- currents in the absence of cyclic AMP in CFTR+ (n = 42) but not in CFTR- cells (n = 4). Similar Cl- current was also activated by high intracellular I- concentration. These results indicate that the CFTR-induced Cl- conductance in LTK- cells can be activated by either cyclic AMP or high intracellular halide concentrations.

Right-angle light scattering to assay basal and regulated plasma membrane Cl- conductances.

We describe a simple and rapid technique for assaying both constitutive and regulated plasma membrane Cl- conductances. The method uses right-angle light scattering to measure the rate of swelling of cells in suspension, in which the anion conductance is rate limiting for swelling, due to introduction of high plasma membrane cation conductance using gramicidin. The technique was verified using Chinese hamster ovary cells and mouse L cells, both stably transfected with the cystic fibrosis transmembrane conductance regulator (CFTR), to confer a specific cAMP-activated Cl- conductance not normally present in these cell types. In agreement with results obtained using other methods for assaying Cl- permeability in these cells, forskolin stimulated a significant increase in plasma membrane Cl- conductance in CFTR-expressing cells, as indicated by an increase in light scattering. That the enhanced light scattering by the cells was the result of cell swelling due to NaCl influx was shown by ion substitution experiments, in which no forskolin-induced increase in light scatter occurred in N-methyl-D-glucamine Cl- or Na+ gluconate medium. Enhanced light scattering was also observed in both CFTR-expressing and control cells stimulated with the Ca2+ ionophore, ionomycin. Extracellular anion substitution, to exploit the inwardly directed halide gradient utilized in this protocol, enabled determination of the anion selectivities of both the cAMP- and Ca(2+)-activated Cl- channels. Thus this technique provides a simple optical method for rapidly assaying not only constitutive and regulated Cl- conductance pathways but also their anion selectivities.

Purinergic receptor activation of Cl- secretion in T84 cells.

The regulation by ATP of Cl- secretion in T84 cells grown on filters was investigated by measuring short-circuit current (Isc = net Cl- secretion). ATP (greater than or equal to 10 microM) added to the basolateral side markedly stimulated Isc both in the presence and absence of forskolin-activated Isc. Fluorescence microscopy of cells loaded with the Ca2+ indicator fura-2 showed that ATP stimulated a transient increase in intracellular free Ca2+ concentration [Ca2+]i. The augmentation of forskolin-stimulated Isc by ATP was at least partly caused by mobilization of Ca2+ from an internal store because prior depletion of the store using ionomycin prevented the response. The activity sequence for stimulation of Isc in the presence of forskolin was adenosine 5'-O-(3-thiotriphosphate) = 5'-adenylylimidodiphosphate (AMP-PNP) greater than ATP greater than ADP greater than AMP, suggesting the presence of a P2 purinergic receptor. Neither beta, gamma-methyleneadenosine 5'-triphosphate nor alpha, beta-methyleneadenosine 5'-triphosphate increased the Isc. Stimulation of Isc by ATP in the absence of forskolin was at least partly due to the breakdown of ATP to AMP and adenosine, which act at P1 receptors to stimulate Isc, since 1) inhibition of the ecto-phosphohydrolase 5'-nucleotidase by alpha, beta-methylene-ADP partially inhibited stimulation of Isc by ATP, 2) the adenosine receptor antagonists caffeine and 8-phenyltheophylline markedly inhibited the ATP-stimulated Isc, and 3) AMP-PNP, a weakly hydrolyzable analogue of ATP, caused a much smaller increase in Isc compared with ATP. Adenosine had no effect on [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator.

A cAMP-inducible chloride permeability has been detected in mouse fibroblast (L cell) lines upon stable integration of a full-length cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR). As indicated by a Cl(-)-indicator dye, the Cl- permeability of the plasma membrane increases by 10- to 30-fold within 2 min after treatment of the cells with forskolin, an activator of adenylyl cyclase. The properties of the conductance are similar to those described in secretory epithelial cells; the whole-cell current-voltage relationship is linear and there is no evidence of voltage-dependent inactivation or activation. In contrast, this cAMP-dependent Cl- flux is undetectable in the untransfected cells or cells harboring defective cDNA constructs, including one with a phenylalanine deletion at amino acid position 508 (delta F508), the most common mutation causing cystic fibrosis. These observations are consistent with the hypothesis that the CFTR is a cAMP-dependent Cl- channel. The availability of a heterologous (nonepithelial) cell type expressing the CFTR offers an excellent system to understand the basic mechanisms underlying this CFTR-associated ion permeability and to study the structure and function of the CFTR.

