A host-specific diaminobutyrate aminotransferase contributes to symbiotic performance, homoserine metabolism, and competitiveness in the Rhizobium leguminosarum/Pisum sativum system.

Rhizobium leguminosarum bv. viciae (Rlv) UPM791 effectively nodulates pea and lentil, but bacteroids contain a number of proteins differentially expressed depending on the host. One of these host-dependent proteins (C189) is similar to a diaminobutyrate-2-oxoglutarate aminotransferase (DABA-AT). DABA-AT activity was demonstrated with cell extracts and with purified protein, so C189 was renamed as Dat. The dat gene was strongly induced in the central, active area of pea nodules, but not in lentil. Mutants defective in dat were impaired in symbiotic performance with pea plants, exhibiting reduced shoot dry weight, smaller nodules, and a lower competitiveness for nodulation. In contrast, there were no significant differences between mutant and wild-type in symbiosis with lentil plants. A comparative metabolomic approach using cell-free extracts from bacteroids induced in pea and lentil showed significant differences among the strains in pea bacteroids whereas no significant differences were found in lentil. Targeted metabolomic analysis revealed that the dat mutation abolished the presence of 2,4-diaminobutyrate (DABA) in pea nodules, indicating that DABA-AT reaction is oriented toward the production of DABA from L-aspartate semialdehyde. This analysis also showed the presence of L-homoserine, a likely source of aspartate semialdehyde, in pea bacteroids but not in those induced in lentil. The dat mutant showed impaired growth when cells were grown with L-homoserine as nitrogen source. Inclusion of DABA or L-homoserine as N source suppressed pantothenate auxotropy in Rlv UPM791, suggesting DABA as source of the pantothenate precursor ß-alanine. These data indicate that Rlv UPM791 Dat enzyme is part of an adaptation mechanism of this bacterium to a homoserine-rich environment such as pea nodule and rhizosphere.

Computational analyses, molecular dynamics, and mutagenesis studies of unprocessed form of [NiFe] hydrogenase reveal the role of disorder for efficient enzyme maturation.

Biological production and oxidation of hydrogen is mediated by hydrogenases, key enzymes for these energy-relevant reactions. Synthesis of [NiFe] hydrogenases involves a complex series of biochemical reactions to assemble protein subunits and metallic cofactors required for enzyme function. A final step in this biosynthetic pathway is the processing of a C-terminal tail (CTT) from its large subunit, thus allowing proper insertion of nickel in the unique NiFe(CN)2CO cofactor present in these enzymes. In silico modelling and Molecular Dynamics (MD) analyses of processed vs. unprocessed forms of Rhizobium leguminosarum bv. viciae (Rlv) hydrogenase large subunit HupL showed that its CTT (residues 582-596) is an intrinsically disordered region (IDR) that likely provides the required flexibility to the protein for the final steps of proteolytic maturation. Prediction of pKa values of ionizable side chains in both forms of the enzyme's large subunit also revealed that the presence of the CTT strongly modify the protonation state of some key residues around the active site. Furthermore, MD simulations and mutant analyses revealed that two glutamate residues (E27 in the N-terminal region and E589 inside the CTT) likely contribute to the process of nickel incorporation into the enzyme. Computational analysis also revealed structural details on the interaction of Rlv hydrogenase LSU with the endoprotease HupD responsible for the removal of CTT.

Bradyrhizobium algeriense sp. nov., a novel species isolated from effective nodules of Retama sphaerocarpa from Northeastern Algeria.

We have characterized genetic, phenotypic and symbiotic properties of bacterial strains previously isolated from nitrogen-fixing nodules of Retama sphaerocarpa from Northern Algeria. Phylogenetic analyses of 16S rRNA genes and three concatenated housekeeping genes, recA, atpD and glnII, placed them in a new divergent group that is proposed to form a new Bradyrhizobium species, Bradyrhizobium algeriense sp. nov. (type strain RST89T, LMG 27618 and CECT 8363). Based on these phylogenetic markers and on genomic identity data derived from draft genomic sequences, Bradyrhizobium valentinum LmjM3T, Bradyrhizobium lablabi CCBAU 23086T, Bradyrhizobium retamae Ro19T, and Bradyrhizobium jicamae PAC68T are the closest relatives of B. algeriense RST89T, with sequence identities of 92-94% and Average Nucleotide Identities (ANIm) under 90%, well below the 95-96% species circumscription threshold. Likewise, a comparison of whole-cell proteomic patterns, estimated by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF) mass spectrometric analysis, yielded almost identical spectra between B. algeriense strains but significant differences with B. valentinum, Bradyrhizobium paxllaeri, Bradyrhizobium icense, B. lablabi, B. jicamae and B. retamae. A phylogenetic tree based on symbiotic gene nodC revealed that the B. algeriense sequences cluster with sequences from the Bradyrhizobium symbiovar retamae, previously defined with B. retamae strains isolated from Retama monosperma. B. algeriense strains were able to establish effective symbioses with Retama raetam, Lupinus micranthus, Lupinus albus and Genista numidica, but not with Lupinus angustifolius or Glycine max.

