Widespread use of Lactobacillus OppA, a surface located protein, as an adhesin that recognises epithelial cell surface glycosaminoglycans.

Specific adherence is the first requisite that a microorganism has to fulfil to become established onto a mucosal surface. It was previously shown that the OppA surface protein of Lactobacillus salivarius Lv72 bound HeLa cell cultures through interaction with glycosaminoglycans (GAGs). To determine whether this is a peculiarity of that strain or whether it can be extended to other lactobacilli, 12 strains, belonging to six species, were confronted with HeLa-cell cultures in the presence of soluble GAGs. Interference was observed to six of them, heparan sulphate and chondroitin sulphate C being more interfering than chondroitin sulphate A or chondroitin sulphate B. Furthermore, inhibition of the biosynthesis of GAGs or their elimination from the cell surface with specific enzymes also resulted in reduced adherence. Analysis of the surface proteome of Lactobacillus crispatus Lv25 and of Lactobacillus reuteri RC14 revealed single proteins that immunoreacted with antibodies raised against OppA, the main adhesin of L. salivarius Lv72. Upon MALDI-TOF-TOF analysis, they were identified as OppA-like proteins, thus indicating that these proteins participate as adhesins in attachment of diverse lactobacilli to the surface of human epithelial cells.

Two alkaline motifs in the Lactobacillus salivarius Lv72 OppA surface are important to its adhesin function.

Glycosaminoglycans are involved in the attachment of Lactobacillus salivarius Lv72, a strain of vaginal origin, to HeLa cell cultures, indicating that they play a fundamental role in the attachment of mutualistic bacteria to the epithelium lining cavities where the normal microbiota thrives. The bacterial OppA protein has been proposed as an adhesin involved in this adherence since, once purified, it significantly interferes with attachment of the lactobacilli to HeLa cell cultures. In this article, the role of OppA is confirmed through the determination of its location at the cell surface and its ability to promote Lactobacillus casei and Lactococcus lactis adherence to eukaryotic cell cultures upon cloning and expression of oppA in these bacteria. The OppA sequence showed five potential domains for glycosaminoglycan-binding, and structural modelling of the protein showed that two of them were located in the vicinity of an OppA superficial groove whose width approached the diameter of the helical form of heparin in solution. Their involvement in the binding was demonstrated through substitution of critical basic amino acids by acidic ones, which resulted in loss of affinity for heparan sulphate and chondroitin sulphate depending on the domain mutated, suggesting that there might be a certain degree of specialisation. In addition, circular dichroism analysis showed that the spectrum changes induced by OppA-heparan sulphate binding were attenuated by the variant proteins, indicating that these motifs are the OppA recognition domains for the eukaryotic cell surface.

Análisis de decisión: coste-efectividad en el tratamiento quirúrgico de la neuralgia del trigémino / No disponible

Introducción: La neuralgia trigeminal es un síndrome de dolor facial conocido y caracterizado por dolores severos, intermitentes, eléctricos y como de sacudidas en la cara, para el cual han sido aplicados diversos tratamientos quirúrgicos, hasta la fecha, no hay un tratamiento ideal que sea invasivo en grado mínimo y aceptable para el paciente, como consecuencia, los pacientes y los especialistas se enfrentan a una incertidumbre considerable al hacer decisiones sobre la conducta terapéutica. Objetivo: Identificar cuál de los tratamientos quirúrgicos para la NT brinda la mejor oportunidad en términos de coste-efectividad. Material y método: Se realizó un estudio con un diseño de Análisis de Decisión: coste-efectividad considerando cinco alternativas quirúrgicas en el tratamiento de la NT: termocoagulación por radiofrecuencia, microcompresión con glicerol, microcompresión con balón, radiocirugía, y microdescompresión vascular. Los datos se obtuvieron de la literatura. Se revisó la base de datos MEDLINE desde el año 2000 hasta el 2010, a través de PubMed Central. Se seleccionaron los estudios que abordaran la neuralgia trigeminal primaria y mostraran resultados relacionados con el alivio del dolor, la tasa de mortalidad o la presencia de complicaciones. Se identificaron 196 estudios pero solo 22 (11,22 %) fueron elegibles para el estudio. Se utilizo el programa DATA 3.5 for Health Care, versión 3.5.5 de TreeAge Software Inc. Resultados: La microdescompresión vascular y la termocoagulación son las técnicas que ofrecen mayores probabilidades de efectividad para el alivio del dolor con valores esperados de 0,8946 y 0,8863. Para la relación que se establece entre coste y resultado se consideró los días libres de dolor. La mejor elección costeefectividad es el tratamiento quirúrgico con termocoagulación con menos valor en la razón coste-efectividad marginal, $171.58 para una efectividad de la cirugía de 89 % por cada paciente tratado. Conclusión: Considerando que la termocoagulación por radiofrecuencia y la microdescompresión vascular constituyen las variantes quirúrgicas más utilizadas por la alta efectividad, concluimos que la termocoagulación por radiofrecuencia es más beneficiosa, ofrece escasas complicaciones y es menos costosa (AU)

