Effect of apolipoprotein E and butyrylcholinesterase genotypes on cognitive response to cholinesterase inhibitor treatment at different stages of Alzheimer's disease.

Factors that influence response to drug treatment are of increasing importance. We report an analysis of genetic factors affecting response to cholinesterase inhibitor therapy in 165 subjects with Alzheimer's disease (AD). The presence of apolipoprotein E ε4 (APOE ε4) allele was associated with early and late cognitive response to cholinesterase inhibitor treatment in mild AD (Mini-Mental State Examination (MMSE) ≥21) (P<0.01). In moderate-to-severe AD (MMSE ≤15), presence of the BCHE-K variant was associated with late response to cholinesterase inhibitor treatment (P=0.02). Testing for APOE and BCHE genotypes may be useful in therapeutic decision making.

Interferon-induced guanylate-binding proteins lack an N(T)KXD consensus motif and bind GMP in addition to GDP and GTP.

The primary structures of interferon (IFN)-induced guanylate-binding proteins (GBPs) were deduced from cloned human and murine cDNAs. These proteins contained only two of the three sequence motifs typically found in GTP/GDP-binding proteins. The N(T)KXD motif, which is believed to confer guanine specificity in other nucleotide-binding proteins, was absent. Nevertheless, the IFN-induced GBPs exhibited a high degree of selectivity for binding to agarose-immobilized guanine nucleotides. An interesting feature of IFN-induced GBPs is that they strongly bound to GMP agarose in addition to GDP and GTP agaroses but failed to bind to ATP agarose and all other nucleotide agaroses tested. Both GTP and GMP, but not ATP, competed for binding of murine GBP-1 to agarose-immobilized GMP. The IFN-induced GBPs thus define a distinct novel family of proteins with GTP-binding activity. We further demonstrate that human and murine cells contain at least two genes encoding IFN-induced GBPs. The cloned murine cDNA codes for GBP-1, an IFN-induced protein previously shown to be absent from mice of Gbp-1b genotype.

Developmental regulation of FKBP65. An ER-localized extracellular matrix binding-protein.

FKBP65 (65-kDa FK506-binding protein) is a member of the highly conserved family of intracellular receptors called immunophilins. All have the property of peptidyl-prolyl cis-trans isomerization, and most have been implicated in folding and trafficking events. In an earlier study, we identified that FKBP65 associates with the extracellular matrix protein tropoelastin during its transport through the cell. In the present study, we have carried out a detailed investigation of the subcellular localization of FKBP65 and its relationship to tropoelastin. Using subcellular fractionation, Triton X-114 phase separation, protease protection assays, and immunofluorescence microscopy (IF), we have identified that FKBP65 is contained within the lumen of the endoplasmic reticulum (ER). Subsequent IF studies colocalized FKBP65 with tropoelastin and showed that the two proteins dissociate before reaching the Golgi apparatus. Immunohistochemical localization of FKBP65 in developing lung showed strong staining of vascular and airway smooth muscle cells. Similar areas stained positive for the presence of elastic fibers in the extracellular matrix. The expression of FKBP65 was investigated during development as tropoelastin is not expressed in adult tissues. Tissue-specific expression of FKBP65 was observed in 12-d old mouse tissues; however, the pattern of expression of FKBP65 was not restricted to those tissues expressing tropoelastin. This suggests that additional ligands for FKBP65 likely exist within the ER. Remarkably, in the adult tissues examined, FKBP65 expression was absent or barely detectable. Taken together, these results support an ER-localized FKBP65-tropoelastin interaction that occurs specifically during growth and development of tissues.

Regulation of fetal lung disaturated phosphatidylcholine synthesis by de novo palmitate supply.

Lung surfactant disaturated phosphatidylcholine (PC) is highly dependent on the supply of palmitate as a source of fatty acid. The purpose of this study was to investigate the importance of de novo fatty acid synthesis in the regulation of disaturated PC production during late prenatal lung development. Choline incorporation into disaturated PC and the rate of de novo fatty acid synthesis was determined by the relative incorporation of [14C]choline and 3H2O, respectively, in 20-day-old fetal rat lung explants and in 18-day-old explants which were cultured 2 days. Addition of exogenous palmitate (0.15 mM) increased (26%) choline incorporation into disaturated PC but did not inhibit de novo fatty acid synthesis, as classically seen in other lipogenic tissue. Even in the presence of exogenous palmitate, de novo synthesis accounted for 87% of the acyl groups for disaturated PC. Inhibition of fatty acid synthesis by agaric acid or levo-hydroxycitrate decreased the rate of choline incorporation into disaturated PC. When explants were subjected to both exogenous palmitate and 60% inhibition of de novo synthesis, disaturated PC synthesis was below control values and 75% of disaturated PC acyl moieties were still provided by de novo synthesis. These data show that surfactant disaturated PC synthesis is highly dependent on the supply of palmitate from de novo fatty acid synthesis.

