Structure of human uropepsin at 2.45 angstrom resolution

The molecular structure of human uropepsin, an aspartic proteinase from the urine produced in the form of pepsinogen A in the gastric mucosa, has been determined by molecular replacement using human pepsin as the search model. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.99, b = 75.56, c = 89.90 Angstrom. Crystallographic refinement led to an R factor of 0.161 at 2.45 Angstrom resolution. The positions of 2437 non-H protein atoms in 326 residues have been determined and the model contains 143 water molecules. The structure is bilobal, consisting of two predominantly beta -sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo-twofold axis. A model of the uropepsin-pepstatin complex has been constructed based on the high-resolution crystal structure of pepsin complexed with pepstatin.

OCCURRENCE OF MACRO-B CHROMOSOMES IN ASTYANAX-SCABRIPINNIS PARANAE (PISCES, CHARACIFORMES, CHARACIDAE)

B chromosomes occur in several Neotropical fish species. Cytogenetic analysis of 27 specimens (15 females and 12 males) of Astyanax scabripinnis paranae from the Araqua river (a small headwater tributary of the Tiete river) shows that this population has 2n = 50 chromosomes (4M + 30 SM + 4ST + 12A), two chromosome pairs with NORs and conspicuous C-band positive blocks in the terminal position of the long arm of four chromosome pairs. In this population, eight females presented 2n = 51 chromosomes and the extra chromosome was a large metacentric similar in size and morphology to the first chromosome pair in the karotype. This accessory chromosome is entirely heterochromatic in C-banded metaphases and shows a late replication pattern evidenced by BrdU incorporation. There was no significant correlation between the presence of B chromosomes and increased NOR activity at the P >0.05 level. Some aspects related to these B chromosomes are discussed.

EFFECT OF PRETREATMENT WITH VENOM OF APIS-MELLIFERA BEES ON THE YIELD OF GAMMA-RAY INDUCED CHROMOSOME-ABERRATIONS IN HUMAN BLOOD-LYMPHOCYTES

Venom of the honey bee Apis mellifera induced a protective effect against the induction of dicentric chromosomes by gamma radiation (2.0 Gy) in human peripheral blood lymphocytes which the cultures were treated with 0.00015 mul venom/1 ml medium 6 h before irradiation. In cultures to which the venom was added immediately before irradiation with 0.25, 1.0 and 2.0 Gy, no significant differences in number of dicentric chromosomes induced was observed when compared to cultures submitted to irradiation only. The venom did not induce clastogenic effects nor did it increase the frequency of sister chromatid exchanges.

Prostaglandin E-2 inhibits Paracoccidioides brasiliensis killing by human monocytes

Human monocytes lacked fungicidal activity against high virulence strain of Paracoccidioides brasiliensis, even after IFN-gamma activation. However, monocytes treated with indomethacin exhibited an effective killing against this fungus, suggesting a role of prostaglandin E-2 (PGE(2)) in the inhibition process. Thus, the purpose of this work was to determine whether the effect of PGE2 in fungicidal activity was related with decrease on H2O2 release, the metabolite involved in P. brasiliensis killing, and changes in the levels of TNF-alpha, IL-6 and IL-10. Human monocytes challenged with the fungus produced high PGE(2) levels, which in turn inhibited the fungicidal activity of these cells by reducing H2O2 and TNF-alpha production. (C) 2007 Elsevier Masson SAS. All rights reserved.

IDENTIFICATION OF COMMON FRAGILE SITES IN CHROMOSOMES OF 2 SPECIES OF BAT (CHIROPTERA, MAMMALIA)

In the karyotypes of the bat species Molossus ater and M molossus, spontaneous and bromodeoxyuridine (BrdU)- or aphidicolin (APC)-sensitive fragile sites were located. Four chromosome regions harbored APC-sensitive fragile sites: 1q9 and 8q4 in both M ater and M molossus, 3q3 in M ater, and 1p7 in M molossus. The fragile sites in 1q9 and 8q4 were also observed without induction in M molossus. BrdU-sensitive fragile sites were not detected. Despite observations in several other species, the fragile sites detected in Molossus are not coincident with the breakpoints involved in the chromosome rearrangements occurring in the evolution of 7 species of the Molossidae family.

