Extracellular proteinases of Candida species pathogenic yeasts.

The increased incidence of severe disseminated infections caused by the opportunistic yeast-like fungi Candida spp. highlights the urgent need for research into the major virulence factors of these pathogens-extracellular aspartic proteinases of the candidapepsin and yapsin families. Classically, these enzymes were considered to be generally destructive factors that damage host tissues and provide nutrients for pathogen propagation. However, in recent decades, novel and more specific functions have been suggested for extracellular candidal proteinases. These include contributions to cell wall maintenance and remodeling, the formation of polymicrobial biofilms, adhesion to external protective barriers of the host, the deregulation of host proteolytic cascades (such as the complement system, blood coagulation and the kallikrein-kinin system), a dysregulated host proteinase-inhibitor balance, the inactivation of host antimicrobial peptides, evasion of immune responses and the induction of inflammatory mediator release from host cells. Only a few of these activities recognized in Candida albicans candidapepsins have been also confirmed in other Candida species, and characterization of Candida glabrata yapsins remains limited.

Charge Stabilized Silver Nanoparticles Applied as Antibacterial Agents.

Monodisperse silver nanoparticle sols were synthesized via chemical reduction processes in aqueous environment without using polymeric stabilizing agents or surfactants. The sols obtained using various reducing agents; inorganic cell permeabilizers and organic phenolic compounds; inter alia gallic acid (GA) and tannin (TA) were thoroughly characterized by various physicochemical methods such as TEM, SEM, AFM, DLS and micro-electrophoresis. The antibacterial activity of the sols against two E. coli strains was characterized via the determination of the Minimum Bactericidal Concentration (MBC). All sols exhibited a pronounced bactericidal effect against the standard K12 strain, especially the GA and TA sols showing MBC concentration as low as 1-5 mg L(-1). In the case of the antibiotic resistant strain the highest activity (MBC of 10 mg L(-1)) was observed for the sol synthesized using sodium hypophosphate and sodium tripolyphosphate. Additionally, interactions of silver nanoparticles with bacteria cell were studied using TEM and AFM imaging. It was shown that the silver particles attach to the bacteria surface inducing disintegration, which enables their penetration inside the bacteria. Our measurements confirmed that the surface chemistry of silver nanoparticles can play a decisive role.

Moonlighting proteins as virulence factors of pathogenic fungi, parasitic protozoa and multicellular parasites.

The delicate balance between eukaryotic pathogens and their human hosts during the initiation and development of infection is a complex process involving many diverse interactions. Different infectious agents, including pathogenic fungi, parasitic protozoa and multicellular parasites, directly interact through their cell surface with epithelial or endothelial cells of the human host as well as various proteinaceous host ligands such as extracellular matrix or plasma proteins. Eukaryotic pathogens possess a number of virulence factors but a relatively recently recognized and particularly interesting group of factors capable of enhancing virulence is the set of so-called 'moonlighting proteins'. This term was coined for a relatively large collection of housekeeping enzymes lacking special targeting motifs that would determine their extracellular localization, but that are often present at the cell surface of pathogen. Several such enzymes with key metabolic functions in glycolysis, the pentose phosphate cycle or other fundamental intracellular processes perform entirely new, non-catalytic roles often associated with adhesion to host ligands. Our current study summarizes some of the current knowledge of interesting moonlighting proteins which play putative or confirmed roles as virulence factors in pathogenic fungi, parasitic protozoa and multicellular parasites.

A high-density, integrated genetic linkage map of lettuce (Lactuca spp.).

An integrated map for lettuce comprising of 2,744 markers was developed from seven intra- and inter-specific mapping populations. A total of 560 markers that segregated in two or more populations were used to align the individual maps. 2,073 AFLP, 152 RFLP, 130 SSR, and 360 RAPD as well as 29 other markers were assigned to nine chromosomal linkage groups that spanned a total of 1,505 cM and ranged from 136 to 238 cM. The maximum interval between markers in the integrated map is 43 cM and the mean interval is 0.7 cM. The majority of markers segregated close to Mendelian expectations in the intra-specific crosses. In the two L. saligna x L. sativa inter-specific crosses, a total of 155 and 116 markers in 13 regions exhibited significant segregation distortion. Data visualization tools were developed to curate, display and query the data. The integrated map provides a framework for mapping ESTs in one core mapping population relative to phenotypes that segregate in other populations. It also provides large numbers of markers for marker assisted selection, candidate gene identification, and studies of genome evolution in the Compositae.

