ASSOCIATION OF COL1A1 SP1 AND FOK-I VDR GENETIC POLYMORPHISMS IN YOUNG MALE IDIOPATHIC OSTEOPOROSIS.

INTRODUCTION: Primary osteoporosis during childhood and adolescence represents an uncommon condition, and secondary forms are more likely to manifest at this age due to chronic disease and adverse effects of medical treatment. CASE REPORT: The authors report the case of a young male patient with a history of multiple idiopathic non-vertebral fragility fractures in addition to a family history of maternal osteoporosis and fracture, in whom osteoporosis was confirmed according to 2013 International Society for Clinical Densitometry (ISCD) criteria. Bone markers indicated low bone formation marker osteocalcin. Genetic testing revealed homozygosity for Sp1 COL1A1 gene polymorphism in combination to Fok-I vitamin D receptor (VDR) heterozygous polymorphism, to contribute to low bone mass and increased fracture risk. Severe premenopausal osteoporosis was present in the patient's mother, who was also tested positive for both gene polymorphisms. CONCLUSION: This case report highlights the association between COL1A1 and VDR candidate gene polymorphisms and fragility fractures in a family. Individual genetic testing might be of clinical value in idiopathic osteoporosis in young patients, identifying subjects at increased fracture risk.

New and old microbial communities colonizing a seventeenth-century wooden church.

The North of Romania is known for its wooden churches dating from the seventeenth and eighteenth centuries. Their deterioration constitutes a major problem due to their value for the cultural heritage. The microbial community from a seventeenth-century wooden church (Nicula, Romania) was investigated by characterization of uncultivated and cultivated bacteria using 16S rDNA sequence analysis. The study revealed not only the prevalence of the Bacillus thuringiensis strain IAM 12077 but also the presence of new microbial communities of Planomicrobium and Variovorax that were not previously reported in paintings or on wood. The identification of fungi showed the presence of seven common genera found on the walls and icon surfaces. Common bacteria from the human oral microbiota were not identified in the bacterial community.

In vitro evaluation of the effects of yttria-alumina-silica microspheres on human keratinocyte cells.

The behavior of yttria-alumina-silica spray-dried microspheres was investigated in vitro on a human keratinocyte cell line, first to exclude their cytotoxicity. The HaCaT cells were chosen due to their well-characterized phenotype and their phagocytic ability. Microscopic analysis and cell viability tests showed no negative effect of the microspheres on cells morphology and behavior. Scanning electron microscopy and transmission electron microscopy results evidenced the cellular internalization of the microspheres at 48 h after their incubation with cultured cells. The shape, size distribution, structure, composition, and chemical states of the elements on samples surface were analyzed by SEM, transmission electron microscopy, x-ray diffraction, and x-ray photoelectron spectroscopy, because these properties could influence their internalization by cells. The yttrium distribution on the microspheres surface was indicated by fluorescence microscopy imaging. The microspheres dimension and shape inside the cells was in accordance with their dimension and shape before incubation. The microspheres seemed captured and engulfed by the cells in native form and appeared resistant to degradation over the first 48 h. Most of the analyzed cells took up more microspheres, suggesting that the microspheres were actively phagocytosed by the cells and accumulated within the cytoplasm. X-ray photoelectron spectroscopy results on Al and Si atomic environments denoted Al-O-Si crosslinks, which improve the surface protection to corrosion.

