Platelet-delivered therapeutics.

We have proposed that modified platelets could potentially be used to correct intrinsic platelet defects as well as for targeted delivery of therapeutic molecules to sights of vascular injury. Ectopic expression of proteins within α-granules prior to platelet activation has been achieved for several proteins, including urokinase, factor (F) VIII, and partially for FIX. Potential uses of platelet-directed therapeutics will be discussed, focusing on targeted delivery of urokinase as a thromboprophylactic agent and FVIII for the treatment of hemophilia A patients with intractable inhibitors. This presentation will discuss new strategies that may be useful in the care of patients with vascular injury as well as remaining challenges and limitations of these approaches.

The similar effects of verbal and non-verbal intervening tasks on word recall in an elderly population.

Vulnerability to retroactive interference has been shown to increase with cognitive aging. Consistent with the findings of memory and aging literature, the authors of the California Verbal Learning Test-II (CVLT-II) suggest that a non-verbal task be administered during the test's delay interval to minimize the effects of retroactive interference on delayed recall. The goal of the present study was to determine the extent to which retroactive interference caused by non-verbal and verbal intervening tasks affects recall of verbal information in non-demented, older adults. The effects of retroactive interference on recall of words during Long-Delay recall on the California Verbal Learning Test-II (CVLT-II) were evaluated. Participants included 85 adults age 60 and older. During a 20-minute delay interval on the CVLT-II, participants received either a verbal (WAIS-III Vocabulary or Peabody Picture Vocabulary Test-IIIB) or non-verbal (Raven's Standard Progressive Matrices or WAIS-III Block Design) intervening task. Similarly to previous research with young adults (Williams & Donovick, 2008), older adults recalled the same number of words across all groups, regardless of the type of intervening task. These findings suggest that the administration of verbal intervening tasks during the CVLT-II do not elicit more retroactive interference than non-verbal intervening tasks, and thus verbal tasks need not be avoided during the delay interval of the CVLT-II.

Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine.

Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.

MIP-3alpha induces human eosinophil migration and activation of the mitogen-activated protein kinases (p42/p44 MAPK).

The CC chemokine macrophage inflammatory protein-3alpha (MIP-3alpha) is the product of recent electronic cloning efforts, however, little characterization of its spectrum of biological effects has been undertaken. Human eosinophils exhibited pertussis-toxin-sensitive migration in response to human recombinant (hr)MIP-3alpha. Messenger RNA for the MIP-3alpha receptor, CCR-6, and low levels of surface expression were demonstrated by reverse transcriptase-polymerase chain reaction and FACS analysis. Analyses of cell signaling revealed dose-dependent increases in intracellular calcium mobilization, calcium transients that were, however, greatly reduced when compared with MCP-3-induced responses. Further investigations of MIP-3alpha-induced signal transduction revealed time- and dose-dependent, partially pertussis toxin-dependent, increases in phosphorylation of the p42/p44 mitogen-activated protein kinases (MAPK) that occurred at 10- to 100-fold lower concentrations, and that were linked to a phosphoinositide 3-kinase pathway. These results suggest that MIP-3alpha can regulate multiple, parallel signal transduction pathways in eosinophils, and suggest that MAPK activation by MIP-3alpha in eosinophils is a significant signaling pathway for migration induction.

Pharmacological and signaling analysis of human chemokine receptor CCR-7 stably expressed in HEK-293 cells: high-affinity binding of recombinant ligands MIP-3beta and SLC stimulates multiple signaling cascades.

The chemokine receptor CCR-7 is expressed in T, NK, and dendritic cells in a time-ordered and stimulus-dependent manner. Thorough analyses of the pharmacological profiles of the recombinant ligands for CCR-7, MIP-3beta/ELC/CK-beta 11, and SLC/Exodus-2/TCA4/6C-kine, using CCR-7-expressing HEK-293E transfectants determine that ligands both bind with a K(d) in the 100 pM range-10- to 100-fold greater affinities than published K(d) values. High-affinity binding of each ligand is associated with rapid mobilization of intracellular calcium and cell migration as predicted for chemokine GPCRs, and in keeping with more recent evidence, robust activation of mitogen-activated protein kinase (MAPK).

Activation of mitogen-activated protein kinase regulates eotaxin-induced eosinophil migration.