Wavelet mapping of sleep spindles in young patients with epilepsy.

Using the wavelet mapping of sleep spindles we investigated influence of focal epilepsy on spindle generation. We found that the maximum of sleep spindle intensity is usually localized away from the epileptic focus. We discuss the possibility of the application of wavelet mapping for localization of epileptic foci prior to epileptic neurosurgery.

GenomePixelizer--a visualization program for comparative genomics within and between species.

GenomePixelizer is a visualization tool that generates custom images of the physical or genetic positions of specified sets of genes in whole genomes or parts of genomes. Multiple sets of genes can be shown simultaneously with user-defined characteristics displayed. It allows the analysis of duplication events within and between species based on sequence similarities. The program is written in Tcl/Tk and works on any platform that supports the Tcl/Tk toolkit. GenomePixelizer generates HTML ImageMap tags for each gene in the image allowing links to databases. Images can be saved and presented on web pages.

[Monitoring of renal function in epileptic children and teenagers treated with valproic acid or carbamazepine in concomitant therapy with tiagabine]. / Monitorowanie czynnosci nerek u dzieci i mlodziezy chorych na padaczke leczonych kwasem walproinowym lub karbamazepina w terapii skojarzonej z tiagabina.

The aim of the study was the evaluation of the influence of 4-month concomitant tiagabine (TGB) and valproic acid (VPA) or carbamazepine (CBZ) therapy on renal function of epileptic children and teenagers. Initial parameter values, indicated on renal disfunction, were compared with these obtained after VPA and TGB or CBZ and TGB therapy and with values in healthy children and teenagers. Investigation group was composed of 22 children and teenagers with drug-resistant focal epilepsy. We observed that in the time of concomitant VPA and TGB therapy increased the NAG/g creatinine activity index. In spite the fact of statistical significance of these changes, they were not outside the normal range. beta 2-microglobulin concentrations in urine of epileptic children treated with VPA in monotherapy before concomitant therapy with TGB were higher than in control group. That difference was statistically significant. Addition of TGB to the therapy normalized this parameter. During concomitant VPA and TGB or CBZ and TGB therapy we didn't observe statistically significant changes of parameters indicating on glomerular disfunction. In the VPA therapy before concomitant treatment with tiagabine the disfunction of tubules and glomerules was observed. On the other side in the concomitant VPA and TGB therapy the disfunction of tubules and glomerules didn't occurred. We can conclude that concomitant therapy VPA or CBZ with tiagabine don't affect the renal function in clinical significant manner. Therapy with VPA could leads to minimal disfunction of tubules what is represented by increasing of beta 2-microglobulin level in urine.

[Interactions of antiepileptic drugs]. / Interakcje leków przeciwpadaczkowych.

Because antiepileptic drugs have narrow therapeutic ranges these drug interactions has received much attention. In the paper the main problems concerning interactions of old and novel antiepileptic drugs are presented.

Some molecular and enzymatic properties of a homogeneous preparation of thiaminase I purified from carp liver.