Novel allosteric conformation of human HB revealed by the hydration and anion effects on O-2 binding

The effect of anions on the stability of different functional conformations of Hb is examined through the determination of the dependence of O-2 affinity on water activity (a(w)). The control of a(w) is effected by varying the sucrose osmolal concentration in the bathing solution according to the osmotic stress method. Thus, the hydration change following Hb oxygenation is determined as a function of Cl- and of DPG concentration. We find that only similar to 25 additional water molecules bind to human Hb during the deoxy-to-oxy conformation transition in the absence of anions, in contrast with similar to 72 that bind in the presence of more than 50 mM Cl- or more than 15 mu M DPG. We demonstrate that the increase in the hydration change linked with oxygenation is coupled with anion binding to the deoxy-Hb. Hence, we propose that the deoxy-Hb coexists in two allosteric conformations which depend on whether anion is bound or not: the tense T-state, with low oxygen affinity and anion bound, or a new allosteric P-state, with intermediate oxygen affinity and free of bound anions. The intrinsic oxygen affinity of this unforeseen P-state and the differential binding of Cl-, DPG, and H2O between states P and T and P and R are characteristics which are consistent with those expected for a putative intermediate allosteric state of Hb. These findings represent a new opportunity to explore the structure-function relationships of hemoglobin regulation.

RH maps of bovine chromosomes 15 and 29: conservation of human chromosomes 11 and 5

Comparative mapping data on evolutionary conserved coding sequences and synteny maps between human and cattle are insufficient to define the extent and distribution of conserved segments between these two species, because the order of loci is often rearranged. A 5000-rad cattle whole-genome radiation hybrid (WG-RH) panel was constructed to provide high-resolution comparative maps and also to integrate linkage maps of microsatellites with evolutionary conserved genes and transcripts in a single ordered map. We used the WG-RH panel to construct radiation hybrid maps of bovine Chromosomes (Chrs) 15 and 29 (BTA15 and BTA29), integrating microsatellites from published linkage maps with selected genes. The comprehensive map of BTA15 consists of 24 markers. 13 of which were placed in the framework map. Eleven molecular markers compose the comprehensive map of BTA29. seven of which were placed in the framework map. We identified the homologous regions between bovine Chr 15 (BTA15) and human Chrs 5 and 11 (HSA5 and HSA11), as well as between BTA29 and HSA11, the present study demonstrates that WG-RH mapping is an efficient method for integrating multiple genetic maps into one map and for incorporating monomorphic Type I loci into ordered maps for comparison between species.

Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis

Patients with paracoccidioidomycosis (PCM) display a certain degree of immunecompromise characterized by lymphocyte hyporesponsiveness to the main Paracoccidioides brasiliensis antigen (gp43). To determine whether cytokines are involved in this state, we evaluated the secretion of IL-2, IL-10 and IFN-gamma by peripheral blood mononuclear cells (PBMC) from patients with the acute (AF) and chronic (CF) forms of PCM and from healthy, P. brasiliensis-sensitized subjects. gp43-stimulated PBMC from healthy subjects produced substantial amounts of IL-2, IFN-gamma and IL-10, whereas PBMC from AF and CF patients produced low levels of IL-2 and IFN-gamma but substantial amounts of IL-10, Phytohaemagglutinin-induced cytokine secretion was comparable among AF and CF patients and healthy subjects, suggesting integrity of non-specific cellular immune mechanisms in PCM. gp43-pulsed adherent cells, but not non-adherent cells, mere the main source of IL-10, Moreover, IL-2 and IFN-gamma secretion correlated inversely with the amount of specific antibodies produced by patients and healthy subjects. Our results suggest that the imbalance in cytokine production of patients with PCM plays a role in the gp43-hyporesponsiveness and the marked (non-protective) antibody production of these patients. (C) 2001 Academic Press.

Cytotaxonomy of the crickets Endecous Saussure, 1878 with an overview of the chromosomes of Phalangopsinae Group (Orthoptera: Phalangopsinae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Inhibitory effect of PGE(2) on the killing of Paracoccidioides brasiliensis by human monocytes can be reversed by cellular activation with cytokines

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

Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-Å resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin β-turn of the second inner loop pivots at Val64 and Asp70 by 60°. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 β-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle. © 1993 American Chemical Society.

