7alpha- and 12alpha-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid

We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7alpha- and 12alpha-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12alpha-HSDH-mediated total oxidation of sodium cholate followed by the Wolf-Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7alpha-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield).     

Rha1, a new mutant of Arabidopsis disturbed in root slanting,gravitropism and auxin physiology

A new Arabidopsis mutant is characterized (rha1) that shows, in the roots, reduced right-handed slanting, reduced gravitropism and resistance to 2,4-D, TIBA, NPA and ethylene. It also shows reduced length in the shoot and root, reduced number of lateral roots and shorter siliques. The gene was cloned through TAIL-PCR and resulted in a HSF. Because none of the known gravitropic and auxinic mutants result from damage in a HSF, rha1 seems to belong to a new class of this group of mutants. Quantitative PCR analysis showed that the expression of the gene is increased by heat and cold shock, and by presence of 2,4-D in the media. Study of the expression through the GUS reporter gene revealed increased expression in clinostated and gravistimulated plants, but only in adult tissues, and not in the apical meristems of shoots and roots.Key words: auxin, ethylene, slanting, gravitropism, HSFsArabidopsis primary roots, and especially those from some ecotypes (Ws, Landsberg), when grown on an agar dish, tilted on the vertical, show a wavy pattern, and a clear slanting towards a direction that has been considered the right-hand.^1–4 In the case of the mutant rha1, the right-handed slanting is notably reduced, its primary roots growing partly to the right-hand, partly straight down and partly to the left-hand, even though a slight preference for the right-hand is apparent.In addition, its roots show resistance to the inhibitory action of the auxin 2,4-D, ethylene (ACC), and the auxin transport inhibitors TIBA and NPA. These characteristics qualify the mutant as an auxinic one, and therefore a connection between the reduced slanting and the auxinic disturbances could be imagined. It is not known, however, what controls the slanting process itself, even though it appears as the consequence of a chiral circumnutational process. As reported,⁴ it seems the result of a chiral circumnutation with preference for the right-hand, transformed in a lateral slanting movement, because of the impact of the helix with the hard agar surface. This process results in the formation of waves, when the circumnutation helix impactig the agar reverses direction at every half turn, or the formation of large loops and strict loops (coils) when there is no reversion. The latter case seems to be a consequence of the fact that gravity is no longer “felt”. This has been previously noted in some mutants, or sometimes in old roots.rha1 is not the only mutant known to show reduction or increase of slanting, because other mutants were reported by Rutherford and Masson,³ and subsequent publications from the same group. Almost all show an increase of slant, with the exception of rhd3 and its alleles that show a complete suppression of the process.⁵ The mutated gene in rha1 was cloned through TAIL-PCR and shown to be a HSF. No other auxinic mutant, among those for which the gene was cloned, is known to be mutated in a HSF.HSFs, that are characteristically involved in the activation of the HSPs (heat shock proteins), which protect the cells from damage arising from high temperature and other stresses, have been shown to be involved also in different processes.^6,7 Hence, also in the case of rha1, we can well imagine other different functions, beside that of counteracting the heat shock. In particular, since the connection with auxin regulated processes is evident, we can suppose that the action could be on the PP2A phosphatase, as in the case of the rcn1 mutant, or of the human HSF2. The RCN1 protein corresponds to one unit of the PP2A,⁸ and the HSF2⁹ has been shown to substitute itself for the C subunit and alter the function of the phosphatase (Fig. 1). Experiments directed to see if a heat shock can modify the slanting of the roots in the wild-type and rha1, gave negative results, even though these experiments will need to be repeated under more widely ranging conditions. These results seem to indicate that the mechanism which induces asymmetric growth in roots is complex, and it is not controlled by a single gene.Open in a separate windowFigure 1Model proposed for the regulation of the PP2A activity by the RHA1 protein. (modified after Hong and Sarge, 1999).On the other hand, another puzzling characteristic of rha1 is the fact that its roots are resistant only to the auxin 2,4-D, and not to NAA and IAA. Differences in the response of the primary roots to different auxins have already been reported, and it was suggested that the response to NAA should be different, because this substance can penetrate passively the cell membranes.¹⁰ In the case of rha1, however, it seems that IAA can also penetrate the cells passively. This is in line with the chemiosmotic hypothesis,¹¹ but seems in contrast with the previous supposition. The resistance to ethylene, however, could indicate that the reduced inhibitory effect of 2,4-D is a consequence of the ethylene production induced by the synthetic auxin. On the other hand, the resistance to the auxin transport inhibitors TIBA and NPA, cannot be explained so easily. Possibly, in the mutant rha1, there is a reduced level of receptors for the considered substances.Using semiquantitative PCR analysis it was shown that rha1 retains the function of a HSF, the gene being clearly upregulated by heat and cold stress, and also by 2,4-D, but not by rotation on a clinostat or gravistimulation. The upregulation of the expression by 2,4-D was confirmed by a study of GUS expression in a transformed rha1, and with this technique the effects of gravity and simulated microgravity appeared clearly stimulatory too. No GUS expression however was apparent in the shoot and root meristems, and consequently we propose that the gene does not influence the first part of the graviresponse, but possibly the general transport of auxin through the plant.Thus, the mutant seems to be disturbed in root gravitropism, as well as in responses to the auxines and circumnutation. However not in the general circumnutation process, but in its chiral aspect, which is the cause of the slanting to the right-hand. Gravitropism, circumnutation and auxin physiology, thus, seem to be in some way connected in a complex integrated process, that, hopefully, will be gradually revealed in all its different aspects, through the future efforts of plant scientists.     

