Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.

Sample purity is the key for a successful in-depth analysis of any given subcellular proteome. The suitability of free-flow electrophoresis to assist conventional, centrifugation-based techniques in the preparation of plant mitochondria from green and non-green tissue was assessed by various means, including functional assays, immunoblots, electron microscopy and differential gel electrophoresis. Results indicated a significant increase in purity of the mitochondrial samples, highlighted specific contaminants previously reported as mitochondrial proteins, and also pointed to new means for separating plastids and peroxisomes from mitochondria in plant organellar extracts by exploiting differences in surface charge. This approach has the potential to allow a deeper and more comprehensive investigation of the Arabidopsis organellar proteomes, by providing a second dimension of separation based on surface charge in addition to conventional centrifugation purification protocols relying on size and density.

New proteins can evolve by duplication and divergence or de novo, from previously noncoding DNA. A recently observed mechanism is for peptides to evolve within a "host" protein and emerge by proteolytic processing. The first examples of such interstitial peptides were ones hosted by precursors for seed storage albumin. Interstitial peptides have also been observed in precursors for seed vicilins, but current evidence for vicilin-buried peptides (VBPs) is limited to seeds of the broadleaf plants pumpkin and macadamia. Here, an extensive sequence analysis of vicilin precursors suggested that peptides buried within the N-terminal region of preprovicilins are widespread and truly ancient. Gene sequences indicative of interstitial peptides were found in species from Amborellales to eudicots and include important grass and legume crop species. We show the first protein evidence for a monocot VBP in date palm seeds as well as protein evidence from other crops including the common tomato, sesame and pumpkin relatives, cucumber, and the sponge loofah ( Luffa aegyptiaca). Their excision was consistent with asparaginyl endopeptidase-mediated maturation, and sequences were confirmed by tandem mass spectrometry. Our findings suggest that the family is large and ancient and that based on the NMR solution structures for loofah Luffin P1 and tomato VBP-8, VBPs adopt a helical hairpin fold stapled by two internal disulfide bonds. The first VBPs characterized were a protease inhibitor, antimicrobials, and a ribosome inactivator. The age and evolutionary retention of this peptide family suggest its members play important roles in plant biology.

Aims: Oxidative stress is important in the pathogenesis of liver fibrosis through its induction of hepatic stellate cell (HSC) proliferation and enhancement of collagen synthesis [1, 2]. Reactive oxygen species have been found to be essential second messengers in the signaling of both major fibrotic growth factors, platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), in cultured HSC and liver fibrosis. The non-toxic aminothiol N-acetyl-L-cysteine (NAC) inhibits cellular activation and attenuates experimental fibrosis in liver. We have previously shown that NAC is capable of reducing the effects of TGF-β in cultured HSC through its direct reducing activity upon TGF-β molecules and monomerisation of its type III receptor endoglin [3, 4]. Methods: We here analyzed the effects of NAC on PDGF integrity, receptor binding, and downstream signaling in culture-activated HSC. The PDGF-BB signaling pathways were evaluated by autophosphorylation of the Tyr residues of PDGF-activated PDGFR, phosphorylated ERK1/2 MAPK and phospho Akt of PI3K. Results: We found that NAC dose-dependently induces disintegration of PDGF in vitro. However, even high doses (>20 mM) were not sufficient to prevent the phosphorylation of the PDGF receptor type β, extracellular signal-regulated kinases (ERK1/2), or protein kinase B (PKB/Akt). Conclusions: We conclude that NAC inhibits PDGF signaling in cultured HSC through a mechanism involving the cellular redox status, rather than through a direct reducing effect on PDGF or its signaling components. Furthermore, antifibrogenic effects induced by NAC and other thiols in experimental liver fibrosis might be the result of intracellular redox status that inhibits several signaling pathways, especially in both major growth factors PDGF and TGF-β.

