Ribosomal DNA evidence for the diversification of Tropaeolum sect. Chilensia (Tropaeolaceae)

Maximum parsimony and Bayesian likelihood analysis of ribosomal DNA internal transcribed spacer (ITS) sequences from 55 samples representing 25 species, subspecies, and/or hybrids of Tropaeolum sect. Chilensia yielded a tree with a large proportion of well-supported nodes at the interspecific and intersubspecific level. However, the tree shows samples from two species, T.azureum and T. brachyceras, arising in two different positions. Samples representing subspecies of T. hookerianum and T. leptophyllum suggest that these species are polyphyletic. The data corroborate evidence for hybridization between T. brachyceras and T. tricolor. Consideration of interfertility data, past and present Chilean ecology, and empirical evidence for the behavior of phenotypic and genotypic characters in known hybrids suggest a high likelihood that reticulate evolution has played a role in the diversification of T. sect. Chilensia. This reticulate evolution may explain the discordance between the ITS tree and the conventional taxonomy. High divergence in ITS sequences between T. sect. Chilensia and other members of Tropaeolum prohibits reliable outgroup-rooting, but midpoint rooting places the root at a partition comprising taxa whose distribution conforms to a relictual eastern-western South American disjunction described for other taxa. Within the Chilean taxa, the analysis suggests that biogeographic diversification has been from the mesophytic southern habitats northward to central mediterranean and northern arid zones.     

Spatial and temporal variation of photosynthesis in intertidal Mazzaella laminarioides (Bory)Fredericq (Rhodophyta, Gigartinales)

The red alga Mazzaella laminarioides is an economically important species with an extended latitudinal distribution along the Chilean coast. Its populations form mid-intertidal stands, several meters wide, and therefore are differentially exposed to environmental variables that result in temporal and spatial variability in productivity. We evaluated the effect of latitude and intertidal height on productivity by in situ measurement of photosynthetic performance. Daily and seasonal variations of O₂-evolution rate and maximal quantum yield (F _v/F _m) were determined in plants from the upper and lower intertidal zone at two localities 1500,km apart. Results suggest that plant responses were mainly affected by irradiation, temperature and desiccation. At local level, upper intertidal plants showed a reduced photosynthetic rate and quantum efficiency as compared to those displayed by plants from the lower intertidal, indicating their higher level of excitation energy acclimation. Stronger acclimation differences between upper and lower intertidal plants were observed in spring and summer. Differences in photosynthetic parameters between reproductive phases were recorded in autumn and winter, regardless of the position of the individuals in the intertidal zone. The effects of tidal elevation on seasonal patterns of photosynthesis were also influenced by latitude. Seasonal variation in photosynthetic efficiency was observed in plants from the northern population at both intertidal elevations, but only at the upper intertidal level in the southern population. This study shows that production variability in M. laminarioides results from differences in the intensity of environmental factors observed seasonally at local (intertidal) and latitudinal scales.     

PP2A regulates BCL-2 phosphorylation and proteasome-mediated degradation at the endoplasmic reticulum

Anti-apoptotic activity of BCL-2 is mediated by phosphorylation at the endoplasmic reticulum (ER), but how this phosphorylation is regulated and the mechanism(s) by which it regulates apoptosis are unknown. We purified macromolecular complexes containing BCL-2 from ER membranes and found that BCL-2 co-purified with the main two subunits of the serine/threonine phosphatase, PP2A. The association of endogenous PP2A and BCL-2 at the ER was verified by co-immunoprecipitation and microcystin affinity purification. Knock down or pharmacological inhibition of PP2A caused degradation of phosphorylated BCL-2 and led to an overall reduction in BCL-2 levels. We found that this degradation was due to the action of the proteasome acting selectively at the ER. Conversely, overexpression of PP2A caused elevation in endogenous BCL-2. Most importantly, we found that PP2A knock down sensitized cells to several classes of death stimuli (including ER stress), but this effect was abolished in a genetic background featuring knock in of a non-phosphorylatable BCL-2 allele. These studies support the hypothesis that PP2A-mediated dephosphorylation of BCL-2 is required to protect BCL-2 from proteasome-dependent degradation, affecting resistance to ER stress.     

Norepinephrine induces calcium spikes and proinflammatory actions in human hepatic stellate cells

Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through alpha1-adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigated the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory, and fibrogenic actions in the injured liver. Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular Ca2+ concentration ([Ca2+]i) was studied in fura-2-loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC II) phosphorylation. Cell proliferation and collagen-alpha1(I) expression were assessed by [3H]thymidine incorporation and quantitative PCR, respectively. NF-kappaB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Normal human livers expressed alpha(1A)-adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed alpha(1A)-adrenoceptors. NE induced multiple rapid [Ca2+]i oscillations (Ca2+ spikes). Prazosin (alpha1-blocker) completely prevented NE-induced Ca2+ spikes, whereas propranolol (nonspecific beta-blocker) partially attenuated this effect. NE caused phosphorylation of MLC II and cell contraction. In contrast, NE did not affect cell proliferation or collagen-alpha1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. NE stimulated NF-kappaB activation. BAY 11-7082, a specific NF-kappaB inhibitor, blocked NE-induced chemokine secretion. We conclude that NE stimulates NF-kappaB and induces cell contraction and proinflammatory effects in human HSC. Catecholamines may participate in the pathogenesis of portal hypertension and liver fibrosis by targeting HSC.     

