Benzyloxycarbonyl-proline-prolinal (ZPP): Dual complementary roles for neutrophil inhibition.

Neutrophil influx and activation contributes to organ damage in several major lung diseases. This inflammatory influx is initiated and propagated by both classical chemokines such as interleukin-8 and by downstream mediators such as the collagen fragment cum neutrophil chemokine Pro-Gly-Pro (PGP), which share use of the ELR + CXC receptor family. Benzyloxycarbonyl-proline-prolinal (ZPP) is known to suppress the PGP pathway via inhibition of prolyl endopeptidase (PE), the terminal enzyme in the generation of PGP from collagen. However, the structural homology of ZPP and PGP suggests that ZPP might also directly affect classical glutamate-leucine-arginine positive (ELR+) CXC chemokine signaling. In this investigation, we confirm that ZPP inhibits PE in vitro, demonstrate that ZPP inhibits both ELR + CXC and PGP-mediated chemotaxis in human and murine neutrophils, abrogates neutrophil influx induced by murine intratracheal challenge with LPS, and attenuates human neutrophil chemotaxis to sputum samples of human subjects with cystic fibrosis. Cumulatively, these data demonstrate that ZPP has dual, complementary inhibitory effects upon neutrophil chemokine/matrikine signaling which make it an attractive compound for clinical study of neutrophil inhibition in conditions (such as cystic fibrosis and chronic obstructive pulmonary disease) which evidence concurrent harmful increases of both chemokine and matrikine signaling.

The impact of the climate on the epidemiology of Dirofilaria immitis in the pet population of the Canary Islands.

Cardiopulmonary dirofilariosis (heartworm) is a zoonotic vector borne disease caused by Dirofilaria immitis which affects domestic dogs and cats. Two of the seven Canary Islands are historically hyperendemic areas of dirofilariosis, although no epidemiological study has ever been carried out which includes the other islands. The aim of the study was to complete the epidemiological status of cardiopulmonary dirofilariosis in the canine and feline population throughout all the Canary Islands. 1643 client-owned dogs and 707 client-owned cats were tested for D. immitis antigens (dogs), and anti-D. immitis and anti-Wolbachia antibodies (cats). The prevalence of canine dirofilariosis in the Canary Islands was 15.7%, and the seroprevalence of feline dirofilariosis was 18.1%. A remarkable disparity was found when evaluating the results by island separately, which ranged from from 0% in Lanzarote and El Hierro, low prevalences and seroprevalences in Fuerteventura (1.8% and 2.5% in dogs and cats, respectively), to higher prevalences on the other 4 islands; ranging between 15.7% (dogs) and 14.3% (cats) in La Palma 22.5% (dogs) and 24.1% (cats) in Tenerife. In addition, prevalences and seroprevalences were very variable within each island, these differences being associated to local climate conditions. The distribution and prevalence of dirofilariosis in the Canary Islands is heterogeneous and related to climate, demographic factors and management of pets in the studied areas. Dirofilariosis remains hyperendemic in 4 of the 7 Islands. Since D. immitis is a zoonosis, veterinary and health authorities should be aware of the current prevalence and seroprevalence of animal dirofilariosis. The results show the need for awareness raising campaigns to promote the implementation of prophylactic measures in pets, in order to achieve a decrease in the prevalence of animal dirofilariosis in the Canary Islands.

Changes in digestive enzymes through developmental and molt stages in the spiny lobster, Panulirus argus.

Changes in major digestive enzymes through developmental and molt stages were studied for the spiny lobster Panulirus argus. There were significant positive relationships between specific activity of trypsin and amylase enzymes and lobster size, whereas esterase and lipase specific activities decreased as lobsters aged. No relationship was found between amylase/trypsin ratio and lobster size. Positive trends were found, however, for trypsin/lipase and amylase/lipase ratios. Results suggest that changes in enzyme activity respond to the lobsters' physiological needs for particular dietary components although multivariate analysis suggested that enzyme activities could be not totally independent of diet. On the other hand, the pattern of changes of major enzyme activities through molt cycle was similar for most enzymes studied. Following molt, trypsin, chymotrypsin, amylase, and lipase activities gradually increased to maximal levels at late intermolt (C4) and premolt (D). There were no variations in the electrophoretic pattern of digestive enzymes through developmental and molt stages and thus, it is demonstrated that regulation is exerted quantitatively rather than qualitatively. Further studies on the effect of other intrinsic and extrinsic factors on digestive enzyme activities are needed to fully understand digestive abilities and regulation mechanisms in spiny lobsters.