The combined action of the electron transport chain (ETC) and ATP synthase is essential in determining energy efficiency in plants, and so is important for cellular biosynthesis, growth and development. Owing to the sessile nature of plants, mitochondria must operate over a wide temperature range in the environment, necessitating a broad temperature tolerance of their biochemical reactions. We investigated the temperature response of mitochondrial respiratory processes in isolated mitochondria and intact plants of Arabidopsis thaliana and considered the effect of instantaneous responses to temperature and acclimation responses to low temperatures. We show that at 4°C the plant mitochondrial ATP synthase is differentially inhibited compared with other elements of the respiratory pathway, leading to decreased ADP : oxygen ratios and a limitation to the rate of ATP synthesis. This effect persists in vivo and cannot be overcome by cold-temperature acclimation of plants. This mechanism adds a new element to the respiratory acclimation model and provides a direct means of temperature perception by plant mitochondria. This also provides an alternative explanation for non-phosphorylating ETC bypass mechanisms, like the alternative oxidase to maintain respiratory rates, albeit at lower ATP synthesis efficiency, in response to the sensitivity of ATP synthase to the prevailing temperature.

Planned communities are one way that people have attempted to influence land use patterns and living situations to accommodate specific sociocultural, economic, and environmental needs and desires from the very start of settlement. Columbia, Maryland, a planned community, will celebrate its 50th anniversary in 2017. At its beginning, James Rouse and his planning team used a sociological approach to develop Columbia, and help achieve its original goals, including: the creation of a fully, self-sustaining city where residents could both live and work, respecting and integrating the natural environment into the built environment, sustainably accommodating the future growth of the community, integrating mixed income and racially diverse families, and making a profit. Almost 50 years on, Columbia, via Columbia Association, is interested in understanding the staying power of the original goals as it looks forward and plans for Columbia’s future. In Fall 2015, Columbia Association, PALS (Partnership for Action Learning in Sustainability), and the Department of Anthropology at the University of Maryland collaborated to collect oral histories from Columbia’s residents on their experiences of living in the community over the past 50 years. Nine undergraduate students and one graduate student taking ANTH 468O/689O: Researching Environment & Culture, under the direction of Dr. L. Jen Shaffer in the Department of Anthropology, interviewed 28 men and women residents of Columbia. An additional four interviews from Columbia Association’s archives were added to the transcripts of the collected oral histories for further analysis. The analysis of the interview texts explored residents’ experiences of economic, demographic, sociocultural and environmental change over the past 50 years and examined responses to such change in the effort to identify ideas for developing sustainable plans to respond to future changes in Columbia. All audio and video recordings of the oral history interviews are archived at Columbia Archives at Columbia Association.

Aims: One of the key cytokines regulating hepatic stellate cell (HSC) function in the course of liver fibrogenesis is TGF-β [1]. In endothelial cells, TGF-β activates two interrelated Smad-dependent signaling cascades [2]. We recently found that these pathways are also present in HSC (unpublished). The "classical" pathway involves the ALK5 type I receptor which activates Smads2/3 and the "accessory" pathway transmits signals via ALK1 and Smads1/5/8 [3]. Although HSC express both type III receptors, betaglycan and endoglin, their contribution to the signaling by the ALK1 pathway is discussed controversially [4, 5]. To elucidate the role of betaglycan and endoglin in these two TGF-β signaling pathways, we utilized the myoblast cell line L6E9 as a model system [6].

