Changes in blood parameters of hybrid tambacu fish parasitized by Dolops carvalhoi (Crustacea, Branchiura), a fish louse

Measurement of blood parameters has been used for many years as a tool for monitoring the health offish. The present study investigated the effects of a natural infestation of Dolops carvalhoi Lemos de Castro, 1949, on red blood cells, thrombocytes and white blood cell counts, as well as plasma glucose and serum electrolyte levels in hybrid tambacu (Piaractus mesopotamicus × Colossoma macropomum). Parasitized fish showed low haematocrit and magnesium levels and increases in MCHC, plasma glucose levels, serum protein, sodium and chloride levels, number of monocytes and PAS-positive granular leukocytes (PAS-GL), when compared with values in control fish. This study is the first to report changes in fish physiology caused by D. carvalhoi infestation, and the results obtained indicate that a mild infection can lead to important osmoregulatory disturbances in hosts.

Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio)

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene. © 2008 The Authors.

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

Background: Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results: In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Conclusions: Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication. © 2013 Morais et al.; licensee BioMed Central Ltd.

Effects of the acute exposition to glyphosate-based herbicide on oxidative stress parameters and antioxidant responses in a hybrid Amazon fish surubim (Pseudoplatystoma sp)

The aim of this study was to investigate the effects of acute glyphosate (active ingredient) exposure on the oxidative stress biomarkers and antioxidant defenses of a hybrid surubim (Pseudoplatystoma sp). The fish were exposed to different herbicide concentrations for 96 h. The thiobarbituric acid-reactive substances (TBARS), protein carbonyls and antioxidant responses were verified. The 15 mg a.p L-1 of herbicide resulted in the death of 50% of the fish after 96 h. An increase in liver and muscle TBARS levels was observed when fish were exposed to the herbicide. The protein carbonyl content was also increased in the liver (4.5 mg a.p L-1 concentration) and brain (2.25 mg a.p L-1 concentration). The antioxidant activities decreased in the liver and brain after exposure to herbicide. Levels of ascorbic acid in the liver (2.25 mg a.p L-1 and 4.5 mg a.p L-1 concentrations) and brain (2.25 mg a.p L-1 concentration) were increased post-treatment. Levels of total thiols were increased in the liver and brain (2.25 mg L-1 and 7.5 mg a.p L-1, respectively). Glyphosate exposure, at the tested concentrations affects surubim health by promoting changes that can affect their survival in natural environment. Some parameters as TBARS and protein carbonyl could be early biomarkers for Roundup exposure in this fish species. (C) 2014 Elsevier Inc. All rights reserved.

New ligands of the cellular retinoic acid-binding protein 2 (CRABP2) suggest a role for this protein in chromatin remodeling

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hybrid Scaffolds Built From PET and Collagen as a Model For Vascular Graft Architecture

A hybrid material with excellent mechanical and biological properties is produced by electrospinning a co-solution of PET and collagen. The fibers are mapped using SEM, confocal Raman microscopy and collagenase digestion assays. Fibers of different compositions and morphologies are intermingled within the same membrane, resulting in a heterogeneous scaffold. The collagen distribution and exposure are found to depend on the PET/collagen ratio. The materials are chemically and mechanically characterized and biologically tested with fibroblasts (3T3-L1) and a HUVEC culture in vitro. All of the hybrid scaffolds show better cell attachment and proliferation than PET. These materials are potential candidates to be used as vascular grafts.