Short communication: influence of Staphylococcus aureus intramammary infection on serum copper, zinc, and iron concentrations.

The goal of the present study was to characterize changes in serum trace mineral concentrations in cattle with experimentally induced Staphylococcus aureus mastitis. Nine primiparous Holstein-Friesian cattle were challenged with approximately 150 cfu of Staph. aureus ATCC29740 by intramammary infusion on d 6, 7, and 8 of lactation. Serum Cu, Zn, and Fe concentrations were determined immediately before and at 24, 48, and 72 h after the final intramammary infusion of Staph. aureus. Infection status (cfu/mL of Staph. aureus), milk somatic cell count, and mastitis score were also determined at these times. Infection resulted in a decrease in mean serum Cu, Zn, and Fe concentrations to 89, 83, and 81% of preinfection concentrations at 24 h postchallenge. One-way analysis of variance for repeated measures demonstrated a significant change in serum zinc concentration. The reductions in trace mineral concentrations were of less magnitude than observed following experimental E. coli mastitis.

Protein nitration, metabolites of reactive nitrogen species, and inflammation in lung allografts.

This study investigated nitration and chlorination of epithelial lining fluid (ELF) proteins in patients (n = 29) who had undergone lung allotransplantation. We assayed lung lavage nitrotyrosine (NT) and chlorotyrosine (CT) by HPLC. We measured NT, nitrate (NO(3)(-)), and nitrate (NO(2)(-)) in bronchoalveolar lavage fluid (BALF) and total nitrite (NO(2)(-) + NO(3)(-)) in serum of another group of lung transplant patients (n = 82). In the first group (n = 29), percent nitration of tyrosines (Tyr) (NT/total Tyr x 100) in BALF proteins was: patients, 0.01 (0.00-0.12)%; median (25th-75th% confidence interval), and control subjects 0.01 (0.00-0.02)%. CT (CT/ total Tyr x 100) occurred only in the patients' BALF: 0.01 (0. 00- 0.02)%. In the second group (n = 82), nitrotyrosine (NT) was detected by ELISA in the BALF of patients: 9 (0-41) pmol/mg pro and control subjects: 28 (26-33). Total nitrite (NO(2)(-) + NO(3)(-)) in BALF of the patients: 3.3 (1.9-5.1) microM significantly exceeded that in control subjects: 1.3 (0.8-1.3) microM; p = 0.0133. Serum nitrite also was significantly higher in patients: 37 (26-55) microM than control subjects: 19 (17-20) microM; p = 0.0037. Airway inflammation in transbronchial biopsies (B score) correlated with NT in BALF (p = 0.0369). Lung transplants have increased airway concentrations of reactive nitrogen species (RNS) metabolites. NT, a marker of peroxynitrite (ONOO(-)), is related to the degree of airway inflammation in lung transplants.

Nitric oxide and peroxynitrite in the perinatal period.

Many of the actions of nitric oxide are not due to nitric oxide itself, but rather by the secondary formation of oxidants like peroxynitrite. Peroxynitrite leaves a footprint in the nitration of tyrosine, which helps track the formation of reactive nitric oxide-derived species in diseases and even normal development.

Anti-nitrotyrosine antibodies for immunohistochemistry.

Nitrotyrosine is an important marker for the formation of peroxynitrite and possibly other reactive nitrogen species derived from nitric oxide in vivo (1). Pathological conditions can substantially increase the production of nitric oxide, yet this molecule itself does not generally yield nitration of tyrosine residues in proteins when added to biological samples (1,2). However nitric oxide reacts at near diffusion-limited rates with superoxide (O(2) (-)) to form the strong oxidant peroxynitrite (ONOO(-)) (3). Nitration on the 3-position of tyrosine is a major product of peroxynitrite attack on proteins (4,5). Certainly, small amounts of nitrotyrosine can be produced in vivo by other mechanisms (6), but peroxynitrite is by far the most efficient mechanism for nitrating tyrosine under biologically relevant conditions with natural antioxidants and alternative targets present.

