Contrasting Patterns of Functional Diversity in Coffee Root Fungal Communities Associated with Organic and Conventionally Managed Fields.

The structure and function of fungal communities in the coffee rhizosphere are influenced by crop environment. Because coffee can be grown along a management continuum from conventional application of pesticides and fertilizers in full sun to organic management in a shaded understory, we used coffee fields to hold host constant while comparing rhizosphere fungal communities under markedly different environmental conditions with regard to shade and inputs. We characterized the shade and soil environment in 25 fields under conventional, organic, or transitional management in two regions of Costa Rica. We amplified the internal transcribed spacer 2 (ITS2) region of fungal DNA from coffee roots in these fields and characterized the rhizosphere fungal community via high-throughput sequencing. Sequences were assigned to guilds to determine differences in functional diversity and trophic structure among coffee field environments. Organic fields had more shade, a greater richness of shade tree species, and more leaf litter and were less acidic, with lower soil nitrate availability and higher soil copper, calcium, and magnesium availability than conventionally managed fields, although differences between organic and conventionally managed fields in shade and calcium and magnesium availability depended on region. Differences in richness and community composition of rhizosphere fungi between organic and conventionally managed fields were also correlated with shade, soil acidity, and nitrate and copper availability. Trophic structure differed with coffee field management. Saprotrophs, plant pathogens, and mycoparasites were more diverse, and plant pathogens were more abundant, in organic than in conventionally managed fields, while saprotroph-plant pathogens were more abundant in conventionally managed fields. These differences reflected environmental differences and depended on region.IMPORTANCE Rhizosphere fungi play key roles in ecosystems as nutrient cyclers, pathogens, and mutualists, yet little is currently known about which environmental factors and how agricultural management may influence rhizosphere fungal communities and their functional diversity. This field study of the coffee agroecosystem suggests that organic management not only fosters a greater overall diversity of fungi, but it also maintains a greater richness of saprotrophic, plant-pathogenic, and mycoparasitic fungi that has implications for the efficiency of nutrient cycling and regulation of plant pathogen populations in agricultural systems. As well as influencing community composition and richness of rhizosphere fungi, shade management and use of fungicides and synthetic fertilizers altered the trophic structure of the coffee agroecosystem.

Taxonomic shifts in arbuscular mycorrhizal fungal communities with shade and soil nitrogen across conventionally managed and organic coffee agroecosystems.

The composition of arbuscular mycorrhizal fungal (AMF) communities should reflect not only responses to host and soil environments, but also differences in functional roles and costs vs. benefits among arbuscular mycorrhizal fungi. The coffee agroecosystem allows exploration of the effects of both light and soil fertility on AMF communities, because of the variation in shade and soil nutrients farmers generate through field management. We used high-throughput ITS2 sequencing to characterize the AMF communities of coffee roots in 25 fields in Costa Rica that ranged from organic management with high shade and no chemical fertilizers to conventionally managed fields with minimal shade and high N fertilization, and examined relationships between AMF communities and soil and shade parameters with partial correlations, NMDS, PERMANOVA, and partial least squares analysis. Gigasporaceae and Acaulosporaceae dominated coffee AMF communities in terms of relative abundance and richness, respectively. Gigasporaceae richness was greatest in conventionally managed fields, while Glomeraceae richness was greatest in organic fields. While total AMF richness and root colonization did not differ between organic and conventionally managed fields, AMF community composition did; these differences were correlated with soil nitrate and shade. OTUs differing in relative abundance between conventionally managed and organic fields segregated into four groups: Gigasporaceae associated with high light and nitrate availability, Acaulosporaceae with high light and low nitrate availability, Acaulosporaceae and a single relative of Rhizophagus fasciculatus with shade and low nitrate availability, and Claroideoglomus/Glomus with conventionally managed fields but uncorrelated with shade and soil variables. The association of closely related taxa with similar shade and light availabilities is consistent with phylogenetic trait conservatism in AM fungi.

Newborn screening for homocystinurias: Recent recommendations versus current practice.

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.

The GlpF residue Trp219 is part of an amino-acid cluster crucial for aquaglyceroporin oligomerization and function.