Fatty acid synthesis in the fetal lung: relationship to surfactant lipids.

The aims of this study were to investigate the control of fatty acid synthesis and its relationship to surfactant production in the fetal lung during alteration of hormonal and substrate conditions. Lung explants from 18 day fetuses (term = 22 days) which were cultured 2 days in the presence of 10 mM lactate showed parallel acceleration of de novo fatty acid synthesis (3H2O incorporation) and [14C]choline incorporation into disaturated phosphatidylcholine (DSPC) compared to culture of explants in glucose. Both the cultured and fresh explants were resistant to the classical short term (4 h) cAMP inhibition of fatty acid synthesis with 3 mM dibutyryl cAMP or 0.5 mM aminophylline. In the cultured explants short term cAMP elevation increased DSPC production, and long term (2 day) cAMP elevation caused a further increase in DSPC synthesis and also stimulated fatty acid synthesis. In cultured explants from 17 day fetuses, dexamethasone (0.1 microM) caused a synergistic increase with aminophylline in both fatty acid synthesis and DSPC production whereas, in explants from 18 day fetuses, dexamethasone inhibited both processes and reduced the level of stimulation of DSPC and fatty acid synthesis seen with aminophylline alone. Dexamethasone also reduced the stimulation of both DSPC and fatty acid synthesis produced in the culture of 18 day explants with bacitracin (0.5 mg/ml), whereas the combination of bacitracin and aminophylline resulted in a synergistic increase in DSPC production. Culture with glucagon (0.1 microM) also stimulated DSPC synthesis but at physiological levels insulin had no effect on either DSPC or fatty acid synthesis. These data show that lung fatty acid synthesis exhibits unique features of fatty acid synthesis regulation compared to other lipogenic tissues and also suggest a link between de novo fatty acid synthesis and surfactant production during the critical period of accelerated lung maturation.

Update on pulmonary edema: the role and regulation of endothelial barrier function.

Discovery of the pathophysiologic mechanisms leading to pulmonary edema and identification of effective strategies for prevention remain significant clinical concerns. Endothelial barrier function is a key component for maintenance of the integrity of the vascular boundary in the lung, particularly since the gas exchange surface area of the alveolar-capillary membrane is large. This review is focused on new insights in the pulmonary endothelial response to injury and recovery, reversible activation by edemagenic agents, and the biochemical/structural basis for regulation of endothelial barrier function. This information is discussed in the context of fundamental concepts of lung fluid balance and pulmonary function.

Regulation of endothelial barrier function by the cAMP-dependent protein kinase.

Elevation of cAMP promotes the endothelial cell (EC) barrier and protects the lung from edema development. Thus, we tested the hypothesis that both increases and decreases in PKA modulate EC function and coordinate distribution of regulatory, adherence, and cytoskeletal proteins. Inhibition of PKA activity by RpcAMPS and activation by cholera toxin was verified by assay of kemptide phosphorylation in digitonin permeabilized EC. Inhibition of PKA by RpcAMPS or overexpression of the endogenous inhibitor, PKI, decreased monolayer electrical impedance and exacerbated the decreases produced by agonists (thrombin and PMA). RpcAMPS directly increased F-actin content and organization into stress fibers, increased co-staining of actin with both phosphatase 2B and myosin light chain kinase (MLCK), caused reorganization of focal adhesions, and decreased catenin at cell borders. These findings are similar to those evoked by thrombin. In contrast, cholera toxin prevented the agonist-induced resistance decrease and protein redistribution. Although PKA activation attenuated thrombin-induced myosin light chain (MLC) phosphorylation, PKA inhibition per se did not cause MLC phosphorylation or affect [Ca2+]i. These studies indicate that a decrease in PKA activity alone can produce disruption of barrier function via mechanisms not involving MLCK and support a central role for cAMP/PKA in regulation of cytoskeletal and adhesive protein function in EC which correlates with altered barrier function.

Regulation of interleukin-1-stimulated GMCSF mRNA levels in human endothelium.