Binding of avian IgY to type VII collagen does not activate complement and leucocytes and fails to induce subepidermal blistering in mice.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a severe autoimmune skin disease characterized by tissue-bound and circulating autoantibodies to type VII collagen, the major component of anchoring fibrils. When passively transferred into mice, rabbit IgG against type VII collagen induces Fc-dependent activation of complement, the recruitment of leucocytes into the skin, and subepidermal blistering. In addition to these inflammatory mechanisms, clinical and experimental evidence suggests that antibodies against type VII collagen might induce blisters by disrupting the ligand function of type VII collagen by an Fc-independent mechanism. OBJECTIVES: To study noninflammatory mechanisms of blister formation in experimental EBA. METHODS: We generated chicken IgY antibodies directed to recombinant type VII collagen and examined their pathogenic activity using ex vivo and animal models. RESULTS: Mice injected with these chicken IgY antibodies showed binding of the antibodies to the dermal-epidermal junction of skin sections. However, IgY antibodies did not fix complement C3 in enzyme-linked immunosorbent assay and immunofluorescence complement-binding assays. In addition, IgY antibodies did not induce dermal-epidermal separation ex vivo. Following their passive transfer into Balb/c mice, chicken IgY antibodies against type VII collagen bound at the dermal-epidermal junction, but, in contrast to rabbit IgG, did not fix complement C3, recruit granulocytes, or induce skin blisters. CONCLUSIONS: These findings demonstrate that binding of avian IgY to type VII collagen is not pathogenic in vivo and strongly suggest that in experimental EBA, antibodies to type VII collagen induce blisters not by direct disruption of the ligand function of type VII collagen, but rather by an Fc-dependent engagement of humoral and cellular inflammatory factors.

NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.

The contribution of phagocyte-derived reactive oxygen species to tissue injury in autoimmune inflammatory diseases is unclear. Here we report that granulocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase crucially contributes to tissue injury in experimental models of the antibody-mediated autoimmune disease epidermolysis bullosa acquisita. Neutrophil cytosolic factor 1-deficient mice lacking functional NADPH oxidase were resistant to skin blistering by the passive transfer of antibodies against type VII collagen. Pharmacological inhibition or deficiency of human NADPH oxidase abolished dermal-epidermal separation caused by autoantibodies and granulocytes ex vivo. In addition, recruitment of granulocytes into the skin was required for tissue injury, as demonstrated by the resistance to experimental blistering of wild-type mice depleted of neutrophils and of CD18-deficient mice. Transfer of neutrophil cytosolic factor 1-sufficient granulocytes into neutrophil cytosolic factor 1-deficient mice demonstrated that granulocytes provide the NADPH oxidase required for tissue damage. Our findings identify granulocyte-derived NADPH oxidase as a key molecular effector engaged by pathogenic autoantibodies and provide relevant targets for prevention of tissue damage in granulocyte-mediated autoimmune diseases.

[Endothelium-derived hyperpolarizing factor]. / Factorul hiperpolarizant derivat din endoteliu.

Endothelial cells control the tone of the underlying smooth muscle by releasing relaxing factors, such as nitric oxide (NO), prostacycline, endothelium-derived hyperpolarizing factor (EDHF) and contracting factors such as thromboxane A2, endothelins, endoperoxides and superoxide anion. The term EDHF should be restricted to a relaxing factor(s) that differs from NO and prostacyclin. The nature of EDHF is as yet unknown. EDHF might be a cytochrome P-450 metabolite of arachidonic acid or an endogenous cannabinoid--anandamide. The EDHF component of the relaxation is more important in smaller than larger arteries. In various models of disease and in aging animals endothelium-dependent hyperpolarization is diminished. The identification of EDHF and the understanding of its physiological role could lead to the design of new therapies în several cardiovascular diseases.

Trace analysis of triazines and organophosphorus pesticides in water.

A method for the determination of trace levels of triazines and organophosphorus pesticides in water is presented. The extraction method is based on a solid-phase extraction on C-18 bound silica SPE cartridges. A precolumn filled with Merck C-18 bound silica and home-made C-18 bound silica have been tested at a flow-rate of 3 ml/min with comparable preconcentration yields. A SIM-MS method using a (15)N labelled internal standard has been developed for the organophosphorus pesticides. Detection limits lower than 1 microg/L have been obtained. Separations have been carried out on a conventional GC column OV 17 (1 m) and a capillary column OV 17 (25 m) with a temperature program from 150 degrees C (2 min) to 300 degrees C (rate of 6 degrees C/min).

Calmodulin binding of a peptide derived from the regulatory domain of Bordetella pertussis adenylate cyclase.