The vestibule loop regions of aquaglyceroporins are involved in accumulation of glycerol inside the channel pore. Even though most loop regions do not show high sequence similarity among aquaglyceroporins, loop E is highly conserved in aquaglyceroporins, but not in members of the homologous aquaporins. Specifically, a tryptophan residue is extremely conserved within this loop. We have investigated the role of this residue (Trp219) that deeply protrudes into the protein and potentially interacts with adjacent loops, using the E. coli aqualgyeroporin GlpF as a model. Replacement of Trp219 affects the activity of GlpF and impairs the stability of the tetrameric protein. Furthermore, we have identified an amino acid cluster involving Trp219 that stabilizes the GlpF tetramer. Based on our results we propose that Trp219 is key for formation of a defined vestibule structure, which is crucial for glycerol accumulation as well as for the stability of the active GlpF tetramer.

The Hemoglobin Homolog Cytoglobin in Smooth Muscle Inhibits Apoptosis and Regulates Vascular Remodeling.

OBJECTIVE: The role of hemoglobin and myoglobin in the cardiovascular system is well established, yet other globins in this context are poorly characterized. Here, we examined the expression and function of cytoglobin (CYGB) during vascular injury. APPROACH AND RESULTS: We characterized CYGB content in intact vessels and primary vascular smooth muscle (VSM) cells and used 2 different vascular injury models to examine the functional significance of CYGB in vivo. We found that CYGB was strongly expressed in medial arterial VSM and human veins. In vitro and in vivo studies indicated that CYGB was lost after VSM cell dedifferentiation. In the rat balloon angioplasty model, site-targeted delivery of adenovirus encoding shRNA specific for CYGB prevented its reexpression and decreased neointima formation. Similarly, 4 weeks after complete ligation of the left common carotid, Cygb knockout mice displayed little to no evidence of neointimal hyperplasia in contrast to their wild-type littermates. Mechanistic studies in the rat indicated that this was primarily associated with increased medial cell loss, terminal uridine nick-end labeling staining, and caspase-3 activation, all indicative of prolonged apoptosis. In vitro, CYGB could be reexpressed after VSM stimulation with cytokines and hypoxia and loss of CYGB sensitized human and rat aortic VSM cells to apoptosis. This was reversed after antioxidant treatment or NOS2 (nitric oxide synthase 2) inhibition. CONCLUSIONS: These results indicate that CYGB is expressed in vessels primarily in differentiated medial VSM cells where it regulates neointima formation and inhibits apoptosis after injury.

The Escherichia coli Envelope Stress Sensor CpxA Responds to Changes in Lipid Bilayer Properties.

The Cpx stress response system is induced by various environmental and cellular stimuli. It is also activated in Escherichia coli strains lacking the major phospholipid, phosphatidylethanolamine (PE). However, it is not known whether CpxA directly senses changes in the lipid bilayer or the presence of misfolded proteins due to the lack of PE in their membranes. To address this question, we used an in vitro reconstitution system and vesicles with different lipid compositions to track modulations in the activity of CpxA in different lipid bilayers. Moreover, the Cpx response was validated in vivo by monitoring expression of a PcpxP-gfp reporter in lipid-engineered strains of E. coli. Our combined data indicate that CpxA responds specifically to different lipid compositions.

When two turn into one: evolution of membrane transporters from half modules.

The recently increasing number of atomic structures for active transporters has not only revealed strong conservation in the architecture of sequence-unrelated transporter families, but also identified a unifying element called the 'inverted repeat topology,' which is found in nearly all transporter folds to date. Indeed, most membrane transporters consist of two or more domains with similar structure, so-called repeats. It is tempting to speculate that transporters have evolved by duplication of one repeat followed by gene fusion and modification events. An intriguing question is, whether recent genes encoding such a 'half-transporter' still exist as independent folding units. Although it seems likely that the evolution of membrane transport proteins, which harbor internal repeats, is linked to these minimal structural building blocks, their identification in the absence of structural data represents a major challenge, as sequence homology is not an issue. In this review we discuss two protein families, the DedA family and the SWEET family, being potential half-transporters and putative ancestors for two of the most abundant secondary transporter families, the MFS family and the LeuT-fold family.

Orai3 is an estrogen receptor α-regulated Ca²âº channel that promotes tumorigenesis.