The regulation of interleukin-1 (IL-1)-mediated increases in GMCSF mRNA levels in human endothelium was examined and determined to occur in a time- and protein kinase C (PKC)-dependent manner. IL-1beta induced the early activation and translocation of PKC isotypes alpha and beta2 to the nucleus and PKC inhibition attenuated the IL-1-mediated increase in GMCSF mRNA levels. PKC activation by PMA alone, in the absence of IL-1beta activation, however, was insufficient to allow GMCSF mRNA detection. Increasing cyclic adenosine nucleotide (cAMP) levels suppressed IL-1beta-induced increases in GMCSF mRNA levels. In contrast, botulinum toxin C, which mediates the ADP ribosylation of a 21 kD ras-related G protein, augmented IL-1beta-induced GMCSF mRNA expression. Inhibition of protein synthesis (with cycloheximide) raised basal GMCSF mRNA transcripts to detectable levels, augmented IL-1-induced increases in GMCSF mRNA levels, and exhibited negative regulation by cAMP. Finally, disruption of either microtubules (with colchicine) or microfilaments (with cytochalasin B) resulted in reduced GMCSF mRNA expression in response to IL-1beta. These results are compatible with a model wherein IL-1-mediated increases in human endothelial cell GMCSF mRNA may be linked to both nuclear protein kinase C activation and activation of a low molecular weight G-protein, although neither activity alone is sufficient to increase the levels of GMCSF mRNA.

Role of tyrosine phosphorylation in thrombin-induced endothelial cell contraction and barrier function.

Thrombin-induced endothelial cell (EC) barrier dysfunction is highly dependent upon phosphorylation of serine and threonine residues present on myosin light chains (MLC) catalyzed by a novel EC myosin light chain kinase (MLCK) isoform. In this study, we examined the participation of tyrosine protein phosphorylation in EC contraction, gap formation and barrier dysfunction. We first determined that thrombin significantly increases protein tyrosine kinase activity and protein tyrosine phosphorylation in bovine pulmonary artery EC. Tyrosine kinase inhibitors, genistein and 2,5 DHC, reduced EC tyrosine kinase activities, however, only genistein significantly attenuated thrombin-mediated increases in albumin clearance and reductions in transendothelial electrical resistance. Similarly, genistein but not 2,5 DHC, decreased basal and thrombin-induced Ca2+ increases and MLC phosphorylation in the absence of alterations in Type 1 or 2A serine/threonine phosphatase activities. Immunoprecipitation of the EC MLCK isoform revealed a 214 kD immunoreactive phosphotyrosine protein and genistein pretreatment significantly reduced MLCK activity in MLCK immunoprecipitates. Although thrombin induced the translocation of p60src from the cytosol to the EC cytoskeleton, a detectable increase in the level of MLCK tyrosine phosphorylation was not noted after thrombin challenge. Taken together, our data suggest that genistein-sensitive tyrosine kinase activities are involved in thrombin-mediated EC MLCK activation, MLC phosphorylation, and barrier dysfunction.

Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung.

Determination of protein transfer across the endothelial barrier or the entire alveolar capillary membrane is critical for investigation of mechanisms leading to pulmonary edema. The purpose of this study was to evaluate Evans blue dye for determination of protein clearance across cultured bovine pulmonary artery endothelial cell monolayers and as a quantitative marker for albumin leakage to the air spaces in isolated perfused rat lungs. Evans blue dye bound tightly to albumin (EBA) as determined by lack of transfer through dialysis membranes and specific elution with albumin from a molecular exclusion column. EBA was equivalent to 125I-labeled albumin for calculation of albumin clearance rates (Calb) across intact and challenged monolayers [Calb (+ vehicle) = 0.12 microliters/min; Calb (+10 nM alpha-thrombin) = 0.47 microliters/min; Calb (+5 mg/ml trypsin) = 1.29 microliters/min]. Transfer of EBA was linear with time in both the endothelial cell monolayer model and the perfused lung. EBA was a sensitive marker for early edema in the perfused lung (before detectable weight gain) as well as for severe edema in the oxidant-injured lung (marked EBA accumulation in lavage fluid) and was a more specific marker for protein transfer than lavage fluid protein. EBA transfer is a convenient, reproducible, and accurate means to assess alterations in vascular permeability.

Diperoxovanadate alters endothelial cell focal contacts and barrier function: role of tyrosine phosphorylation.

