Adenosine and ATPγS protect against bacterial pneumonia-induced acute lung injury.

Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, disrupts the alveolar-capillary barrier, triggering pulmonary vascular leak thus inducing acute lung injury (ALI). Extracellular purines, adenosine and ATP, protected against ALI induced by purified LPS. In this study, we investigated whether these purines can impact vascular injury in more clinically-relevant E.coli (non-sterile LPS) murine ALI model. Mice were inoculated with live E. coli intratracheally (i.t.) with or without adenosine or a non-hydrolyzable ATP analog, adenosine 5'-(γ-thio)-triphosphate (ATPγS) added intravenously (i.v.). After 24 h of E. coli treatment, we found that injections of either adenosine or ATPγS 15 min prior or adenosine 3 h after E.coli insult significantly attenuated the E.coli-mediated increase in inflammatory responses. Furthermore, adenosine prevented weight loss, tachycardia, and compromised lung function in E. coli-exposed mice. Accordingly, treatment with adenosine or ATPγS increased oxygen saturation and reduced histopathological signs of lung injury in mice exposed to E. coli. Lastly, lung-targeting gene delivery of adenosine or ATPγS downstream effector, myosin phosphatase, significantly attenuated the E. coli-induced compromise of lung function. Collectively, our study has demonstrated that adenosine or ATPγS mitigates E. coli-induced ALI in mice and may be useful as an adjuvant therapy in future pre-clinical studies.

A liquid chromatography/mass spectrometry-based method for the selection of ATP competitive kinase inhibitors.

The currently approved kinase inhibitors for therapeutic uses and a number of kinase inhibitors that are undergoing clinical trials are directed toward the adenosine triphosphate (ATP) binding site of protein kinases. The 5'-fluorosulfonylbenzoyl 5'-adenosine (FSBA) is an ATP-affinity reagent that covalently modifies a conserved lysine present in the nucleotide-binding site of most kinases. The authors have developed a liquid chromatography/mass spectrometry-based method to monitor binding of ATP competitive protein kinase inhibitors using FSBA as a nonselective activity-based probe for protein kinases. Their method provides a general, rapid, and reproducible means to screen and validate selective ATP competitive inhibitors of protein kinases.

Triatoma infestans apyrases belong to the 5'-nucleotidase family.

Apyrases are nucleoside triphosphate-diphosphohydrolases (EC 3.6.1.5) present in a variety of organisms. The apyrase activity found in the saliva of hematophagous insects is correlated with the prevention of ADP-induced platelet aggregation of the host during blood sucking. Purification of apyrase activity from the saliva of the triatomine bug Triatoma infestans was achieved by affinity chromatography on oligo(dT)-cellulose and gel filtration chromatography. The isolated fraction includes five N-glycosylated polypeptides of 88, 82, 79, 68 and 67 kDa apparent molecular masses. The isolated apyrase mixture completely inhibited aggregation of human blood platelets. Labeling with the ATP substrate analogue 5'-p-fluorosulfonylbenzoyladenosine showed that the five species have ATP-binding characteristic of functional apyrases. Furthermore, tandem mass spectroscopy peptide sequencing showed that the five species share sequence similarities with the apyrase from Aedes aegypti and with 5'-nucleotidases from other species. The complete cDNA of the 79-kDa enzyme was cloned, and its sequence confirmed that it encodes for an apyrase belonging to the 5'-nucleotidase family. The gene multiplication leading to the unusual salivary apyrase diversity in T. infestans could represent an important mechanism amplifying the enzyme expression during the insect evolution to hematophagy, in addition to an escape from the host immune response, thus enhancing acquisition of a meal by this triatomine vector of Chagas' disease.

The nucleotide-binding site of human sphingosine kinase 1.

Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly(82) of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an approximately 45-fold higher K(m)((ATP)). We have also shown that Lys(103) is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX(17-21)K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.

Antinociceptive effect of a new P(2Z)/P2X7 antagonist, oxidized ATP, in arthritic rats.

The neurotransmitter adenosine triphosphate (ATP) is released from sensory nerve endings during inflammation and acts at the level of P2X receptors. We used the irreversible inhibitor of P2z/P2X7 receptor, designated oxidized ATP (oATP), to test its possible antinociceptive activity in arthritic rats. We induced unilateral inflammation of the rat hind paw by local injection of Freund's complete adjuvant. Administration of the adjuvant resulted in a significant reduction of paw pressure threshold (PPT). Injection of oATP into inflamed paws significantly increased, in a dose-dependent manner, PPT values to levels comparable with or higher than those evaluated in control uninflamed paws. The data indicate that the P2z/P2X7 receptor system exerts a role in nociception and that oATP, by inhibiting such a receptor, reduces the nociceptive signal in the course of peripheral inflammation.

