The research of new antimicrobial compounds has an impact on public health and economy of many countries. Given the great problem of bacterial resistance, the study of new molecules that bypass this mechanism is of great importance. Trypsin is an enzyme necessary for gut physiology and the peptides it forms could be of great interest to the pharmaceutical industry. In this study the antibacterial activity of undigested and trypsin-hydrolyzed iron-depleted form of lactoferrin, (apo-bLf) and undigested and diferric bovine lactoferrin (bLf) were evaluated against different bacterial species. Apo-bLf was less susceptible to trypsin hydrolysis compared to the diferric form and its tryptic fragments with molecular weight lower than 5000 Da had greater activity than those obtained from the diferric-bLf. It is plausible that the antimicrobial activity is exerted mainly by the interaction of the N-terminal moiety of the protein with the bacterial cell. The in silico analysis of the interdomain movements, showed that the conformation of the active N-terminal part of apo-bLf is more open than that of the diferric form. The increased accessibility of the N-terminal region seems to be responsible for the antimicrobial activity of the apo-bLf and its tryptic fragments.

Rationale: Limited knowledge exists on phenotypes associated with the D1152H cystic fibrosis transmembrane conductance regulator (CFTR) mutation.Objectives: To characterize phenotypes in subjects with a D1152H allele in trans with another CFTR mutation.Methods: D1152H subjects were identified using the French Cystic Fibrosis (CF) Registry.Phenotypic characteristics were compared with those of CF subjects in the Registry (Controls).Main Results: Forty two subjects with D1152H alleles were identified.Diagnostic features included chronic sinopulmonary disease (n = 25), congenital absence of the vas deferens (n = 11), systematic neonatal screening (n = 4), and genetic counseling (n = 2).Excluding systematic neonatal diagnosis, median age at diagnosis was 33 (interquartile range, [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41]) yr in D1152H subjects and was 0.58 [0.17-3.67]yr in Controls (P < 0.001).Median sweat chloride concentrations were 43.5 [39-63] mmol/l in D1152H subjects vs. 100 mmol/l in Controls (P < 0.001).Only 10/42 (24%) D1152H subjects had classic CF.Nonclassic CF (sweat chloride 60 mmol/l and compatible clinical manifestations) was present in 26/42 (61.9%)D1152H subjects; 6/42 (14.3%)D1152H subjects were asymptomatic.Bronchiectasis was present in 67% of D1152H subjects, but Pseudomonas aeruginosa colonization and pancreatic insufficiency were present in <30% of subjects.Estimated rates of decline in FEV1 and in FVC were lower in D1152H subjects vs. Controls (P < 0.001).None the D1152H subjects identified since 1999 died or had lung transplantation despite an elevated age at last evaluation.Conclusions: D1152H is a mutation that when present in trans with a CF-causing mutation causes variable phenotypes, often characterized by nonclassic (mild) cystic fibrosis.Supported by: Vaincre la Mucoviscidose 18 Cystic Fibrosis and mitochondrial gene mutation MT-RNRI

The neonatal screening protocol for cystic fibrosis (CF) is based on a first determination of blood immunoreactive trypsin (IRT1), followed by a first level genetic test that includes the 31 worldwide most common mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (DNA31), and a second determination of blood immunoreactive trypsin (IRT2). This approach identifies, in addition to affected subjects, a high proportion of newborns with hypertrypsinaemia at birth, in whom only one mutation is identified and who have a negative or borderline sweat test and pancreatic sufficiency. Although it has been suggested that hypertrypsinaemia may be caused by a single CFTR mutation, whether such neonates should be merely considered as healthy carriers remains a matter of debate as hypertrypsinaemia at birth may be a biochemical marker of a CFTR malfunction because of a second mild mutation. We analyzed, by means of an extended sequencing protocol, 32 newborns who tested positive at an IRT1/DNA31/IRT2 screening protocol and in whom only one CFTR mutation was found. The results obtained demonstrate that 62.5% of these newborns were also carrying a second mild CFTR mutation. The high proportion of compound heterozygous subjects, combined with the results of a 4‐year follow‐up in nine of these subjects all of whom displaying initial CF clinical symptoms, suggest that it may be possible to use the IRT1/DNA31/IRT2 protocol of neonatal screening to identify newborns with atypical forms of CF. In view of these findings, an extended genetic search for subjects with compound heterozygosity and a periodic clinical assessment should be considered.