Eotaxin is a potent eosinophil chemoattractant that plays an important role in regulating eosinophil tissue levels both in healthy individuals and in diseases associated with significant eosinophil infiltrates, such as the allergic inflammation observed in asthma. Here, we demonstrate that treatment of eosinophils with eotaxin induces the phosphorylation of the mitogen-activated protein kinases (MAPKs) p42 and p44, leading to kinase activation. Blockade of MAPK activation by the MAPK kinase inhibitor PD98059 leads to a dramatic decrease in eotaxin-induced eosinophil rolling in vivo and chemotaxis in vitro. This blockade in the leukocyte migration process is consistent with the observed inhibition of actin polymerization and rearrangement within the eosinophil following treatment with MAPK inhibitor. It is suggested, therefore, that the intrinsic mechanism of eotaxin-induced eosinophil rolling and migration involves activation of the p42/p44 MAPK, possibly through regulation of the cytoskeletal rearrangements necessary for chemotaxis.

Human thymocytes express CCR-3 and are activated by eotaxin.

Eotaxin has been characterized as a chemokine involved in eosinophil activation; however, mRNA for this C-C chemokine has been shown to be constitutively expressed in thymus. Immunohistochemical analysis showed a punctate distribution pattern, with eotaxin expression localized mainly in the medulla and in Hassle's corpuscles. Moreover, the receptor for eotaxin, CCR-3, was detected on thymocytes, with the highest level of expression being on the CD8 single-positive population. Equilibrium binding analyses on unfractionated thymocytes demonstrated specific 125I-eotaxin binding profiles comparable with CCR-3 transfectants. Eotaxin induced cell migration and mobilization of intracellular calcium in all thymocytes except the immature CD4(-)/CD8(-) population. Eotaxin also induced the secretion of the chemokines interleukin-8, RANTES, and macrophage inflammatory protein-1beta from thymocyte cultures in vitro. These results suggest that eotaxin-induced thymocyte activation may have important physiological implications for lymphocyte mobilization within and from this lymphoid organ.

Novel subunit composition of a renal epithelial KATP channel.

Unique ATP-inhibitable K+ channels (KATP) in the kidney determine the rate of urinary K+ excretion and play an essential role in extracellular K+ balance. Here, we demonstrate that functionally similar low sulfonylurea affinity KATP channels are formed by two heterologous molecules, products of Kir1.1a and cystic fibrosis transmembrane conductance regulator (CFTR) genes. Co-injection of CFTR and Kir1.1a cRNA into Xenopus oocytes lead to the expression of K+ selective channels that retained the high open probability behavior of Kir1.1a but acquired sulfonylurea sensitivity and ATP-dependent gating properties. Similar to the KATP channels in the kidney but different from KATP channels in excitable tissues, the Kir1.1a/CFTR channel was inhibited by glibenclamide with micromolar affinity. Since the expression of Kir1.1a and CFTR overlap at sites in the kidney where the low sulfonylurea affinity KATP are expressed, our study offers evidence that these native KATP channels are comprised of Kir1.1a and CFTR. The implication that Kir subunits can interact with ABC proteins beyond the subfamily of sulfonylurea receptors provides an intriguing explanation for functional diversity in KATP channels.

Mechanisms of MARCKS gene activation during Xenopus development.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a high affinity cellular substrate for protein kinase C. The MARCKS gene is under multiple modes of transcriptional control, including cytokine- and transformation-dependent, cell-specific, and developmental regulation. This study evaluated the transcriptional control of MARCKS gene expression during early development of Xenopus laevis. Xenopus MARCKS was highly conserved with its mammalian and avian homologues; its mRNA and protein were abundant in the maternal pool and increased after the mid-blastula transition (MBT). The Xenopus MARCKS gene was similar to those of other species, except that a second intron interrupted the 5'- untranslated region. By transiently transfecting XTC-2 cells and microinjecting Xenopus embryos with reporter gene constructs containing serial deletions of 5'-flanking MARCKS sequences, we identified a 124-base pair minimal promoter that was critical for promoter activity. Developmental gel shift assays revealed that a CBF/NF-Y/CP-1-like factor and an Sp1-like factor bound to this region in a manner correlating with the onset of Xenopus MARCKS transcription at MBT. Mutations in the promoter that abolished binding of these two factors also completely inhibited transcriptional activation of the MARCKS gene at MBT. The binding sites for these two factors are highly conserved in the human and mouse MARCKS promoters, suggesting that these elements might also regulate MARCKS transcription in other species. These studies not only increase our knowledge of the transcriptional regulation of the MARCKS genes but also have implications for the mechanisms responsible for zygotic activation of the Xenopus genome at MBT.