The fatty acid profiles and energetic substrates of two Nile tilapia (Oreochromis niloticus, Linnaeus) strains, Red-Stirling and Chitralada, and their hybrid

Protein and lipid content as well as the fatty acid (FA) composition of storage tissues were analysed in two varieties of Oreochromis niloticus (Red-Stirling and Chitralada) and their hybrid. The animals were maintained in cages for 11 months. The samples were taken when the animals weighed 10, 50, 100, 250 and 500 g. The results showed that changes in the metabolic processes occur during an increase in body mass in both varieties of tilapia and also their hybrid, but that these differences are not found in animals collected at the commercial weight. The protein content of the fillet and liver decreased with growth and the same protein content associated with growth was found for fillet lipid content. The genetic variety did not influence the FA profile of the fillet, but different genotypes had different hepatic FA compositions. Even with the same lipid content, the hepatocytes of Chitralada accumulated higher levels of polyunsaturated fatty acids (PUFA) n6 in triglycerides and increased C22:6n3 in the hepatocyte membranes. The higher n6PUFA content was compensated by a lower fraction of saturated FA in the hepatocyte triglycerides. The skin of Chitralada also had higher n6PUFA and C22:6n3 contents, suggesting a higher ability to deposit PUFA in the skin due to alterations in the liver synthetic pathway.

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

Multicelled Behaviour in E. coli: Design and experimental characterization of an engineered library of hybrid promoters.

Synthetic biology has recently had a great development, many papers have been published and many applications have been presented, spanning from the production of biopharmacheuticals to the synthesis of bioenergetic substrates or industrial catalysts. But, despite these advances, most of the applications are quite simple and don’t fully exploit the potential of this discipline. This limitation in complexity has many causes, like the incomplete characterization of some components, or the intrinsic variability of the biological systems, but one of the most important reasons is the incapability of the cell to sustain the additional metabolic burden introduced by a complex circuit. The objective of the project, of which this work is part, is trying to solve this problem through the engineering of a multicellular behaviour in prokaryotic cells. This system will introduce a cooperative behaviour that will allow to implement complex functionalities, that can’t be obtained with a single cell. In particular the goal is to implement the Leader Election, this procedure has been firstly devised in the field of distributed computing, to identify the process that allow to identify a single process as organizer and coordinator of a series of tasks assigned to the whole population. The election of the Leader greatly simplifies the computation providing a centralized control. Further- more this system may even be useful to evolutionary studies that aims to explain how complex organisms evolved from unicellular systems. The work presented here describes, in particular, the design and the experimental characterization of a component of the circuit that solves the Leader Election problem. This module, composed of an hybrid promoter and a gene, is activated in the non-leader cells after receiving the signal that a leader is present in the colony. The most important element, in this case, is the hybrid promoter, it has been realized in different versions, applying the heuristic rules stated in [22], and their activity has been experimentally tested. The objective of the experimental characterization was to test the response of the genetic circuit to the introduction, in the cellular environment, of particular molecules, inducers, that can be considered inputs of the system. The desired behaviour is similar to the one of a logic AND gate in which the exit, represented by the luminous signal produced by a fluorescent protein, is one only in presence of both inducers. The robustness and the stability of this behaviour have been tested by changing the concentration of the input signals and building dose response curves. From these data it is possible to conclude that the analysed constructs have an AND-like behaviour over a wide range of inducers’ concentrations, even if it is possible to identify many differences in the expression profiles of the different constructs. This variability accounts for the fact that the input and the output signals are continuous, and so their binary representation isn’t able to capture the complexity of the behaviour. The module of the circuit that has been considered in this analysis has a fundamental role in the realization of the intercellular communication system that is necessary for the cooperative behaviour to take place. For this reason, the second phase of the characterization has been focused on the analysis of the signal transmission. In particular, the interaction between this element and the one that is responsible for emitting the chemical signal has been tested. The desired behaviour is still similar to a logic AND, since, even in this case, the exit signal is determined by the hybrid promoter activity. The experimental results have demonstrated that the systems behave correctly, even if there is still a substantial variability between them. The dose response curves highlighted that stricter constrains on the inducers concentrations need to be imposed in order to obtain a clear separation between the two levels of expression. In the conclusive chapter the DNA sequences of the hybrid promoters are analysed, trying to identify the regulatory elements that are most important for the determination of the gene expression. Given the available data it wasn’t possible to draw definitive conclusions. In the end, few considerations on promoter engineering and complex circuits realization are presented. This section aims to briefly recall some of the problems outlined in the introduction and provide a few possible solutions.