Calcium opens the dialogue between plants and arbuscular mycorrhizal fungi

Calcium ion is considered a ubiquitous second messenger in all eukaryotic cells. Analysis of intracellular Ca²⁺ concentration dynamics has demonstrated its signalling role in plant cells in response to a wide array of environmental cues. The implication of Ca²⁺ in the early steps of the arbuscular mycorrhizal symbiosis has been frequently claimed, mainly by analogy with what firmly demonstrated in the rhizobium-legume symbiosis. We recently documented transient Ca²⁺ changes in plant cells challenged with diffusible molecules released by arbuscular mycorrhizal fungi. Ca²⁺ measurements by the recombinant aequorin method provided new insights into the molecular communications between plants and these beneficial fungi.Key words: legume symbioses, arbuscular mycorrhiza, calcium signalling, fungal signal, plant cell cultures, aequorinIn the rhizosphere plants meet a wide array of microorganisms. In favorable interactions, such as arbuscular mycorrhizal (AM) and nitrogen fixing symbioses, a dialogue is progressively established between the two interacting organisms to make the appropriate partner choice. These two-way communications rely on the interchange of signals released by both potential symbionts. After perception of the signalling molecules, a signal transduction pathway is induced, leading to the activation of the proper genetic and developmental program in both partners.Variations in intracellular free Ca²⁺ concentration occur as one of the initial steps in signalling pathways activated in plants when they encounter pathogens,¹ fungal biocontrol agents² and nitrogen-fixing bacteria.³ Molecules secreted by microorganisms, after binding to specific receptors, trigger in plant cells transient changes in cytosolic Ca²⁺ level, due to the influx of the ion from the extracellular environment and/or the release from internal Ca²⁺ storage compartments.^4,5 Ca²⁺ messages delivered to plant cells are at least partly deciphered on the basis of their spatial and temporal features. The occurrence of different Ca²⁺ signatures guarantees the specificity of the ensuing physiological responses.In the legume-rhizobium symbiosis a definite pattern of Ca²⁺ oscillations has been reported to occur in response to the rhizobial signalling molecule, the Nod factor, in the nucleus and perinuclear cytoplasm of the root hair.⁶ The Ca²⁺ spike number has been recently demonstrated to regulate nodulation gene expression.⁷Legumes are able to engage in a dual symbiotic interaction, with rhizobia and AM fungi. Components of the Ca²⁺-mediated signalling pathway are shared by the two symbioses.⁸ In the mycorrhizal signal transduction pathway the involvement of Ca²⁺ has long been speculated, based on the observed similarities with symbiotic nitrogen fixation.³To evaluate the possible participation of Ca²⁺ in the early steps of the AM symbiosis, we have used a simplified experimental system given by plant cell suspension cultures stably expressing the bioluminescent Ca²⁺-sensitive reporter aequorin.⁹ The use of cultured cells circumvents the problem posed by multilayered organs: in aequorin-transformed seedlings, possible Ca²⁺ changes occurring in rhizodermal cells—the first place where the AM fungal signals are perceived and transduced—can be misrecorded due to luminescence calibration over all root cell layers, resulting in an underestimation of the Ca²⁺ signal in the responsive cells. An experimental design based on challenging host plant cells with the culture medium of different AM fungi (Gigaspora margarita, Glomus mosseae and intraradices) provided the first firm evidence that Ca²⁺ is involved as intracellular messenger during mycorrhizal signalling, at least in a pre-contact stage. Cytosolic Ca²⁺ changes, characterized by specific kinetic parameters, were triggered by diffusates obtained from AM resting and germinating spores,⁹ and extraradical mycelium.¹⁰ Cultured plant cells demonstrated to be competent to perceive the diffusible signal released by AM fungi and to decode the message in a Ca²⁺-dependent pathway. Based on these experiments, it seems that AM fungi announce their presence to the plant through the constitutive release of a chemical signal, even before experiencing the proximity of the plant or its AM symbiotic signals. The notion that the secreted fungal molecules herald, through Ca²⁺, a beneficial message which can be acknowledged only by competent receivers, is supported by: (1) the lack of defense response induction and the upregulation of some genes essential for the AM symbiosis initiation in host plant cells; (2) the unresponsiveness of cultured cells from the nonhost plant Arabidopsis thaliana.Ca²⁺-mediated perception of both AM fungal and rhizobial signals by plant cells unifies the signalling pathways activated in the two symbioses. However, the actual occurrence of Ca²⁺ spiking in AM symbiosis remains to be ascertained, due to limitations of the recombinant aequorin method, when applied to an asynchronous cell population. Contribution of internal Ca²⁺ stores, in particular the nucleus, to the observed Ca²⁺ changes will be a future research goal to be achieved through a pharmacological approach and/or targeting of Ca²⁺ indicators to intracellular compartments.The identification of the plant-derived mycorrhizal signal as strigolactones¹¹ and their inducing activity on AM fungi¹² have represented a major breakthrough in the AM symbiosis research field. Elucidation of the chemical nature of the AM fungal factor, which plays several effects on host plants,^9,13–15 is eagerly awaited.Understanding how AM fungi and rhizobia select compatible plant hosts, thus activating the appropriate symbiotic program, is another facet to be considered in the future to get a complete overview of early signaling events in legume symbioses. Analysis of Ca²⁺ signalling implication in the microbial partner would require the delivery of reliable and sensitive Ca²⁺ probes (such as aequorinor GFP-based¹⁶) for Ca²⁺ measurements in living microorganisms. The recombinant aequorin method has been successfully applied to monitor dynamic changes in intracellular Ca²⁺ levels in the bacteria Anabaena sp.,¹⁷ E. coli,¹⁸ and recently by us in rhizobial strains.¹⁹ Unfortunately, AM fungi have proved not to be amenable to stable transformation, being coenocytic, multinucleate and heterokaryotic,^20,21 and only transient transformants have been obtained so far.^22,23 Further development of the transformation technologies may provide in the future a valuable tool to analyse, from the fungal side, signal perception and transduction during arbuscular mycorrhiza establishment.     