Subcellular distribution and targeting of the intracellular chloride channel p64.

p64 is an intracellular chloride channel originally identified in bovine kidney microsomes. Using a combination of immunofluorescent and electron microscopic technique, we demonstrate that p64 resides in the limiting membranes of perinuclear dense core vesicles which appear to be regulated secretory vesicles. Heterologous expression of p64 in PancI cells, a cell type which does not normally express p64, results in targeting to a similar compartment. Mutagenesis experiments demonstrate that both the N- and C-terminal domains of the protein independently contribute to subcellular distribution of the protein. The C-terminal domain functions to prevent expression of p64 on the plasma membrane and the N-terminal domain is necessary to deliver p64 to the appropriate membrane compartment.

Estimating stand water use of large mountain ash trees and validation of the sap flow measurement technique.

Mountain ash (Eucalyptus regnans F.J. Muell.) forest catchments exhibit a strong relationship between stand age and runoff, attributed inter alia to differences in tree water use. However, the tree water use component of the mountain ash forest water balance is poorly quantified. We have used the sap flow technique to obtain estimates of daily water use in large mountain ash trees. First, the sap flow technique was validated by means of an in situ cut tree experiment. Close agreement was obtained between the sap flow estimate of water use and the actual uptake of water by the tree from a reservoir. Second, we compared the variability in sap velocity between a symmetric and an asymmetric tree by using multiple sap flow loggers. In the symmetric tree, velocity was fairly uniform throughout the xylem during the day, indicating that accurate sap flow estimates can be obtained with a minimal number of sampling points. However, large variations in sap velocity were observed in the asymmetric tree, indicating that much larger sampling sizes are required in asymmetric stems for an accurate determination of mean sap velocity. Finally, we compared two procedures for scaling individual tree sap flow estimates to the stand level based on stem diameter and leaf area index measurements. The first procedure was based on a regression between stem diameter and tree water use, developed on a small sample of trees and applied to a stand-level census of stem diameter values. Inputs to the second procedure were tree water use and leaf area of a single tree and the leaf area index of the stand. The two procedures yielded similar results; however, the first procedure was more robust but it required more sampling effort than the second procedure.

Identification and partial characterization of a domain in CFTR that may bind cyclic nucleotides directly.

BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is activated by cAMP-dependent phosphorylation. CFTR channel activity is also stimulated by cGMP-dependent protein kinase and protein kinase C. RESULTS: Here, we show that CFTR channel activation by cGMP may also occur directly. In oocytes from one-third of Xenopus donors, the activation of CFTR by cGMP averaged 87% of the level achieved by cAMP. The currents activated by either cyclic nucleotide displayed similar current-voltage relationships, kinetics, pharmacology and halide selectivity. Sequential stimulation by cAMP and cGMP was not additive, suggesting that both cyclic nucleotides activate the same channel; cGMP was one order of magnitude more potent than cAMP, and its action was insensitive to protein kinase inhibitors. Analysis of the amino-acid sequence of CFTR revealed a domain in the amino-terminal portion of the third cytoplasmic loop that resembles a class of cyclic-nucleotide-binding domains related to that of the catabolite-gene activator protein, CAP. Two CFTR residues in this domain--Val397 and Lys420--were identified which, when changed to alanine, altered the response to cGMP independently of the response to cAMP. CONCLUSIONS: We conclude that direct cyclic nucleotide binding may play a role in channel gating of CFTR. The cGMP-binding domain may provide a useful target for pharmacologic intervention in cystic fibrosis.

Relationships between stem diameter, sapwood area, leaf area and transpiration in a young mountain ash forest.

We examined relationships between stem diameter, sapwood area, leaf area and transpiration in a 15-year-old mountain ash (Eucalyptus regnans F. Muell.) forest containing silver wattle (Acacia dealbata Link.) as a suppressed overstory species and mountain hickory (Acacia frigescens J.H. Willis) as an understory species. Stem diameter explained 93% of the variation in leaf area, 96% of the variation in sapwood area and 88% of the variation in mean daily spring transpiration in 19 mountain ash trees. In seven silver wattle trees, stem diameter explained 87% of the variation in sapwood area but was a poor predictor of the other variables. When transpiration measurements from individual trees were scaled up to a plot basis, using stem diameter values for 164 mountain ash trees and 124 silver wattle trees, mean daily spring transpiration rates of the two species were 2.3 and 0.6 mm day(-1), respectively. The leaf area index of the plot was estimated directly by destructive sampling, and indirectly with an LAI-2000 plant canopy analyzer and by hemispherical canopy photography. All three methods gave similar results.