Hepoxilin A3 induces changes in cytosolic calcium, intracellular pH and membrane potential in human neutrophils.

The effects of hepoxilin A3 (HxA3), a 12-lipoxygenase metabolite of arachidonic acid, on cytosolic calcium ([Ca2+]i), intracellular pH (pHi), transmembrane potential and right-angle light scattering in human neutrophils were investigated. A rapid, transient elevation of [Ca2+]i was observed with HxA3 which was dependent on the concentration used. The effect of HxA3 on [Ca2+]i was blocked by pertussis toxin, suggesting involvement of receptors coupled to GTP-binding proteins. Experiments in Ca2(+)-free medium and using intracellular Ca2+ chelators indicated that HxA3 mobilized Ca2+ from intracellular stores. At similar concentrations, HxA3 altered pHi, producing an initial acidification followed by an alkalinization. The initial acidification was decreased in cells loaded with a Ca2+ chelator. In the presence of N-ethyl-N-(1-methylethyl)amino amiloride, an inhibitor of the Na+/H+ antiport, HxA3 induced a greater acidification but failed to elicit the recovery phase, suggesting that the latter is due to activation of the antiport. HxA3 also depolarized the membrane potential, although this effect was small. A decrease in right-angle light scattering, qualitatively similar to that observed with chemotactic peptides, was seen with HxA3, indicating that the 12-lipoxygenase metabolite can induce shape changes in neutrophils. At the concentrations used for the above effects, HxA3 was unable to generate a respiratory burst. These findings suggest that hepoxilins, which are formed by stimulated neutrophils, may have a role as messengers in neutrophil activation.

Influence of thyroid status on Ca2+ mobilization and amylase secretion in rat pancreatic acini.

Thyroid hormones influence Ca2+ homeostasis in both skeletal and cardiac muscle. Since secretory cells, like muscle cells, store and use Ca2+ in stimulus-response coupling, we have studied the effects of thyroid status on Ca2+ mobilization and secretion in a model secretory tissue, the pancreatic acinar cell. Hyperthyroidism was induced by rats by daily, subcutaneous injections of triiodothyronine for 8 days and hypothyroidism by adding 6-n-propyl-2-thiouracil to the drinking water for 14 days. Pancreatic acini were prepared by collagenase digestion of pancreatic tissue from hyper- and hypo-thyroid animals and from euthyroid controls. Ca2(+)-mobilization was assessed using Quin-2 fluorescence and secretion by assaying amylase release. The data indicate that the amount of Ca2+ mobilized by the muscarinic agonist carbachol or by cholecystokinin octapeptide increases with increasing thyroid hormone concentrations. Only in hypothyroidism was this change in Ca2+ homeostasis reflected by a parallel change in amylase secretion. This implies the existence of some compensatory mechanism which stabilizes secretory rate in the face of stimulus-evoked increases in intracellular Ca2+ concentration.

Calcium concentration and amylase secretion in guinea pig pancreatic acini: interactions between carbachol, cholecystokinin octapeptide and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate.

The effects of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) on amylase secretion and cytoplasmic free calcium concentration ([Ca2+]i) were investigated in dispersed guinea pig pancreatic acini. Carbachol evoked dose-dependent increases in amylase secretion and [Ca2+]i with half-maximal responses at 2.5 and 5 microM, respectively. Carbachol-induced calcium transients could be blocked by atropine. In the presence of a maximal effective dose of carbachol, cholecystokinin octapeptide caused no further increase in [Ca2+]i, suggesting that both agonists act on the same pool of trigger calcium. TPA (10(-9)-10(-6) M) stimulated amylase secretion with no change in [Ca2+]i. Maximum amylase secretion occurred at 0.5 microM TPA. Preincubation of acini in the presence of TPA resulted in a time- and dose-dependent inhibition (IC50 = 30 nM) of the carbachol-induced rise in [Ca2+]i, the maximal effect being observed within 3 min. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate was ineffective in inhibiting the carbachol-stimulated rise in [Ca2+]i. These findings suggest that, in addition to stimulating amylase secretion, probably through protein kinase C, TPA may also exert a negative feedback control over secretagogue-induced calcium transients.