Phylogenetic relationships of the lamprologine cichlid genus Lepidiolamprologus (Teleostei: Perciformes) based on mitochondrial and nuclear sequences, suggesting introgressive hybridization

Using sequences of the mitochondrial NADH dehydrogenase subunit 2 gene (ND2, 1047bp) and a segment of the non-coding mitochondrial control region, as well as nuclear sequences including two introns from the S7 ribosomal protein and the loci TmoM25, TmoM27, and UME002, we explore the phylogenetic relationships of Lepidiolamprologus, one of seven lamprologine cichlid genera in Lake Tanganyika, East Africa. Analyses consisted of direct optimization using POY, including a parsimony sensitivity analysis, and maximum likelihood and Bayesian inference for comparison. With respect to Lepidiolamprologus, the results based on the mitochondrial dataset were robust to parameter variation in POY. Lepidiolamprologus cunningtoni was resolved in a large clade sister to ossified group lamprologines, among which the remaining Lepidiolamprologus were nested. In addition to L. attenuatus, L. elongatus, L. kendalli, and L. profundicola, Neolamprologus meeli, N. hecqui, N. boulengeri, N. variostigma, and two undescribed species were resolved in a two-pore Lepidiolamprologus clade sister to Lamprologus callipterus and two species of Altolamprologus. Lepidiolamprologus nkambae, in marked conflict with morphological and nuclear DNA evidence, nested outside of the two-pore Lepidiolamprologus clade, suggesting that the mtDNA signal has been convoluted by introgressive hybridization.     

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.     

Actions of gonadotropin-releasing hormone analogues in pituitary gonadotrophs and their modulation by ovarian steroids

Recently, GnRH antagonists (GnRHant) like cetrorelix and ganirelix have been introduced in protocols of controlled ovarian hyperstimulation for assisted reproductive techniques to prevent premature luteinizing hormone (LH) surges. Here we tested, whether the actions of cetrorelix and the GnRH agonist (GnRHag) triptorelin in gonadotrophs are dependent on the steroid milieu. Furthermore, we characterized the actions of cetrorelix and triptorelin on LH secretion and the total LH pool. Female rat pituitary cells were treated either with 0.1 nM triptorelin for 1, 2, 4 and 6 days or for 1, 3, 5 and 6 h or with 1, 10 or 100 nM cetrorelix for 1, 2, 3 and 5 h or for 10 min. Cells were stimulated for 3h with different concentrations of GnRH (10 pM-1 microM). For analysis of the total LH pool, which is composed of stored and released LH, cells were lysed with 0.1% Triton X-100 at -80 degrees C overnight. To test, whether the steroid milieu affects the actions of cetrorelix and triptorelin, cells were incubated for 52 h with 1 nM estradiol (E) alone or with combinations of 100 nM progesterone (P) for 4 or 52 h, respectively. Cells were then treated with 0.1 nM triptorelin for 9 h or 1 nM cetrorelix for 3 h and stimulated for 3 h with different concentrations of GnRH (10 pM-1 microM). The suppressive effect of triptorelin on LH secretion was fully accomplished after 3 h of treatment, for cetrorelix only 10 min were sufficient. The concentration of cetrorelix must be at least equimolar to GnRH to block LH secretion. Cetrorelix shifted the EC50s of the GnRH dose-response curve to the right. Triptorelin suppressed total LH significantly (from 137 to 36 ng/ml) after 1 h in a time-dependent manner. In contrast, only high concentrations of cetrorelix increased total LH. In steroid treated cells the suppressive effects of triptorelin were more distinct. One nanomolar cetrorelix suppressed GnRH-stimulated LH secretion of cells not treated with steroids from 10.1 to 3.5 ng/ml. In cells, additionally treated with estradiol alone or estradiol and short-term progesterone, LH levels were higher (from 3.5 to 5.4 or 4.5 ng/ml, respectively). In cells co-treated with estradiol and progesterone for 52 h LH secretion was only suppressed from 10.1 to 9.5 ng/ml. Steroid treatments diminished the suppressive effect of cetrorelix on LH secretion. In conclusion, the depletion of the total LH pool contributes to the desensitizing effect of triptorelin. The actions of cetrorelix and triptorelin are dependent on the steroid milieu.     

