Lysophosphatidic acid is detectable in human bronchoalveolar lavage fluids at baseline and increased after segmental allergen challenge.

BACKGROUND: Lysophosphatidic acid (LPA) is a biologically active lysophospholipid and a component of normal plasma. LPA binds to receptors expressed on circulating and structural lung cells and affects cell growth and activation. Whether LPA is present in the lung has not been previously reported. OBJECTIVE: To develop an assay to measure LPA in bronchoalveolar lavage (BAL) fluids, and to study the association between LPA and allergic airway inflammation. METHODS: Seventeen allergic subjects underwent bronchoscopy and segmental allergen challenge, followed 18 h later by BAL. Supernatants were analysed for LPA content using liquid chromatography and mass spectroscopy. Expression of LPA receptors on primary bronchial epithelial cells was analysed by immunolabelling, and the effects of LPA on epithelial cell barrier function was investigated by measuring transepithelial resistance. RESULTS: LPA was detectable in BAL from control lung segments, and significantly increased 18 h after allergen challenge. Polyunsaturated species of LPA were especially increased following segmental allergen challenge. LPA levels did not strongly correlate with the number or percentages of eosinophils, neutrophils of lymphocytes, whereas MIP-3alpha (CCL20) levels correlated significantly with the allergen-driven influx of lymphocytes. The levels of LPA from control sites correlated inversely with BAL protein content, suggesting that LPA promoted epithelial barrier integrity at baseline. Experiments using primary human bronchial epithelial cells confirmed that LPA tightened the epithelial cell barrier. CONCLUSION: Lysophosphatidic acid is detectable in human BAL fluids at baseline and its expression increases during allergic inflammation. LPA does not appear to be a dominant chemoattractant for eosinophils or lymphocytes during allergic airway inflammation. In the absence of ongoing inflammation, LPA may promote epithelial barrier integrity.

[Effect of pantogam on visual function and hemodynamic of eyes in patients with primary open-angle glaucoma]. / Vliianie pantogama na zritel'nye funktsii i gemodinamiku glaz bol'nykh pervichnoi otkrytougol'noi glaukomoi.

The complex therapy in patients with primary open-angle glaucoma, with normalized intraocular pressure but with declined visual functions included the use of the Russian neurometabolic preparation pantogam. The improvement of the visual function appeared as enlargement of the borders of visual fields, decreased area and sensitivity deficit (according to computer perimetric data). Doppler ultrasonography has demonstrated an increase in the mean blood velocity in the orbital vessels and a decrease in eyeball blood supply defects after pantogam therapy which is a favourable sign of a glaucomatous process. The authors consider the use of pantogam to be rational in the complex therapy of patients with glaucoma.

[Psychophysiological effects of combined administration of pantogam and potassium orotate in patients with neurotic disorders]. / Psikhofiziologicheskie éffekty kombinirovannogo primeneniia pantogama i orotata kaliia u bol'nykh s nevroticheskimi rasstroistvami.

The paper presents a complex psychophysiological analysis of the effect of a combined administration of pantogam and potassium orotate (kalii orotas) on the dynamics of cognitive function in patients with neurotic disorders. The investigation was conducted in an 8-stage consecutive cycle and employed computer-aided diagnostic system. It was established that the combined use of pantogam and potassium orotate produces a positive effect upon the dynamics of restoration of the attention and memory mechanisms in neurotic patients.

[Testing domestic and imported medical engineering products by the interference stability requirements]. / Ispytaniia izdelii meditsinskoi tekhniki otechestvennogo i importnogo proizvodstva po trebovaniiam pomekhoustoichivosti.

The paper presents some conclusions on about the interference stability of medical engineering products, which have been made on the tests for requirements for electromagnetic compatibility of different kinds of products--from the simplest to the most elaborate life-support systems. It gives a list of basic standards for interference stability, as well as criteria for the quality of operation of the products tested for the stability.

Sorting of mannose 6-phosphate receptors mediated by the GGAs.