Characterization of gamma-aminobutyrate type A receptors with atypical coupling between agonist and convulsant binding sites in discrete brain regions.

Gamma-ainobutyric acid type A (GABA(A)) receptor ionophore ligand t-[35S]butylbicyclophosphorothionate ([35S]TBPS) was used in an autoradiographic assay on brain cryostat sections to visualize and characterize atypical GABA-insensitive [35S]TBPS binding previously described in certain recombinant GABA(A) receptors and the cerebellar granule cell layer. Picrotoxinin-sensitive but 1-mM GABA-insensitive [35S]TBPS binding was present in the rat cerebellar granule cell layer, many thalamic nuclei, subiculum and the internal rim of the cerebral cortex, amounting in these regions up to 6% of the basal binding determined in the absence of exogenous GABA. Similar binding properties were detected also in human and chicken brain sections. Like the GABA-sensitive [35S]TBPS binding, GABA-insensitive binding was profoundly decreased by pentobarbital, pregnanolone, loreclezole and Mg2+. The binding was reversible and apparently dependent on Cl- ions. Localization of the GABA-insensitive [35S]TBPS binding was not identical to that of high-affinity [3H]muscimol binding and diazepam-insensitive [3H]Ro 15-4513 binding, two previously established receptor subtype-dependent binding heterogeneities in the rat brain. The present study reveals a component of the GABA-ionophore enriched in the thalamus and cerebellar granule cells, possibly representing poorly desensitized or desensitizing receptors.

Preferential coassembly of alpha4 and delta subunits of the gamma-aminobutyric acidA receptor in rat thalamus.

Pharmacological study of rat thalamic gamma-aminobutyric acidA (GABAA) receptors revealed the presence of two distinct populations, namely, diazepam-sensitive and diazepam-insensitive [3H]Ro15-4513 binding sites accounting for 94 +/- 2% (1339 +/- 253 fmol/mg protein) and 6 +/- 2% (90 +/- 44 fmol/mg protein) of total sites, respectively. Thalamic diazepam-insensitive sites exhibited a pharmacology that was distinct from diazepam-sensitive sites but comparable to that of the alpha4beta3gamma2 subtype of the GABAA receptor stably expressed in L(tk-) cells. Immunoprecipitation experiments with a specific anti-alpha4-antiserum immunoprecipitated 20 and 7% of total thalamic [3H]muscimol and [3H]Ro15-4513 sites, respectively. Combinatorial immunoprecipitation using antisera against the alpha4, gamma2, and delta subunit revealed that alpha4delta- and alpha4gamma2-containing receptors account for 13 +/- 2 and 8 +/- 3% of [3H]muscimol sites from thalamus, respectively. It also indicated that all delta subunits coexist with an alpha4 subunit in this brain region. In conclusion, our results show that in rat thalamus both alpha4betagamma2 and alpha4betadelta subtypes are expressed but alpha4betadelta is the major alpha4-containing GABAA receptor population.

Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells.

CHO-K1 cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1-P2X6 subunits. DbATP (EC50 approximately equal to 100 microM) evoked non-desensitizing inward currents which reversed at approximately equal to 0 mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO-PRO-1, in CHO-KI cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca2+ accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgammaS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and (alphabeta)methylene ATP were inactive at concentrations up to 100 microM. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min. These data suggest that CHO-K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.

P2Y2 receptors are expressed by human osteoclasts of giant cell tumor but do not mediate ATP-induced bone resorption.

Extracellular nucleotides acting through P2 receptors elicit a range of responses in many cell types. Previously, we have cloned the G-protein coupled P2Y2 receptor from a human osteoclastoma complementary deoxyribonucleic acid (cDNA) library and demonstrated its expression by reverse transcription linked (RT)-PCR and Southern analysis in a number of skeletal tissues, including a purified population of giant cells. In this study we have localized the expression of P2Y2 receptor transcripts to osteoclasts of giant cell tumor of bone by in situ hybridization. In osteoblasts and other cell types, the P2Y2 receptor is coupled to Ins(1,4,5)P3-mediated Ca2+ release from intracellular stores. In this study, the P2Y2 receptor agonists adenosine triphosphate (ATP) and uridine triphosphate (UTP) did not increase cytosolic free calcium concentration ([Ca2+]i) in giant cells isolated from osteoclastoma, while the G-protein coupled calcium sensing receptor agonist, Ni2+, elevated [Ca2+]i in the same cells. These data indicate that P2Y2 receptor transcripts expressed by giant cells are not presented at the surface of cells as functional receptors, or alternatively, functional receptors are coupled to an effector other than [Ca2+]i. ATPgammaS (10 micromol/L), but not UTP (10 micromol/L), significantly stimulated resorption by an enriched giant cell population. These results indicate that ATP-induced effects on resorption, following direct osteoclastic activation, are mediated by a P2 receptor other than the P2Y2 subtype. Nucleotides, released locally in the bone microenvironment in response to acute trauma or transient physical stress, will interact with a complement of P2 receptors expressed by both osteoclasts and osteoblasts to influence the remodeling process.