Diperoxovanadate (DPV), a potent tyrosine kinase activator and protein tyrosine phosphatase inhibitor, was utilized to explore bovine pulmonary artery endothelial cell barrier regulation. DPV produced dose-dependent decreases in transendothelial electrical resistance (TER) and increases in permeability to albumin, which were preceded by brief increases in TER (peak TER effect at 10-15 min). The significant and sustained DPV-mediated TER reductions were primarily the result of decreased intercellular resistance, rather than decreased resistance between the cell and the extracellular matrix, and were reduced by pretreatment with the tyrosine kinase inhibitor genistein but not by inhibition of p42/p44 mitogen-activating protein kinases. Immunofluorescent analysis after DPV challenge revealed dramatic F-actin polymerization and stress-fiber assembly and increased colocalization of tyrosine phosphoproteins with F-actin in a circumferential pattern at the cell periphery, changes that were abolished by genistein. The phosphorylation of focal adhesion and adherens junction proteins on tyrosine residues was confirmed in immunoprecipitates of focal adhesion kinase and cadherin-associated proteins in which dramatic dose-dependent tyrosine phosphorylation was observed after DPV stimulation. We speculate that DPV enhances endothelial cell monolayer integrity via focal adhesion plaque phosphorylation and produces subsequent monolayer destabilization of adherens junctions initiated by adherens junction protein tyrosine phosphorylation catalyzed by p60(src) or Src-related tyrosine kinases.

Role of histamine in acute oleic acid-induced lung injury.

The action of histamine in oleic acid (OA)-induced injury was investigated using the isolated guinea pig lung perfused with blood-free media. OA infusion caused a significant increase in pulmonary arterial pressure, airway inspiratory pressure, lung weight, and protein flux across the alveolar-capillary barrier. These changes were dose dependent and caused injury regardless of the chemical form of OA (salt or free acid). Triolein (a neutral fat) infused at comparable emulsion particle size did not alter lung weight or bronchoalveolar lavage protein concentration in the perfused lung, suggesting that mechanical obstruction or emboli per se is not responsible for initiating early events in OA-induced injury. Infusion of OA caused a significant early histamine release into the venous effluent in the presence of aminoguanidine, a histamine catabolism inhibitor. Pretreatment with H1-receptor antagonists significantly attenuated OA-induced increase in lung weight and protein leak. These data support the link between OA-induced mast cell degranulation, histamine release, and OA-induced edema.

Role of microvascular pressure in reactive oxygen-induced lung edema.

O2 radicals are important in the pathogenesis of acute lung injury. The purpose of this investigation was to determine the role that microvascular pressure plays in edema induced by reactive O2 species generated by xanthine oxidase. In isolated rat lungs perfused with Krebs buffer plus 4% albumin, 5 mM glucose, and 2 mM xanthine at constant flow (13 ml/min), addition of xanthine oxidase (0.02 U/ml) caused a progressive increase in both pulmonary arterial and microvascular pressure (double occlusion method), which preceded the onset of edema. Both the pressure rise and edema formation were blocked by catalase, suggesting that vascular injury was related to H2O2 production. Lungs not exposed to free radicals that had microvascular pressure elevated to match that of the xanthine oxidase-perfused lungs showed only a small, reversible (nonedematous) weight gain. Lungs exposed to xanthine oxidase but perfused at constant microvascular pressure (5 Torr, similar to control lungs) showed a significant delay in protein-rich edema formation. These data indicate that reactive O2 metabolites induced lung injury, which is accompanied by increased microvascular pressure. Although the rise in microvascular pressure was shown not to be essential for edema formation, it does play a role in acceleration of the rate of transvascular fluid loss.

Regulation of thrombin-induced endothelial cell activation by bacterial toxins.