Store-operated Ca(2+) entry (SOCE) encoded by Orai1 proteins is a ubiquitous Ca(2+)-selective conductance involved in cellular proliferation and migration. We recently described up-regulation of Orai3 channels that selectively mediate SOCE in estrogen receptor α-expressing (ERα(+)) breast cancer cells. However, the connection between ERα and Orai3 and the role of Orai3 in tumorigenesis remain unknown. Here, we show that ERα knockdown decreases Orai3 mRNA (by ∼63%) and protein (by ∼44%) with no effect on Orai1. ERα knockdown decreases Orai3-mediated SOCE (by ∼43%) and the corresponding Ca(2+) release-activated Ca(2+) (CRAC) current (by ∼42%) in ERα(+) MCF7 cells. The abrogation of SOCE in MCF7 cells on ERα knockdown can be rescued by ectopic expression of Orai3. ERα activation increased Orai3 expression and SOCE in MCF7 cells. Epidermal growth factor (EGF) and thrombin stimulate Ca(2+) influx into MCF7 cells through Orai3. Orai3 knockdown inhibited SOCE-dependent phosphorylation of extracellular signal-regulated kinase (ERK1/2; by ∼44%) and focal adhesion kinase (FAK; by ∼46%) as well as transcriptional activity of nuclear factor for activated T cells (NFAT; by ∼49%). Significantly, Orai3 knockdown selectively decreased anchorage-independent growth (by ∼58%) and Matrigel invasion (by ∼44%) of ERα(+) MCF7 cells with no effect on ERα(-) MDA-MB231 cells. Moreover, Orai3 knockdown inhibited ERα(+) cell tumorigenesis in immunodeficient mice (∼66% reduction in tumor volume). These data establish Orai3 as an ERα-regulated channel and a potential selective therapeutic target for ERα(+) breast cancers.

Mercury bioaccumulation in Southern Appalachian birds, assessed through feather concentrations.

Mercury contamination in wildlife has rarely been studied in the Southern Appalachians despite high deposition rates in the region. From 2006 to 2008 we sampled feathers from 458 birds representing 32 species in the Southern Appalachians for total mercury and stable isotope δ (15)N. Mercury concentrations (mean ± SE) averaged 0.46 ± 0.02 µg g(-1) (range 0.01-3.74 µg g(-1)). Twelve of 32 species had individuals (7 % of all birds sampled) with mercury concentrations higher than 1 µg g(-1). Mercury concentrations were 17 % higher in juveniles compared to adults (n = 454). In adults, invertivores has higher mercury levels compared to omnivores. Mercury was highest at low-elevation sites near water, however mercury was detected in all birds, including those in the high elevations (1,000-2,000 m). Relative trophic position, calculated from δ (15)N, ranged from 2.13 to 4.87 across all birds. We fitted linear mixed-effects models to the data separately for juveniles and year-round resident adults. In adults, mercury concentrations were 2.4 times higher in invertivores compared to omnivores. Trophic position was the main effect explaining mercury levels in juveniles, with an estimated 0.18 ± 0.08 µg g(-1) increase in feather mercury for each one unit rise in trophic position. Our research demonstrates that mercury is biomagnifying in birds within this terrestrial mountainous system, and further research is warranted for animals foraging at higher trophic levels, particularly those associated with aquatic environments downslope from montane areas receiving high mercury deposition.

Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site-specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins.

Structural basis for two-component system inhibition and pilus sensing by the auxiliary CpxP protein.

Bacteria are equipped with two-component systems to cope with environmental changes, and auxiliary proteins provide response to additional stimuli. The Cpx two-component system is the global modulator of cell envelope stress in gram-negative bacteria that integrates very different signals and consists of the kinase CpxA, the regulator CpxR, and the dual function auxiliary protein CpxP. CpxP both inhibits activation of CpxA and is indispensable for the quality control system of P pili that are crucial for uropathogenic Escherichia coli during kidney colonization. How these two essential biological functions of CpxP are linked is not known. Here, we report the crystal structure of CpxP at 1.45 Å resolution with two monomers being interdigitated like "left hands" forming a cap-shaped dimer. Our combined structural and functional studies suggest that CpxP inhibits the kinase CpxA through direct interaction between its concave polar surface and the negatively charged sensor domain on CpxA. Moreover, an extended hydrophobic cleft on the convex surface suggests a potent substrate recognition site for misfolded pilus subunits. Altogether, the structural details of CpxP provide a first insight how a periplasmic two-component system inhibitor blocks its cognate kinase and is released from it.

