Circulating autoantibodies against the NR2 peptide of the NMDA receptor are associated with subclinical brain damage in hypertensive patients with other pre-existing conditions for vascular risk.

Arterial hypertension (HT) and other vascular pre-existing conditions (PEC) generate asymptomatic brain damage which increases the occurrence of stroke and cognitive decline. The aim of this work was to explore if serum antibodies against the NR2 subunit of the NMDA receptor (NR2Ab) could predict subclinical brain damage (SBD) in hypertensive patients with PEC. Forty seven neurologically asymptomatic hypertensive subjects were classified according to the number of PEC (retinopathy, overweight/obesity, diabetes mellitus and dyslipidemia). NR2A/B Ab were measured in serum employing an ELISA method. 3.0-T Brain MRI imaging was performed, and visual ratings of white matter hyperintensities (WMH) and counts of dilated Virchow-Robin spaces (DVRS) and lacunes were obtained. Brain atrophy was evaluated with cortical thickness measurements and linear measures. Higher levels of NR2Ab were associated with more severe periventricular WMH (PWMH), more DVRS and more severe SBD; while greater frontal interhemispheric fissure width (IHFW), as a linear measure of frontal atrophy, was inversely related with NR2Ab. Overall and regional cortical thickness were not significantly associated with NR2 Ab. A multivariate analyses showed that IHFW and PWMH were the only variables independently associated with serum NR2Ab concentration. ROC analysis revealed that NR2Ab (cutoff: 1.7ng/ml) predicted PWMH with a sensitivity and specificity of 65% and 87% respectively. CONCLUSIONS: Serum NR2Ab levels may reflect SBD in HT subjects with PEC, especially in younger populations at risk, where age-related cortical atrophy has not yet been fully established.

Factores genéticos de riesgo relacionados con la enfermedad pulmonar del recién nacido / Genetics markers of risk for respiratory distrees in newborn

Las causas de distrés respiratorio en el neonato son muy diversas: hay factores nutricionales e inflamatorios y trastornos del desarrollo embrionario que pueden contribuir al problema; pero en los últimos años ha cobrado importancia la consideración de los factores genéticos de riesgo. Entre las causas genéticas de distrés respiratorio, la mejor documentada es el déficit hereditario de la proteína del surfactante pulmonar SPB, que da lugar a un fracaso respiratorio agudo, crónico y potencialmente irreversible. La disponibilidad de marcadores genéticos de utilidad clínica para evaluar el riesgo de distrés respiratorio en el neonato permitirá el desarrollo de estrategias racionales para el tratamiento pulmonar y la posibilidad de ofrecer consejo genético a las familias de riesgo (AU)

Esclerosis Múltiple en la infancia / Childhood multiple sclerosis

La esclerosis múltiple (EM) es una enfermedad que suele presentarse en la edad adulta, aunque algunos casos pueden debutar en la infancia. En niños, esta entidad resulta de especial interés por varias razones: en primer lugar, es relativamente infrecuente; en segundo lugar, los datos clínicos y los hallazgos de laboratorio son a menudo diferentes de los que se observan en la población adulta, y por último, dada la dificultad de la evaluación clínica, sobre todo en la edad preescolar, es muy importante la integración de todas las determinaciones paraclínicas para obtener un diagnóstico diferencial precoz y para el correcto tratamiento del paciente pediátrico (AU)

G protein-coupled receptor kinase 2 (GRK2): mechanisms of regulation and physiological functions.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in determining the rate and extent of G protein-coupled receptor (GPCR) desensitization and resensitization. Recent data indicate that GRK2 activity, subcellular distribution and expression are tightly regulated. The important physiological function of GRK2 as a modulator of the efficacy of GPCR signal transduction systems is exemplified by its relevance in cardiovascular physiopathology as well as by its emerging role in the regulation of chemokine receptors.

Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called beta-adrenergic kinase 1 or betaARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein beta-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant betaARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines.

Analysis of the particle size distribution and internal volume of liposomal preparations.

In this work we studied the particle size distribution of three liposomal preparations by quasi-elastic light scattering spectroscopy. Sized unilamellar vesicles of small diameter (s-SUV) were prepared by ultrasonication and subsequent centrifugation followed by extrusion through polycarbonate membranes of 0.2-micron pore size. Large unilamellar vesicles were obtained by reversed-phase evaporation (REV) and extrusion through polycarbonate filters with or without preliminary freezing-thawing cycles (VETI and VETII, respectively). After preparation, REV were sized to small diameter REV (s-REV) by extrusion through 0.4- and 0.2-micron polycarbonate membranes. According to the results, the s-SUV preparations were made up of two subpopulations, the major of which consisted of vesicles that were 26 nm in mean diameter and accounted for 95% of the overall s-SUV population. The s-REV dispersions always resolved into two populations centered at 120 and 380 nm, the relative proportions of which depended on the pore size of the filters used. VET structures were composed of a single population of vesicles that were approximately 100 nm in mean diameter. Cholesterol inclusion into the bilayer composition extended the distribution without altering its mean value. On the other hand, the internal volumes calculated from mean diameters or assuming a Gaussian distribution were inconsistent with experimental data obtained by usual techniques.

Comparison of particle size and encapsulation parameters of three liposomal preparations.