The delivery of soluble hydrolases to lysosomes is mediated by the cation-independent and cation-dependent mannose 6-phosphate receptors. The cytosolic tails of both receptors contain acidic-cluster-dileucine signals that direct sorting from the trans-Golgi network to the endosomal-lysosomal system. We found that these signals bind to the VHS domain of the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). The receptors and the GGAs left the trans-Golgi network on the same tubulo-vesicular carriers. A dominant-negative GGA mutant blocked exit of the receptors from the trans-Golgi network. Thus, the GGAs appear to mediate sorting of the mannose 6-phosphate receptors at the trans-Golgi network.

HIV-1 reverse transcriptase interaction with model RNA-DNA duplexes.

HIV-1 reverse transcriptase (HIV-1 RT) is a multifunctional enzyme responsible for converting viral RNA into preintegrative DNA during the early stages of viral infection. DNA polymerase and RNase H activities are required, and several conformationally distinct primer-templates must be accommodated by the enzyme during the process. Parameters of interaction between model substrates (ligands) and HIV-1 RT (wild type p66/p51 and the RNase H-deficient mutant p66(E478Q)/p51) (analytes) were estimated by surface plasmon resonance at 25 degrees C, pH 8.0. Binding of RT to the ligands is specific and can be analyzed using a conventional 1:1 binding algorithm. RNA-DNA hybrids with 5'-template overhangs of 6 and 12 nucleotides bind to RT approximately one order of magnitude stronger than the corresponding 36-mer with blunt ends due to slower dissociation. Immobilization of the latter through either the 5'-end of RNA or DNA strand does not change the equilibrium constant (K(D)) for wild-type RT but the values of kinetic constants of association and dissociation differ significantly. For the p66(E478Q)/p51 enzyme, orientation effects are notable even altering the K(D) value. Binding of the p66(E478Q)/p51 to any RNA-DNA hybrids is slightly stronger compared with wild type. Data can be interpreted in terms of the mechanism of reverse transcription.

Halenaquinol, a natural cardioactive pentacyclic hydroquinone, interacts with sulfhydryls on rat brain Na(+),K(+)-ATPase.

Halenaquinol inhibited the partial reactions of ATP hydrolysis by rat brain cortex Na(+),K(+)-ATPase, such as [3H]ATP binding to the enzyme, Na(+)-dependent front-door phosphorylation from [gamma-(33)P]ATP, and also Na(+)- and K(+)-dependent E(1)<-->E(2) conformational transitions of the enzyme. Halenaquinol abolished the positive cooperativity between the Na(+)- and K(+)-binding sites on the enzyme. ATP and sulfhydryl-containing reagents (cysteine and dithiothreitol) protected the Na(+),K(+)-ATPase against inhibition. Halenaquinol can react with additional vital groups in the enzyme after blockage of certain sulfhydryl groups with 5,5'-dithio-bis-nitrobenzoic acid. Halenaquinol inhibited [3H]ouabain binding to Na(+),K(+)-ATPase under phosphorylating and non-phosphorylating conditions. Binding of fluorescein 5'-isothiocyanate to Na(+),K(+)-ATPase and intensity of fluorescence of enzyme tryptophanyl residues were decreased by halenaquinol. We suggest that interaction of halenaquinol with the essential sulfhydryls in/or near the ATP-binding site of Na(+),K(+)-ATPase resulted in a change of protein conformation and subsequent alteration of overall and partial enzymatic reactions.

The distribution of free sterols, polyhydroxysteroids and steroid glycosides in various body components of the starfish Patiria (=Asterina) pectinifera.

The distribution of free sterols, polyhydroxysteroids and steroid glycosides in different body components of the Far-eastern starfish Patiria (=Asterina) pectinifera has been studied. It was shown that free sterol fractions from aboral and oral body walls, gonads, stomach and pyloric ceca contained Delta(7) sterols with a preponderance of 5alpha-cholest-7-en-3beta-ol. All these body components had also toxic steroid oligoglycosides. However, polyhydroxysteroids and related low molecular weight steroid glycosides were found in stomach and pyloric ceca only. In pyloric ceca, the sulfated monoside 'asterosaponin' P(1) was identified as a main polar steroid, whereas 6-sodium sulfate of cholestane-3beta,4beta,6alpha,7alpha,8,15beta,16beta,26-octaol predominated in the stomach. Probable biological functions of polar steroids and free sterols in this starfish were discussed. It was suggested that some polyhydroxysteroids and related monoglycosides play the same biological role as bile alcohols and bile acids do in vertebrates.

