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.

Regulation of IL-2 expression by transcription factor BACH2 in umbilical cord blood CD4+ T cells.

On activation, umbilical cord blood (UCB) CD4(+) T cells demonstrate reduced expression of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), whereas maintaining equivalent interleukin-2 (IL-2) levels, as compared with adult peripheral blood (PB) CD4(+) T cells. Nuclear factor of activated T cells (NFAT1) protein, a transcription factor known to regulate the expression of IL-2, TNF-alpha and IFN-gamma, is reduced in resting and activated UCB CD4(+) T cells. In contrast, expression of Broad-complex-Tramtrack-Bric-a-Brac and Cap'n'collar homology 1 bZip transcription factor 2 (BACH2) was shown by gene array analyses to be increased in UCB CD4(+) T cells and was validated by qRT-PCR. Using chromatin immunoprecipitation, BACH2 was shown binding to the human IL-2 proximal promoter. Knockdown experiments of BACH2 by transient transfection of UCB CD4(+) T cells with BACH2 siRNA resulted in significant reductions in stimulated IL-2 production. Decreased IL-2 gene transcription in UCB CD4(+) T cells transfected with BACH2 siRNA was confirmed by a human IL-2 luciferase assay. In summary, BACH2 maintains IL-2 expression in UCB CD4(+) T cells at levels equivalent to adult PB CD4(+) T cells despite reduced NFAT1 protein expression. Thus, BACH2 expression is necessary to maintain IL-2 production when NFAT1 protein is reduced, potentially impacting UCB graft CD4(+) T-cell allogeneic responses.

Cyclosporin A effects during primary and secondary activation of human umbilical cord blood T lymphocytes.

OBJECTIVE: Cyclosporin A (CsA), effective in prophylaxis and treatment of graft-vs-host disease (GVHD) after human allogeneic transplantation, blunts T-cell responses by inhibiting nuclear factor of activated T cells-1 (NFAT1) activation. This laboratory has shown that NFAT1 protein expression is severely reduced in human UCB (umbilical cord blood) T cells. Since UCB is increasingly used as a hematopoietic stem cell source in allogeneic transplantation, it is important to determine whether CsA sensitivity in UCB differs from that of adult T cells. METHODS: Surface flow cytometric analysis, intracellular cytokine staining, flow cytometric analysis of cell death, and thymidine incorporation were used in this study to determine T-cell activation and effector functions during primary and secondary stimulation in the presence of CsA. RESULTS: Although we observed differential CsA sensitivity of T-cell activation marker (CD69, CD45RO, CD25) upregulation comparing UCB and adult, we did not observe any significant difference in CsA sensitivity of T-cell effector functions. Importantly, we observed reduced IFN-gamma and TNF-alpha expression in UCB T cells both in primary and secondary stimulation, as well as increased rates of activation-induced cell death (AICD). CONCLUSION: Thus, our studies do not support the previous hypothesis that reduced GVHD observed after UCB transplantation is attributable to increased CsA sensitivity of UCB T cells. Rather, reduced UCB T-cell cytokine production and increased AICD may be important cellular mechanisms underlying these favorable rates of GVHD in UCB transplant recipients.

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.

[Huntington chorea in a boy aged 14 years]. / Plasawica Huntingtona u 14-letniego chlopca.

The authors present a case of Huntington disease in a 14 year old boy. The basis of genetic, pathoetiology, clinical course of disease, significance of molecular analysis of DNA are discussed.

Mechanism of ligand-protein interaction in plant seed thiamine-binding proteins. Preliminary chemical identification of amino acid residues essential for thiamine binding to the buckwheat-seed protein.

Thiamine-binding protein, isolated from buckwheat seeds, was chemically modified in an attempt to identify amino acid residues involved in protein-thiamine interaction. No evidence was found in support of specific roles of arginine residues, sulfhydryl groups, amino groups and tyrosine residues. Under carefully controlled reaction conditions (Tris pH 5-6), the modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide caused a complete loss of thiamine-binding capacity. Thus, the carboxyl groups seemed to be essential for binding, possibly for ionic interaction with protein-bound thiamine cation. A selective modification of histidine residues using diethylpyrocarbonate correlated with a loss of thiamine-binding capacity; the modification and the loss of binding capacity could be reversed with hydroxylamine; some ligand-protection against modification was observed. From Tsou analysis of diethylpyrocarbonate modification and resulting loss of thiamine-binding it was suggested that 1-2 of 20 histidine residues of the protein were essential for thiamine binding. The essential histidine(s) might be present in the binding site and possibly were involved in hydrogen bonding(s) with protein-bound thiamine molecule.

Mechanism of ligand-protein interaction in plant seed thiamin-binding proteins. Probing the binding site of protein isolated from buckwheat seeds with a series of thiamin-related compounds.