Liposomes can be produced by using various techniques such as mechanical agitation, ultrasonication, ether injection, detergent dialysis, reversed-phase evaporation, extrusion through polycarbonate filters of appropriate pore size, etc. Each procedure provides liposomes with their own perculiar features so the choice of a given one to obtain suitable vesicles for use as drug delivery systems should be based on careful experimentation. In this work we tested three liposome preparation methods that provide unilamellar structures with high yields. In fact, unilamellar vesicles of small diameters (SUV) were prepared by ultrasonication and large unilamellar vesicles (LUV) were obtained by (a) reversed-phase evaporation (REV) and (b) extrusion through polycarbonate filters--with or without preliminary freezing-thawing (VETI and VETII, respectively). According to the results obtained, subsequent filtration of the SUV and REV liposomal preparations results in dramatically decreased average diameters and polydispersity indices, as well as in marked changes in their encapsulation parameters and in lipid losses. Among extruded liposomes, those obtained with preliminary freezing-thawing (VETII) resulted in a higher lipid recovery, even though their average hydrodynamic diameter and polydispersity index as measured by quasi-elastic light-scattering spectroscopy (QELSS) were quite similar to those of the VETI counterparts.

Phenylbutazone action on dimiristoyl phosphatidylcholine liposome phase transition and 8-anilino-1-naphthalene sulfonate binding.

Phenylbutazone (PhB), a powerful anti-inflammatory drug, is able to modify the phase transition of phospholipid bilayers without changing its calorimetric enthalpy (delta Hcal), as can be shown by differential scanning calorimetry (DSC) experiments. Under PhB interaction, dimiristoyl phosphatidylcholine (DMPC) multilamellar liposomes (MLV) undergo lateral phase separation as a result of immiscibility in the bilayer plane. On the other hand, the binding of the anionic fluorescent probe 8-anilino-1-naphthalene sulfonate (ANS) to the surface of DMPC liposomes is altered by PhB. Even though the quantum efficacy of the probe fluorescence emission remains unaffected, the negative cooperativity of the binding process disappears, with the intrinsic dissociation constant showing only a minor variation. From these results it is concluded that PhB would be most likely located close to the lipid:water interface.

A hisT::Tn5 mutation affects production of microcins B17, C7, and H47 and colicin V.

A Tn5 insertion decreasing the production of microcin B17 was mapped to 50.2 min on the Escherichia coli chromosome map. Sequence analysis showed that the insertion disrupted hisT, the gene encoding pseudouridine synthase I, a tRNA-modifying enzyme. hisT::Tn5 mutant cells were also shown to be defective for the production of other antibiotic peptides, such as microcin C7, microcin H47, and colicin V.

An unusual mechanism for resistance to the antibiotic coumermycin A1.

Bacterial DNA gyrases are type II topoisomerases made up of two A subunits and two B subunits. Coumarins are carbohydrate-containing antibiotics that inhibit topoisomerases II by competing with ATP for binding to the enzymes. High resistance to coumarins is produced in bacterial species by mutations in gyrB, the gene encoding subunit B. We have found an unusual mechanism of resistance to coumarins in Escherichia coli. This mechanism is exhibited by cells containing the wild-type gyrB, or its 5' half, in high copy number. Since homologous mutant gyrB (coumermycin resistant) truncated genes did not confer drug resistance at all under the same conditions, we propose that this mechanism of resistance is due to drug sequestration by the overproduced wild-type GyrB polypeptides. A corollary of this is that the amino half of GyrB is required and sufficient to fashion the ATP-binding domain of DNA gyrase, a conclusion that was further supported by mapping three independent coumarin-resistant mutations at Arg-136 of GyrB. Just upstream of this residue there is a glycine-rich sequence highly conserved in all topoisomerases II, which seems to be a good candidate for the actual ATP-binding site.

Molecular characterization of pmbA, an Escherichia coli chromosomal gene required for the production of the antibiotic peptide MccB17.

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains harbouring plasmid pMccB17. We have isolated two mutations that strongly reduce the production of MccB17. These mutations, which map at 96 min on the E. coli chromosome, define a new gene that we have called pmbA. A chromosomal DNA fragment of about 13 kb, including the wild-type pmbA allele, was cloned into a mini-Mu plasmid vector. pmbA was located within the cloned DNA fragment by insertional mutagenesis and deletion analysis. The nucleotide sequence of a 1.7 kb DNA region containing the gene was determined. pmbA encodes a hydrophilic protein of 450-amino-acid residues with a predicted molecular size of 48375D, which was visualized in polyacrylamide gels. Protein profiles of cellular envelope and soluble fractions from cells with plasmids overproducing PmbA indicated that it is cytoplasmic. Physiological experiments suggested that pmbA mutants synthesize a molecule (pro-MccB17) able to inhibit DNA replication but unable to be released from cells. We propose that PmbA facilitates the secretion of the antibiotic by completing its maturation.

Complete recovery of hemopoiesis following bone marrow transplant in a patient with unresponsive congenital hypoplastic anemia (Blackfan-Diamond syndrome).

Allogeneic bone marrow transplantation (BMT) was carried out on a 5-year-old boy with congenital hypoplastic anemia (CHA), who did not respond to corticosteroids and who was displaying signs of progressive hemosiderosis. Pretransplant preparation had to be modified because respiratory failure and cerebral edema supervened. This preparatory regimen consisted of busulfan (2 mg/kg for four days), cyclophosphamide (50 mg/kg for one day), and total body irradiation (750 rad). Hemopoiesis was completely restored and is still maintained 650 days after transplantation. This is the second published report on the use of BMT to treat a patient with CHA, and it is the first time it has resulted in long-term survival. BMT should be considered for patients with CHA who do not respond to corticosteroids.