Bicarbonate secretion in rabbit ileum: electrogenicity, ion dependence, and effects of cyclic nucleotides.

BACKGROUND: Ileal HCO3- secretion is not well understood. The aim of this study was to examine its Na+ and Cl- dependencies, electrogenicity, and responses to amiloride, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonate (SITS), and cyclic nucleotides. METHODS: The serosa to mucosa HCO3- flux (Jsm) across rabbit ileal mucosa mounted between HCO(3-)-free mucosal solution and HCO(3-)-containing serosal solutions was determined by titration. RESULTS: In SO4(2-)-containing Ringer's solution, Jsm varied with [Na+] in two phases, one with a high and one with a low affinity for Na+; amiloride inhibited the high- and SITS inhibited the low-affinity phase. Switching from SO4(2-)- to Cl(-)-containing Ringer's solution caused a SITS-inhibitable 42% increase in Jsm. Changes in Jsm were coupled 3:2 with changes in short-circuit current. Cyclic nucleotide effects on Jsm were as follows. In SO4(2-)-containing Ringer's solution at 141 (but not 80) mmol/L Na+, theophylline caused equal increases in Jsm and short-circuit current that equaled the combined effects of 8-Br-5'-cyclic guanosine monophosphate (cGMP) and 8-Br-5'-cyclic adenosine monophosphate (cAMP). Serosal SITS blocked these effects, but amiloride did not. In Cl(-)-containing Ringer's solution, theophylline and bumetanide together (but not separately) increased Jsm. CONCLUSIONS: (1) Basolateral HCO3- entry occurs via Na+/H exchange and a SITS-inhibitable process (Na(+)-HCO3- cotransport?). (2) Most HCO3- exit across the brush border occurs by a Cl(-)-independent process and some by Cl-/HCO3- exchange. (3) At low cellular [Cl-], HCO3- can be secreted via anion channels activated by cAMP and cGMP. (4) Ileal HCO3- secretion is electrogenic.

cAMP-activated C1 conductance is expressed in Xenopus oocytes by injection of shark rectal gland mRNA.

Development of reliable expression systems for use in identification and functional characterization of proteins required for secretory Cl channel activity is key to understanding the molecular basis of cystic fibrosis (CF). Until now, heterologous expression of epithelial Cl channels had not been accomplished. We show here that Xenopus oocytes express an adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl conductance after injection of mRNA from shark rectal gland. Current through this conductance was rapidly activated by intracellular application of cAMP, reversed near the chloride equilibrium potential (ECl), blocked by the Cl channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoate, and was not affected by preincubation with the intracellular calcium buffer bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, a condition that prohibits activation of the endogenous Ca-activated Cl conductance.

Epithelial K channel expressed in Xenopus oocytes is inactivated by protein kinase C.

K homeostasis is maintained in higher animals by epithelia of the kidney and intestine. Little is known regarding the molecular regulation of K secretion. We injected Xenopus oocytes with mRNA from teleost intestine, a K-secreting epithelium with apical membrane K channels. Oocytes expressed a conductance that displayed whole-cell current properties with the following characteristics: marked selectivity for K over Na and Cl, voltage-independent kinetics, Ca insensitivity, tonic activation, and inward rectification in symmetrical K. Barium, quinine, and tetraethylammonium blocked the conductance, whereas apamin, charybdotoxin, and 4-aminopyridine did not. The K conductance was rapidly (t1/2 = 10 min) and completely inactivated by 4 beta-phorbol 12-myristate 13-acetate but not by 4 alpha-phorbol 12,13-didecanoate. Sucrose density gradient fractionation revealed that mRNA required for expression is in the 1- to 2-kilobase size range, suggesting the possibility that a single subunit encodes the channel. The K conductance expressed from injection of size-fractionated mRNA was identical in all respects to that seen using unfractionated mRNA, including response to 4 beta-phorbol 12-myristate 13-acetate. The results suggest that protein kinase C regulates K secretion in epithelia by modulation of apical K channels.

Bicarbonate secretion by rabbit proximal colon.