Survival, lung injury, and lung protein nitration in heterozygous MnSOD knockout mice in hyperoxia.

This study tested whether a strain of heterozygous Mn superoxide dismutase (SOD) knockout mice differed from wild types in response to lethal (100 or 85%) or sublethal (50 or 75%) oxygen exposures. Lung MnSOD activity was significantly (-40%) less in the heterozygous mice, and lung catalase activity was also significantly decreased. Total SOD activity, glutathione peroxidase, and glutathione reductase did not differ between heterozygous (+/-) and wild-type (+/+) mice. We exposed both heterozygous and wild-type mice to hyperoxia (50, 75, 85, or 100% oxygen) until death or for 48 hours to assess sublethal lung injury. Survival of the heterozygous and wild-type mice did not differ significantly in 100 or 85% oxygen. No mice of either genotype died in 50 or 75% oxygen (14-day exposures). Hyperoxia exposures significantly increased (by two-way ANOVA) the alveolar lavage protein concentration, percent neutrophils, and lung wet-dry/dry weight ratios. No significant differences occurred between the heterozygous and wild-type mice for any marker of injury at any oxygen level. Lavage fluid total nitrite concentrations did not differ at any oxygen level. Hyperoxia caused a similar degree of nitration of lung structural proteins detected by immunohistochemistry in both groups.

Immunohistochemical methods to detect nitrotyrosine.

The immunohistochemical detection of nitrotyrosine is a robust method for detecting peroxynitrite and other reactive nitrogen species. Success depends on optimizing conditions for the particular tissue and experimental design under investigation and the use of positive and negative controls to verify specificity. The two controls of dithionite reduction and blocking with nitrotyrosine are a powerful combination to demonstrate specificity. The pathological significance of tyrosine nitration in proteins can also be approached. Generally, nitrated proteins can be isolated from diseased tissues by immunoprecipitation and Western blotting. The sites of nitration on specific proteins can be determined by mass spectrometry, which has revealed surprising specificity in which tyrosines and/or proteins are nitrated in vivo. This provides important evidence concerning the functional consequences of peroxynitrite formation in vivo.

Peroxynitrite irreversibly decreases diastolic and systolic function in cardiac muscle.

Much of the damaging action of nitric oxide in heart may be due to its diffusion-limited reaction with superoxide to form peroxynitrite. Direct infusion of peroxynitrite into isolated perfused hearts fails to model the effects of in situ formation because the bulk of peroxynitrite decomposes before reaching the myocytes. To examine the direct effects of peroxynitrite on the contractile apparatus of the heart, we exposed intact and skinned rat papillary muscles to a steady state concentration of 4-microM peroxynitrite for 5 min, followed by a 30-min recovery period to monitor irreversible effects. In intact muscles developed force fell immediately to 26% of initial force, recovering to 43% by 30 min. Resting tension increased by 600% immediately, and was still elevated 500% by 30 min. Nitrotyrosine immunochemistry showed that peroxynitrite can induce tyrosine nitration at low concentrations and is capable of penetrating 200-380 microm into the papillary muscle after a 5-min infusion. Decomposed peroxynitrite had no effect on either intact or skinned muscle developed force or resting tension. Our results show that peroxynitrite directly damages both developed force and resting tension of isolated heart muscle, which can be extrapolated to systolic and diastolic injury in intact hearts.

Antisphingolipid antibodies in the sera of leprosy patients.

Earlier we reported the presence of significant levels of antigalactocerebroside (GalC) antibodies in the sera of leprosy patients. This study corroborates the above result and also gives evidence for the presence of antibodies to the nonpolar ceramide (Cer) moiety of GalC. AntiCer antibody titres were higher as compared to antiGalC antibodies in all categories of leprosy. The specificity of antibodies directed to the Cer moiety was confirmed using Lactosyl-BSA and neutralization assays. Statistically significant and positive correlations were observed between antiGalC and antiCer antibodies. Responsiveness factors were computed using natural logarithmic transformation of the variables.