A homogeneous preparation of thiaminase I (thiamine:base 2-methyl-4-aminopyrimidine-5-methenyl transferase, EC 2.5.1.2) was obtained from carp liver, for the first time from a nonbacterial source. Its molecular mass was 55 kDa by gel filtration and by SDS-PAGE regardless the presence of the reducing agent, indicating that the native enzyme consists of a single polypeptide chain. The determined sequence of 20 residues at the N-terminal of carp thiaminase I seemed to be unique. The enzyme was tested for ability to decompose a number of thiamine analogues. Even very extensive modifications of the thiazolium fragment were well tolerated, but around the pyrimidine fragment the active center seemed to exert steric restrictions against 1' (N)- and 2' (C)- atoms, while the 4'-amino group and untouched 6'-carbon atom were absolutely essential for the enzyme action. Numerous nucleophiles could be used by the enzyme as cosubstrates, aniline, pyridine, and 2-mercaptoethanol being the best among compounds tested. Protein chemical modification experiments indicated that histidine residues, carboxyl groups, and sulfhydryl groups may play specific roles in the thiaminase I-catalyzed reaction. Like in the bacterial enzyme, a sulfhydryl group may be a catalytically critical active-site nucleophile. The histidine residues and carboxyl groups may be essential for thiamine binding to the active site.

Fast, isotope-free methods for the assay of thiamine-binding proteins and for the determination of their affinities to thiamine-related compounds.

A fast, isotope-free method for the determination of parameters for the interactions of proteins with thiamine and related compounds was developed. The free and bound forms of a ligand (thiamine or a fluorogenic analogue) were separated by ultrafiltration using commercially available centrifugal protein microconcentrators (Nanosep, Pall Filtron). The free thiamine concentration in the filtrate was analysed by (i) a pre-column derivatisation of thiamine to thiochrome with the use of alkaline potassium hexacyanoferrate(III) followed by reverse-phase HPLC (isocratic, analytical ODS column, 10 mM potassium phosphate, pH 7.8, 5% tetrahydrofuran) with fluorometric detection (excitation at 365 nm, emission at 430 nm), or (ii) an ion-pair reverse-phase HPLC (isocratic, ODS column, 0.08% trifluoroacetic acid-0.08% sodium octanesulfonate-25% tetrahydrofuran) with post-column derivatisation and fluorometric detection. The 'saturation-binding' version (single ligand added in increasing doses to the protein samples) of this method allowed the determination of low micromolar concentrations of thiamine-binding proteins and of the dissociation constants of their complexes with thiamine or fluorogenic thiamine analogues in the range of 0.3-10 microM. Using the other, 'competitive displacement' version (constant amount of thiamine plus increasing doses of a competing ligand), dissociation constants at least one order of magnitude higher could successfully be determined.

[The use of lamotrigine monotherapy in children with newly diagnosed partial epilepsy]. / Zastosowanie lamotriginy w monoterapii dzieci z nowo zdiagnozowana padaczka czesciowa.

Lamotrigine (Lamictal, Glaxo Wellcome) is a drug which is used as add-on therapy in patients with refractory epilepsy. Several previous studies have demonstrated the efficacy of lamotrigine monotherapy, but only few have been done in pediatric patients. The aim of our study was the assessment of efficacy and tolerability of lamotrigine monotherapy in children with newly diagnosed partial epilepsy. Lamictal was used in 19 children (11 boys and 8 girls), aged 3-16 years. 17 patients demonstrated complex partial seizures (with or without secondarily generalisation), 2 children had simplex partial seizures. Symptomatic epilepsy was diagnosed in 10 patients and cryptogenic epilepsy in 9 cases. The drug was administered at the dose of 3.87 +/- 1.02 mg/kg/day during 24 weeks. Three children withdrew from the study because of adverse events: one patient developed rash, two ones seizure exacerbation. Lamictal produced of at least 50% reduction in seizure frequency in 12 (63.15%) children, included 10 seizure-free patients. One third patients experienced EEG improvement. The most common adverse effects were gastrointestinal and sleep disturbances, infections, dizziness, all of them were mild and transient and observed more often in children under 12 years of age. Lamotrigine monotherapy is effective and safe for treatment of newly diagnosed cryptogenic and symptomatic epilepsy with partial seizures but further studies are necessary specially in young children.

[Vigabatrin in the treatment of intractable focal epilepsy in children and adolescents. Two-year study]. / Wigabatryna w leczeniu lekoopornych padaczek ogniskowych u dzieci i mlodziezy. Badania dwuletnie.