The structure of the T127L/S128A mutant of cAMP receptor protein facilitates promoter site binding.

The x-ray crystal structure of the cAMP-ligated T127L/S128A double mutant of cAMP receptor protein (CRP) was determined to a resolution of 2.2 A. Although this structure is close to that of the x-ray crystal structure of cAMP-ligated CRP with one subunit in the open form and one subunit in the closed form, a bound syn-cAMP is clearly observed in the closed subunit in a third binding site in the C-terminal domain. In addition, water-mediated interactions replace the hydrogen bonding interactions between the N(6) of anti-cAMP bound in the N-terminal domains of each subunit and the OH groups of the Thr(127) and Ser(128) residues in the C alpha-helix of wild type CRP. This replacement induces flexibility in the C alpha-helix at Ala(128), which swings the C-terminal domain of the open subunit more toward the N-terminal domain in the T127L/S128A double mutant of CRP (CRP*) than is observed in the open subunit of cAMP-ligated CRP. Isothermal titration calorimetry measurements on the binding of cAMP to CRP* show that the binding mechanism changes from an exothermic independent two-site binding mechanism at pH 7.0 to an endothermic interacting two-site mechanism at pH 5.2, similar to that observed for CRP at both pH levels. Differential scanning calorimetry measurements exhibit a broadening of the thermal denaturation transition of CRP* relative to that of CRP at pH 7.0 but similar to the multipeak transitions observed for cAMP-ligated CRP. These properties and the bound syn-cAMP ligand, which has only been previously observed in the DNA bound x-ray crystal structure of cAMP-ligated CRP by Passner and Steitz (Passner, J. M., and Steitz, T. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2843-2847), imply that the cAMP-ligated CRP* structure is closer to the conformation of the allosterically activated structure than cAMP-ligated CRP. This may be induced by the unique flexibility at Ala(128) and/or by the bound syn-cAMP in the hinge region of CRP*.

Signal-binding specificity of the mu4 subunit of the adaptor protein complex AP-4.

The medium (mu) chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (YXXphi) sorting signals found within the cytoplasmic domains of integral membrane proteins. Here, we describe the signal-binding specificity and affinity of the medium subunit mu4 of the recently described adaptor protein complex AP-4. To elucidate the determinants of specificity, we screened a two-hybrid combinatorial peptide library using mu4 as a selector protein. Statistical analyses of the results revealed that mu4 prefers aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at Y-1 and Y+3 (phi). In addition, we examined the interaction of mu4 with naturally occurring YXXphi signals by both two-hybrid and in vitro binding analyses. These experiments showed that mu4 recognized the tyrosine signal from the human lysosomal protein LAMP-2, HTGYEQF. Using surface plasmon resonance measurements, we determined the apparent dissociation constant for the mu4-YXXphi interaction to be in the micromolar range. To gain insight into a possible role of AP-4 in intracellular trafficking, we constructed a Tac chimera bearing a mu4-specific YXXphi signal. This chimera was targeted to the endosomal-lysosomal system without being internalized from the plasma membrane.

Probing the lipid-free structure and stability of apolipoprotein A-I by mutation.

To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipoprotein A-I (apoA-I) and its role in protein stability, recombinant wild-type and seven site-specific mutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescence spectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering the secondary structure, suggesting that G185 and G186 are located in a loop/disordered region. A triple substitution (L222K, F225K, F229K) leads to a small increase in the alpha-helical content and stability, indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. The C-terminal truncation Delta(209-243) does not change the alpha-helical content but reduces the protein stability. Truncation of a larger segment, Delta(185-243), does not affect the secondary structure or stability. In contrast, an intermediate truncation, Delta(198-243), leads to a significant reduction in the alpha-helical content, stability, and unfolding cooperativity. The internal 11-mer deletion Delta(187-197) has no significant effect on the conformation or stability, whereas another internal 11-mer deletion, Delta(165-175), dramatically disrupts and destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial for proper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminal region 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactions in the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located in the N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to the N-terminus.

RNA polymerase-cNMP-ligated cAMP receptor protein (CRP) mutant interactions in the enhancement of transcription by CRP mutants.