Cycloactive, aneugenic and clastogenic effects of Paracoccidioides brasiliensis exoantigen in human lymphocyte cultures

The in vitro effect of Paracoccidioides brasiliensis exoantigen on the human lymphocytes cell cycle and chromosomes was studied. Human peripheral blood lymphocyte cultures from ten healthy, white, non-smoking, non-related adult males (mean age 31·3 ± 8·2 years) were studied. Blood cultures were treated with three exoantigen concentrations (0·25, 2·50 and 10·00 μg ml -1). At least 1000 metaphases were analysed at each concentration, for evaluation of numerical and structural chromosome aberrations (cA) and 30 000 for mitotic index (MI). Among the treated cultures, statistically significant differences in the frequencies of MI and cA were not observed. Nevertheless, when compared with control cultures, they all showed a significantly lower frequency of MI and higher frequency of cA. It is suggested that the detected alterations were caused by the exoantigen, its fractions or its metabolites. © 1996 Informa UK Ltd All rights reserved.

Determination of neutral polymorphisms (frameworks) of the human beta globin gene in beta thalassaemias by PCR/DGGE

Purpose: Considering the importance of type beta thalassaemias as hereditary syndromes of high significance in different populations of Mediterranean origin and, by extension, in the Brazilian population, the objective of the present study was to determine by PCR/DGGE the gene structures responsible for neutral polymorphisms (frameworks) observed in the human beta globin gene associated with the mutations responsible for type beta thalassaemias in a sample of the Brazilian population and, more specifically, of the population of the State of São Paulo. Patients and methods: Thirty individuals with beta thalassaemic mutations were analyzed: 22 mutations were in codon 39 (C->T), 5 in IVS1-110 (G->A), 2 in IVS1-6 (T->C) and 1 in IVS1-1 (G->A). DNA was extracted and selective amplification was performed by PCR extending from position IVS1 nt 46 to IVS2 nt 126 (474 pb). The product was then analyzed by polyacrylamide gel electrophoresis on a denaturing 10-60% urea/formamide gradient. Results: The results demonstrated that, as expected, the mutations responsible for type beta thalassaemia observed in this population are of Mediterranean origin, with 73% distribution represented by codon 39,17% by IVS1-110, 7% by IVS1-6 and 3% by IVS1-1. In turn, framework distribution seems to indicate a higher frequency of Fr 1-1 in codon 39 and IVS1-110, of Fr 1-3 in IVS1-6 and of Fr 1-2 in IVS1-1. Conclusions: These results permit us to conclude that gene amplification by PCR followed by DGGE is an appropriate method for the separation of DNA molecules that differ even by a single base change and therefore can be utilized to detect the alterations observed in the human beta globin gene. This methodology shows that, using only a pair of primers, it is possible to define the frameworks that are observed in the beta globin gene.

Evaluating the karyotypic diversity in species of Hyla (Anura, Hylidae) with 2n = 30 chromosomes based on the analysis of ten species

Ten species of Hyla with 2n = 30 from Brazilian fauna were analysed cytogenetically. Hyla minuta is the unique presenting all bi-armed metacentric or submetacentric chromosomes in the karyotype, therefore, with the highest FN = 60. The remaining species have a variable number of uni-armed telocentric or subtelocentric chromosomes: H. cruzi, H. elianeae, and H. rubicundula with three pairs (FN = 54), H. berthalutzae, H. elegans, H. microps, and H. nana with four pairs (FN = 52), and H. nahdereri and H. sanborni with five pairs (FN = 50). The uni-armed elements are among pairs 5, 6, 7, 11, 14, and 15, which also appeared with metacentric or submetacentric morphology. The remaining chromosome pairs 1, 2, 3, 4, 8, 9, 10, 12, and 13 were never found to be telocentric or subtelocentric. AgNOR patterns are species-specific, the majority of the species exhibiting a single pair with AgNORs, with the exception of H. elegans and H. nana with more than one chromosome pair bearing this cytological marker. C banding was obtained in H. berthalutzae, H. cruzi, H. elegans, H. elianeae, H. microps, H. minuta, H. nahdereri, and H. nana, which showed positively stained centromeric heterochromatin. Our analysis confirms the great karyotypic diversity in the species of Hyla with 2n = 30, with no species sharing identical karyotypes.