UNLABELLED: Vigabatrin VGB is the drug of III generation applied for a long time to patients in Poland. In Polish literature there were many reports published concerning the efficacy of VGB, however, most are short-term examinations. The AIM OF THE STUDY WAS: 1. the analysis of VGB efficacy in long term treatment of intractable focal epilepsies in children and adolescents; 2. stating if efficacy of VGB treatment depends on localization of epileptic focus; 3. answering if the effect of tolerance is developed in relation to VGB. MATERIALS AND METHODS: There were 50 patients between 4 to 20 years of age (average 12.7 +/- 4.8) with intractable focal epilepsy in the examined group. partial complex seizures were the dominant type of seizures at 74% of the examined patients. Time of observation amounted to 24 months. The patients were administered with VGB as add-on therapy. 37 patients (74%) were diagnosed with temporal lobe epilepsy, 11 (22%) with frontal lobe epilepsy, 1 patient (2%) with parietal lobe epilepsy and 1 (2%) with occipital lobe epilepsy. Patients who did not complain of seizures for a period of at least one year were classified as those with "seizure free". RESULTS: The examination was completed for 36 patients (76%). The treatment was discontinued in 12 patients (24%) as there was not a sufficient seizure control. One patient with temporal lobe epilepsy stopped the drug as three occurred the epileptic state of atypic generalised absence seizures induced by VGB. The final evaluation comprised all 50 examined patients. 9 patients (18%) achieved complete seizure control, 24 (48%) reduction to 50-99%. There was no change in 17 patients (34%). The examined patients with frontal localisation of epileptic focus presented significantly worse results of treatment, i.e. (reduction in the number of seizures over 35.3-36.5%) than the patients with temporal lobe epilepsy (reduction over 63.8 +/- 37.2%--p < 0.001). No significant difference in the average number of seizures in the 5th and 24th month of treatment was reported in the whole examined group. This contradicts the existence of the effect of tolerance in relation to VGB. CONCLUSIONS: 1. Vigabatrin is a very efficient drug in long-term treatment of patients with intractable focal epilepsies. 2. Patients with temporal lobe epilepsy present statistically better treatment results than patients with frontal lobe epilepsy. 3. No effect of tolerance to VGB was recorded during a 2-year observation.

Identification and characterization of a bovine sperm protein that binds specifically to single-stranded telomeric deoxyribonucleic acid.

Telomere DNA at the physical termini of chromosomes forms a single-stranded 3' overhang. In lower eukaryotes, e.g., ciliated protozoa, this DNA extension is capped by specific proteins that have been structurally and functionally characterized. Much less is known about single-stranded telomere DNA-binding proteins in vertebrates. Here we describe a new protein from bovine sperm designated bsSSTBP that specifically interacts with single-stranded (TTAGGG)(N) DNA. The bsSSTBP was extracted from nuclei by 0.6 M KCl. The native size of this protein, estimated by gel filtration, was 20-40 kDa. SDS-PAGE of the UV cross-linked complex between bsSSTBP and telomere DNA indicated that several polypeptides are involved in complex formation. Bovine sSSTB had high specificity toward nucleotide sequence, since single nucleotide substitutions in the (TTAGGG)(4) substrate suppressed binding. The minimal number of (TTAGGG) repeats required for binding of bsSSTBP was 3, and the protein recognized linear but not folded DNA structures. We propose that the bsSSTBP participates in telomere-telomere interactions and the telomere membrane localization observed in mature sperm. In mammals, somatic telomere-binding proteins are apparently substituted by sperm-specific ones that may lead to a structural reorganization of telomere domains to fulfill functions important during meiosis and fertilization.

[Long-term safety of using tiagabine in epilepsy]. / Bezpieczenstwo dlugoterminowego stosowania tiagabiny w padaczce.