Transforming growth factor-ß1-induced transcript 1 protein, a novel marker for smooth muscle contractile phenotype, is regulated by serum response factor/myocardin protein.

Serum response factor (SRF) plays a central role in regulating expression of smooth muscle-specific genes partly by associating with the potent tissue-specific cofactor myocardin. Previous studies have shown that transforming growth factor-ß1-induced transcript 1 (TGFB1I1, also known as Hic-5) is a TGF-ß-responsive gene and is involved in the cellular response to vascular injury, but the regulation of TGFB1I1 expression remains elusive. In this report, we demonstrated that TGFB1I1 is a novel marker for the smooth muscle contractile phenotype and is regulated by SRF/myocardin. We found that TGFB1I1 is specifically expressed in smooth muscle cells (SMCs) and in smooth muscle-rich tissues. Furthermore, TGFB1I1 expression is significantly down-regulated in a variety of models for smooth muscle phenotypic modulation. The TGFB1I1 promoter contains an evolutionarily conserved CArG element, and this element is indispensible for myocardin-induced transactivation of TGFB1I1 promoter. By oligonucleotide pulldown and chromatin immunoprecipitation assays, we found that SRF binds to this CArG element in vitro and in vivo. Ectopic expression of myocardin is sufficient to induce endogenous TGFB1I1 expression in multiple cell lines whereas knocking-down myocardin or SRF significantly attenuated TGFB1I1 expression in SMCs. Furthermore, our data demonstrated that SRF is essential for TGF-ß-mediated induction of TGFB1I1. Finally, silencing of TGFB1I1 expression significantly promotes SMC proliferation. Collectively, this study provides the first evidence that TGFB1I1 is not only an SRF/myocardin-regulated smooth muscle marker but also critical for maintaining smooth muscle contractile phenotype by inhibiting smooth muscle proliferation.

Hic-5 is required for fetal gene expression and cytoskeletal organization of neonatal cardiac myocytes.

Cardiac costameres link the extracellular matrix to the sarcomere at the z-disc and contain proteins such as integrins and other signaling molecules implicated in the regulation of pathological hypertrophy. Paxillin family members, hic-5 and paxillin, are scaffolding proteins associated with the integrin complex that have been shown to mediate numerous protein interactions in other cell types. While paxillin has been described in postnatal heart, hic-5 has not been identified. Our results provide evidence of hic-5 in neonatal cardiac myocytes co-localized with paxillin and alpha-actinin at the z-discs and the ends of actin filaments. Treatment with the hypertrophic agonist phenylephrine resulted in increased hic-5 expression while having no effect on paxillin levels. To see if increased hic-5 expression was sufficient to induce changes in cytoskeletal organization, hic-5 was overexpressed in myocytes by adenoviral infection. Hic-5 overexpression significantly increased the number of cells with organized cytoskeleton. Using siRNA mediated knockdown, we examined the requirement of hic-5 and paxillin in regulation of phenylephrine induced gene expression and cytoskeletal organization. Our results indicate that hic-5, not paxillin is required for upregulation of ANF and alpha-skeletal actin genes as well as in cytoskeletal reorganization. Finally, we demonstrated that hic-5 upregulation occurs downstream of MEK1/2-ERK1/2 signaling as inhibition of MEK1/2 using U0126 inhibitor completely inhibited hic-5 upregulation by PE. In a complimentary study, we showed that hic-5 knockdown had no effect on PE induced ERK1/2 phosphorylation. These findings demonstrate a novel role for hic-5 in the regulation of actin cytoskeleton and fetal gene expression.

Racial/Ethnic Disparities in Morbidity and Mortality for Preterm Neonates Admitted to a Tertiary Neonatal Intensive Care Unit.