Changes in the optical properties of liposome dispersions in relation to the interlamellar distance and solute interaction.

The changes in absorbance produced when liposomes are subject to increasing osmotic pressures were correlated with the distance at which the undulation, hydration and steric repulsions dominate. It is found that at low pressures, when the bilayers are apart by more than 1 nm, the absorbance decreases with the decrease in the bilayer distance. However, at higher pressures where the bilayer are in contact within 0.7 nm the absorbance increases with the increase in pressure. This is well explained by the scattering law for particles of diameter comparable to the wavelength and fits with the empirical Bangham's law used for permeability assays. At much higher pressures, a break in the absorbance at 0.5 nm of the interbilayer distance denotes that absorbance is sensitive to the perturbation when steric forces dominate. These effects were compared to those obtained with solutes that may replace water at the membrane interface by hydrogen bonding. The results indicate that the membrane approach produces a similar effect to sucrose on both calorimetric and optical properties, suggesting that the bilayer interaction promotes a partial dehydration or reorganization of the water at the interface. The relevance of these findings on the permeation assays done with vesicles and cells by means of light scattering in which the bilayers are considered unperturbed is discussed.

Physical mapping of 49 microsatellite markers on chromosome 19 and correlation with the genetic linkage map.

We have regionally localized 49 microsatellite markers developed by Généthon using a panel of previously characterized somatic cell hybrids that retain fragments from chromosome 19. The tight correlation observed between the physical and the genetic orders of the microsatellites provide cytogenetic anchorages to the genetic map data. We propose a position for the centromere just above D19S415, from the study of two hybrids, each of which retains one of the two derivatives of a balanced translocation t(1;19)(q11;q11). Microsatellites, which can be identified by a standard PCR protocol, are useful tools for the localization of disease genes and for the establishment of YAC or cosmid contigs. These markers can also judiciously be used for the characterization of new hybrid cell line panels. We report such a characterization of 11 clones, 8 of which were obtained by irradiation-fusion. Using the whole hybrid panel, we were able to define the order of 12 pairs of genetically colocalized microsatellites. As examples of gene mapping by the combined use of microsatellites and hybrid cell lines, we regionally assigned the PVS locus between the 19q13.2 markers D19S417 and D19S423 and confirmed the locations of fucosyltransferase loci FUT1, FUT2, and FUT5.

Properties of gel phase lipid-trehalose bilayers upon rehydration.

When dipalmitoylphosphatidylcholine bilayers dried under vacuum in different concentrations of trehalose are rehydrated in buffer without the sugar they show different physicochemical properties in the gel state in comparison to the normal gel state. Dry DPPC/trehalose mixtures are readily dispersed in buffer below the phase transition showing by electron microscopy a morphology similar to liposomes prepared by dispersing the lipids in buffer above the phase transition temperature. In these conditions, an increase in the peak at 570 nm of merocyanine after the dehydration-rehydration process in the presence of the sugar is observed and the water permeation increases to values comparable to those found in the fluid state as indicated by the activation energy values and the osmotic volume. The trehalose-dried liposomes rehydrated in buffer show a similar osmotic response to hypertonic gradient as DPPC liposomes without sugar near the phase transition temperature. In accordance with this behavior the trehalose-dried liposomes are lysed below the phase transition temperature by lysoderivatives. These modifications of the gel state of hydrated phospholipids by trehalose can only be achieved if a drastic dehydration is performed in the presence of the sugar. After rehydration the changes in the gel state can be detected after dyalizing the rehydrated membranes in media without trehalose during at least 24 h. These results suggest that trehalose is still intercalated between the phospholipids after restoring water to the dried liposomes either at temperatures below or above the phase transition.

Different functional pools of acetylcholinesterase induce changes in rat locus coeruleus noradrenaline metabolism.

