ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

Influence of acute pancreatitis on the in vitro responsiveness of rat mesenteric and pulmonary arteries

Background: Acute pancreatitis is an inflammatory disease characterized by local tissue injury and systemic inflammatory response leading to massive nitric oxide (NO) production and haemodynamic disturbances. Therefore, the aim of this work was to evaluate the vascular reactivity of pulmonary and mesenteric artery rings from rats submitted to experimental pancreatitis.Male Wistar rats were divided into three groups: saline (SAL); tauracholate (TAU) and phospholipase A(2) (PLA(2)). Pancreatitis was induced by administration of TAU or PLA(2) from Naja mocambique mocambique into the common bile duct of rats, and after 4 h of duct injection the animals were sacrificed. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP) and phenylephrine (PHE) in isolated mesenteric and pulmonary arteries were obtained. Potency (pEC(50)) and maximal responses (E(MAX)) were determined. Blood samples were collected for biochemical analysis.Results: In mesenteric rings, the potency for ACh was significantly decreased from animals treated with TAU (about 4.2-fold) or PLA(2) (about 6.9-fold) compared to saline group without changes in the maximal responses. Neither pEC(50) nor E(MAX) values for Ach were altered in pulmonary rings in any group. Similarly, the pEC(50) and the E(MAX) values for SNP were not changed in both preparations in any group. The potency for PHE was significantly decreased in rat mesenteric and pulmonary rings from TAU group compared to SAL group (about 2.2- and 2.69-fold, for mesenteric and pulmonary rings, respectively). No changes were seen in the E(MAX) for PHE. The nitrite/nitrate (NO(x)(-)) levels were markedly increased in animals submitted to acute pancreatitis as compared to SAL group, approximately 76 and 68% in TAU and PLA(2) protocol, respectively.Conclusion: Acute pancreatitis provoked deleterious effects in endothelium-dependent relaxing response for ACh in mesenteric rings that were strongly associated with high plasma NO(x)(-) levels as consequence of intense inflammatory responses. Furthermore, the subsensitivity of contractile response to PHE in both mesenteric and pulmonary rings might be due to the complications of this pathological condition in the early stage of pancreatitis.

Interaction between Advanced Glycation End Products Formation and Vascular Responses in Femoral and Coronary Arteries from Exercised Diabetic Rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Non-newtonian flow and pressure drop of pineapple juice in a plate heat exchanger

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An ultraviolet photoacoustic spectroscopy study of the interaction between Lys49-phospholipase A(2) and amphiphilic molecules

We have used near ultraviolet photoacoustic spectroscopy (PAS) over the wavelength range 240-320 nm to investigate the complex formed between the homodimeric bothropstoxin-I, a lysine-49-phospholipase A(2) from the venom of Bothrops jararacussu (BthTx-I), with the anionic amphiphile sodium dodecyl sulfate (SDS). At molar ratios > 10, the complex developed a significant light scatter, accompanied by a decrease in the intrinsic tryptophan fluorescence intensity emission (ITFE) of the protein, and an increase in the near UV-PAS signal. Difference PAS spectroscopy at SDS/BthTx-I ratios < 8 were limited to the region 280-290 nm, suggesting initial SDS binding to the tryptophan 77 located at the dimer interface. At SDS/BthTx-I ratios > 10, the intensity between 260 and 320 nm increases demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interaction. PAS signal phase changes at wavelengths specific for each aromatic residue suggest that the Trp77 becomes more buried on SDS binding, and that protein structural changes and dehydration may alter the microenvironments of Tyr and Phe residues. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scatter is significant. (c) 2006 Elsevier B.V. All rights reserved.