OBJECTIVE: The objective of this study was to assess whether in-hospital morbidity or mortality differed by race/ethnicity for preterm neonates admitted to the neonatal intensive care unit (NICU). STUDY DESIGN: In a retrospective cohort study, preterm infants, < 37 weeks, were admitted to the NICU from 1994 to 2009. Exclusions included structural anomalies and aneuploidy. Primary outcome was in-hospital mortality (IHM). Secondary outcomes were respiratory distress syndrome (RDS), interventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), and retinopathy of prematurity (ROP). Sub-analysis of very preterm (VPT) infants, < 28 weeks, was performed. Five racial/ethnic groups (REGs) were compared: White, Black, Hispanic, Asian, and Mixed. Associations were modeled by logistic regression. White neonates (WNs) were the referent group. Unadjusted and adjusted odds ratios and 95% confidence intervals for remaining REGs were reported. p value was significant at 5% for overall tests and at Bonferroni-corrected level < 0.0125 for between-race comparisons with WNs. RESULTS: Four thousand nine hundred fifty-five preterm neonates were identified; 153 were excluded leaving 4802 for analysis. After controlling covariates that were chosen a priori, there was no difference across REGs for IHM (all between-race comparison p values > 0.0125). There was a significant difference in RDS among Black neonates (BNs) (aOR 0.57, 95% CI 0.45-0.73; p < 0.001) and Hispanic neonates (HNs) (aOR 0.67, 95% CI 0.50-0.89; p = 0.005) compared to WNs. The risk of ROP was significantly different across REGs with HNs having a 70% increase in ROP (aOR 1.70, 95% CI 1.15-2.49; p = 0.008) and Mixed neonates (MNs) experiencing a 55% reduction (aOR 0.45, 95% CI 0.29-0.68; p < 0.001) compared to WNs. There was no difference in IVH or NEC across REGs (all p values > 0.0125). In the VPT cohort sub-analysis, BNs experienced a significant 59% reduction in IHM compared to WNs (BNs aOR 0.41, 95% CI 0.22-0.73; p = 0.003). MNs experienced a 46% reduction in ROP compared to WNs (aOR 0.54, 95% CI 0.35-0.81; p = 0.004). There was no difference in RDS, IVH, or NEC in very preterm infants across REGs (all between comparison p values > 0.0125). CONCLUSION: In preterm neonates, in-hospital mortality does not significantly differ across racial and ethnic groups. However, in very preterm infants, in-hospital mortality for Black neonates is improved. There are morbidity differences (RDS, ROP) seen among racial/ethnic groups.

Use of resveratrol to improve the effectiveness of cisplatin and doxorubicin: study in human gynecologic cancer cell lines and in rodent heart.

OBJECTIVE: The purpose of this study was to investigate whether resveratrol adds to the growth inhibitory effects of cisplatin and doxorubicin on ovarian and uterine cancer cells and to evaluate whether resveratrol diminishes the cardiac toxicity of doxorubicin in rodent heart. STUDY DESIGN: Human ovarian (OVCAR-3) and uterine (Ishikawa) cancer cells in culture were treated with cisplatin and doxorubicin, respectively, with and without resveratrol; and cell growth and viability were evaluated. Neonatal rat ventricular myocytes received doxorubicin in the presence and absence of resveratrol, and cell viability was evaluated. Mice received doxorubicin +/- resveratrol, and electrocardiograms were evaluated. Data were analyzed with analysis of variance and Scheffe's test. RESULTS: Resveratrol combined with cisplatin or with doxorubicin demonstrated an additive growth-inhibitory anticancer effect with a left shift of the cisplatin and doxorubicin dose/response curves. Resveratrol increased the viability of neonatal rat ventricular myocytes that were treated with doxorubicin and reduced doxorubicin-induced bradycardia and QTc interval prolongation in mice. CONCLUSION: Resveratrol adds to the growth inhibitory/anticancer activity of cisplatin and doxorubicin in vitro and protects against doxorubicin-induced cardiac toxicity both in vitro and in mice.

Toxoplasmosis, Parvovirus, and Cytomegalovirus in Pregnancy.

There are several infections in adults that warrant special consideration in pregnant women given the potential fetal consequences. Among these are toxoplasmosis, parvovirus B19, and cytomegalovirus. These infections have an important impact on the developing fetus, depending on the timing of infection. This article reviews the modes of transmission as well as maternal and neonatal effects of each of these infections. In addition, the article outlines recommended testing, fetal surveillance, and treatment where indicated.

Seronegative Maternal Ocular Myasthenia Gravis and Delayed Transient Neonatal Myasthenia Gravis.