We investigated the changes in the hepatic proteome in murine models for toxic-induced fibrogenesis and sclerosing cholangitis. A comprehensive comparison of protein changes observed is made and the mechanistical basis of the expression changes is discussed. Hepatic fibrosis was induced by repetitive intraperitoneal CCl4 treatment of BALB/c mice or developed spontaneously in BALB/c-ATP-binding cassette, subfamily B, member 4 (Abcb4) knock out mice. Fibrosis was verified by a morphometric score and assessment of hydroxyproline content of liver tissue, respectively. The innovative difference in-gel electrophoresis (DIGE) technique was used to analyse protein expression levels of the mouse proteome. Results were confirmed by Western blotting and real-time RT-PCR. In CCl4-induced fibrosis 20 out of 40 and in BALB/c-Abcb4(-/-) mice 8 out of 28 differentially expressed proteins were identified utilizing DIGE. Only two proteins, selenium-binding protein (Sbp2) and carbonic anhydrase 3, have been unidirectionally expressed (i.e. down-regulated) in both models. Relevant differences in the pathogenesis of toxically induced liver fibrosis and sclerosing cholangitis exist. The only novel protein with regard to liver fibrosis depicting a unidirectional expression pattern in both animal models was Sbp2. An explicit protein function could not be clarified yet.

Exposure to adverse environmental conditions causes oxidative stress in many organisms, leading either to disease and debilitation or to response and tolerance. Mitochondria are a key site of oxidative stress and of cellular response and play important roles in cell survival. We analyzed the response of mitochondria in pea (Pisum sativum) plants to the common stresses associated with drought, cold, and herbicides. These treatments all altered photosynthetic and respiratory rates of pea leaves to various extents, but only herbicides significantly increased lipid peroxidation product accumulation. Mitochondria isolated from the stressed pea plants maintained their electron transport chain activity, but changes were evident in the abundance of uncoupling proteins, non-phosphorylating respiratory pathways, and oxidative modification of lipoic acid moieties on mitochondrial proteins. These data suggest that herbicide treatment placed a severe oxidative stress on mitochondria, whereas chilling and particularly drought were milder stresses. Detailed analysis of the soluble proteome of mitochondria by gel electrophoresis and mass spectrometry revealed differential degradation of key matrix enzymes during treatments with chilling being significantly more damaging than drought. Differential induction of heat shock proteins and specific losses of other proteins illustrated the diversity of response to these stresses at the protein level. Cross-species matching was required for mass spectrometry identification of nine proteins because only a limited number of pea cDNAs have been sequenced, and the full pea genome is not available. Blue-native separation of intact respiratory chain complexes revealed little if any change in response to environmental stresses. Together these data suggest that although many of the molecular events identified by chemical stresses of mitochondria from a range of model eukaryotes are also apparent during environmental stress of plants, their extent and significance can vary substantially.

Background/Aims: We analyzed the expression of the novel identified platelet-derived growth factor-C and -D (PDGF-C, PDGF-D) in an experimental bile duct-ligated rat model and compared them to those of PDGF-A, PDGF-B and their receptors, and assessed the biological function of these novel cytokines in cultured hepatic stellate cells (HSC).

Adiponectin is a member of adipose tissue-secreted hormones, so-called adipokines, and has wide implications in glucose and lipid metabolism. It is also found in liver tissue and in NAFLD/NASH it provides protective features on liver tissue like antiinflammatory and antifibrotic effects. There are two specific receptors for adiponectin: AdipoR1 with abundant expression and AdipoR2 with expression especially on hepatocytes. Yet, little is known about the role of adiponectin in chronic HCV-infection. Recently, genetic studies of single nucleotide polymorphisms (SNP) of the adiponectin gene, e.g. +276G>T, have shown that genetic polymorphisms are implicated in altered protein function. Methods: From a total of 480 cases of chronic HCV-infection 99 paraffin-embedded, formalin fixed liver tissues including S0 (n=2), S1 (n=27), S2 (n=27), S3 (n=27) and S4 (n=16) were taken for immunohistological investigation of adiponectin. We performed immunostaining of adiponectin and counted positively stained portal tracts. For genetic analysis of SNP +276G>T in the adiponectin gene 183 cases of chronic HCV-infection, containing fibrosis stages S1, S2, S3 and S4, were examined by Real-Time PCR using SNP-specific TaqMan probes. Results: Immunofluorescent double-staining showed adiponectin reactivity in endothelial cells, especially, of portal vessels. In addition, moderate adiponectin immunostaining was observed in smooth muscle cells of portal arteries and in much lesser extend in sinusoidal cells. Nevertheless, adiponectin mRNA was not detectable in liver biopsies. In the panel of specimens with high grade fibrosis we found a slightly increasing number of adiponectin-positive portal tracts. Thus, we propose a modulating effect of adiponectin on the progression of liver fibrosis in chronic HCV-infection. Next we analyzed the SNP +276G>T, which is known to alter adiponectin function. We found the mutated T allele in position +276 to be more frequent in higher fibrosis stages. SUMMARY AND Conclusion: Adiponectin is mainly found in endothelial cells of the portal tract. In liver tissue with chronic HCV-infection showing progressive fibrosis adiponectin staining is slightly increased. Analysis of the SNP +276G>T of the adiponectin gene indicates that the 276T substitution might be involved in fibrosis progression and should be tested as a putative prognostic marker in chronic hepatitis C.