Tiagabine (TGB) is a novel antiepileptic drug efficacious for the treatment of partial epilepsies. The aim of that work is short presentation of current data concerning long-term safety of TGB. Tolerance to TGB does not develop with long-term therapy. Idiosyncratic reaction and changes in haematology and chemistry values have not been associated with TGB therapy. The most common adverse effects are dizziness, asthenia, nervousness, tremor, diarrhoea and depression. The current data do not show any evidence of relationship between visual field constriction and TGB treatment. No adverse effects on cognitive abilities have been found. There are contradictory data concerning tiagabine-induced nonconvulsive status epilepticus. Because of high safety and efficacy TBG is an important new antiepileptic drug for the treatment of intractable partial epilepsies.

[Open multicenter study of the effectiveness and safety of gabitril in epileptic patients with partial seizures]. / Otwarte wieloosrodkowe badanie skutecznosci i bezpieczenstwa stosowania Gabitrilu u chorych z padaczka z napadami czesciowymi.

The paper presents the results obtained by 53 investigators implementing the first Polish multicentre study of the effectiveness and safety of tiagabine (Gabitril). The study included 81 patients with refractory epilepsy with partial seizures. The duration of the study was 16 weeks. For the initial 6 weeks, Gabitril was gradually introduced till a dose of 30 mg/day was achieved. Within the subsequent 10 weeks the treatment effectiveness was observed and monitored, with the provision that the dose could be increased. The final analysis included 62 patients, while in 12 subjects the treatment was discontinued in less than 16 weeks. The results indicate a very beneficial effect of Gabitril on the frequency of seizures in patients with drug-resistant epilepsy. Almost 1 of the analyzed patients were seizure free. The most beneficial effects with respect to seizure number and intensity reduction were noted in subjects with partial complex and partial seizures with secondary generalization. The dynamic character of the decrease in seizure frequency was best observed between the first and third month of therapy. In 2/3 of patients the recommended dose was achieved and maintained. Less than 15% of subjects were excluded from the study, mainly due to lack of therapeutic effects. The number and character of adverse effects observed in the course of the present study did not differ from these noted in long-term Gabitril trials. The drug was demonstrated to exert no effect on vital functions and laboratory parameters. The results confirm the high effectiveness of Gabitril in treatment of patients with partial seizures and a good tolerance of this agent.

Comparative cleavage sites within the reactive-site loop of native and oxidized alpha1-proteinase inhibitor by selected bacterial proteinases.

Human alpha1-proteinase inhibitor (alpha1-PI) is responsible for the tight control of neutrophil elastase activity which, if down regulated, may cause local excessive tissue degradation. Many bacterial proteinases can inactivate alpha1-PI by hydrolytic cleavage within its reactive site, resulting in the down regulation of elastase, and this mechanism is likely to contribute to the connective tissue damage often associated with bacterial infections. Another pathway of the inactivation of alpha1-PI is reversible and involves oxidation of a critical active-site methionine residue that may influence inhibitor susceptibility to proteolytic inactivation. Hence, the aim of this work was to determine whether this oxidation event might affectthe rate and pattern of the cleavage of the alpha1-PI reactive-site loop by selected bacterial proteinases, including thermolysin, aureolysin, serralysin, pseudolysin, Staphylococcus aureus serine proteinase, streptopain, and periodontain. A shift of cleavage specificity was observed after alpha1-PI oxidation, with a preference for the Glu354-Ala355 bond by most of the proteinases tested. Only aureolysin and serralysin cleave the oxidized form of alpha1-PI faster than the native inhibitor, suggesting that bacteria which secrete these metalloproteinases may specifically take advantage of the host defense oxidative mechanism to accelerate elimination of alpha1-PI and, consequently, tissue degradation by neutrophil elastase.

Ligand-protein interaction in plant seed thiamine-binding proteins. Binding of various thiamine analogues to the sepharose-immobilized buckwheat-seed protein.