The enhancement of the transcription of three synthetic promoters by cNMP-ligated cAMP receptor protein (CRP)/mutant complexes was determined from the transcription yields of a short AAUU transcript in an abortive initiation in vitro transcription assay. The cNMP-ligated CRP and mutants were cAMP, cGMP, and cIMP ligated with CRP, T127L CRP, S128A CRP, and T127L/S128A CRP. The transcriptional activation of a 152-base pair lacUV5 promoter (synlac promoter) with a CRP consensus binding site sequence (syncon promoter) was enhanced by an average factor of 12.3 +/- 0.5 with the cAMP-ligated complexes of CRP/mutants and cGMP-ligated T127L, although their promoter binding site affinities varied by a factor of 5. However, in the presence of bound RNA polymerase, the binding affinities only ranged from 0.8 +/- 0.2 x 10(7) m(-)(1) for cAMP-ligated CRP* to 1.8 +/- 0. 3 x 10(7) m(-)(1) for cAMP-ligated CRP, indicating that the CRP/mutant interacts with the bound RNA polymerase, which would account for the near constancy of the enhancement factors. The corresponding enhancement factors for the synlac promoter and a promoter with a different CRP binding site sequence (syngal promoter) were also nearly the same, 7.2 +/- 0.7 and 6 +/- 1, respectively. The binding reaction of the syncon promoter to the RNA polymerase is exothermic, with a binding constant (K(b)) = 2.1 +/- 0. 2 x 10(7) m(-1).

[Interference testing in certification of medical equipment]. / Ispytaniia na pomekhoustoichivost' pri sertifikatsii meditsinskoi tekhniki.

The paper considers requirements for noise immunity of medical equipment, types of electromagnetic disturbances, and a list of medical apparatuses intended for obligatory certification testing for noise immunity. The paper presents experimental data on the impact of electromagnetic disturbances on different types of medical apparatuses.

Inhibition of membrane transport ATPases by halenaquinol, a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata.

Halenaquinol, a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata, was found to be a powerful inhibitor of the rat brainstem and of the rat brain cortex Na+, K(+)-ATPases and the rabbit muscle sarcoplasmic reticulum Ca(2+)-ATPase with I50 values of 7.0 x 10(-7), 1.3 x 10(-6) and 2.5 x 10(-6) M, respectively. Halenaquinol also inhibited K(+)-phosphatase activity of the rat brain cortex Na+, K(+)-ATPase with an I50 value of 3 x 10(-6) M. Ouabain-insensitive Mg(2+)-ATPase activity of the microsomal fraction of the rat brain cortex was weakly inhibited by halenaquinol. Inhibition was irreversible, dose- and time-dependent. Naphthohydroquinone fragment in structures of halenaquinol, related natural and model compounds was very important for an inhibiting effect.

Two different modes of inhibition of the rat brain Na+, K(+)-ATPase by triterpene glycosides, psolusosides A and B from the holothurian Psolus fabricii.

Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na+, K(+)-ATPase (Na, K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na+, K(+)-ATPase with I50 values of 1 x 10(-4) M and 3 x 10(-4) M, respectively. PsA significantly stimulated [3H]ATP binding to Na+, K(+)-ATPase, weakly increased [3H]ouabain binding to the enzyme, and inhibited K(+)-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [3H]ATP binding to Na+, K(+)-ATPase, and had no effect on [3H]ouabain binding to the enzyme. K(+)-Phosphatase activity was inhibited by PsB in parallel with Na+, K(+)-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na+, K(+)-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.

Stimulatory effects of starfish sapogenins (Asterias amurensis and Lethasterias nanimensis chelifera) on molluscan heart (Spisula sachalinensis).