Aims: Liver biopsy is the gold standard to assess whether or not fibrosis in CHC is advancing, stable or regressing and to provide prognostic and therapeutic information. A promising approach to non-invasive biomarker discovery referring to diagnose CHC and differentiating mild from moderate/advanced stage of liver disease is protein profiling using surface enhanced laser desorption/ionisation (SELDI)-based ProteinChip® technology.

‘Lucky’, a multiple-leaflet red clover (Trifolium pratense L.) cultivar (29-L-38-1801) (Reg. no. CV-28, PI 638509) was developed by the Kentucky Agricultural Experiment Station and released in 2004. It is an increase with further selection of the GP-9 multiple-leaflet gene marker registered by Crop Science in 1982 (Taylor, 1982). During 1998 to 2002, five cycles of selection were conducted in greenhouses and fields to increase the expression of the character. The 2002 population consisting of 76% multiple-leafleted plants was interpollinated under field conditions to produce the seeds being released. Leaves of plants of the cultivar vary from three to eight leaflets (rarely more) and occasionally produce “cornucopia-shaped” leaflets varying up to three (rarely more). Plants vary in number of multiple-leafleted leaves from 0 to 10 depending on size and number of leaves per plant. If multiple-leaflets as small as 1 cm are desired they may be expressed in month-old seedlings spaced in stands of approximately one plant per 2.5 cm2 At this spacing 50% of plants have multiple-leaflets and 33% have two or more multiple-leaflets per plant. This cultivar is intended for ornamental plantings and for four-leaf clover charms. Seeds of ‘Lucky’ will be maintained by the Foundation Seed Project, Agricultural Experiment Station, and the University of Kentucky. Commercial companies interested in purchasing this cultivar should apply to the corresponding author.

Mitochondrial respiration and tricarboxylic acid (TCA) cycle activity are required during salt stress in plants to provide ATP and reductants for adaptive processes such as ion exclusion, compatible solute synthesis and reactive oxygen species (ROS) detoxification. However, there is a poor mechanistic understanding of how salinity affects mitochondrial metabolism, particularly respiratory substrate source. To determine the mechanism of respiratory changes under salt stress in wheat leaves, we conducted an integrated analysis of metabolite content, respiratory rate and targeted protein abundance measurements. Also, we investigated the direct effect of salt on mitochondrial enzyme activities. Salt-treated wheat leaves exhibit higher respiration rate and extensive metabolite changes. The activity of the TCA cycle enzymes pyruvate dehydrogenase complex and the 2-oxoglutarate dehydrogenase complex were shown to be directly salt-sensitive. Multiple lines of evidence showed that the γ-aminobutyric acid (GABA) shunt was activated under salt treatment. During salt exposure, key metabolic enzymes required for the cyclic operation of the TCA cycle are physiochemically inhibited by salt. This inhibition is overcome by increased GABA shunt activity, which provides an alternative carbon source for mitochondria that bypasses salt-sensitive enzymes, to facilitate the increased respiration of wheat leaves.