Introduction: Trigeminal neuralgia is a facial pain syndrome known and characterized by severe, intermittent, electric shocklike, shooting pain in the face for which a number of surgical therapies have been used. To date no ideal, minimally invasive, patient-acceptable treatment is available, hence both patients and specialists face substantial uncertainty when making decisions regarding therapeutic action. Objective: To identify which surgical treatment for TN offers the best opportunity in terms of cost-effectiveness. Material and method: A study was performed using a decision analysis/cost-effectiveness analysis design considering five surgical alternatives in the management of TN: radiofrequency thermocoagulation, glycerol microcompression, balloon microcompression, radiosurgery, and microvascular decompression. Data were obtained from the literature. A review was carried out of the MEDLINE database from 2000 to 2010 via PubMed Central. Studies were selected that addressed primary trigeminal neuralgia and demonstrated pain relief and reduced mortality and complication rates. In all 196 studies were identified but only 22 (11.22 %) were eligible for the study. The software package used was the DATA 3.5 for Health Care, version 3.5.5, program by TreeAge Software Inc. Results: Microvascular decompression and thermocoagulation are the techniques more likely to provide effective pain relief, with expected values of 0.8946 and 0.8863, respectively. For the relationship between cost and outcome pain-free days were considered; the best choice in terms of cost-effectiveness is surgery and thermocoagulation, with a lower value in the marginal cost-effectiveness ratio: $171.58 for an effectiveness of 89 % per treated patient. Conclusion: Considering that both radiofrequency thermocoagulation and microvascular decompression are the most commonly used surgical procedures because of their high effectiveness, we conclude that radiofrequency thermocoagulation is more beneficial, has few complications, and is less costly (AU)

Termocoagulación por radiofrecuencia en la neuralgia occipital de Arnold / Radiofrequency thermocoagulation in Arnold's occipital neuralgia

Se presenta a 47 pacientes con neuralgia occipital operados en el Hospital Clínico Quirúrgico Hermanos Ameijeiras entre los años 2006 y 2008, mediante la técnica percutánea de termocoagulación por radiofrecuencia convencional. El objetivo fue determinar la efectividad del proceder quirúrgico a través de los resultados obtenidos. Se describen las variables sexo, lado del dolor afectado, etiología y resultados quirúrgicos. El sexo femenino predominó en el 79% de los casos. El lado occipital más afectado fue el izquierdo (70,2%). La causa idiopática ocupó el primer lugar con el 81%, seguida de la traumática con el 14,8%. Se obtuvieron buenos resultados en el 91,4%. La termocoagulación por radiofrecuencia convencional es un método de mínima invasión con carácter lesivo al nervio, fácil de realizar, con nula morbimortalidad y su gran efectividad lo convierte en una de las alternativas terapéuticas en el alivio de este dolor (AU)

We present a series of 47 patients with occipital neuralgia operated on between 2006 and 2008 in the Hospital Clínico Quirúrgico Hermanos Ameijeiras, using the conventional percutaneous radiofrequency thermocoagulation technique. The aim was to determine effectiveness of the procedure through the results obtained. Variables, including gender, side affected by pain, aetiology and surgical results were recorded. The majority were female (79%). The left occipital was the side more affected (70.2%). Idiopathic was the main cause (81%) followed by trauma with 14.8%. The technique had a 91.4% success rate. Conventional radiofrequency thermocoagulation is a minimally invasive method which damages the nerve, easy to use, with no morbidity or mortality, and its increased efficacy makes it one of the alternative therapies in the relief of this pain (AU)

Termocoagulación facetaria lumbar. Experiencia en 252 pacientes / Thermocoagulation of lumbar facet joints. Experience in 252 patients