Soluble thiamine-binding proteins occur in microorganisms, some animal tissues, and plant seeds. Their representative, the buckwheat-seed protein, was chosen as a model for chemical studies on the mechanism of ligand-protein interaction in these systems. In this work, in order to refine a concept of the chemical topography of the thiamine-binding center, the buckwheat seed protein was immobilized in Sepharose gel and probed with a new set of thiamine-related compounds. In terms of the standard change of Gibbs free energy on the complex formation, the following energetic contributions were specifically assigned to major structural features of the thiamine molecule: (i) 35-45% to the specific electronic structure of planar, unsaturated thiazolium ring with positive charge asymmetrically delocalized, one half of that contribution being attributable to the S(1) atom, (ii) 11-18% to nitrogen atoms and their electronic coupling within the pyrimidine ring, (iii) 15% to the 4'-amino group, and (iv) less than 10% to the hydroxyethyl chain.

Effect of oxidation of beta-amyloid precursor protein on its beta-secretase cleavage. A model study with synthetic peptides and candidate beta-secretases.

As the amyloidogenic processing of beta-amyloid precursor protein (betaAPP) proceeds under conditions of oxidative stress, the methionine-596 residue at the beta-secretase cleavage point is likely in an oxidized state. In the present work, possible consequences of the oxidation of Met-596 for the generation of the N-terminus of amyloid beta protein were modeled using synthetic peptide substrates, matching 587-606 sequence fragment of betaAPP and containing either intact methionine or methionine sulfoxide. Patterns and rates for the cleavage of these substrates by purified mast cell chymase, cathepsin G, cathepsin D, matrix metalloproteinase-3 and neutrophil elastase, were compared. Only the three first proteases, all previously suggested as candidate beta-secretases, preferentially cleaved the "intact" substrate after Met-596. For chymase and cathepsin G, the specificity of this cleavage increased upon a shift from optimal alkaline pH to acidic pH, which is also more compatible with the plausible intracellular localization of amyloidogenic betaAPP processing. The substitution of methionine sulfoxide for methionine in the substrate slowed down the cleavage rate for all the enzymes tested, by a factor of 6-15. This was associated with shifts of cleavage preferences to points of minor importance for the "intact" peptide, suggesting a specific resistance of the peptide bond after MetSO-596 against proteolysis.

A novel mechanism for bradykinin production at inflammatory sites. Diverse effects of a mixture of neutrophil elastase and mast cell tryptase versus tissue and plasma kallikreins on native and oxidized kininogens.

Coprocessing of kininogens by a mixture of human mast cell tryptase and neutrophil elastase was explored as a potential substitute for the kallikrein-dependent pathway for kinin generation during inflammation. Tryptase easily excised bradykinin from the synthetic heptadecapeptide, ISLMKRPPGFSPFRSSR, but was unable to produce significant amounts of kinin by proteolysis of kininogens. However, a mixture of tryptase and elastase released bradykinin from each protein with a yield comparable to that of human plasma kallikrein. Significantly, neither plasma nor tissue kallikrein was able to effectively process N-chlorosuccinimide-oxidized high molecular weight kininogen, an effect attributed to the oxidation of a methionine residue upstream from the N terminus of the kinin domain. In support of these results the model heptadecapetide, ISL(MO)KRPPGFSPFRSSR, was also resistant to hydrolysis by either kallikrein. In contrast, the release of bradykinin from oxidized peptide or protein substrates by the tryptase/elastase mixture was not altered. Because kininogen modification may occur at inflammatory sites, as a result of the oxidative burst of recruited neutrophils and macrophages, these results suggest an alternative pathway for kinin production and the necessity for the novel utilization of two specific proteinases known to be released from these cells during inflammatory episodes.

Increased telomere size in sperm cells of mammals with long terminal (TTAGGG)n arrays.

An increase in the length of telomeres in human sperm compared to somatic cells has long been noted and considered within a popular hypothesis involving telomere shortening and cell aging. In the present study we determined telomere length in two species with long terminal TTAGGG arrays--bovine and porcine. Using several independent methods we demonstrate that the telomeres in the sperm of human, porcine and bovine are elongated by 69%, 24%, and 14%, respectively, in comparison with somatic tissues. Therefore, increased sperm telomere length is a feature preserved throughout mammalian evolution. The biological role of this phenomenon is discussed in the context of telomere functions in meiosis and fertilization.