Climatic niche and neutral genetic diversity of the six Iberian pine species: a retrospective and prospective view

Quaternary climatic fluctuations have left contrasting historical footprints on the neutral genetic diversity patterns of existing populations of different tree species. We should expect the demography, and consequently the neutral genetic structure, of taxa less tolerant to particular climatic extremes to be more sensitive to long‐term climate fluctuations. We explore this hypothesis here by sampling all six pine species found in the Iberian Peninsula (2464 individuals, 105 populations), using a common set of chloroplast microsatellite markers, and by looking at the association between neutral genetic diversity and species‐specific climatic requirements. We found large variation in neutral genetic diversity and structure among Iberian pines, with cold‐enduring mountain species (Pinus uncinata, P. sylvestris and P. nigra) showing substantially greater diversity than thermophilous taxa (P. pinea and P. halepensis). Within species, we observed a significant positive correlation between population genetic diversity and summer precipitation for some of the mountain pines. The observed pattern is consistent with the hypotheses that: (i) more thermophilous species have been subjected to stronger demographic fluctuations in the past, as a consequence of their maladaptation to recurrent glacial cold stages; and (ii) altitudinal migrations have allowed the maintenance of large effective population sizes and genetic variation in cold‐tolerant species, especially in more humid regions. In the light of these results and hypotheses, we discuss some potential genetic consequences of impending climate change.     

Description of Paratetrahymena parawassi n. sp. using Morphological and Molecular Evidence and a Phylogenetic Analysis of Paratetrahymena and Other Taxonomically Ambiguous Genera in the Order Loxocephalida (Ciliophora,Oligohymenophorea)

ABSTRACT. The marine scuticociliate Paratetrahymena parawassi n. sp. is described on the basis of morphology, especially infraciliature, and the sequence of its small subunit (SSU) rRNA gene to become the second known member of its genus. Paratetrahymena and other ciliates in the order Loxocephalida possess a mixture of morphological and morphogenetic features characteristic of the subclasses Hymenostomatia and Scuticociliatia. Accordingly, we used SSU rRNA sequences to analyze the phylogeny of Paratetrahymena and three other loxocephalid genera. Paratetrahymena and Cardiostomatella vermiformis formed a moderately well‐supported clade that diverged at a deep level from all other scuticociliates, supporting separation of loxocephalids from other scuticociliates as a suprafamilial taxon. Sathrophilus holtae was a sister taxon to Paratetrahymena and Cardiostomatella in a poorly supported, unresolved relationship; nevertheless, association of all three genera into a single clade was supported by an approximately unbiased (AU) test. Any association of these genera singly or as a group with the Hymenostomatia was rejected decisively by AU tests and by a complete absence in the loxocephalids of the unique nucleotide identities that distinguish hymenostomes. Therefore, the morphological and morphogenetic similarities of loxocephalids to hymenostomes may be plesiomorphies, and the conflicting mix of scuticociliate and hymenostome characteristics seen in loxocephalids may result from differing rates of character evolution. Dexiotrichides pangi and Urocentrum, which is currently classified as a peniculid, formed a small clade that associated with hymenostomes and peritrichs. Monophyly of the Loxocephalida with Dexiotrichides and/or Urocentrum included was not rejected by AU; however, inclusion of Urocentrum in the Peniculia was rejected by AU tests. A hypothesis is offered to explain the lack of resolution of loxocephalid ciliates and Urocentrum in phylogenetic trees, namely that their phylogenetic positions are influenced by a combination of heterogeneous data and long‐branch attraction caused by poor representation of taxa in analyses. The well‐known genus Cyclidium, a member of the order Pleuronematida, was revealed to be polyphyletic as a byproduct of our analyses of loxocephalids. In particular, Cyclidium porcatum appears to fall outside the clade containing typical members of the subclass Scuticociliatia and thus invites investigation as a possible member of the order Loxocephalida.     

Study of nitrate stress in Desulfovibrio vulgaris Hildenborough using iTRAQ proteomics.