Se presentan 252 pacientes con el diagnóstico de dolorlumbar facetario a los que se le realizó la técnica quirúrgicade termocoagulación percutánea de la faceta articular.Nuestro propósito principal fue aliviar el dolor en estos pacientes,evaluamos la eficacia de la técnica con un 74,7%de resultados quirúrgicos satisfactorios, así como la descripciónde diferentes aspectos como: edad, sexo, causas yla topografía segmentaria del dolor

Two hundred fifty two patients with diagnosis of lumbarfacet joint pain underwent the surgical technique ofpercutaneous thermocoagulation of the facet joint. Ourmajor aim was to relief pain in those patients. We assessthe effectiveness of the technique, with 74.7% ofsurgical success, and we also describe different aspectssuch as: age, sex, causes and segmentary pain topography

Estudio comparativo entre la termocoagulación retrogasseriana y la microcompresión con balón catéter en la neuralgia trigeminal / Gasserian thermocoagulation and microcompression with balloon catheter in trigeminal neuralgia

Se presentan 180 pacientes con el diagnóstico de neuralgiatrigeminal, a 90 se les realizó termocoagulación percutáneadel ganglio de Gasser por radiofrecuencia y a los otros 90 microcompresión percutánea retrogasseriana conbalón catéter de Fogarty. Nuestro propósito principal fuealiviar el dolor facial en pacientes portadores de neuralgiatrigeminal, evaluamos los resultados de ambas técnicas, asícomo la descripción de diferentes aspectos que comprendieron:clasificación clínico-topográfica de las ramas afectadas,evolución, resultados quirúrgicos inmediatos y a largoplazo (tomando como límite mínimo el año de operado),además de las complicaciones y ventajas de ambos procederesquirúrgicos. En ambos procederes los buenos resultadosquirúrgicos sobrepasaron el 90%

We present 180 patients diagnosed of trigeminal neuralgia,90 of which underwent percutaneous thermocoagulationof the Gasser’s ganglion with radiofrequency and theother 90 underwent retrogasserian percutaneous microcompressionwith Fogarty balloon catheter. Our main purposewas to relief facial pain in patients with trigeminalneuralgia. We review the results of both techniques anddescribe different aspects such as clinicotopographic classificationof the branches involved, evolution, short-term andlong-term surgical results (taking as lower limit one year afterthe surgery), as well as complications and benefits ofthe two surgical procedures. In both cases, good surgicalresults were obtained in excess of 90%

Deficiencia de vitamina B12 y tratamiento por vía oral. Una opción tan eficaz como (todavía) poco utilizada / B12 vitamin deficiency and oral treatment. An option as efficacious as it is (still) little used

No disponible

Analysis of the morphogenetic cluster and genome of the temperate Lactobacillus casei bacteriophage A2.

The genes that encode the morphogenetic proteins of bacteriophage A2 are clustered and expressed as a single operon which originates a late transcript of more than 20 kb. This DNA stretch is analyzed in the context of the whole phage genome, which presents the following peculiarities: a) the head presents two major proteins that share their NH(2) termini, i.e.: both are translated from a single gene (orf5), b) these two proteins suffer a proteolytic maturation process before being incorporated into the capsid, rendering a 123 NH(2) terminal putative polypeptide that is postulated to be the scaffolding protein of the phage, c) similar maturation processes occur at the portal and tail length determination proteins, having all in common a Pho-Pho-Arg downward arrow sequence (where Pho stands for any hydrophobic amino acid) at the processing point, d) the genes encoding the subunits of the terminase (orf61 and orf2) are separated by the cohesive ends, e) two genes that might mediate lysogenic conversion ( orf19 and orf22) have been identified and f) the genome presents a dispensable region (which covers at least 10 orfs, as judged from analysis of deletion mutants) that might be involved in maintaining its size between the packaging limits of the capsid.

Plasmodium falciparum circumsporozoite (CS) protein peptides specifically bind to HepG2 cells.