Sapogenins from the starfish Asterias amurensis and Lethasterias nanimensis chelifera, 5 alpha-pregn-9(11)-ene-3 beta,6 alpha-diol-20-one, 5 alpha-cholest-9(11)-ene-3 beta,6 alpha-diol-23-one, 5 alpha-cholesta-9(11),24(25)-diene-3 beta,6 alpha-diol-23-one, (20E)-5 alpha-cholesta-9(11),20(22)-diene-3 beta,6 alpha-diol-23-one and 24 zeta-methyl-5 alpha-cholesta-9(11),20(22)-diene-3 beta,6 alpha-diol-23-one, stimulated the contractile force of the heart of the mollusk Spisula sachalinensis at concentration of 5 x 10(-5) M. Ouabain, a specific inhibitor of Na+,K(+)-ATPase, at concentration of 5 x 10(-5) M had no effect on this physiological model. Starfish sapogenins of the cholestane series moderately inhibited rat brain cortex Na+,K(+)-ATPase and decreased Ca2+ influx into Ehrlich carcinoma cells. In contrast, pregnane asterogenin asterone did not inhibit Na+,K(+)-ATPase and increased the influx of Ca2+ into cells. These effects were not the result of cell membrane damage, because none of the compounds tested have hemolytic activity.

Determination of the conformations of cAMP receptor protein and its T127L,S128A mutant with and without cAMP from small angle neutron scattering measurements.

Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRP*) in D2O K3PO4 buffer containing 0.5 M KCl, in the absence and presence of 3',5' cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively "closed" form where the alpha-helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively "open" form where the alpha-helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form. Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement with in vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription.

Effect of cAMP binding site mutations on the interaction of cAMP receptor protein with cyclic nucleoside monophosphate ligands and DNA.

Although cAMP binding to wild type cAMP receptor protein (CRP) induces specific DNA binding and activates transcription, cyclic nucleoside monophosphate (cNMP) binding to the CRP mutant Ser128 --> Ala does not, whereas the double CRP mutant Thr127 --> Leu/Ser128 --> Ala activates transcription even in the absence of cNMP. Isothermal titration calorimetry measurements on the cNMP binding reactions to the S128A and T127L/S128A mutants show that the reactions are mainly entropically driven as is cAMP binding to CRP. In contrast to cAMP binding to CRP, the binding reactions are noncooperative and exothermic with binding enthalpies (DeltaHb) ranging from -23.4 +/- 0.9 kJ mol-1 for cAMP binding to S128A at 39 degrees C to -4.1 +/- 0.6 kJ mol-1 for cAMP binding to T127L/S128A at 24 degrees C and exhibit enthalpy-entropy compensation. To account for the inactivity of the S128A mutant, in vitro and in vivo DNA binding experiments were performed on the cAMP-ligated S128A mutant. The cAMP-ligated S128A mutant binds to the consensus DNA binding site with approximately the same affinity as that of cAMP-ligated CRP but forms a different type of complex, which may account for loss of transcriptional activity by the mutant. Energy minimization computations on the cAMP-ligated S128A mutant show that amino acid conformational differences between S128A and CRP occur at Ser179, Glu181, and Thr182 in the center of the DNA binding site, implying that these conformational changes may account for the difference in DNA binding.

Alterations in the physiochemical characteristics of low and high density lipoproteins after lipolysis with phospholipase A2. A spin-label study.

Human low and high density lipoproteins (LDL and HDL, respectively) were treated with porcine pancreatic phospholipase A2 (PLA2) in the presence of albumin resulting in hydrolysis of 40-84% of the lipoproteins phospholipids. The resulting PLA2-treated LDL and HDL and concurrent control lipoproteins incubated without PLA2 were reisolated by ultracentrifugation and labelled with 5-doxyl- and 16-doxyl-stearic acid, and with a spin-labelled analogue of maleimide. Analysis of ESR spectra showed that phospholipid hydrolysis both of LDL and HDL resulted in an increase in order, micro-viscosity and polarity of lipid regions in the surface monolayer of the particles. In the temperature range from 3 degrees C to 50-60 degrees C, Arrhenius plots of a spectral parameter of LDL and HDL labelled with 5-doxyl-stearate exhibited alterations which suggest an increase in free cholesterol content near the surface of the lipoproteins after PLA2-treatment. ESR spectra of the maleimide analogue bound covalently to the protein moiety of the lipoproteins have demonstrated that, following phospholipid hydrolysis, the conformation of the apoproteins became more condensed, with more masked domains. The possible implications of the revealed alterations for enhanced delivery of LDL and HDL cholesterol to cells after phospholipolysis of the lipoproteins are discussed.