Fluorogenic peptide substrates for carboxydipeptidase activity of cathepsin B

Cathepsin B is a lysosomal cysteine protease exhibiting mainly dipeptidyl carboxypeptidase activity, which decreases dramatically above pH 5.5, when the enzyme starts acting as an endopeptidase. Since the common cathepsin B assays are performed at pH 6 and do not distinguish between these activities, we synthesized a series of peptide substrates specifically designed for the carboxydipeptidase activity of cathepsin B. The amino-acid sequences of the P(5)-P(1) part of these substrates were based on the binding fragments of cystatin C and cystatin SA, the natural reversible inhibitors of papain-like cysteine protease. The sequences of the P'(1)-P'(2) dipeptide fragments of the substrates were chosen on the basis of the specificity of the S'(1)-S'(2) sites of the cathepsin B catalytic cleft. The rates of hydrolysis by cathepsin B and papain, the archetypal cysteine protease, were monitored by a continuous fluorescence assay based on internal resonance energy transfer from an Edans to a Dabcyl group. The fluorescence energy donor and acceptor were attached to the C- and the N-terminal amino-acid residues, respectively. The kinetics of hydrolysis followed the Michaelis-Menten model. Out of all the examined peptides Dabcyl-R-L-V-G-F- E(Edans) turned out to be a very good substrate for both papain and cathepsin B at both pH 6 and pH 5. The replacement of Glu by Asp turned this peptide into an exclusive substrate for cathepsin B not hydrolyzed by papain. The substitution of Phe by Nal in the original substrate caused an increase of the specificity constant for cathepsin B at pH 5, and a significant decrease at pH 6. The results of kinetic studies also suggest that Arg in position P(4) is not important for the exopeptidase activity of cathepsin B, and that introducing Glu in place of Val in position P(2) causes an increase of the substrate preference towards this activity.     

Prereplicative purine methylation and postreplicative demethylation in each DNA duplication of the Escherichia coli replication cycle

Escherichia coli plasmid DNA activated for initiation of duplication is in a stable low linking number supercoiled conformation. Low linking number DNA is methylated at the internal purines of a frequent 5'-Pyr-Pyr-Pur-Pur tetramer with a 5'-Pyr-Pur-3' axis of symmetry and is cut at the axis of symmetry by pneumococcal restriction enzyme DpnI when methylated in both strands. Purine methylation is of adenine in one strand and guanine in the other. Methylation of one of the two purines is removed during the cell cycle, presumably before the reverse shift to the B-supercoiled conformation. The topological transition was reconstituted in vitro only with DNA unmethylated at purines. Methylation-restriction analyses coupled with the chemical properties of low-linking number DNA and B-DNA respectively, suggest that removal of guanine methylation is essential for the low-linking number to B-DNA transition and hence for the deactivation of replication. Demethylation of methylguanine could explain the presence in E. coli of the two-member inducible operon known as ada. Characteristics of ada suggest a cascade of chemical DNA modifications that reverse prereplicative guanine methylation. Guanine demethylation could provide a model for the pivotal role played by de novo methylation in replication and for the essential role of "repair" enzyme ExoIII in demethylation leading to the reversal of replicative DNA activation and other processes that affect DNA function.     

Pulsed magnetic fields applied to a transferred arterial loop support the rat groin composite flap

Pulsed magnetic fields have been shown to stimulate neovascularization in the authors' laboratory. The rat groin composite flap was used to create a prospective randomized trial to test the effectiveness of these pulsed magnetic fields. The skin paddle to this flap is highly consistent, and the authors proposed using the flap to study how pulsed magnetic fields affect composite flap survival when the dominant vessel to the flap is divided and flap survival becomes dependent on a transferred vessel loop. Forty-three rats had the tail artery microsurgically anastomosed to the femoral artery and placed between the groin musculature and the abdominal skin. Pulsed magnetic energy of 1 gauss was applied for 8 (n = 14) or 12 (n = 8) weeks to the experimental groups. Control groups were treated in a comparable manner for 8 (n = 16) or 12 (n = 5) weeks. After the 8 or 12 weeks, all groups had an 8 x 4-cm skin flap raised, and the superficial epigastric artery, the main feeding vessel, was ligated. After 5 days, the total area of the flap and the area of necrosis were traced onto velum paper for each rat. The percent survival was calculated per rat, and a mean survival percentage was calculated per group. The experimental animals treated with pulsed magnetic fields for 8 weeks had statistically significant improved flap survival over the control animals. The study provides evidence that pulsed magnetic energy stimulates angiogenesis and suggests a possible use of this modality to create island vascular flaps in otherwise random vascular territories.     