Hepatocyte invasion by malaria parasites is mediated by specific molecular interactions. Several lines of evidence suggest the importance of the surface plasmodial circumsporozoite (CS) protein in the sporozoite invasion of hepatocytes. Identification of the sequences involved in binding to hepatocytes is an important step towards understanding the structural basis for the sporozoite-hepatocyte interaction. In this study, binding assays between Plasmodium falciparum CS peptides and HepG2 cells were performed. Fifteen overlapping residue 20 mer long peptides, spanning the entire CS sequence, were tested in HepG2 cell binding assays. Five High Binding Activity Peptides (HBAPs) to HepG2 cells were identified: 4593, (NANPNANPNANP); 4383, (NSRSLGENDDGNNEDNEKLR); 4388, (GNGQGHNMPNDPNRNVDENA); 4389, (HNMPNDPNRNVDENANANSA) and 4390, (DPNRNVDENANANSAVKNNN). The HBAP HepG2 interaction is independent of charge and amino-acid composition, but sequence dependent. Four HBAPs (4383, 4388, 4389 and 4390) are bound with similar affinity to a 50 kDa molecule. These HBAPs define three Hepatocyte Binding Sequences (HBSs): HBS-1, located between residues 68 and 87 (HBAP 4383); HBS-11, the repeat NANP region (HBAP 4593), for which anti repeat antibodies are able to specifically inhibit sporozoite invasion of hepatocytes have been reported; and HBS-111, between residues 286 and 315 (HBAPs 4388, 4388 and 4390), respectively. Interestingly, HBS 111 carries two earlier-reported B-epitopes (underlined) in peptides 4388, 4389 and 4390 (GNGQGHNMPNDPNRNVD ENANANSAVKNN) in its sequence. The HBSs reported here show lesser interspecie-variability than the entire protein in species invading the same kind of hepatic cells. This data supports these HBSs' important role in CS-protein function; they could be used as ligand by the sporozoite to invade hepatic cells.

Plasmodium falciparum acid basic repeat antigen (ABRA) peptides: erythrocyte binding and biological activity.

Non overlapping 20-mer peptides, covering the complete sequence of acid basic repeat antigen (ABRA) of Plasmodium falciparum, were synthesised and tested in binding assays to erythrocytes. Five peptides localised in the N-terminal region coded 2148 (121LQSHKKLIKALKKNIESYQN(140)), 2149 (141KKHLIYKNKSYNPLLLSCVK(160)), 2150 (161KMNMLKENVDYIQKNQNLFK(180)), 2152 (201YKSQGHKKETSQNQNENNDN(220)) and 2153 (221QKYQEVNDEDDVNDEEDTND(240)) specifically bind to erythrocytes. These peptides bind independently of the peptide and erythrocyte charge, with high affinity (Kd between 70 and 180 nM) and the hydrophobic interaction is important for this binding ( approximately 30% hydrophobic critical residues). These results allow us define a specific erythrocyte binding region (residues 121-240), which may bound to at least three different binding sites on erythrocytes. Peptide 2153 shares the underlined sequence 221QKYQEVNDEDDVNDEEDTND(240) with an earlier 18-mer peptide recognised by human exposed sera. Peptides number 2148 and 2149 in vitro inhibit erythrocyte invasion by merozoites. We found that 2149 peptide and some of its glycine analogues show specific haemolytic and/or antimicrobial activity. We discuss a possible role of ABRA or its regions in the merozoite invasion of erythrocyte.

Nucleotide sequence and analysis of pBL1, a bacteriocin-producing plasmid from Lactococcus lactis IPLA 972.

The complete sequence of the 10.9-kbp bacteriocinogenic plasmid pBL1 from Lactococcus lactis subsp. lactis IPLA 972 has been determined. Thirteen ORFs were encountered, of which 5 were incomplete. pBL1 proved to be a narrow-host-range plasmid which replicates neither in Bacilus subtilis nor in Lactobacillus spp. The structural organization of the pBL1 replication region was highly similar to other well-known theta-replicating plasmids of lactococci, at both the untranslated (the replication origin) and the translated (repB and orfX) sequences. As in other plasmids, the product of orfX was not necessary for plasmid replication. However, it was shown to be involved in plasmid stability. Three genes organized in an operon-like structure encompassed, most likely, the bacteriocin-encoding region. Upstream of the origin of replication a nicking site (oriT) was found. This oriT sequence proved to be functional by mobilization of plasmids wearing it. One complete and several partial IS elements were identified on pBL1.

Plasmodium falciparum AMA-1 erythrocyte binding peptides implicate AMA-1 as erythrocyte binding protein.

