3H]BHDP as a novel and selective ligand for sigma1 receptors in liver mitochondria and brain synaptosomes of the rat

The binding profile of [(3)H]BHDP ([(3)H]N-benzyl-N'-(2-hydroxy-3,4-dimethoxybenzyl)-piperazine) was evaluated. [(3)H]BHDP labelled a single class of binding sites with high affinity (K(d)=2-3 nM) in rat liver mitochondria and synaptic membranes. The pharmacological characterization of these sites using sigma reference compounds revealed that these sites are sigma receptors and, more particularly, sigma1 receptors. Indeed, BHDP inhibited [(3)H]pentazocine binding, a marker for sigma1 receptors, with high affinity in a competitive manner. BHDP is selective for sigma1 receptors since it did not show any relevant affinity for most of the other receptors, ion channels or transporters tested. Moreover, in an in vitro model of cellular hypoxia, BHDP prevented the fall in adenosine triphosphate (ATP) levels caused by 24 h hypoxia in cultured astrocytes. Taken together, these results demonstrate that [(3)H]BHDP is a potent and selective ligand for sigma1 receptors showing cytoprotective effects in astrocytes.     

Hormonal regulation of oestrogen formation by Leydig cells in vitro: immunocytochemical approach

The ability of the testis to convert androgens into oestrogens is related to the presence of a microsomal enzyme, aromatase, in testicular cells. The aim of this study was to show whether the supplementation of culture media with LH or an aromatase inhibitor could affect the process of aromatisation in Leydig cells of the bank vole in vitro. This was investigated by means of immunocytochemistry and radioimmunological assays. In control cultures of Leydig cells, both steroid hormones secretion as well as immunoreactivities for aromatase and oestrogen receptor were weaker than in those treated with LH. On the contrary, the addition of aromatase inhibitor into the culture medium resulted in a decreased intensity of immunocytochemical stainings in comparison with the control. Concomitantly, the androgen level was slightly higher, whereas that of oestrogen significantly lower than in the control cultures. Additionally, to check whether steroid hormones are able to regulate aromatase or oestrogen receptor immunoexpressions, some of the Leydig cell cultures were enriched with testosterone or oestradiol, respectively. Strong immunoreactivities for both aromatase and oestrogen receptor were observed. This suggests that Leydig cells in vitro are able to regulate directly the secretion of oestrogens by active aromatase. Finally, it is concluded that oestrogen formation in bank vole Leydig cells in vitro can be influenced by various factors. It should be stressed, however, that the effect of hormone stimulation or aromatase inhibitor action appeared to be dependent on the length of light cycles that bank voles were exposed prior to the isolation of Leydig cells.     

Multilocus enzyme electrophoresis analysis of Trypanosoma cruzi isolates from a geographically restricted endemic area for Chagas' disease in Argentina

A set of 65 Trypanosoma cruzi stocks from dogs, opossums, insect vectors and humans was isolated in a geographically restricted endemic area for Chagas' disease in Argentina and was analysed by multilocus enzyme electrophoresis for 15 loci. The results show that at least five multilocus genotypes (clonets) circulate in the study area, one belonging to T. cruzi IIe, one to T. cruzi IId and three clonets belonging to T. cruzi I; and they confirm the presence of these lineages in the country. The three clonets attributed to T. cruzi I were identical to each other for all loci except for Sod-2, where three different patterns were identified. These patterns suggest the presence of two homozygous genotypes and one heterozygous genotype. Our results also suggest association of clonet IIe with dogs, clonet IId with humans and the three T. cruzi I clonets with Didelphis albiventris. On the other hand, there was no significant association between Triatoma infestans and any particular clonet circulating in the area. These findings are consistent with the hypothesis of natural selection, from mixed populations of T. cruzi in vectors, toward more restricted populations in mammals. The epidemiological implications of the possible selection of different clonets by different mammal hosts and the significance of two homozygous genotypes and one heterozygous genotype for the Sod-2 locus are discussed.     

Suppression of temperature-sensitive chromosome replication of an Escherichia coli dnaX(Ts) mutant by reduction of initiation efficiency

Temperature sensitivity of DNA polymerization and growth of a dnaX(Ts) mutant is suppressible at 39 to 40 degrees C by mutations in the initiator gene, dnaA. These suppressor mutations concomitantly cause initiation inhibition at 20 degrees C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX(Ts). One dnaA(Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA(Cs,Sx) suppression of dnaX, which codes for both the tau and gamma subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX(Ts) mutant at the somewhat intermediate temperature of 39 to 40 degrees C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA(Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX(Ts) strains growing at 39 and 34 degrees C. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX(Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA(Cs,Sx) dnaX(Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39 degrees C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39 degrees C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38 degrees C. Fourth, the dnaX(Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA(Cos) allele, which causes overinitiation, into the dnaX(Ts) mutant exacerbated its temperature sensitivity.     