Polycyclic aromatic hydrocarbons from wood pyrolyis in charcoal production furnaces

Polycyclic aromatic hydrocarbons (PAH) were measured in smoke samples from wood carbonization during charcoal production, in both particulate matter (PM) and gaseous phases. Samples were acquired using a medium-volume air sampler at 1.5 m distance from the furnace. Particle-bound PAH were collected on Fluoropore polytetrafluoroethylene filters and gas-phase PAH were collected into sorbent tubes with XAD-2 resin. PAH were extracted with dichloromethane-methanol and analyzed using gas chromatography-mass spectrometry. The results showed total emission from the furnace of 26 mu g/m(3) for the 16 PAH and 2.8 mu g/m(3) for the 10 genotoxic PAH (from fluoranthene to benzo[g,h,i]perylene). High emission of 16 PAH in the first 8 h of wood carbonization was detected (64 mu g/m(3); 56% of the total emission). Associated with PM, 11% of the total emission of 16 PAH (in both phases) and 60% of 10 genotoxic PAH were found. Relative ratios (for example, [Phe]/[Phe] + [Ant]) for the PAH of the same molecular weight were obtained and compared with the published data. The concentrations of benzo[a]pyrene equivalent (BaPeq) were estimated using the list of toxic equivalent factors suggested by Nisbet and LaGoy, 1992. The values of 0.30 and 0.06 mg/m3 were obtained for the total concentrations of BaPeq in PM and gaseous phase, respectively. (c) 2006 Elsevier B.V. All rights reserved.

Effects of central alpha-adrenergic agonists on hormone-induced 3% NaCl and water intake

Male rats received intracerebroventricular (ICV) renin (600 ng) or daily subcutaneous injections of deoxycorticosterone (5 mg) to induce 3% NaCl and water intake. Noradrenaline (NOR; 40-160 nmol) and clonidine (CLO; 5-20 nmol) injected ICV. induced 70 to 100% inhibition of the intakes. Phenylephrine (PHE; 40-160 nmol) injected ICV induced 60 to 95% inhibition of the intakes. NOR and PHE induced a stronger inhibition on the 3% NaCl intake induced by renin than on the intake induced by deoxycorticosterone (DOC), and CLO did the opposite. CLO was always more effective than PHE to induce inhibition of the intakes. The results suggest that NOR inhibits hormone (angiotensin II, aldosterone)-induced NaCl intake by acting mainly on alpha(2)-adrenergic receptors.

Molecular characterization of hemoglobin alpha-D chains from Geochelone carbonaria and Geochelone denticulata land turtles

In order to help elucidate the evolution of alpha-globins, the complete cDNA and amino acid sequences of Geochelone carbonaria and Geochelone denticulata land turtles alpha-D chains have been described. In G. carbonaria, the cDNA is 539 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 520. In G. denticulata, the cDNA is 536 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 517. Both cDNAs codify 141 amino acid residues, differing from each other in only four amino acid residues. When comparing with human Hb alpha-chain, alterations in important regions can be noted: alpha110 Ala-Gly, alpha114 Pro-Gly, alpha117 Phe-Tyr and alpha122 His-Gln. There is a high homology between the amino acids of these turtles when compared with chicken alpha-D chains, progressively decreasing when compared with human, crocodile, snake, frog and fish alpha-chains. Phylogenetic analysis of alpha-D chains shows that those of turtles are closer to those of birds than to snakes and lizards. (C) 2002 Elsevier B.V. All rights reserved.

Polycyclic aromatic hydrocarbons and other selected organic compounds in ambient air of Campo Grande City, Brazil

Fourteen samples of particulate matter and semi-volatile organic compounds were collected during 6 months in the city of Campo Grande, South Mato Grosso State, Brazil. Particle-bound polycyclic aromatic hydrocarbons (PAHs) were collected on Fluoropore PTFE filters and gas-phase PAHs were collected into sorbent tubes with XAD-2 resin. Both types of samples were extracted with a dichloromethane/methanol mixture (4:1 v/v), then the extracts were subjected to gas chromatography-mass spectrometry (GC-MS) analysis. PAHs, oxidized PAH (oxy-PAHs), phenols and methoxyphenols were identified by use of GC retention indices and MS files. The average value obtained for the sum of 15 PAHs was 21.05 ng m(-3) (range: 8.94-62.5 ng m(-3)). The presence of specific tracers and calculations of characteristic ratios (e.g. [Phe]/[Phe] + [Ant]) were used to identify the sources of the emissions of PAHs in the atmospheric samples. Levoglucosan (the anhydride of beta-glucose), retene (1-methyl-7-isopropylphenanthrene) and methoxyphenols (derivatives of syringol and guaiacol) and tracers for wood burning were identified. This study demonstrates that biomass burning from the rural zone is the main source of PAHs and emissions of other substances in the investigated site of Campo Grande. (c) 2004 Elsevier Ltd. All rights reserved.