The response of Desulfovibrio vulgaris Hildenborough (DvH), a sulphate-reducing bacterium, to nitrate stress was examined using quantitative proteomic analysis. DvH was stressed with 105 mM sodium nitrate (NaNO(3)), a level that caused a 50% inhibition in growth. The protein profile of stressed cells was compared with that of cells grown in the absence of nitrate using the iTRAQ peptide labelling strategy and tandem liquid chromatography separation coupled with mass spectrometry (quadrupole time-of-flight) detection. A total of 737 unique proteins were identified by two or more peptides, representing 22% of the total DvH proteome and spanning every functional category. The results indicate that this was a mild stress, as proteins involved in central metabolism and the sulphate reduction pathway were unperturbed. Proteins involved in the nitrate reduction pathway increased. Increases seen in transport systems for proline, glycine-betaine and glutamate indicate that the NaNO(3) exposure led to both salt stress and nitrate stress. Up-regulation observed in oxidative stress response proteins (Rbr, RbO, etc.) and a large number of ABC transport systems as well as in iron-sulphur-cluster-containing proteins, however, appear to be specific to nitrate exposure. Finally, a number of hypothetical proteins were among the most significant changers, indicating that there may be unknown mechanisms initiated upon nitrate stress in DvH.     

Model to assess muscle protein turnover: domain of validity using amino acyl-tRNA vs. surrogate measures of precursor pool

Current models to measure protein turnover across muscle bed are based on many surrogate measures of amino acyl-tRNA. We measured muscle protein turnover based on tracer-to-tracee ratios of the stable isotopes of leucine, phenylalanine, and ketoisocaproate (KIC) in artery and vein and muscle amino acyl-tRNA and muscle tissue fluid (TF) in 26 healthy subjects. A three-compartment model calculation based on arteriovenous and tRNA measurements was first performed and its domain of validity assessed. The results were then compared with those using simpler approaches based on surrogate measures of tRNA such as those of TF and KIC and a one-compartment model based on arteriovenous amino acids. In 96% of cases, the model using tRNA was applicable, but only in a lower percentage of cases were the results using surrogate measures applicable. Protein breakdown, protein synthesis, and shunting of amino acids from artery to vein were consistently underestimated, and fluxes of amino acid from artery to intracellular compartment and from intracellular compartment to vein were overestimated, when surrogate measures were used. The one-compartment model also underestimated protein breakdown and synthesis. Measurements using tissue fluid gave results closer to those based on tRNA. In conclusion, a three-compartment model using arteriovenous samples and amino acyl-tRNA provides measurements of muscle protein turnover of acceptable precision in 96% of cases. The precision was unacceptable in a substantial percentage of cases, and the accuracy of the estimation of protein fluxes was significantly affected when surrogate measures were used.     

Molecular cloning of the cell surface antigen identified by the osteoprogenitor-specific monoclonal antibody,HOP-26

Bone is a highly organized structure comprising a calcified connective tissue matrix formed by mature osteoblasts, which develop from the proliferation and differentiation of osteoprogenitor cells. The osteogenic cell lineage is thought to arise from a population of uncommitted multipotential stromal precursor cells (SPC) which reside close to all bone surfaces, in the bone marrow spaces and the surrounding connective tissue. These SPC also give rise to related cell lineages which form cartilage, smooth muscle, fat, and fibrous tissue. Due to the lack of well defined cell surface markers, little is known of the precise developmentally regulated changes in phenotype which occur during the differentiation and maturation of human osteoprogenitor cells into functional osteoblasts and ultimately, terminally differentiated osteocytes. In order to identify antibody reagents with greater specificity for osteoprogenitors we generated a series of antibodies following immunization with freshly isolated human bone marrow stromal fibroblasts. One such antibody, HOP-26, reacts with a cell surface antigen expressed by SPC and developing bone cells. We now demonstrate that this mAb identifies a member of the tetraspan family of cell surface glycoproteins, namely CD63. Western blot analysis of human bone marrow stromal cells (HBMSC) has revealed that like a well defined CD63 mAb 12F12, HOP-26 interacts with a heavily glycosylated cell surface protein with an apparent molecular weight of 50-60 kD.     

Influence of increased central venous pressure on baroreflex control of sympathetic activity in humans