Genomic Diversity in the Endosymbiotic Bacterium Rhizobium leguminosarum.

Rhizobium leguminosarum bv. viciae is a soil α-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

A short history of the 5-HT2C receptor: from the choroid plexus to depression, obesity and addiction treatment.

This paper is a personal account on the discovery and characterization of the 5-HT2C receptor (first known as the 5-HT1C receptor) over 30 years ago and how it translated into a number of unsuspected features for a G protein-coupled receptor (GPCR) and a diversity of clinical applications. The 5-HT2C receptor is one of the most intriguing members of the GPCR superfamily. Initially referred to as 5-HT1CR, the 5-HT2CR was discovered while studying the pharmacological features and the distribution of [3H]mesulergine-labelled sites, primarily in the brain using radioligand binding and slice autoradiography. Mesulergine (SDZ CU-085), was, at the time, best defined as a ligand with serotonergic and dopaminergic properties. Autoradiographic studies showed remarkably strong [3H]mesulergine-labelling to the rat choroid plexus. [3H]mesulergine-labelled sites had pharmacological properties different from, at the time, known or purported 5-HT receptors. In spite of similarities with 5-HT2 binding, the new binding site was called 5-HT1C because of its very high affinity for 5-HT itself. Within the following 10 years, the 5-HT1CR (later named 5-HT2C) was extensively characterised pharmacologically, anatomically and functionally: it was one of the first 5-HT receptors to be sequenced and cloned. The 5-HT2CR is a GPCR, with a very complex gene structure. It constitutes a rarity in the GPCR family: many 5-HT2CR variants exist, especially in humans, due to RNA editing, in addition to a few 5-HT2CR splice variants. Intense research led to therapeutically active 5-HT2C receptor ligands, both antagonists (or inverse agonists) and agonists: keeping in mind that a number of antidepressants and antipsychotics are 5-HT2CR antagonists/inverse agonists. Agomelatine, a 5-HT2CR antagonist is registered for the treatment of major depression. The agonist Lorcaserin is registered for the treatment of aspects of obesity and has further potential in addiction, especially nicotine/ smoking. There is good evidence that the 5-HT2CR is involved in spinal cord injury-induced spasms of the lower limbs, which can be treated with 5-HT2CR antagonists/inverse agonists such as cyproheptadine or SB206553. The 5-HT2CR may play a role in schizophrenia and epilepsy. Vabicaserin, a 5-HT2CR agonist has been in development for the treatment of schizophrenia and obesity, but was stopped. As is common, there is potential for further indications for 5-HT2CR ligands, as suggested by a number of preclinical and/or genome-wide association studies (GWAS) on depression, suicide, sexual dysfunction, addictions and obesity. The 5-HT2CR is clearly affected by a number of established antidepressants/antipsychotics and may be one of the culprits in antipsychotic-induced weight gain.

Oceanic island biogeography through the lens of the general dynamic model: assessment and prospect.

The general dynamic model of oceanic island biogeography (GDM) has added a new dimension to theoretical island biogeography in recognizing that geological processes are key drivers of the evolutionary processes of diversification and extinction within remote islands. It provides a dynamic and essentially non-equilibrium framework generating novel predictions for emergent diversity properties of oceanic islands and archipelagos. Its publication in 2008 coincided with, and spurred on, renewed attention to the dynamics of remote islands. We review progress, both in testing the GDM's predictions and in developing and enhancing ecological-evolutionary understanding of oceanic island systems through the lens of the GDM. In particular, we focus on four main themes: (i) macroecological tests using a space-for-time rationale; (ii) extensions of theory to islands following different patterns of ontogeny; (iii) the implications of GDM dynamics for lineage diversification and trait evolution; and (iv) the potential for downscaling GDM dynamics to local-scale ecological patterns and processes within islands. We also consider the implications of the GDM for understanding patterns of non-native species diversity. We demonstrate the vitality of the field of island biogeography by identifying a range of potentially productive lines for future research.