Preferential locomotion of leukemic cells towards laminin isoforms 8 and 10.

To identify the laminin isoforms of the basement membranes that could be implicated in the extravasation process of neoplastic lymphocytes, a number of purified laminins and one native renal laminin complex were comparatively investigated for their ability to promote migration of neoplastic lymphocytes in vitro. The identity/composition of a human placental laminin complex was asserted by combining immunochemical assays, sequence determination of tryptic peptides, and ultrastructural analysis to be composed predominantly of laminin-10 in which the coiled-coil C-terminal regions and the G globular domain of the alpha5 chain were preserved intact despite the enzymatic treatment used for its isolation. Lymphoma and leukemic cell lines failed to migrate towards laminin-4, -9, -11, moved poorly in response to laminin-1, -2/4, -5 and the renal laminin complex, but markedly locomoted towards the subendothelial laminin-8 and -10. The motility-promoting interaction with these latter laminins was interchangeably mediated by the alpha3beta1 and alpha6beta1 integrins. Lymphocyte locomotion on laminins assayed in the presence of cytokines was either reduced or enhanced suggesting that local cytokine milieu could further influence motility response.     

Gape coloration reliably reflects immunocompetence of barn swallow (Hirundo rustica) nestlings

In some passerines, parents allocate more food to offspringwith the brightest red gapes, but the function of parental decisionsbased on offspring gape coloration is unknown. We hypothesizethat gape coloration is part of a communication system wherenestlings reveal their condition to attending parents, whichmay thus base their decisions on reliable signals of offspringreproductive value. We analyze the effects of brood size manipulation,injection with an immunogen and food deprivation, on gape coloration,morphology, and T-cell–mediated immunocompetence of nestlingbarn swallows (Hirundo rustica). For each gape we measured threecomponents of coloration (hue, saturation, and brightness) andobtained an overall color score by principal component analysis.Enlargement of brood size and injection with an antigen resultedin less red and less saturated and brighter gape color. Nestlingsin enlarged broods had smaller body mass and T-cell–mediatedimmunocompetence compared to those in reduced broods. A positivecovariation existed between redness and saturation of gape colorand T-cell–mediated immunocompetence. Gape color siblingsraised in different nests did not depend on parentage. Thus,condition-dependent gape coloration can reveal different componentsof nestling state on which parents may base their adaptive decisionsabout allocation of care to the offspring.     