Stripped segments of proximal colon (1-6 cm distal to the ampulla caecalis coli) were studied in vitro in Ussing chambers under short-circuit conditions using the pH-stat technique. With glucose and HCO3-CO2 present in the serosal bathing solution only, proximal colon alkalinizes the luminal bathing solution at a rate of 2.1 +/- 0.2 mu eq X h-1 X cm-2 (n = 36). With HCO3-CO2 present in the luminal bathing solution alone, proximal colon does not significantly acidify or alkalinize the serosal bathing solution. Addition of glucose (10 mM) to the luminal bathing solution abolished luminal alkalinization. Removal of HCO3 and CO2 from the serosal bathing solution or replacement of O2 with N2 also abolished luminal alkalinization. Acetazolamide (0.1 mM) added to both bathing solutions did not alter the rate of luminal alkalinization. Ion-replacement studies revealed that the alkalinization process was highly dependent on the presence of Na in the bathing solutions and much less dependent on the presence of Cl. Furthermore, ouabain (0.1 mM) significantly reduced luminal alkalinization. As in rabbit ileum, serosal epinephrine (0.1 mM) did not alter luminal alkalinization but increased serosal alkalinization by a Na-dependent mechanism. These results suggest that luminal alkalinization results from a Na-dependent, active transcellular HCO3 transport process and that a Na-dependent HCO3 absorptive process is activated by adrenergic stimuli.

Active potassium secretion by rabbit proximal colon.

Fluxes of K from mucosa to serosa or serosa to mucosa have been examined in stripped preparations of rabbit proximal and distal colon in vitro under short-circuit conditions in Ussing chambers. Results from these studies demonstrate that steady-state radioisotopic fluxes of K are achieved after 90 min and remain constant for at least 2 h. Determination of the K concentration dependence of the serosal-to-mucosal K flux revealed that this flux contains both saturable and nonsaturable components. Addition of ouabain (0.1 mM) abolished the saturable component of the serosal-to-mucosal K flux. The mucosal-to-serosal K flux is a linear function of K concentration between 1 and 20 mM under basal conditions. In paired tissues, serosal-to-mucosal K flux is always greater than mucosal-to-serosal flux under basal conditions resulting in net K secretion. However, addition of barium (2 mM) to the mucosal or serosal bathing solution had no significant effect on either unidirectional or net K fluxes. In addition, mucosal bumetanide (0.1 mM) or removal of Cl from both bathing solutions had no significant effect on unidirectional or net K fluxes. In rabbit distal colon, Cl removal from the bathing solutions significantly reduced serosal-to-mucosal K flux, resulting in net K absorption. These results indicate that rabbit proximal colon like rabbit distal colon actively secretes K. However, unlike distal colon the proximal colon does not possess an active K uptake mechanism at the apical cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentration-dependent effects of disulfonic stilbenes on colonic chloride transport.

Stripped rabbit colonic mucosa was studied in vitro in Ussing chambers to determine effects of the disulfonic stilbenes 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and the diuretic furosemide on unidirectional and net Cl fluxes. Results from these studies reveal that SITS (1 mM) added to either the serosal or mucosal bathing solution reduced both unidirectional Cl fluxes with no significant change in net Cl flux. The effects of SITS do not appear to be mediated by an effect on the shunt permeability since SITS (1 mM) did not alter either the intercept or slope of the Na concentration dependence of the serosal-to-mucosal Na flux. Furosemide (1 mM) decreased the serosal-to-mucosal Cl flux without altering short-circuit current (Isc) when added to the luminal bathing solution and reduced both unidirectional fluxes and increased Isc when added to the serosal bathing solution. DIDS (0.5 mM) added to the luminal bathing solution did not alter unidirectional Cl fluxes or Isc. However, serosal addition of DIDS produced dose-dependent changes in Cl transport. At 5 microM DIDS reduced the mucosal-to-serosal Cl flux without altering the serosal-to-mucosal flux or Isc. At 50 microM DIDS reduced the mucosal-to-serosal Cl flux and increased Isc, and at 0.5 mM DIDS increased the serosal-to-mucosal Cl flux, reduced the mucosal-to-serosal Cl flux, and increased Isc and transepithelial conductance. The effect of 0.5 mM DIDS on Isc was reduced by Ca removal from the serosal bathing solution and by the loop diuretics furosemide and bumetanide.(ABSTRACT TRUNCATED AT 250 WORDS)