Diversity of Bradyrhizobium strains nodulating Lupinus micranthus on both sides of the Western Mediterranean: Algeria and Spain.

Lupinus micranthus is a lupine distributed in the Mediterranean basin whose nitrogen fixing symbiosis has not been described in detail. In this study, 101 slow-growing nodule isolates were obtained from L. micranthus thriving in soils on both sides of the Western Mediterranean. The diversity of the isolates, 60 from Algeria and 41 from Spain, was addressed by multilocus sequence analysis of housekeeping genes (16S rRNA, atpD, glnII and recA) and one symbiotic gene (nodC). Using genomic fingerprints from BOX elements, 37 different profiles were obtained (22 from Algeria and 15 from Spain). Phylogenetic analysis based on 16S rRNA and concatenated atpD, glnII and recA sequences of a representative isolate of each BOX profile displayed a homogeneous distribution of profiles in six different phylogenetic clusters. All isolates were taxonomically ascribed to the genus Bradyrhizobium. Three clusters comprising 24, 6, and 4 isolates, respectively, accounted for most of the profiles. The largest cluster was close to the Bradyrhizobium canariense lineage, while the other two were related to B. cytisi/B. rifense. The three remaining clusters included only one isolate each, and were close to B. canariense, B. japonicum and B. elkanii species, respectively. In contrast, phylogenetic clustering of BOX profiles based on nodC sequences yielded only two phylogenetic groups. One of them included all the profiles except one, and belonged to symbiovar genistearum. The remaining profile, constituted by a strain related to B. elkanii, was not related to any well-defined symbiotic lineage, and may constitute both a new symbiovar and a new genospecies.

Serotonin receptors in brain revisited.

UNLABELLED: In the early 1980's, the dispute on the existence of a multiplicity of receptors for neurotransmitter was at its height. Several subtypes of serotonin (5-HT) receptors were proposed on the basis of radioligand binding assays. In order to provide further support to the existence of these receptors we performed quantitative autoradiographic mapping of the binding of several ligands for the 5-HT1 receptor labeling the subtypes 5-HT1A, 5-HT1B and 5-HT1C, and characterized pharmacologically these different receptors. The results demonstrated differential localization of the subtypes of 5-HT1 receptors indicating that they were expressed by different cell populations, probably neurons, in the brain and further supporting their reality. Shortly afterwards, the cloning of the genes coding for these 5-HT receptors, and many others, ended the dispute by demonstrating that they were different proteins. The advent of Molecular Biology provided new methodologies for the study of the chemical and molecular anatomy of 5-HT receptors in brain, by visualizing cells expressing their mRNA by in situ hybridization and showed that the family of mammalian 5-HT receptors has 14 members, a figure much larger than ever suspected at that time. ORIGINAL ARTICLE ABSTRACT: QUANTITATIVE AUTORADIOGRAPHIC MAPPING OF SEROTONIN RECEPTORS IN THE RAT BRAIN. I. SEROTONIN-1 RECEPTORS: The distribution of serotonin-1 (5-HT1) receptors in the rat brain was studied by light microscopic quantitative autoradiography. Receptors were labeled with [(3)H]serotonin (5-[(3)H]HT), 8-hydroxy-2-[H-dipropylamino-(3)H]tetralin (8-OH-[(3)H]DPAT), [(3)H]LSD and [(3)H]mesulergine, and the densities quantified by microdensitometry with the aid of a computer-assisted image-analysis system. Competition experiments for 5-[(3)H]HT binding by several serotonin-1 agonizts led to the identification of brain areas enriched in each one of the three subtypes of 5-HT1 recognition sites already described (5-HT1A, 5-HT1B, 5-HT1C). The existence of these׳selective׳ areas allowed a detailed pharmacological characterization of these sites to be made in a more precise manner than has been attained in membrane-binding studies. While 5-[(3)H]HT labeled with nanomolar affinity all the 5-HT1 subtypes, the other (3)H-labeled ligands labeled selectively 5-HT1A (8-OH-[(3)H]DPAT), 5-HT1C ([(3)H]mesulergine) and both of them ([(3)H]LSD). Very high concentrations of 5-HT1 receptors were localized in the choroid plexus, lateroseptal nucleus, globus pallidus and ventral pallidum, dentate gyrus, dorsal subiculum, olivary pretectal nucleus, substantia nigra, reticular and external layer of the entorhinal cortex. The different fields of the hippocampus (CA1-CA4), some nuclei of the amygdaloid complex, the hypothalamic nuclei and the dorsal raphé, among others, also presented high concentrations of sites. Areas containing intermediate densities of 5-HT1 receptors included the claustrum, olfactory tubercle, accumbens, central gray and lateral cerebellar nucleus. The nucleus caudate-putamen and the cortex, at the different levels studied, presented receptor densities ranging from intermediate to low. Finally, in other brain areas-pons, medulla, and spinal cord-only low or very low concentrations of 5-HT1 receptors were found. From the areas strongly enriched in 5-HT1 sites, dentate gyrus and septal nucleus contained 5-HT1A sites, while globus pallidus, dorsal subiculum, substantia nigra and olivary pretectal nucleus were enriched in 5-HT1B. The sites in the choroid plexus, which presented the highest density of receptors in the rat brain, were of the 5-HT1C subtype. The distribution of 5-HT1 receptors reported here is discussed in correlation with the distribution of serotoninergic neurons and fibers, the related anatomical pathways and the effects which appear to be mediated by these sites. © 1985.This article is part of a Special Issue entitled SI:50th Anniversary Issue. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