In situ hybridization of hepatitis C virus RNA in liver cells of an experimentally infected rhesus macaque

The liver tissue of a rhesus macaque inoculated with hepatitis C virus (HCV) has been analyzed for the presence of HCV RNA using the technique of in situ hybridization, both at light and electron microscopy levels. The animal was inoculated by the intrasplenic route using a HCV infected autogenic hepatocyte transplant. The serum sample used to infect the hepatocyte cells was characterized by polymerase chain reaction technique and shown to be positive for HCV RNA, genotype 3 with 10(7) RNA copies/ml. In situ hybridization was performed using a complementary negative strand probe made with the specific primer. We were able to detect and localize viral RNA in altered membranes of the rough endoplasmic reticulum of infected liver cells, showing evidence of virus replication in vivo.     

Chromosome 17p12-q11 harbors susceptibility loci for systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against intracellular components, the formation of immune complexes, and inflammation in various organs, typically the skin and kidney glomeruli. The etiology of the disease is not well understood but is most likely the result of the interaction between genetic and environmental factors. In order to identify susceptibility loci for SLE, we have performed genome scans with microsatellite markers covering the whole genome in families from Argentina, Italy, and Europe. The results reveal a heterogeneous disease with different susceptibility loci in different family sets. We have found significant linkage to chromosome 17p12-q11 in the Argentine set of families. The maximum LOD score was given by marker D17S1294 in combination with D17S1293, when assuming a dominant inheritance model (Z=3.88). We also analyzed a repeat in the promoter region of the NOS2A gene, a strong candidate gene in the region, but no association was found. The locus on chromosome 17 has previously been identified in genetic studies of multiple sclerosis families. Several other interesting regions were found at 1p35, 1q31, 3q26, 5p15, 11q23 and 19q13, confirming previously identified loci for SLE or other autoimmune diseases.A list of members of the Collaborative Group on the Genetics of SLE and the Argentine Collaborative Group is given in the AppendixBernardo Pons-Estel is the coordinator of the Argentine Collaborative Group     

Duplex-Doppler ultrasonography in the detection of lower extremities deep venous thrombosis and in the detection of alternative findings

The diagnoses observed in patients referred for the Doppler ultrasonographic examination of peripheral and iliac veins for suspected deep venous thrombosis (DVT) are presented in this study. During 48 months 2,610 patients were examined by duplex Doppler ultrasonography (US). Among these, 1,879 were women (72%) and 731 men (28%), with the age-range 16-91 (mean 56, 2) years. Ultrasonic scanners Acuson 128 XP 10, ATL HDI 5000, GE Logiq 7, and GE Logiq 9 were used, with transducers in the frequency range from 2.5-14 MHz. Findings were categorized into four main categories: (1) deep venous thrombosis (DVT); (2) pathology predominantly related to superficial veins without DVT, (3) pathology of adjacent structures; (4) normal findings. 562 patients had DVT (21.5%). 1,108 patients (42.5%) had predominant pathology of superficial veins: postthrombotic syndrome, superficial thrombophlebitis and varicose veins. 390 patients (14.9%) had pathology of surrounding structures, unrelated to veins, the most common pathology being popliteal cysts and muscular hematomas. These lesions must be properly diagnosed by US to avoid erroneous anticoagulant treatment.     