The role of AMA-1 during merozoite invasion has not yet been determined. However, reported experimental evidence suggests that this protein can be used, in particular as erythrocyte-binding protein, since, Fab fragments against this protein are able to block merozoite invasion. Using a previously described methodology, eight peptides with high binding activity to human erythrocyte, scattered along the different domains and having around 130 nM affinity constants, were identified in the Plasmodium falciparum AMA-1 protein. Their binding activity was sialic acid independent. Some of these peptides showed homology with the erythrocyte binding domains of one of the apical organelle protein family, MAEBL, identified in rodent malarial parasites. One of these peptides shares amino acid sequence with a previously reported B-cell epitope which induces antibodies to block parasite growth. The critical residues were identified for erythrocyte binding conserved peptides 4313 (DAEVAGTQYRLPSGKCPVFG), 4321 (VVDNWEKVCPRKNLQNAKFG), 4325 (MIKSAFLPTGAFKADRYKSH) and 4337 (WGEEKRASHTTPVLMEKPYY). All conserved peptides were able to block merozoite invasion of new RBC and development, suggesting that these peptides are involved in P. falciparum invasion.

Characterization of the DNA replication module of bacteriophage A2 and use of its origin of replication as a defense against infection during milk fermentation by Lactobacillus casei.

Adjacent to the lysis/lysogeny cassette of the A2 phage genome lies a stretch of over 8 kb, which contains a series of genes probably involved in DNA replication. Fifteen open reading frames (orfs) were identified, 13 of which are encoded on the main coding strand and only two on the complementary strand. Database searches and comparative analyses allowed the identification of an open reading frame (orf455) that shows similarity with DNA helicases and contains a variant zinc-finger motif known from the phage T7 helicase/primase. Orf770 showed similarity to putative plasmid and phage DNA primases. Downstream of orf770 is a noncoding 258-bp region rich in direct and inverted repeats, which specifically binds to proteins whose synthesis is induced during phage infection. When present in a plasmid, this region can direct a partial bacteriophage resistance phenotype due to interference with phage DNA replication, both under laboratory conditions and during milk fermentation. It is deduced that this stretch contains the origin of replication of phage A2.

A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro.

The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.

Generation of food-grade recombinant lactic acid bacterium strains by site-specific recombination.

The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (beta-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding beta-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the beta-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation.

Solution structure of plantaricin C, a novel lantibiotic.

Plantaricin C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin, is a lantibiotic. One dehydroalanine, one lanthionine and three beta-methyl-lanthionine residues were found in its 27 amino acid sequence. The plantaricin C structure has two parts: the first comprises the six NH2-terminal residues, four of which are lysines, which confer a strong positive charge to this stretch. The amino acids in positions 7 and 27 form the lanthionine bridge, giving a globular conformation to the rest of the molecule. The beta-methyl-lanthionine bridges are established between residues 12-15, 13-18 and 23-26. This central region has a charge distribution compatible with an amphipathic alpha-helix, through which plantaricin C would become inserted into the membrane matrix of sensitive organisms, provoking the opening of pores and leakage of the cytoplasmic content.

A2 cro, the lysogenic cycle repressor, specifically binds to the genetic switch region of Lactobacillus casei bacteriophage A2.

Lysogenic induction of temperate bacteriophage A2 of Lactobacillus casei is controlled by the action of its cI and cro products at the phage operator region. Three 20-bp inverted repeated DNA segments (subsites O1, O2, and O3) and the two divergent (PL and PR) promoters were mapped within the 153-bp operator region. The A2-encoded Cro product is shown to be the functional homolog of lambda Cro. The binding of Cro to the three operator subsites is noncooperative and yields two discrete protein-DNA complexes of retarded migration in mobility shift assays. The Kapp value for the Cro-PL-PR DNA complex was estimated to be 6 nM. Cro shows a slightly higher affinity for O3 than for O1 and O2 subsites. The O3 subsite overlaps the -35 hexamer of the PL promoter, which directs cI expression. A Cro mutant protein, devoid of the last 12 residues (Cro*), allowed the assignment of the DNA-binding domain to the NH2 end of Cro. The C end enhances its affinity for the DNA and probably stabilizes bending induced by Cro.

Duplication of the beta-galactosidase gene in some Lactobacillus plantarum strains.

Curing of a plasmid that encoded a beta-galactosidase gene (beta-gal) from the Lactobacillus plantarum strain of dairy origin LL441 was not accompanied by complete loss of the lactose utilization phenotype. DNA-DNA hybridization, using an internal fragment of the beta-gal gene as a probe, revealed a second determinant located on the chromosome of the cured derivatives. The chromosomal copy was present in all of a series of beta-Gal+ L. plantarum and Lactobacillus pentosus strains from different origins. In addition, four other L. plantarum strains harboured plasmid encoded beta-gal genes as well. Since both sequences cross-hybridized and present a similar genetic organization, it is postulated that the plasmid copy was generated through gene duplication and, probably, selected by growth of the strains in lactose rich environments.