How is leghemoglobin involved in peribacteroid membrane degradation during nodule senescence?

An increase in the rate of succinate and glutamate uptake by isolated symbiosomes from French bean nodules was observed in the presence of iron plus H2O2. The lipid bilayer, and not proteins involved in transport, seems to be the major target of radical attack. Leghemoglobin in the presence of a 6-fold excess of H2O2 (where heme breakdown and iron release occurred) provoked also an increase in peribacteroid membrane permeability. In contrast, this hemoprotein in the presence of a 2-fold excess of H2O2 (where a protein radical was generated) was without effect. We suggest that in vivo the release of heme iron may constitute the major process concerning the involvement of leghemoglobin in the degradation of the peribacteroid membrane during nodule senescence.

Vertebrate development in the environment of space: models, mechanisms, and use of the medaka.

With the advent of space travel, it is of immediate interest and importance to study the effects of exposure to various aspects of the altered environment of space, including microgravity, on Earth-based life forms. Initial studies of space travel have focused primarily on the short-term effects of radiation and microgravity on adult organisms. However, with the potential for increased lengths of time in space, it is critical to now address the effects of space on all phases of an organism's life cycle, from embryogenesis to post-natal development to reproduction. It is already possible for certain species to undergo multiple generations within the confines of the Mir Space Station. The possibility now exists for scientists to consider the consequences of even potentially subtle defects in development through multiple phases of an organism's life cycle, or even through multiple generations. In this discussion, we highlight a few of the salient observations on the effects of the space environment on vertebrate development and reproductive function. We discuss some of the many unanswered questions, in particular, in the context of the choice of appropriate models in which to address these questions, as well as an assessment of the availability of hardware already existing or under development which would be useful in addressing these questions.

Characterization of L-plastin interaction with beta integrin and its regulation by micro-calpain.

Recent evidences suggest that plastin/fimbrin is more than a simple actin cross-linking molecule. In this context and based on the fact that other members of the same family interact with transmembrane proteins, such as integrins, we have investigated a possible interaction between L-plastin and integrins. By combining coimmunoprecipitation of endogenous proteins and in vitro techniques based on solid phase and solution assays, we demonstrate that L-plastin is an additional binding partner for the beta-chain of integrin and confirmed that both proteins display some colocalization. We then show that L-plastin binds to the cytoplasmic domain of beta1 integrin and to beta1 and beta2 peptides. Using recombinant L-plastin domains, we demonstrate that the integrin-binding sites are not located in NH(2) terminal part of L-plastin but rather in the two actin-binding domains. Using pull-down, cross-linking experiments, and enzyme-linked immunosorbent assay, we show that the L-plastin/integrin complex is regulated by mu-calpain cleavage and is not directly dissociated by calcium. Indeed, despite the ability of calpain to cleave both proteins, only the cleavage of beta integrin hindered the formation of the L-plastin/integrin complex. We discuss these results in the light of the three-dimensional structure of the actin-binding domains of L-plastin.

A non-covalent peptide-based strategy for siRNA delivery.

The major obstacle to clinical development of siRNAs (short interfering RNAs), like for most of the nucleic-acid-based strategies, is their poor cellular uptake and bioavailability. Although several viral and non-viral strategies have been proposed to improve siRNA delivery, their applications in vivo remain a major challenge. We have developed a new strategy, based on a short amphipathic peptide, MPG, that is able to form stable nanoparticles with siRNA. MPG-based particles enter the cell independently of the endosomal pathway and can efficiently deliver siRNA in a fully biologically active form into a variety of cell lines and in vivo. This short review will discuss the mechanism and the potency of the MPG strategy for siRNA delivery both in vitro and in vivo.

A novel family of putative pheromone receptors in mammals with a topographically organized and sexually dimorphic distribution.

Mammals have retained two functionally and anatomically independent collections of olfactory neurons located in the main olfactory epithelium and in the vomeronasal organ (VNO). Pheromones activate the VNO in order to elicit fixed action behaviors and neuroendocrine changes involved in animal reproduction and aggression. Differential screening of cDNA libraries constructed from individual rat VNO neurons has led to the isolation of a novel family of approximately 100 genes encoding seven transmembrane receptors with sequence similarity with Ca2+-sensing and metabotropic glutamate receptors. These genes are likely to encode a novel family of pheromone receptors. Patterns of receptor gene expression suggest that the VNO is organized into discrete and sexually dimorphic functional units that may permit segregation of pheromone signals leading to specific arrays of behaviors and neuroendocrine responses.

delta-Aminolevulinate uptake by Rhizobium bacteroids and its limitation by the peribacteroid membrane in Legume nodules.

Heme is overproduced during Rhizobium-Legume symbiosis and delta-aminolevulinate (ALA) is a common precursor in both bacterial and plant synthesis pathways of this molecule. ALA uptake by bacteroids from French bean and soybean nodules was characterized. The action of several metabolic inhibitors and the competition effect of malate on this uptake were studied. ALA transport appeared to be mediated by the dicarboxylate carrier system. Purified symbiosomes--bacteroids surrounded by the peribacteroid membrane--failed to accumulate significant amount of ALA. These experiments rule out the possibility for the plant cytosol to provide the bacteroid with ALA and strengthen the restrictive role of the peribacteroid membrane for exchanges between the two symbiotic partners.

Lipid peroxidation in peribacteroid membranes from French-bean nodules.