Separation of the N-7 methyltransferase, the key enzyme in caffeine biosynthesis.

Caffeine biosynthesis comprises sequential methylations at N-7, N-3 and N-1 of the xanthine ring catalysed by S-adenosyl-L-methionine (SAM)-dependent methyltransferase activities that, to date, have not been resolved. Enzyme extracts were prepared from young, emerging coffee leaflets and following anion exchange chromatography, chromatofocusing facilitated the clear separation of the N-7-methyltransferase from the N-3- and N-1-methyltransferase activities. All three N-methyltransferases co-eluted when analysed by gel filtration chromatography and their native molecular mass was ca 67 kDa. Photoaffinity labelling with [methyl-3H]SAM followed by SDS-PAGE of a chromatofocusing-purified preparation containing only N-7-methyltransferase activity demonstrated the presence of a single labelled band of 40 kDa. Similar analysis of a gel filtration purified preparation containing all three N-methyltransferase activities revealed the presence of three labelled bands at 49, 43 and 40 kDa. It remains to be determined whether the 49 and 43 kDa bands are associated with the N-3 and N-1-methyltransferases or whether they are unrelated SAM-dependent methyltransferases or other SAM-binding proteins.

Effects of benzodiazepine agonist, antagonist and inverse agonist on ethanol-induced changes in beta-endorphin levels in specific rat brain regions.

The present study was conducted to evaluate and compare effects of the benzodiazepine agonist diazepam, antagonist flumazenil and inverse agonist RO 15-4513 on ethanol-induced changes in beta-endorphin (beta-EN) levels in specific rat brain regions. Male Sprague-Dawley rats (150-200 g) adapted to a 12-hour light:12-hour dark illumination cycle were used in this study. Ethanol (3 g/kg as 22.5% solution in saline), flumazenil (10 mg/kg), RO 15-4513 (10 mg/kg), diazepam (2 mg/kg) or a combination of ethanol (3 g/kg) and flumazenil (10 mg/kg), RO 15-4513 (10 mg/kg) or diazepam (2 mg/kg) were administered intraperitoneally to rats at 7.00 h. Control animals were injected with saline. Animals were sacrificed by decapitation 1 h after injection; the brains were immediately removed; the cortex, hippocampus and hypothalamus were dissected and their beta-EN levels measured by radioimmunoassay. Ethanol administration significantly increased beta-EN levels in the hippocampus and hypothalamus but had no effect on beta-EN levels in the cortex. Similar increases in beta-EN levels in the hippocampus and hypothalamus also occurred following either flumazenil or diazepam. On the other hand, RO 15-4513 significantly increased beta-EN levels in the cortex and hypothalamus. When flumazenil was concurrently administered with ethanol, it completely reversed the ethanol effects in the hippocampus but failed to do so in the hypothalamus. Concurrent administration of RO 15-4513 with ethanol also reversed the ethanol-induced rise of beta-EN in the hypothalamus. However, concurrent administration of diazepam and ethanol did not block the increase in beta-EN levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoreactive, active derivatives of trypsin and chymotrypsin inhibitors from soybeans and chickpeas.

The photoreactive arylsufenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (STI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-STI indicated that only one of the two tryptophans, 93 or 117, present in STI was modified. Amino acid analysis of the two separated CNBr-cleavage products of 2,4-NAPS-STI showed that only tryptophan 93 underwent modification. 2,4-NAPS-STI fully retained its inhibitory activity against trypsin. The covalent attachment of 2,4-NAPS-STI to tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride. Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas were prepared by selective modification of the epsilon-amino groups of 2,4(5)-NAPS-Cl. The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.

Preparation of photoreactive derivatives of trypsin-chymotrypsin inhibitors from soybeans and chick peas by selective modification of lysine residues.

Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas, were prepared by selective modification of the epsilon-amino groups of lysine residues with 2-nitro-4(5)-azidophenylsulfenyl chlorides (2,4(5)-NAPS-C1). The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of the trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.

[Comparative analysis of lectin interaction with blood serum proteins in man, rodents and ungulates]. / Sravnitel'nyi analiz vzaimodeistviia lektinov s belkami syvorotki krovi cheloveka, gryzunov i kopytnykh.

Studies have been made on the interaction of proteins of the blood serum of man, guinea pig, rat, donkey and ram with concanavalin A, peas lectin and phytohaemagglutinin II. It was found that reactions of the lectins with serum proteins exhibit strictly specific pattern. On the basis of the data obtained, a discussion is made of evolutionary conformation of serum proteins and distribution of lectin receptors within the structure of their molecules.