Cartography of 5-HT1A and 5-HT2A Receptor Subtypes in Prefrontal Cortex and Its Projections.

Since the development of chemical neuroanatomical tools in the 1960s, a tremendous wealth of information has been generated on the anatomical components of the serotonergic system, at the microscopic level in the brain including the prefrontal cortex (PFC). The PFC receives a widespread distribution of serotonin (5-hydroxytryptamine, 5-HT) terminals from the median and dorsal raphe nuclei. 5-HT receptors were first visualized using radioligand autoradiography in the late 1980s and early 1990s and showed, in contrast to 5-HT innervation, a differential distribution of binding sites associated with different 5-HT receptor subtypes. Due to the cloning of the different 5-HT receptor subtype genes in the late 1980s and early 1990s, it was possible, using in situ hybridization histochemistry, to localize cells expressing mRNA for these receptors. Double in situ hybridization histochemistry and immunohistochemistry allowed for the chemical characterization of the phenotype of cells expressing 5-HT receptors. Tract tracing technology allowed a detailed cartography of the neuronal connections of PFC and other brain areas. Based on these data, maps have been constructed that reflect our current understanding of the different circuits where 5-HT receptors can modulate the electrophysiological, pharmacological, and behavioral functions of the PFC. We will review current knowledge regarding the cellular localization of 5-HT1A and 5-HT2A receptors in mammalian PFC and their possible functions in the neuronal circuits of the PFC. We will discuss data generated in our laboratory as well as in others, focusing on localization in the pyramidal and GABAergic neuronal cell populations in different mammalian species using molecular neuroanatomy and on the connections with other brain regions.

Maturation of Rhizobium leguminosarum hydrogenase in the presence of oxygen requires the interaction of the chaperone HypC and the scaffolding protein HupK.

[NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. Synthesis of these metalloenzymes involves a complex series of biochemical reactions catalyzed by a plethora of accessory proteins, many of them required to synthesize and insert the unique NiFe(CN)2CO cofactor. HypC is an accessory protein conserved in all [NiFe] hydrogenase systems and involved in the synthesis and transfer of the Fe(CN)2CO cofactor precursor. Hydrogenase accessory proteins from bacteria-synthesizing hydrogenase in the presence of oxygen include HupK, a scaffolding protein with a moderate sequence similarity to the hydrogenase large subunit and proposed to participate as an intermediate chaperone in the synthesis of the NiFe cofactor. The endosymbiotic bacterium Rhizobium leguminosarum contains a single hydrogenase system that can be expressed under two different physiological conditions: free-living microaerobic cells (∼ 12 µm O2) and bacteroids from legume nodules (∼ 10-100 nm O2). We have used bioinformatic tools to model HupK structure and interaction of this protein with HypC. Site-directed mutagenesis at positions predicted as critical by the structural analysis have allowed the identification of HupK and HypC residues relevant for the maturation of hydrogenase. Mutant proteins altered in some of these residues show a different phenotype depending on the physiological condition tested. Modeling of HypC also predicts the existence of a stable HypC dimer whose presence was also demonstrated by immunoblot analysis. This study widens our understanding on the mechanisms for metalloenzyme biosynthesis in the presence of oxygen.