Background Myasthenia gravis (MG) is an autoimmune disorder with fluctuating muscle weakness, divided into generalized and localized (ocular) forms. Maternal antibodies to acetylcholine receptors cross the placenta and may cause transient neonatal myasthenia gravis (TNMG). We present a case of seronegative maternal ocular MG and delayed TNMG. Case A 29-year-old G3P1011 underwent cesarean birth of a male infant who developed oxygen desaturation requiring supplemental oxygen on day of life (DOL) 3. Based on the clinical course and after exclusion of other diagnoses, the infant was diagnosed with TNMG. Infant's condition improved spontaneously and he was weaned off supplemental oxygen and discharged home on DOL 12. Conclusion Infants born to mothers with seronegative localized (ocular) MG are also susceptible to TNMG which may be late in onset.

Vascular endothelial growth factor-mediated activation of p38 is dependent upon Src and RAFTK/Pyk2.

Vascular endothelial growth factor (VEGF) induces activation of p38 mitogen-activated protein kinase (MAPK) in primary endothelial cells and may be critical for VEGF-induced angiogenesis. We investigated the molecular basis for p38 activation in response to VEGF. The expression of a C-terminal splice variant of FAK, FRNK, had no affect on VEGF-induced activation of p38; however, expression of a dominant-negative RAFTK/Pyk2 mutant led to a decrease in the activation of p38, but had no affect on extracellular signal-regulated kinase (ERK). Since calcium regulates RAFTK/Pyk2, we investigated its role in p38 activity. Preincubation with EGTA suppressed p38 activation, while calcium ionophore induced p38 activity. Inhibition of phospholipase C (PLC) resulted in complete inhibition of ERK, while having no affect on p38 activity. These data suggested a bifurcation in the regulation of MAPKs that occurs at the level of PLC and RAFTK/Pyk2 activation. Src family kinases interact with RAFTK/Pyk2. Inhibition of Src by either pharmacological or genetic means decreased p38 activity. Finally, we found that both Src and RAFTK/Pyk2 were essential for endothelial cell migration. These data identified a novel regulatory network involving extracellular calcium, RAFTK/Pyk2, Src and p38. This signaling network appears to be critical for VEGF-induced endothelial cell migration.

Interaction of ACE2 and integrin beta1 in failing human heart.

ACE2 purified from failing human heart was found to form a complex with integrin beta1 by immunoprecipitation, Western blotting, activity assay, and ESI tandem mass spectroscopy. The ACE2/integrin complex showed a Km of 6.8 microM and a Vmax of 2.13 pmol/min/microl purified enzyme. Activity was optimal at pH 7.5 with Ang II substrate.

Increased angiotensin-(1-7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme Homologue ACE2.

BACKGROUND: The formation of angiotensin-(1-7) from either angiotensin (Ang) I or Ang II in failing human hearts is not well understood. METHODS AND RESULTS: Angiotensinase activity in left and right ventricular membranes from 14 idiopathic dilated cardiomyopathy (IDC), 8 primary pulmonary hypertension (PPH), and 13 nonfailing human hearts was measured with either 125I-Ang I or 125I-Ang II as substrate. Ang-(1-7)-forming activity from 125I-Ang I was inhibited by thiorphan. With 125I-Ang II as substrate, Ang-(1-7) formation was inhibited by the ACE2-specific inhibitor C16. Western blotting with an anti-ACE2 antibody confirmed the presence of ACE2. Angiotensinase activity with 125I-Ang I as substrate was increased in failing IDC left ventricles (LVs) compared with nonfailing LVs (P<0.001). Ang-(1-7)-forming activity with 125I-Ang II as substrate was increased in both failing LVs and right ventricles (RVs) of IDC hearts and only in failing RVs of PPH hearts (PPH LV, 51.12+/-5.25; PPH RV, 89.97+/-11.21; IDC LV, 139.7+/-21.96; and IDC RV, 192.7+/-5.43; NF LV, 32.89+/-5.38; NF RV 40.49+/-10.66 fmol/min per milligram (P<0.05 PPH RV versus PPH LV; P<0.05 PPH RV versus NF RV; P<0.001 IDC LV versus NF LV; P<0.001 IDC RV versus NF RV). CONCLUSIONS: Ang-(1-7)-forming activity from both Ang I and Ang II was increased in failing human heart ventricles but was mediated by at least two different angiotensinases. The first, which demonstrated substrate preference for Ang I, was neutral endopeptidase (NEP)-like. The second was ACE2, as demonstrated by Western blotting and inhibition of activity with C16.