Evaluation of a Real-time PCR-based assay using the lightcycler system for detection of Toxoplasma gondii bradyzoite genes in blood specimens from patients with toxoplasmic retinochoroiditis

PCR based methods have advantages over traditional methods for the diagnosis of toxoplasmosis, especially when serology fails and clinical symptoms are not evident. However, current PCR-based assays are often labour-intensive and not readily quantifiable and have the potential for contamination due to a requirement for postamplification sample handling. Real-time PCR can address these limitations. We have developed and evaluated a highly sensitive Real-time PCR (Light-cycler, LC-PCR) to detect and quantify Toxoplasma gondii B1 and bradyzoite specific genes (SAG-4, MAG-1) in serum and peripheral blood mononuclear cells (PBMC) specimens, from five immunocompetent subjects with clinically suspected toxoplasmic retinochoroiditis (TRC) or without a suspected T. gondii infection. A standard curve for quantitation of parasitic load was generated using SYBR Green I fluorescent detection. The results were compared with those obtained with a nested PCR (n-PCR). In TRC patients, both PCR methods confirmed ophtalmoscopy and fluorangiographic findings. Among the TRC patients, the use of LC-PCR was more sensitive than n-PCR for detection and quantification of either B1 gene (P<0.001) or SAG-4/MAG-1 gene (P<0.05). LC-PCR has been shown particularly useful to accurately determine the parasite DNA load in follow-up specimens in whom the performance of either B1 or SAG-4 and MAG-1 in detecting T. gondii loads, varied with respect to specific antitoxoplasmic treatment.     

A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies

The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.     

Development of a microelectronic chip array for high-throughput genotyping of Helicobacter species and screening for antimicrobial resistance

A microelectronic array assay was developed to specifically genotype Helicobacter pylori versus Helicobacter heilmannii and to determine antimicrobial resistance. Helicobacter 16S rRNA and 23S rRNA genes were specifically generated with Helicobacter genus-specific primers, respectively. The single-nucleotide polymorphisms (SNPs) in 16S rRNA, 268T specific in the H. pylori sequence, and 263A specific in H. heilmannii were used as molecular markers for identification of H. pylori and H. heilmannii, respectively. A triple-base-pair resistant mutation, AGA965-967TTC in 16S rRNA, is known to be responsible for H. pylori tetracycline resistance and was detected to identify resistant strains. H. pylori macrolide resistance was determined by the identification of 3 defined mutations in the 23S rRNA gene using the same method. The assay could be directly used to detect H. pylori in feces. The assay performs multiple determinations, including identification of Helicobacter species and antibiotic resistances, on the same microelectronic platform and is highly amenable to the development of other DNA-based assays.     

Conservation of cis prolyl bonds in proteins during evolution

In proteins and peptides, the vast majority of peptide bonds occurs in trans conformation, but a considerable fraction (about 5%) of X-Pro bonds adopts the cis conformation. Here we study the conservation of cis prolyl residues in evolutionary related proteins. We find that overall, in contrast to local, protein sequence similarity is a clear indicator for the conformation of prolyl residues. We observe that cis prolyl residues are more often conserved than trans prolyl residues, and both are more conserved than the surrounding amino acids, which show the same extent of conservation as the whole protein. The pattern of amino acid exchanges differs between cis and trans prolyl residues. Also, the cis prolyl bond is maintained in proteins with sequence identity as low as 20%. This finding emphasizes the importance of cis peptide bonds in protein structure and function.     

Localized vs. systemic inflammation in guinea pigs: a role for prostaglandins at distinct points of the fever induction pathways?

In guinea pigs, dose-dependent febrile responses were induced by injection of a high (100 microg/kg) or a low (10 microg/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. Both LPS doses further induced a pronounced formation of prostaglandin E(2) (PGE(2)) at the site of localized subcutaneous inflammation. Administration of diclofenac, a nonselective cyclooxygenase (COX) inhibitor, at different doses (5, 50, 500, or 5,000 microg/kg) attenuated or abrogated LPS-induced fever and inhibited LPS-induced local PGE(2) formation (5 or 500 microg/kg diclofenac). Even the lowest dose of diclofenac (5 microg/kg) attenuated fever in response to 10 microg/kg LPS, but only when administered directly into the subcutaneous chamber, and not into the site contralateral to the chamber. This observation indicated that a localized formation of PGE(2) at the site of inflammation mediated a portion of the febrile response, which was induced by injection of 10 microg/kg LPS into the subcutaneous chamber. Further support for this hypothesis derived from the observation that we failed to detect elevated amounts of COX-2 mRNA in the brain of guinea pigs injected subcutaneously with 10 microg/kg LPS, whereas subcutaneous injections of 100 microg/kg LPS, as well as systemic injections of LPS (intra-arterial or intraperitoneal routes), readily caused expression of the COX-2 gene in the guinea pig brain, as demonstrated by in situ hybridization. Therefore, fever in response to subcutaneous injection of 10 microg/kg LPS may, in part, have been evoked by a neural, rather than a humoral, pathway from the local site of inflammation to the brain.