Multiple conformations of 5-HT2A and 5-HT 2C receptors in rat brain: an autoradiographic study with [125I](±)DOI.

Earlier autoradiographic studies with the 5-HT2 receptor agonist [(125)I](±)DOI in human brain showed unexpected biphasic competition curves for various 5-HT2A antagonists. We have performed similar studies in rat brain regions with selective 5-HT2A (M100907) and 5-HT2C (SB242084) antagonists together with ketanserin and mesulergine. The effect of GTP analogues on antagonist competition was also studied. Increasing concentrations of Gpp(NH)p or GTPγS resulted in a maximal inhibition of [(125)I](±)DOI-specific binding of approximately 50 %. M100907 competed biphasically in all regions. In the presence of 100 µM Gpp(NH)p, M100907 still displaced biphasically the remaining [(125)I](±)DOI binding. Ketanserin showed biphasic curves in some regions and monophasic curves in others. In the latter, Gpp(NH)p evidenced an additional high-affinity site. SB242084 competed biphasically in brainstem nuclei and monophasically in the other regions. In most areas, SB242084 affinities were not notably altered by Gpp(NH)p. Mesulergine competed monophasically in all regions without alteration by Gpp(NH)p. These results conform with the extended ternary complex model of receptor action: receptor exists as an equilibrium of multiple conformations, i.e. ground (R), partly activated (R*) and activated G-protein-coupled (R*G) conformation/s. Thus, [(125)I](±)DOI would label multiple conformations of both 5-HT2A and 5-HT2C receptors in rat brain, and M100907 and ketanserin would recognise these conformations with different affinities.

The rkpU gene of Sinorhizobium fredii HH103 is required for bacterial K-antigen polysaccharide production and for efficient nodulation with soybean but not with cowpea.

In this work, the role of the rkpU and rkpJ genes in the production of the K-antigen polysaccharides (KPS) and in the symbiotic capacity of Sinorhizobium fredii HH103, a broad host-range rhizobial strain able to nodulate soybean and many other legumes, was studied. The rkpJ- and rkpU-encoded products are orthologous to Escherichia coli proteins involved in capsule export. S. fredii HH103 mutant derivatives were contructed in both genes. To our knowledge, this is the first time that the role of rkpU in KPS production has been studied in rhizobia. Both rkpJ and rkpU mutants were unable to produce KPS. The rkpU derivative also showed alterations in its lipopolysaccharide (LPS). Neither KPS production nor rkpJ and rkpU expression was affected by the presence of the flavonoid genistein. Soybean (Glycine max) plants inoculated with the S. fredii HH103 rkpU and rkpJ mutants showed reduced nodulation and clear symptoms of nitrogen starvation. However, neither the rkpJ nor the rkpU mutants were significantly impaired in their symbiotic interaction with cowpea (Vigna unguiculata). Thus, we demonstrate for the first time to our knowledge the involvement of the rkpU gene in rhizobial KPS production and also show that the symbiotic relevance of the S. fredii HH103 KPS depends on the specific bacterium-legume interaction.

The Tat pathway of plant pathogen Dickeya dadantii 3937 contributes to virulence and fitness.

Protein secretion plays a very important role in the virulence of the bacterium Dickeya dadantii, the causative agent of soft rot disease, in a wide range of plant species. We studied the contribution of the twin-arginine translocation (Tat) protein system to the adaptation of D. dadantii 3937 to different growth conditions and to the interaction with the plant host. First, a list of 44 putative Tat substrates was obtained using bioinformatic programs taking advantage of the availability of the complete sequence of this bacterium. Second, a tatC mutant strain was constructed and analysed. The mutant displayed a pleiotropic phenotype, showing limited growth in an iron-depleted medium, higher sensitivity to copper, reduced motility on soft agar plates and attenuated virulence in witloof chicory leaves. Our results indicate the Tat system as an important determinant of the virulence and fitness of D. dadantii 3937. Potential Tat substrates related to the tatC mutant phenotype are discussed.

Distribution of 5-HT and DA receptors in primate prefrontal cortex: implications for pathophysiology and treatment.