The effects of acetylcholinesterase (AChE) inhibition in the locus coeruleus (LC) were studied in rats utilizing fasciculin (FAS) and BW248c51 (BW). Both inhibitors were stereotaxically injected into the right LC and the animals were sacrificed 24 h later. Similar groups received atropine (30 mg/kg i.p.) every 5 h during 24 h. Another group of FAS-treated rats received naloxone twice (5 mg/kg i.p.) in 24 h. Other groups of FAS-treated rats were sacrificed 3 and 7 days after injection. An inhibition of 70% of LC AChE activity was observed 24 h after FAS or BW injection. Either FAS or BW induced a significant increase in NA levels in the injected LC compared to control values. Atropine treatment failed to block the FAS effect but it was able to counteract the BW-induced NA increase. NA levels were still increased 3 days after FAS treatment and returned to control values at day 7.

Somatostatin prevents acute pancreatitis after pancreatic duct sphincter hydrostatic balloon dilation in patients with idiopathic recurrent pancreatitis.

The purpose of this study was to determine whether prophylactic somatostatin infusion can prevent pancreatitis after hydrostatic balloon dilation of the pancreatic duct sphincter segment in 16 patients with idiopathic recurrent pancreatitis. This study demonstrated that prophylactic administration of somatostatin before, during, and after the procedure diminished the incidence and severity of acute pancreatitis. We recommend consideration of such prophylaxis in patients undergoing this procedure.

Effects of local inhibition of locus coeruleus acetylcholinesterase by fasciculin in rats.

The inhibition of locus coeruleus (LC) acetylcholinesterase (AChE) by Fasciculin II (FAS), a novel anticholinesterase peptide from the green mamba (Dendroaspis angusticeps) venom, was studied in rats. FAS was stereotaxically injected (0.5 microliters of a 1 mg/ml solution) in the right LC. The left LC was taken as control. A group of rats received only saline injected with the same procedure. An inhibition of 80% of LC AChE activity was observed 24 h later. Monoamine and metabolite levels were assessed by high-performance liquid chromatography (HPLC) with electrochemical detection. A significant increase of noradrenaline (NA) levels was found in the injected side when compared with controls 24 h after injection. Neither dopamine, serotonin nor their metabolites or the NA metabolite 4-methoxyhydroxyphenylglycol showed any change after FAS injection. Atropine (30 mg/kg, i.p.) did not prevent the NA increase.

Intrastriatal dendrotoxin injection: behavioral and neurochemical effects.

Unilateral striatal injection of dendrotoxin (DTX), a polypeptide isolated from the venom of the snake Dendroaspis angusticeps, in rats provoked a complex behavioral syndrome characterized by spontaneous circling towards the contralateral side, stereotypic like chewing movements and gnawing, abnormal postures and convulsions. All these symptoms achieved their maximum on the first day, disappearing during the first week after injection. Neurochemical analyses of striatal monoamines and monoamine metabolites showed a significant increase of dopamine and serotonin metabolites 20 hr after DTX injection. A group of animals sacrificed 15 days after toxin administration showed normal levels of monoamines and their metabolites, except for homovanillic acid levels which were still significantly increased. These data indicate that monoamines are involved in the behavioral syndrome elicited by DTX and are possibly related to its excitatory effect upon brain structures in vivo.

Endoscopic cholangiopancreatography in the infant: evaluation of a new prototype pediatric duodenoscope.

The usefulness of a new pediatric duodenoscope PJF in the diagnosis of neonatal cholestasis was studied in 23 infants with ages ranging from 19 to 150 days. In 22 of 23 infants the papilla was cannulated. In 13 of 14 neonates (93%) with neonatal hepatitis, the common bile duct was opacified and biliary atresia was excluded. In one of two neonates with choledochal cyst, the common bile duct was demonstrated. In six of seven neonates (86%) with biliary atresia, only the pancreatic duct was demonstrated and the diagnosis was suspected. Although absence of a common bile duct opacification does not rule out biliary atresia, ERCP with the new duodenoscope proved to be most useful in the diagnosis of normal biliary tree and served to avoid unnecessary surgery in most infants with neonatal cholestasis.