Sema3A and neuropilin-1 expression and distribution in rat white adipose tissue

Semaphorins are cell surface and/or soluble signals that exert an inhibitory control on axon guidance. Sema3A, the vertebrate-secreted semaphorin, binds to neuropilin-1, which together with plexins constitutes the functional receptor. To verify whether Sema3A is produced by white adipocytes and, in that case, to detect its targets in white adipose tissue, we studied the cell production and tissue distribution of Sema3A and neuropilin-1 in rat retroperitoneal and epididymal adipose depots. Sema3A and neuropilin-1 were detected in these depots by Western blotting. The immunohistochemical results showed that Sema3A is produced in, and possibly secreted by, smooth muscle cells of arteries and white adipocytes. Accordingly, neuropilin-1 was found on perivascular and parenchymal nerves. Such a pattern of distribution is in line with a role for secreted Sema3A in the growth and plasticity of white adipose tissue nerves. Indeed, after fasting, when white adipocytes are believed to be overstimulated by noradrenaline and rearrangement of the parenchymal nerve supply may occur, adipocytic expression of Sema3A is reduced. Finally, the presence of neuropilin-1 in some white adipocytes raises the interesting possibility that Sema3A also exerts an autocrine-paracrine role on these cells.     

Enzymatic surface modification and functionalization of PET: A water contact angle,FTIR, and fluorescence spectroscopy study

The purpose of this study was to investigate the changes induced by a lypolytic enzyme on the surface properties of polyethylene terephthalate (PET). Changes in surface hydrophilicity were monitored by means of water contact angle (WCA) measurements. Fourier Transform Infrared spectroscopy (FTIR) in the Attenuated Total Reflectance mode (ATR) was used to investigate the structural and conformational changes of the ethylene glycol and benzene moieties of PET. Amorphous and crystalline PET membranes were used as substrate. The lipolytic enzyme displayed higher hydrolytic activity towards the amorphous PET substrate, as demonstrated by the decrease of the WCA values. Minor changes were observed on the crystalline PET membrane. The effect of enzyme adhesion was addressed by applying a protease after‐treatment which was able to remove the residual enzyme protein adhering to the surface of PET, as demonstrated by the behavior of WCA values. Significant spectral changes were observed by FTIR–ATR analysis in the spectral regions characteristic of the crystalline and amorphous PET domains. The intensity of the crystalline marker bands increased while that of the amorphous ones decreased. Accordingly, the crystallinity indexes calculated as band intensity ratios (1,341/1,410 cm^?1 and 1,120/1,100 cm^?1) increased. Finally, the free carboxyl groups formed at the surface of PET by enzyme hydrolysis were esterified with a fluorescent alkyl bromide, 2‐(bromomethyl)naphthalene (BrNP). WCA measurements confirmed that the reaction proceeded effectively. The fluorescence results indicate that the enzymatically treated PET films are more reactive towards BrNP. FTIR analysis showed that the surface of BrNP‐modified PET acquired a more crystalline character. Biotechnol. Bioeng. 2009;103: 845–856. © 2009 Wiley Periodicals, Inc.     

Conformational studies of the capsular polysaccharide produced by Neisseria meningitidis group A

The effect of different cations on the conformational and morphological properties of the capsular polysaccharide produced by Neisseria meningitidis group A was investigated. Circular dichroism studies showed that the presence of Na⁺, or Ca²⁺ ions induced different local conformations of the polysaccharide chain through interactions with the phosphodiester group bridging the saccharide residues in the polymer chain. Atomic force microscopy experiments confirmed that the morphology of the polysaccharide chains was different depending on the nature of the counterion. Ammonium ions were associated with the presence of single polymer chains in an elongated conformation, whereas sodium ions favored the folding of the chains into a globular conformation. The addition of calcium ions produced the aggregation of a limited number of globular polysaccharide chains to form a ‘toroidal-like’ structure.     

Gene Expression Profiles of APP and BACE1 in Tg SOD1G93A Cortical Cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Aβ peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer’s disease, as APP, β- and γ-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of γ-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.