Two techniques in particular are used to study site-specific DNA methylation: genomic sequencing after bisulfite modification and polymerase chain reaction after digestion by a methylation-sensitive endonuclease (usually HpaII). Only the former methodology assesses the methylation status of all the cytosine residues in the DNA sequence, but it is so complex and time consuming that the latter procedure, though limited to the restriction sites recognized by the endonuclease(s) used, is often preferred at least for a first analysis. In this work we investigate differences between these two techniques in the assessment of DNA methylation and offer some suggestions on how to avoid uncorrected results. Although there is substantial accordance in the results obtained using these different techniques, we observed a general overestimate for methylation levels above 30% and a general underestimate for methylation levels below this value using the HpaII/PCR technique in the study of methylation of the 5′-flanking region of the mouse myogenin gene in cultured muscle cells and mouse tissues.

The endocannabinoid anandamide (AEA) has many neurovascular activities. However, it is not yet clear how AEA can be metabolized at the neurovascular interface, and how it can move through the vascular and the cerebral compartments. The results reported in this article show that isolated bovine brain microvessels, an ex vivo model of the blood-brain barrier, have detectable levels of endogenous AEA and possess the biochemical machinery to bind and metabolize it, i.e. type-1 and type-2 cannabinoid receptors (CB1R and CB2R), a selective AEA membrane transporter (AMT), an AEA-degrading fatty acid amide hydrolase, and the AEA-synthesizing enzymes N-acyltransferase and N-acyl-phosphatidylethanolamines-specific phospholipase D. We also show that activation of CB1R enhances AMT activity through increased nitric oxide synthase (NOS) activity and subsequent increase of NO production. AMT activity is instead reduced by activation of CB2R, which inhibits NOS and NO release. In addition, binding experiments and immunoelectronmicroscopy demonstrate that different endothelial cells vary in the expression of CB1R and CB2R on the luminal and/or abluminal sides. The different localization of CBRs can lead to a diverse effect on AMT activity on the luminal and abluminal membranes, suggesting that the distribution of these receptors may drive AEA directional transport through the blood-brain barrier and other endothelial cells.

OBJECTIVE To evaluate peripheral blood mononuclear cells (PBMC) expressing natural killer (NK) cell surface markers (CD16 and CD56, in both CD3- and CD3+ cells) and g/d T cell receptors (TCR) involved in non-MHC-restricted cytotoxicity, assessing their possible relationship with clinical and laboratory variables in patients with systemic sclerosis (SSc). METHODS We submitted 50 patients with SSc to detailed clinical and laboratory assessment, and also performed PBMC subset analyses by direct dual immunofluorescence and flow cytometry. RESULTS No statistically significant differences were found in the percentages or the absolute numbers of total lymphocytes, of B cells, and of CD4+ T cells. The absolute number of CD8+ cells was lower (p < 0.03), while HLA-DR+ elements were higher in frequency (p < 0.03) in SSc patients than in healthy controls. SSc patients had lower values (both percentage and absolute number) of NK-T cells (p < 0.01 and p < 0.003, respectively) and of T cells expressing g/d TCR (p < 0.01 and p < 0.005, respectively); whereas NK cells were marginally but not significantly decreased. The absolute number of NK-T cells showed an inverse correlation to erythrocyte sedimentation rate values (p < 0.03; rs = -0.306), percentage of g-globulins (p < 0.01; rs = -0.353), and serum concentrations of IgG (p < 0.02; rs = -0.334). CONCLUSION Impairment of NK-T cells and of T cells expressing g/d TCR may lead to downregulation of normal immune response, and seems to be important for immunological and inflammatory aspects of SSc.

Objective: To evaluate a possible correlation between abnormal semen consistency and cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and variant tracts.Design: Study of CFTR mutations and variant tracts in men with high semen viscosity as compared with normospermic men.Setting: University-based centers for andrology, clinical biochemistry, and cystic fibrosis.Patient(s): Forty-six male partners from infertile couples with sine causa high semen viscosity compared with 72 normospermic men.Intervention(s): Semen sample collection.Main Outcome Measure(s): We obtained the (TG)(m)T-n polymorphic tracts and a panel of 31 mutations of CFTR, semen viscosity, and semen variables.Result(s): The frequencies of the (TG)(12) and T-5 variant alleles were statistically significantly higher in men with high semen viscosity (17.4% and 7.6%, respectively) than in the normospermic control group (6.9% and 1.4%, respectively). The frequency of the genotypes carrying (TG)(12) or T-5 was statistically significantly higher in men with high semen viscosity (39.1%) than in the normospermic control group (16.7%). Four men with high semen viscosity showed the variant (TG)(m)T-n haplotype; one of these men presented variant tracts on both alleles. None of the normospermic controls showed a (TG)(12)T-5 haplotype.Conclusion(s): Semen hyperviscosity could be considered a "minimal clinical expression" of cystic fibrosis; CFTR gene sequence variations may constitute the genetic basis for this disease. (C) 2004 by American Society for Reproductive Medicine.