Crystal structure of a fragment of mouse ubiquitin-activating enzyme

Protein ubiquitination requires the sequential activity of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligase (E3). The ubiquitin-transfer machinery is hierarchically organized; for every ubiquitin-activating enzyme, there are several ubiquitin-conjugating enzymes, and most ubiquitin-conjugating enzymes can in turn interact with multiple ubiquitin ligases. Despite the central role of ubiquitin-activating enzyme in this cascade, a crystal structure of a ubiquitin-activating enzyme is not available. The enzyme is thought to consist of an adenylation domain, a catalytic cysteine domain, a four-helix bundle, and possibly, a ubiquitin-like domain. Its adenylation domain can be modeled because it is clearly homologous to the structurally known adenylation domains of the activating enzymes for the small ubiquitin-like modifier (SUMO) and for the protein encoded by the neuronal precursor cell-expressed, developmentally down-regulated gene 8 (NEDD8). Low sequence similarity and vastly different domain lengths make modeling difficult for the catalytic cysteine domain that results from the juxtaposition of two catalytic cysteine half-domains. Here, we present a biochemical and crystallographic characterization of the two half-domains and the crystal structure of the larger, second catalytic cysteine half-domain of mouse ubiquitin-activating enzyme. We show that the domain is organized around a conserved folding motif that is also present in the NEDD8- and SUMO-activating enzymes, and we propose a tentative model for full-length ubiquitin-activating enzyme.     

Complications after primary and secondary transsclerally sutured posterior chamber intraocular lens implantation

This retrospective study analyses and compares early complications during the first month after primary and secondary posterior chamber implantation of transsclerally sutured IOL. The analysis covered medical records of 65 patients who underwent posterior chamber implantation of transsclerally sutured IOL at the Eye Clinic in Rijeka between 1998 and 2003. In 30 patients (group 1) lenses were implanted in one eye during complicated cataract surgery (primary implantation), whereas 35 patients (group 2) had lenses implanted afterwards (secondary implantation). There were 77 early complications, equally represented in both groups, i.e. 40 in (51.9%) the first and 37 (48.1%) in the second group. The most frequent complications were: vitreous hemorrhages 24.7% (14.3% and 10.4%), cystoid macular edema 19.5% (9.1% and 10.4%), keratopathy 14.3% (6.5% and 7.8%), pupil distortion 11.7% (9.1% and 2.6%), IOL decentration and tilt 10.4% (6.5% and 3.9%), high intraocular pressure 9.1% (2.6% and 6.5%), inflammation 6.5% (2.5% and 3.9%). Retinal and choroidal detachment had low incidence: 2.6% (1.3% and 1.3%) and 1.3% (0% and 1.3%) respectively. As concerns early complications, there were no statistically significant differences between the two groups, except for pupil distortion, which was more frequent in primary IOL implantation (p = 0.045). After primary implantation of IOL, the average visual acuity was 0.38 +/- 0.27, whereas after secondary implantation visual acuity was 0.52 +/- 0.21. The difference was not statistically significant.     

Anticonvulsant effects of the wasp Polybia ignobilis venom on chemically induced seizures and action on GABA and glutamate receptors

Venoms of spiders and wasps are well recognized to present high affinity to the central nervous tissue of many mammalian species. Here we describe the effects of direct exposure of rat (Rattus norvegicus) brains to the crude and denatured venom of the Brazilian social wasp Polybia ignobilis. Lower doses of crude venom injected via intracerebroventricular (i.c.v.) inhibited the exploratory activity of animals, while higher doses provoked severe generalized tonic-clonic seizures, with hind limb extension. The status epilepticus lasted for few minutes leading the animals to respiratory depression and death. In contrast, the denatured venom was anticonvulsant against acute seizures induced by the i.c.v. injection of bicuculline, picrotoxin and kainic acid, but it was ineffective against seizures caused by systemic pentylenetetrazole. Moreover, the [3H]-glutamate binding in membranes from rat brain cortex was inhibited by the denatured venom in lower concentrations than the [3H]-GABA binding. The denatured venom contains free GABA and glutamate (34 and 802 pg/microg of venom, respectively), but they are not the major binding inhibitors. These interactions of venom components with GABA and glutamate receptors could be responsible for the anticonvulsant effects introducing the venom from P. ignobilis as a potential pharmacological source of anticonvulsant drugs.     

Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3,5-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). -Nitro-l-arginine methyl ester (l-NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.