The prefrontal cortex (PFC) has attracted a great research interest because of its involvement in the control of executive functions in both health and disease, and particularly in cognitive functions such as working memory. In schizophrenia, alterations in the PFC are documented at many different levels: molecular, cellular and functional. Furthermore, deficits in cognitive abilities are considered a core feature of schizophrenia and remain a major unmet medical need with respect to this disorder. In order to understand the sites of action of currently used drugs, as well as of the new experimental treatments being developed and acting in this brain region, it is important to have a detailed knowledge of the corresponding chemical neuroanatomy. Here we review current knowledge regarding the cellular localization of 5-HT(1A), 5-HT(2A) and dopamine D1, D5, and D2, D4 receptors in primate PFC and their possible functions in the neuronal circuits of the PFC.

Host-dependent expression of Rhizobium leguminosarum bv. viciae hydrogenase is controlled at transcriptional and post-transcriptional levels in legume nodules.

The legume host affects the expression of Rhizobium leguminosarum hydrogenase activity in root nodules. High levels of symbiotic hydrogenase activity were detected in R. leguminosarum bacteroids from different hosts, with the exception of lentil (Lens culinaris). Transcription analysis showed that the NifA-regulated R. leguminosarum hydrogenase structural gene promoter (P(1)) is poorly induced in lentil root nodules. Replacement of the P(1) promoter by the FnrN-dependent promoter of the fixN gene restored transcription of hup genes in lentil bacteroids, but not hydrogenase activity. In the P(fixN)-hupSL strain, additional copies of the hup gene cluster and nickel supplementation to lentil plants increased bacteroid hydrogenase activity. However, the level of activity in lentil still was significantly lower than in pea bacteroids, indicating that an additional factor is impairing hydrogenase expression inside lentil nodules. Immunological analysis revealed that lentil bacteroids contain reduced levels of both hydrogenase structural subunit HupL and nickel-binding protein HypB. Altogether, results indicate that hydrogenase expression is affected by the legume host at the level of both transcription of hydrogenase structural genes and biosynthesis or stability of nickel-related proteins HypB and HupL, and suggest the existence of a plant-dependent mechanism that affects hydrogenase activity during the symbiosis by limiting nickel availability to the bacteroid.

Successful neonatal double switch in symptomatic patients with congenitally corrected transposition of the great arteries.

Anatomic repair is the standard surgical approach to congenitally corrected transposition of the great arteries. However, timing to perform the procedure remains controversial. We present 2 cases of congenitally corrected transposition of the great arteries and Ebstein's-like anomaly of the tricuspid valve presenting with heart failure. Both cases had successful anatomic repair during the neonatal period.

Differential distribution of PDE4B splice variant mRNAs in rat brain and the effects of systemic administration of LPS in their expression.

Phosphodiesterases (PDE) control intracellular cyclic adenosine monophosphate (cAMP) levels, which appear to play an important role in the regulation of inflammation. PDE4B is especially important in this process. Using in situ hybridization histochemistry we first mapped the expression sites of the four PDE4B splicing forms in rat brain. Using the systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) as an inflammation model in rats, we found an increase in PDEB2 mRNA expression in choroid plexus. The differential expression of PDE4B spliced forms and the differential regulation of PDE4B2 in an inflammatory model further supports an involvement of this splicing variant in the inflammatory response.

Symbiotic hydrogenase activity in Bradyrhizobium sp. (Vigna) increases nitrogen content in Vigna unguiculata plants.

Bradyrhizobium sp. (Lupinus) and Bradyrhizobium sp. (Vigna) mutants in which hydrogenase (hup) activity was affected were constructed and analyzed. Vigna unguiculata plants inoculated with the Bradyrhizobium sp. (Vigna) hup mutant showed reduced nitrogenase activity and also a significant decrease in nitrogen content, suggesting a relevant contribution of hydrogenase activity to plant yield.

Rhizobium leguminosarum biovar viciae symbiotic hydrogenase activity and processing are limited by the level of nickel in agricultural soils.

Analysis of levels of hydrogenase processing and activity in Rhizobium leguminosarum biovar viciae bacteroids from pea (Pisum sativum) plants showed that the oxidation of nitrogenase-evolved hydrogen is limited by the availability of nickel in agricultural soils. This limitation was overcome by using an inoculant strain engineered for higher hydrogenase expression.

Gene products of the hupGHIJ operon are involved in maturation of the iron-sulfur subunit of the [NiFe] hydrogenase from Rhizobium leguminosarum bv. viciae.

In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions (deltahupG, deltahupH, deltahupI, and deltahupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The deltahupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2. Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the deltahupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from deltahupH, deltahupI, and deltahupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the deltahupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.