Affinities of 14 thiamin derivatives or antagonists to a thiamin-binding protein isolated from buckwheat seeds were determined. A competitive displacement of radiolabeled thiamin by unlabeled ligand was analysed by a computerized model-fitting procedure. The dissociation constant of the thiamin-protein complex was 0.93 microM. Most modifications in ligand chemical structure weakened the ligand-protein interaction. A model of the thiamin-binding site is suggested. The hydroxyethyl-chain of thiamin while protein-bound appears to be excluded from the binding region. A positively charged quaternary nitrogen atom of the thiazolium ring probably interacts with some negative group(s) of protein. The rest of the thiazolium ring as well as the amino group of the pyrimidine fragment serve as additional anchors. The three structural features of the thiamin molecule accounting for binding contribute equally to overall binding energy by about 11-12 kJ/mol.

[Congenital toxoplasmosis in a 6-week-old infant]. / Toksoplazmoza wrodzona u 6-tygodniowego niemowlecia.

A case of congenital toxoplasmosis was observed in an infant aged 6 weeks with fatal outcome. Histological changes produced by Toxoplasma gondii in the brain are described.

Gaussian free-energy dependence of electron-transfer rates in iridium complexes.

The kinetics of photoinduced electron-transfer (ET) reactions have been measured in a series of synthetic donor-acceptor complexes. The electron donors are singlet or triplet excited iridium(I) dimers (Ir(2)), and the acceptors are N-alkylpyridinium groups covalently bound to phosphinite ligands on the Ir(2) core. Rate constants for excited-state ET range from 3.5 x 10(6) to 1.1 x 10(11) per second, and thermal back ET (pyridinium radical to Ir(2)(+)) rates vary from 2.0 x 10(10) to 6.7 x 10(7) per second. The variation of these rates with driving force is in remarkably good agreement with the Marcus theory prediction of a Gaussian free-energy dependence.

The influence of the antimitotic drug CCNU on the neurosecretion of rat hypothalamo-hypophyseal system.

The purpose of this investigation was to evaluate the influence of the antimitotic drug CCNU on the morphology of the hypothalamo-hypophyseal neurosecretory system of rat. Adult Wistar rats were treated intragastrically with 2.5 mg CCNU once a week during 3 consecutive weeks and 5 mg at the end of the 4th week. The brains and hypophyses were fixed in Zenker-formol solution. Paraffin slices were stained with chromhematoxylin to demonstrate neurosecretory material and with cresyl violet. PAS reaction was also performed. The experiment resulted in disturbances of the neurosecretory function of the hypothalamo-hypophyseal system in form of alteration in the content of neurosecretion in the neuronal cytoplasm and processes within supraoptic and paraventricular nuclei as well as in the neurohypophysis. The morphometric measurements showed enlargement of the cell nuclei and cytoplasm volumes in the nucleus supraopticus of hypothalamus.

[A case of multiple neoplasm metastases to the brain with few clinical symptoms]. / Skapoobjawowy przypadek kilkudziesieciu przerzutów nowotworowych do mózgu.

In the reported case 74 malignant metastases were found in the brain. The primary tumour was a bronchogenic carcinoma. The clinical symptoms were scant and were restricted mainly to those produced by involvement of the pons without evidence of intracranial hypertension. In the discussion the only relative value of laboratory investigations is stressed, including computerized tomography. The case deserves publication in view of rare occurrence of such oligosymptomatic course despite a high number of metastases.

[A case of the acinous form of meningeal cysticercosis]. / Przypadek postaci groniastej wagrzycy opon mózgowych.

A case of chronic cerebrospinal meningitis is described which caused considerable diagnostic difficulties. Postmortem examination demonstrated parasitic character of the disease. Although all cysts were sterile, the authors suggest in the discussion that in the case the cause was infestation with the larval from of the canine tapeworm Taenia multiceps.

Karyometry and cytophotometry of oligodendroglial DNA in the corpus callosum of rats treated with vicristine during the second part of foetal life.

The effect of transplacentally administered vincristinee on postnatal developmental events occurring in the oligodendroglial cell nuclei of the corpus callosum were studied by means of an automatic analyzer of microscopic pictures "Morphoquant" (VEB Carl Zeiss Jena, GDR). Karyometric and cytophotometrically determined developmental changes of the DNA content were studied in both, the vincristine treated and control rats aged 3, 7, 14, 21 and 28 days. vincristine was administered to pregnant rats four times during the second term of gestation, the single dose containing 0.01 mg of Vincristin per kg of body weight. In the Vincristin treated animals we have found an advanced developmental increase of the perimeter, the surface and convexion areas of oligodendroglial cell nuclei as well as statistically significant differences of parameters characterizing the nuclear shape. In comparison with control animals, the intoxicated ones demonstrated a higher DNA content in oligodendroglial cell nuclei both during the premyelinating (7 days postnatal) and during the early stage of myelination (14 and 21 days postnatal).