This paper reports the solution conformation and calmodulin binding of a 43-residue peptide from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. The peptide (P225-267) was synthesized and 15N-labeled at specific amino acids. It binds calmodulin with an equilibrium dissociation constant of 25 nM. Assignment of the NMR spectrum of the free peptide and analysis of the NOE connectivities and secondary shifts of C alpha protons allowed us to identify a 10-amino acid fragment (Arg237 to Arg246) which is in rapid equilibrium between alpha-helical and irregular structures. Titration experiments showed that at substoichiometric molar ratios the two molecules are in intermediate exchange between free and bound conformations. Using 15N-edited methods we assigned a large part of resonances of the labeled residues in the bound peptide. Analysis of the chemical shift differences between free and bound states shows that the fragment Leu240-Ala257 is the most affected by the interaction. The proton spectra of the calmodulin, in the free and complexed states were extensively assigned using homonuclear experiments. Medium- and long-range NOE patterns are consistent with a largely conserved secondary and tertiary structure. The main changes in chemical shift of calmodulin resonances are grouped in six structural regions both in NH2- and COOH-terminal domains. Intermolecular NOE connectivities indicate that the NH2-terminal of the bound peptide fragment is engulfed in the COOH-terminal domain of calmodulin. The interaction geometry appears to be similar to those previously described for myosin light chain kinase or calmodulin kinase II fragments.

Enzymatic synthesis of guanine nucleotides labeled with 15N at the 2-amino group of the purine ring.

GMP and dGMP labeled with 15N at the 2-amino group of the purine ring was obtained enzymatically from NH4Cl (> 99 at.% 15N) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase. The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes. The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at.% 15N. The proton NMR spectrum of [15N]GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 Hz. When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at 6.47 ppm, indicating that the corresponding proton is bound to 15N.

[The interpretation of the telomic distribution of the pulmonary arterial trunk]. / Interpretarea distributiei telomice a trunchiului arterial pulmonar.

The analytical study of the periodicity of organic processes is of great interest as reflected by the literature. This paper aims at describing the functional implications from the viewpoint of pulmonary functional arterial circulation regularity. In the light of morphological aspects, the pressure falls along a symmetric telomic tree were followed up and compared with the experimentally obtained data.

Isotope dilution mass spectrometry for procaine determination in biological samples.

A simple, rapid and sensitive method for procaine determination is described. Isotope dilution mass spectrometry with (15N)procaine as internal standard was used. The analysis was performed at 4000 resolution by selected ion monitoring with temperature programming. The sample was measured in underivatized form in the direct inlet system. The method shows good analytical parameters: linearity between 0 and 40 micrograms ml-1, good precision and accuracy. The method was applied to the in vitro pharmacokinetic study of the metabolism of procaine in liver homogenates of Wistar rats. The method is rapid, permitting about six samples to be run per hour. Sensitivity of the method permits analysis at a signal-to-noise ratio of 5:1.

Serum theophylline analysis by isotope dilution mass spectrometry.

Serum theophylline analysis was attempted using stable isotope dilution mass spectrometry. Theophylline 15N-labelled in position 7 was used as internal standard. The analysis was performed at resolution 3000 by selected ion monitoring with temperature programming. The sample was introduced in underivatized form by means of the direct inlet probe. Good linearity was obtained in the concentration range 5-40 micrograms ml-1 with relative standard deviations less than 6%. The method is simple and permits six samples to be run per hour. Sensitivity permits analysis at 0.5 micrograms ml-1 at a signal to noise ratio 4:1.

[Clinical observations of treatment with Lyorodin-depot (Jenapharm)]. / Observatii clinice asupra tratamentului cu Lyorodin-depôt (Jenapharm).

According to the indications of fluphenazin decanoate in the long-term treatment in schizophrenia the authors analyse the findings of a simple blind study of Lyorodin-depot on a sample of 20 inpatients. The drug was administered at intervals of 21 days for 18 weeks under clinical surveillance. The method consisted of patients' follow-up on an itemized card, psychological checkups and para-clinical investigations. The analysis of clinical findings reveals the amelioration of symptoms at the parameters accounted for, the favourable evolution low incidence of the side effects that confirms its clinical efficiency and tolerance.