Gating of α3β4 neuronal nicotinic receptor can be controlled by the loop M2-M3 of both α3 and β4 subunits

Previous studies have shown that the gating mechanism of alpha3beta4 neuronal nicotinic receptors is affected by a residue in the middle of the M2-M3 loop of the beta4 subunit. We have extended the study of the same location to the alpha3 subunit. Bovine alpha3beta4 receptors were mutated in position 268, substituting the residue present in wild-type receptors, i.e. leucine in alpha3 and asparagine in beta4, for an aspartate. Wild-type and mutated alpha3 and beta4 subunits were combined to form four different receptors. We have measured macroscopic currents in Xenopus oocytes elicited by nicotine, and related them to surface receptor expression measured with an epibatidine-binding essay. We also obtained single-channel recordings of the receptors to study their kinetic behaviour. The results were analysed in terms of an allosteric model with three states. We found that the effect of the mutation in the alpha3 subunit on the gating of the receptor was similar to the corresponding mutation in the beta4 subunit. The effect when both subunits were mutated was additive, suggesting that the contribution of each subunit to the gating mechanism is independent.     

Contribution of kinases and the CD45 phosphatase to the generation of tyrosine phosphorylation patterns in the T-cell receptor complex zeta chain

The zeta subunit of the T-cell receptor complex plays a crucial role in coupling the antigen binding alphabeta and gammadelta heterodimers to the downstream activation pathways. Three tandem amino acid sequence motifs containing pairs of exactly spaced Tyr-X-X-Leu/Ile sequences, designated as Immunoreceptor Tyrosine-based Activation Motifs (ITAMs), control this function. The phosphorylated forms of ITAMs serve as docking sites for several src homology 2 (SH2) domain containing signaling proteins. The composition of the assembled signaling complex and the outcome of cell activation depends on the tyrosine phosphorylation pattern of the zeta polypeptide. The mechanism that conducts the generation of various phosphorylated forms has not yet been well established. In this study we have analyzed the ability of src family tyrosine kinases and the CD45 tyrosine phosphatase in determining the phosphorylation state of the different ITAMs and the individual tyrosine residues of the TCR zeta chain. The intracellular part of the zeta chain was phosphorylated by src family tyrosine kinases, p56lck and p59fyn in vitro. Synthetic oligopeptides representing full-length or half-sized ITAMs with a single tyrosine residue were also phosphorylated by both p56lck and p59fyn. In contrast, an additional membrane proximal tyrosine residue in the human zeta chain, located outside of the ITAMs, was not phosphorylated. We also examined the activity of the CD45 phosphatase, using a panel of ITAM derivatives, in which one or both tyrosines were phosphorylated. The efficiency of ITAM dephosphorylation by CD45 was dependent on the primary sequence of the oligopeptides and the position of the phosphotyrosine residues. The in vitro data suggest that the CD45 phosphatase rather than the tyrosine kinase(s) may control the generation of specific phosphorylation patterns of the zeta chain during cell activation.     

Discrimination of olfactory sexual cues in staggerer mutant male mice

Generally, staggerer male mice do not express any preference between oestrous and anoestrous female odours in a choice test situation. The staggerer ability to discriminate between these olfactory sexual cues was evaluated in an habituation-dishabituation paradigm. In this situation it was found that the staggerer mice discriminate between these two odours. The lack of sexual odour preference in staggerer male mice is discussed through hormonal and neurological interpretation.     

Molecular cloning and sequencing of the aroA gene from Actinobacillus pleuropneumoniae and its use in a PCR assay for rapid identification

The gene (aroA) of Actinobacillus pleuropneumoniae, serotype 2, encoding 5-enolpyruvylshikimate-3-phosphate synthase was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829, and the nucleotide sequence was determined. A pair of primers from the 5' and 3' termini were selected to be the basis for development of a specific PCR assay. A DNA fragment of 1,025 bp was amplified from lysed A. pleuropneumoniae serotypes 1 to 12 of biovar 1 or from isolated DNA. No PCR products were detected when chromosomal DNAs from other genera were used as target DNAs; however, a 1,025-bp DNA fragment was amplified when Actinobacillus equuli chromosomal DNA was used as a target, which could be easily differentiated by its NAD independence. The PCR assay developed was very sensitive, with lower detection limits of 12 CFU with A. pleuropneumoniae cells and 0.8 pg with extracted DNA. Specificity and sensitivity make this PCR assay a useful method for the rapid identification and diagnosis of A. pleuropneumoniae infections.     

Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia

The incidence of meiotic abnormalities and their relationship with different spermatogenic parameters was assessed in 103 male patients with presumably idiopathic severe oligoasthenozoospermia (motile sperm concentration < or = 1.5 x 10(6)/ml). Meiosis on testicular biopsies was independently evaluated by two observers. Meiotic patterns included normal meiosis and two meiotic abnormalities, i.e. severe arrest and synaptic anomalies. A normal pattern was found in 64 (62.1%), severe arrest in 21 (20.4%) and synaptic anomalies in 18 (17.5%). The overall rate of meiotic abnormalities was 37.9%. Most (66.7%) meiotic abnormalities occurred in patients with a sperm concentration < or = 1 x 10(6)/ml. In this group, total meiotic abnormalities were found in 57.8% of the patients; of these, 26.7% had synaptic anomalies. When the sperm concentration was < or = 0.5 x 10(6)/ml, synaptic anomalies were detected in 40% of the patients. In patients with increased follicle stimulating hormone (FSH) concentrations, total meiotic abnormalities occurred in 54.8% (synaptic anomalies in 22.6%). There were statistically significant differences among the three meiotic patterns in relation to sperm concentration (P < 0.001) and serum FSH concentration (P < 0.05). In the multivariate analysis, sperm concentration < or = 1 x 10(6)/ml and/or FSH concentration > 10 IU/l were the only predictors of meiotic abnormalities.     

Periodontal regeneration of intrabony defects using resorbable membranes: determinants of the healing response. An observational clinical study

The safety and bone-regenerative capacity of a resorbable membrane (Resolut) was evaluated by a nonrandomized prospective clinical study of patients with periodontal defects. Prior to surgical management all patients underwent scaling and root planing and were instructed on oral hygiene. The study included 18 patients (31 periodontal defects) who received surgical treatment by guided tissue regeneration (GTR) using resorbable membranes. The results were evaluated 12 months after surgery in terms of Plaque Index, bleeding index, probing depth, gingival recession, clinical attachment level, and dental mobility. The results obtained show that the use of resorbable membranes in GTR causes few complications. The mean gain in clinical attachment level (4.06 +/- 1.91 mm), with an attachment level gain of more than 3 mm in 81.2% of the defects, suggests the presence of "new attachment." This difference was clinically and statistically significant (t = 11.03, P = 0). The multivariate regression study showed that 60% of the observed variability (F = 11.48, P < 0.001) in clinical attachment level gain was accounted for by the variable's initial probing depth, the Plaque Index of the tooth subjected to GTR, and the infrabony component of the defect.     

A pilot study on the antibodies to HHV-6 variants and HHV-7 in CSF of MS patients

In the possible role for human herpesviruses (HHV) in the pathogenesis of multiple sclerosis (MS) neither clear distinction between the two variants of HHV-6, nor the involvement of HHV-7 have been described. Therefore, we quantitated HHV-6 variant specific and HHV-7 reacting antibodies in the CSF of 13 patients with MS or other neurological disorders by ELISA. Predominance in the positivity of IgG (67%) and IgM (44%) to HHV-6B over that of IgG (44%) with no detectable IgM to HHV-6A, and no antibodies to HHV-7 were found in the CSF of MS patients. None of these antibodies were found in the CSF of controls. This suggests that, intrathecal chronic active or primary HHV-6B infection might contribute to MS progression, while the local effects of HHV-6A and HHV-7 seem to be less important.     

Olfactory esthesioneuroblastoma: scintigraphic expression of somatostatin receptors

Esthesioneuroblastoma is an uncommon tumor originating in the upper nasal cavity and constitutes 3% of all intranasal neoplasms. Few references exist about the expression of somatostatin receptors in these tumors. Our case demonstrates a good correlation between the somatostatin receptor scintigraphy and magnetic resonance imaging.