Enriched peribacteroid membranes were prepared from Phaseolus vulgaris nodules and, in the presence of metleghemoglobin and H(2)O(2), membranal lipid peroxidation was observed. The initial rate of the reaction was low and increased with time. Ferrous leghemoglobin was unable to induce this peroxidation with H(2)O(2). Thus, it appears that leghemoglobin (IV) is not the activated species involved in this process. Heme plays a role in this peroxidation and the hydroxyl radical is not an intermediate of the reaction. Lipid peroxidation in peribacteroid membranes was also observed in the presence of iron ions. A mixture of iron (III) and iron (II) produced a maximal peroxidation. Senescing nodule extracts were able to provoke membranal lipid peroxidation; they contained nonprotein-bound iron. Peribacteroid membranes were more sensitive than microsomes to peroxidation, as measured by malonaldehyde formation.

The mouse transcription factor Stat4 is expressed in haploid male germ cells and is present in the perinuclear theca of spermatozoa.

STAT (signal transducer and activator of transcription) proteins have been shown to be essential transcription factors which mediate biological effects of cytokines. Although most of the STATs have been shown to be widely expressed, Stat4 mRNA has been detected in only a few tissues, including the testis. In the present study, immunoblot analysis confirmed that the presence of Stat4 protein was similarly restricted, with the highest level observed in testis. In situ hybridization, immunoblot, and immunohistochemistry analyses revealed that in the testis, Stat4 was abundantly and exclusively expressed in male germ cells which have completed meiosis, at the round and elongating spermatid stages. Cytolocalization at various times of spermatid differentiation showed that the level of Stat4 protein increased in parallel in both cytoplasm and nuclei. No specific nuclear translocation that would have been an indicator of Stat4 activation was observed at any stage of spermatogenic differentiation. Interestingly, the Stat4 transcription factor was localized to the condensing perinuclear theca of spermatids, a localization that was confirmed by selective biochemical extraction of thecal proteins. Since the theca is known to depolymerize in the cytoplasm of the oocyte during the hours following fertilization, we hypothesized that sperm Stat4 would represent an original paternal contribution to the fertilized egg which may be involved in the onset of zygotic transcription.

Solid-phase synthesis of peptide nucleic acid (PNA) monomers and their oligomerization using disulphide anchoring linkers.

A new simple solid-phase method has been developed for synthesizing Boc-protected peptide nucleic acid (PNA) monomers. An immobilized backbone 3 was built on Expansin resin using an ester disulphide handle: 2-hydroxypropyl-dithio-2'-isobutyric acid (HPDI). The base acetic acids of thymine 5, Z-cytosine 9, Z-adenine 12, and 6-O-benzyl guanine 17 were prepared and coupled to the immobilized backbone. The HPDI handle was cleaved under mild conditions by cyanolysis or assisted hydrolysis with tris(2-carboxyethyl)phosphine (TCEP) to give undamaged PNA monomers. These monomers were coupled to form oligomers by solid-phase method with another disulphide linkage: aminoethyldithio-2-isobutyric acid (AEDI) grafted on an amino-functionalized TentaGel resin, using in situ neutralization and TBTU as activating reagent. Final cleavage of the AEDI linker gave PNA bearing a cysteamide residue that could be useful for optimizing PNA properties. Oligomers of up to 16 residues long were assembled.

A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to a retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons.

A peptide nucleic acid (PNA) antisense for the AUG translation initiation region of prepro-oxytocin mRNA was synthesized and coupled to a r etro-inverso peptide that is rapidly taken up by cells. This bioconjugate was internalized by cultured cerebral cortex neurons within minutes, according to the specific property of the vector peptide. The PNA alone also entered the cells, but more slowly. Cell viability was unaffected when the PNA concentrations were lower than 10 microM and incubation times less than for 24 h. Magnocellular neurons from the hypothalamic supraoptic nucleus, which produce oxytocin and vasopressin, were cultured in chemically defined medium. Both PNA and vector peptide-PNA depressed the amounts of the mRNA coding for prepro-oxytocin in these neurons. A scrambled PNA had no effect and the very cognate prepro-vasopressin mRNA was not affected. The antisense PNA also depressed the immunocytochemical signal for prepro-oxytocin in this culture in a dose- and time-dependent manner. These results show that PNAs driven by the retro-inverso vector peptide are powerful antisense reagents for use on cells in culture.

Score de estratificación de riesgo en recuperación cardiovascular / Stratification risk score in the post-operative recovery of heart surgery

Para evaluar el pronóstico en relación con la mortalidad de los pacientes sometidos a cirugía con circulación extracorpórea se diseñó un puntaje compuesto por 21 ítems que incluyen variables pre, intra y posoperatorias. Los pacientes fueron evaluados con dicho puntaje al ingreso y a las 12 horas del posoperatorio inmediato. Se incluyeron 662 pacientes. Tras realizar el análisis estadístico se observó una diferencia significativa en la mortalidad de los pacientes con puntaje menor de 20 puntos y mayor o igual de 21 puntos. La mortalidad de pacientes con puntaje menor o igual a 20 puntos para cirugías programadas fue del 1,7 por ciento y con más de 20 puntos 36 por ciento (p< 0,00001). La mortalidad de pacientes con puntaje menor o igual a 20 puntos para cirugía de urgencia fue del 5,9 por ciento y de pacientes con puntaje superior a 20 puntos 45,5 por ciento (p< 0,01)