It has previously been shown that thrombin effects on endothelial cells can be mediated via G-proteins, which couple the thrombin receptor to several key physiological responses. As G-proteins are known targets of bacterial toxins, specific toxins were used to further characterize G-protein involvement in thrombin activation of bovine pulmonary arterial endothelial cells (BPAEC) and human umbilical vein endothelial cells (HUVEC). Homogenates were exposed to several bacterial toxins in the presence of 32P-NAD and ADP ribosylation of proteins determined by autoradiography of SDS-PAGE gels. Major substrates were a 40 kDa protein for pertussis toxin, 39, 45 and 52 kDa proteins (Gs) for cholera toxin, a 21 kDa protein for botulinum toxin C, and a 43 kDa protein (actin) for botulinum toxin C2a. The increase in either HUVEC or BPAEC PGI2 release induced by thrombin was not altered by pretreatment with any toxin. However, 1 h treatment of BPAEC monolayers with 1 microgram/ml pertussis toxin resulted in dramatic barrier dysfunction, which was synergistic with the albumin permeability induced by 1 microM thrombin. In contrast, pretreatment with 1 microgram/ml cholera toxin completely prevented the thrombin-induced barrier dysfunction. Moreover, contraction and gap formation due to thrombin challenge, observed by phase contrast microscopy, was greatly augmented by pertussis toxin and prevented by cholera toxin. Whereas 5 micrograms/ml botulinum toxin C did not affect either basal or thrombin-induced barrier dysfunction, botulinum toxin C2a increased basal BPAEC permeability over four-fold. Thus, bacterial toxins have specific and divergent effects on thrombin-induced endothelial cell responses. Botulinum toxin C2a appears to interact directly with actin to produce barrier dysfunction. In contrast, cholera toxin promotes barrier function via its known effects on Gs, stimulating adenylate cyclase and increasing cAMP. Because cholera toxin and pertussis toxin (via inhibition of G(i)) both increase cAMP, yet have opposing effects on barrier function, the present results suggest that pertussis toxin produces barrier dysfunction via ADP ribosylation of a novel G-protein other than G(i) or via a novel action of G(i).

Vascular endothelial cell activation and permeability responses to thrombin.

The serine protease, thrombin, evokes numerous endothelial cell responses which regulate hemostasis, thrombosis and vessel wall pathophysiology. One such response, the development of intercellular gap formation and vascular permeability is relevant to each of these processes and is a cardinal features of inflammation. Regulation of endothelial cell gap formation and therefore permeability is a function of a dynamic balance between competing adhesive, barrier-promoting tethering forces and contractile, tension-producing forces which result in barrier dysfunction. The key tethering events governing focal endothelial cell adhesion to the extracellular matrix and cell-cell interactions are poorly understood. In contrast, information is rapidly increasing regarding endothelial-specific contractile processes driven by the actomyosin molecular motor. The level of myosin light chain phosphorylation catalyzed by a unique myosin light chain kinase promotes productive actin-myosin interaction and governs the degree of centripetal tension produced. In this review the signal transducing and contractile mechanisms by which thrombin elicits endothelial cellular activation through its specific receptor are addressed. The pathways by which thrombin may alter the balance between contractile and tethering forces to promote endothelial cell gap formation are discussed.

Molecular characterization of HIV-2 (ROD) protease following PCR cloning from virus infected H9 cells.

A 450 nucleotide sequence corresponding to the nucleotides 1931-2380 of the viral genome (8) was amplified by polymerase chain reaction (PCR) using template DNA prepared from HIV-2 (ROD) infected H9 cells. The sequence codes for HIV-2 protease and its N-terminal flanking peptide. An identical DNA sequence was obtained from three independent PCR amplifications, which differs from the published sequence of HIV-2 (ROD) in 7 nucleotides scattered throughout the region of the cloned DNA. The cloned DNA was expressed in E. coli cells and resulted in the synthesis of a correctly processed HIV-2 protease, which is enzymatically active. Therefore, none of the seven nucleotide changes, which resulted in two amino acid substitutions, affect the autoproteolytic or trans-cleaving activities of the HIV-2 protease.

Protective role of sulfhydryl reagents in oxidant lung injury.

Recently there has been a great deal of interest in exploring possible ways to protect the lung from oxidant damage. Since sulfhydryl compounds are among the most important endogenous antioxidants, their therapeutic use has been proposed. Glutathione (GSH), the main intracellular nonprotein sulfhydryl, plays an important role in the maintenance of cellular proteins and lipids in their functional state. With oxidant stress, GSH acts to protect cell constituents as evidenced by increased turnover to GSSG, formation of mixed disulfides with proteins, utilization of NADPH, and utilization of glucose in the pentose pathway. When GSH is experimentally lowered (e.g., by protein deficiency or with diethylmaleate) the toxic effects of oxidant stress are exacerbated as evidenced by increased membrane and cell damage, pulmonary edema, and mortality. Several recent investigations have shown that sulfhydryl reagents (particularly N-acetyl cysteine, a cell-permeable GSH precursor) can provide significant protection against certain pulmonary toxins. N-acetyl cysteine reduced the lethal effects of 100% O2 in rats by 65%. Therefore, the therapeutic potential of sulfhydryl reagents in the treatment and prevention of oxidant injury and the mechanisms involved are an important direction for lung research.