Lipids in biocalcification: contrasts and similarities between intimal and medial vascular calcification and bone by NMR

Pathomechanisms underlying vascular calcification biogenesis are still incompletely understood. Biomineral from human atherosclerotic intimal plaques; human, equine, and bovine medial vascular calcifications; and human and equine bone was released from collagenous organic matrix by sodium hydroxide/sodium hypochlorite digestion. Solid-state (13)C NMR of intimal plaque mineral shows signals from cholesterol/cholesteryl esters and fatty acids. In contrast, in mineral from pure medial calcifications and bone mineral, fatty acid signals predominate. Refluxing (chloroform/methanol) intimal plaque calcifications removes the cholesterylic but not the fatty acyl signals. The lipid composition of this refluxed mineral now closely resembles that of the medial and bone mineral, which is unchanged by reflux. Thus, intimal and medial vascular calcifications and bone mineral have in common a pool of occluded mineral-entrained fatty acyl-rich lipids. This population of fatty acid may contain methyl-branched fatty acids, possibly representing lipoprotein particle remnants. Cell signaling and mechanistic parallels between physiological (orthotopic) and pathological (ectopic) calcification are also reflected thus in the NMR spectroscopic fingerprints of mineral-associated and mineral-entrained lipids. Additionally the atherosclerotic plaque mineral alone shows a significant independent pool of cholesterylic lipids. Colocalization of mineral and lipid may be coincidental, but it could also reflect an essential mechanistic component of biomineralization.     

Gene Duplication and Positive Selection Explains Unusual Physiological Roles of the Relaxin Gene in the European Rabbit

The relaxin gene family is a group of genes involved in different physiological roles, most of them related to reproduction. In vertebrates the genes in this family are located in three separate chromosomal locations, and have been called relaxin family locus (RFL) A, B, and C. Among mammals the RFLA and RFLC are the most conserved as no gene copy-number variation has been observed thus far. The RFLB locus is also conserved on most mammals other than primates, where there are several gene gains and losses. Interestingly, the relaxin gene found on the RFLB locus in the European rabbit has acquired a novel role. In addition to the classical reproductive roles, this gene is expressed in tracheobronchial epithelial cells and its expression has been linked to squamous differentiation. We reconstructed the evolutionary history of the European rabbit RFLB locus using the tools of comparative genomics and molecular evolution. We found that the European rabbit possess a RFLB locus which is unique among mammals in that there are five tandemly arranged relaxin gene copies, which contrast with the single relaxin copy gene found in most mammals. In addition we also found that the ancestral pre-duplication gene was subject to the action of positive selection, and several amino acid sites were identified under the action of natural selection including the sites B12 and B13 which are part of the receptor recognition and binding site.     

Extracellular production of <Emphasis Type="Italic">Streptomyces exfoliatus</Emphasis> poly(3-hydroxybutyrate) depolymerase in <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. T104: determination of optimal biocatalyst conditions

The phaZ _Sex gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T _m) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine–histidine–carboxylic acid in the active site. High-performance liquid chromatography (HPLC)–mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.     

Complete genome sequence of Bifidobacterium breve CECT 7263, a strain isolated from human milk

Bifidobacterium breve is an actinobacterium frequently isolated from colonic microbiota of breastfeeding babies. Here, we report the complete and annotated genome sequence of a B. breve strain isolated from human milk, B. breve CECT 7263. The genome sequence will provide new insights into the biology of this potential probiotic organism and will allow the characterization of genes related to beneficial properties.     

Generalization of the dynamic clamp concept in neurophysiology and behavior

The idea of closed-loop interaction in in vitro and in vivo electrophysiology has been successfully implemented in the dynamic clamp concept strongly impacting the research of membrane and synaptic properties of neurons. In this paper we show that this concept can be easily generalized to build other kinds of closed-loop protocols beyond (or in addition to) electrical stimulation and recording in neurophysiology and behavioral studies for neuroethology. In particular, we illustrate three different examples of goal-driven real-time closed-loop interactions with drug microinjectors, mechanical devices and video event driven stimulation. Modern activity-dependent stimulation protocols can be used to reveal dynamics (otherwise hidden under traditional stimulation techniques), achieve control of natural and pathological states, induce learning, bridge between disparate levels of analysis and for a further automation of experiments. We argue that closed-loop interaction calls for novel real time analysis, prediction and control tools and a new perspective for designing stimulus-response experiments, which can have a large impact in neuroscience research.