A molecular mechanism for Lys(49)-phospholipase A(2) activity based on ligand-induced conformational change

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)- phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase active site and the C-terminal myotoxic site, justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Non-newtonian heat transfer on a plate heat exchanger with generalized configurations

For the configuration optimization of plate heat exchangers (PHEs), the mathematical models for heat transfer and pressure drop must be valid for a wide range of operational conditions of all configurations of the exchanger or the design results may be compromised. In this investigation, the thermal model of a PHE is adjusted to fit experimental data obtained from non-Newtonian heat transfer for eight different configurations, using carboxymethylcellulose solutions (CMC) as test fluid. Although it is possible to successfully adjust the model parameters, Newtonian and non-Newtonian heat transfer cannot be represented by a single generalized correlation. In addition, the specific heat, thermal conductivity and power-law rheological parameters of CMC solutions were correlated with temperature, over a range compatible with a continuous pasteurization process.

Synthesis and in vitro evaluation of potential antichagasic dipeptide prodrugs of primaquine

American trypanosomiasis (Chagas' disease) is an endemic parasitic disease afflicting more than 20 million people in Latin America. Currently, therapy is unsatisfactory and only two drugs are available. Primaquine, an antimalarial drug, has trypanocidal activity. Dipeptide derivatives of primaquine, Phe-Arg-PQ, Lys-Arg-PQ, and Phe-Ala-PQ, were synthesized. The choice of the peptides was based on the primary specificity of cruzipain, the major cysteine proteinase from T. cruzi. The prodrugs obtained were tested on the LLC-MK2 cell culture infected with trypomastigotes forms of T. cruzi Phe-Arg-PQ, Lys-Arg-PQ, and Phe-Ala-PQ were active in all stages.

Comparative investigation of the cleavage step in the synthesis of model peptide resins: Implications for N-alpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the N-alpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the N-alpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (All, DRVYIHPF) and its [Gly(8)]-All analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%.) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.

CHARACTERISTICS OF ARTERIAL-HYPERTENSION IN RESPONSE TO BOLUS INJECTION OF PHENYLEPHRINE IN ATROPINIZED PATIENTS

The changes of arterial pressure promoted by bolus injection of 50 mg phenylephrine (PHE) were studied in 20 atropinized patients (5 normal subjects, 13 patients with mitral valve disease, 1 patient with essential arterial hypertension and 1 patient with hypertrophic cardiomyopathy) submitted to routine catheterism. Patients with aortic valve disease, left ventricular outflow tract obstruction and intracardiac shunt were excluded from the study. All patients were in sinus rhythm, without heart failure. Arterial pressure started to increase at 14.8 +/- 5.4 s (range, 5.6 to 27 s; mean +/- SD) after PHE. There was an increase of 37.8 +/- 16.7 mmHg (range, 12.5 to 70 mmHg) in systolic pressure and of 26.6 +/- 11.1 mmHg (range, 7.5 to 42.5 mmHg) in diastolic pressure. Peak hypertension was attained at 36.6 +/- 16.4 s (range, 10.8 to 64.9 s) and hypertension continued for 176 +/- 92 s (range, 11 to 365 s). Heart rate was 114 +/- 21 bpm before PHE and 111 +/- 21 bpm (P<0.05) after PHE. There were no adverse events associated with intravenous PHE injection in any patient, in accordance with the general view that bolus injection of PHE is a safe and practical maneuver to promote arterial hypertension.