Cluster of type IV secretion genes in Helicobacter pylori's plasticity zone

Some genes present in only certain strains of the genetically diverse gastric pathogen Helicobacter pylori may affect its phenotype and/or evolutionary potential. Here we describe a new 16.3-kb segment, 7 of whose 16 open reading frames are homologs of type IV secretion genes (virB4, virB7 to virB11, and virD4), the third such putative secretion gene cluster found in H. pylori. This segment, to be called tfs3, was discovered by subtractive hybridization and chromosome walking. Full-length and truncated tfs3 elements were found in 20 and 19%, respectively, of 94 strains tested, which were from Spain, Peru, India, and Japan. A tfs3 remnant (6 kb) was found in an archived stock of reference strain J99, although it was not included in this strain's published genome sequence. PCR and DNA sequence analyses indicated the following. (i) tfs3's ends are conserved. (ii) Right-end insertion occurred at one specific site in a chromosomal region that is varied in gene content and arrangement, the "plasticity zone." (iii) Left-end insertion occurred at different sites in each of nine strains studied. (iv) Sequences next to the right-end target in tfs3-free strains were absent from most strains carrying full-length tfs3 elements. These patterns suggested insertion by a transposition-like event, but one in which targets are chosen with little or no specificity at the left end and high specificity at the right end, thereby deleting the intervening DNA.     

Deamidations in recombinant human phenylalanine hydroxylase. Identification of labile asparagine residues and functional characterization of Asn --> Asp mutant forms

Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 degrees C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis. Asn(32) in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly(33) (Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., and Flatmark, T. (2003) Eur. J. Biochem., in press). Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 degrees C) expressed enzyme has shown Asn(376) and Asn(133) to be labile residues. Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn(32) and Asn(376) are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme. The Asn(32) --> Asp, Asn(376) --> Asp, and the double mutant forms expressed for 2 h at 28 degrees C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH. Moreover, deamidation of Asn(32) in the wt-hPAH (24 h expression at 28 degrees C) and the Asn(32) --> Asp mutation both increase the initial rate of phosphorylation of Ser(16) by cAMP-dependent protein kinase (p < 0.005). By contrast, the substitution of Gly(33) with Ala or Val, both preventing the deamidation of Asn(32), resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression. The other Asn --> Asp substitutions (in the catalytic domain) revealed that Asn(207) and Asn(223) have an important stabilizing structural function. Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied, i.e. Asn(167) --> Ile and Asn(207) --> Asp, and their phenotypes were characterized.     

Taurine levels and localisation in the stratified squamous epithelia

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.     

Endocrine profiles during doe-litter separation and the subsequent pregnancy in rabbits

This study was carried out to determine the effects of a transient doe-litter separation on plasma prolactin, LH, FSH, estradiol-17beta and progesterone concentrations before artificial insemination and during the subsequent pregnancy. Control does (n=12) had free access to nursing, whereas separated does (n=12) were kept away from their litters for 48 hours before artificial insemination. Both groups were inseminated on day 11 after parturition. Teat stimulation by suckling caused a high increase in prolactin concentrations in separated does (p < 0.0001). Basal prolactin concentrations were observed in both groups on days 8 and 18 of pregnancy. No effect of the treatment was detected on LH and FSH concentrations during the sampling period. A rise of estradiol-17beta concentrations was observed 48 hours after doe-litter separation, compared to control does and to previous values (p < 0.003). Both groups showed low progesterone concentrations before artificial insemination. Pregnant rabbits in both groups showed increased progesterone concentrations on days 8 and 18 of pregnancy. Lower estradiol-17beta concentrations were observed in control does on day 18 of pregnancy compared with separated rabbits (p < 0.003). The results suggest that a transient separation of nursing does from their litters before artificial insemination may promote high follicular steroidogenesis activity leading to increased estradiol-17beta concentrations. This hormonal change could be a result of several stimulatory actions probably triggered by the absence of suckling episodes and may affect the luteotrophic function during the subsequent pregnancy.