Volume expansion often ameliorates symptoms of orthostatic intolerance; however, the influence of this increased volume on integrated baroreflex control of vascular sympathetic activity is unknown. We tested whether acute increases in central venous pressure (CVP) diminished subsequent responsiveness of muscle sympathetic nerve activity (MSNA) to rapid changes in arterial pressure. We studied healthy humans under three separate conditions: control, acute 10 degrees head-down tilt (HDT), and saline infusion (SAL). In each condition, heart rate, arterial pressure, CVP, and peroneal MSNA were measured during 5 min of rest and then during rapid changes in arterial pressure induced by sequential boluses of nitroprusside and phenylephrine (modified Oxford technique). Sensitivities of integrated baroreflex control of MSNA and heart rate were assessed as the slopes of the linear portions of the MSNA-diastolic blood pressure and R-R interval-systolic pressure relations, respectively. CVP increased approximately 2 mmHg in both SAL and HDT conditions. Resting heart rate and mean arterial pressure were not different among trials. Sensitivity of baroreflex control of MSNA was decreased in both SAL and HDT condition, respectively: -3.1 +/- 0.6 and -3.3 +/- 1.0 versus -5.0 +/- 0.6 units.beat(-1).mmHg(-1) (P < 0.05 for SAL and HDT vs. control). Sensitivity of baroreflex control of the heart was not different among conditions. Our results indicate that small increases in CVP decrease the sensitivity of integrated baroreflex control of sympathetic nerve activity in healthy humans.     

Developmental differences in L-type calcium current of human atrial myocytes

We investigated differences in L-type Ca2+ current (ICa) between infant (INF, 1-12 mo old), young adult (YAD, 14-18 yr old), and older adult (AD) myocytes from biopsies of right atrial appendages. Basal ICa was smaller in INF myocytes (1.2 +/- 0.1 pA/pF, n = 29, 6 +/- 1 mo old, 11 patients) than in YAD (2.5 +/- 0.2 pA/pF, n = 20, 16 +/- 1 yr old, 5 patients) or AD (2.6 +/- 0.3 pA/pF, n = 19, 66 +/- 3 yr old, 9 patients) myocytes (P < 0.05). Maximal ICa produced by isoproterenol (Iso) was similar in INF, YAD, and AD cells: 8.4 +/- 1.1, 9.6 +/- 1.0, and 9.2 +/- 1.3 pA/pF, respectively. Efficacy (Emax) was larger in INF (607 +/- 50%) than for YAD (371 +/- 29%) or AD (455 +/- 12%) myocytes. Potency (EC50) was 8- to 10-fold higher in AD (0.82 +/- 0.09 nM) or YAD (0.41 +/- 0.14 nM) than in INF (7.6 +/- 3.5 nM) myocytes. Protein levels were similar for Gialpha2 but much greater for Gialpha3 in INF than in AD or YAD atrial tissue. When Gialpha3 activity was inhibited by inclusion of a Gialpha3 COOH-terminal decapeptide in the pipette, basal ICa and the response to 10 nM Iso were increased in INF, but not in YAD, cells. We propose that basal ICa and the response to low-dose beta-adrenergic stimulation are inhibited in INF (but not YAD or AD) cells as a result of constitutive inhibitory effects of Gialpha3.     

Osteopontin inhibits interleukin-1beta-stimulated increases in matrix metalloproteinase activity in adult rat cardiac fibroblasts: role of protein kinase C-zeta

We have shown that osteopontin (OPN), an extracellular matrix protein, plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Interleukin-1beta (IL-1beta), increased in the heart following MI, increases matrix metalloproteinase (MMP) activity in cardiac fibroblasts in vitro. Here, we show that OPN alone has no effect on MMP activity or expression. However, it reduces IL-1beta-stimulated increases in MMP activity and expression in adult rat cardiac fibroblasts. Pretreatment with bovine serum albumin had no effect on MMP activity or protein content, whereas GRGDS (glycine-arginine-glycine-aspartic acid-serine)-pentapeptide (which interrupts binding of RGD-containing proteins to cell surface integrins) and monoclonal antibody m7E3 (a rat beta3 integrins antagonist) inhibited the effects of OPN. Inhibition of PKC using chelerythrine inhibited the activities of both MMP-2 and MMP-9. Stimulation of cells using IL-1beta increased phosphorylation and translocation of PKC to membrane fractions, which was inhibited by OPN. OPN inhibited IL-1beta-stimulated increases in translocation of PKC-zeta from cytosolic to membrane fractions. Furthermore, the levels of phospho-PKC-zeta were lower in the cytosolic fractions of OPN knock-out mice hearts as compared with wild type 6 days post-MI. Inhibition of PKC-zeta using PKC-zeta pseudosubstrate inhibited IL-1beta-stimulated increases in MMP-2 and MMP-9 activities. These observations suggest that OPN, acting via beta3 integrins, inhibits IL-1beta-stimulated increases in MMP-2 and MMP-9 activity, at least in part, via the involvement of PKC-zeta. Thus, OPN may play a key role in collagen deposition during myocardial remodeling following MI by modulating cytokine-stimulated MMP activity.