Charakterisierung spezieller pflanzlicher Gamma-Tubulin-Sequenzbereiche und Detektion potentieller Interaktionspartner mittels Yeast-two-Hybrid-System

Mitotische und postmitotische Vorgänge pflanzlicher Zellen basieren auf der Funktion von Mikrotubuli. Es liegen nur wenige gesicherte Erkenntnisse zur Organisation dieser Multifunktionalität vor. Eine zentrale Bedeutung wird bei der Nukleation der Mikrotubuli an MTOCs durch γ-Tubulin zugeschrieben. Deren Zusammenlagerung an MTOCs ist jedoch noch nicht richtig verstanden. Domänen, die an der Proteinoberfläche exponiert werden, könnten in Interaktionen involviert sein. Hier werden im Besonderen der γ-A und γ-B-Peptivmotiv diskutiert. Es wurde das γ-A- und γ-B-Peptidmotiv des γ-Tubulins hinsichtlich einer Konservierung innerhalb des Pflanzenreiches untersucht. Die beiden Bereiche sind bei den grünen Landpflanzen stark konserviert. Sie divergieren stark zu den einzelligen Grünalgen Chlamydomonas reinhardtii und Chlorella spec. Es wurden daher in der bestehenden phylogentischen Lücke weitere Organismen hinsichtlich des γ-A und γ-B Peptidmotivs untersucht. Auswahlkriterien der Organismen waren Ein-/Mehrzelligkeit, Besitz/Abwesenheit von Centriolen und Besitz/Abwesenheit von Geißeln. Des weiteren wurde mit verschiedenen γ-Tubulin-Konstrukten um das γ-A- und γ-B-Peptidmotiv, gewonnen aus Nicotiana tabacum (BY2) mittels Y2H-System nach Interaktionspartnern gesucht. Bei den Sequenzuntersuchungen des γ-A- und γ-B-Peptidmotivs konnte festgestellt werden, dass die Konservierung innerhalb der Streptophytenlinie erfolgt. Interessant erweist sich die Tatsache, dass dieses Motiv bei den Jochalgen, welche ebenfalls den Streptophyten angehören, nur im γ-A-Peptidmotiv auftritt. Es besteht die Möglichkeit, dass die beiden potentiellen Interaktionspartner verschiedene Proteine als Interaktions-partner besitzen. Durch eine Anwendung eines auf dem GAL4-Protein basierenden Y2H-Systems mit vier unterschiedlichen Konstrukten des γ-Tubulin-A/B-Peptidbereichs als Köder-konstrukt und einer cDNA-Bibliothek als Beutekonstrukt, wurden diverse Sequenzen identifiziert. Identifiziert wurden das Poly(A)-Bindeprotein, Glycerin-aldehyd-3-phosphatdehydrogenase, die S-adenosyl-L-methionine-Synthetase, diverse Proteasom-Untereinheiten, eine sekretorische Peroxidase, eine Ascorbat-Peroxidase, die NtPOX1-Peroxidase und verschiedene Peroxidasen aus Nicotiana tabacum, Sequenzen des Chloroplastengenoms, ein Myosin-ähnliches Protein und eine Sequenz auf dem 5. Chromosom des Medicago truncatula-Klons mth2-16f8 und diverse humane Sequenzen der Proteine DKFZp68 und DKFZp77. Die Ergebnisse weisen auf eine komplexe Funktionsweise der unterschiedlichen Komponenten des pflanzlichen Cytoskeletts und des γ-Tubulins hin. Zur Aufklärung müsste dies in Zukunft mittels anderer genetischer, biochemischer oder funktioneller Methoden untersucht werden. Hypothesen über Interaktionen der Cytoskelettkomponenten können wahrscheinlich nicht allein durch die Anwendung des Y2H-Systems aufgeklärt werden.

Identifizierung und Charakterisierung neuer Proteine des Abwehrsystems der Porifera - ein Apoptoseregulator und ein antimikrobielles Protein aus dem Schwamm Suberites domuncula

Aufgrund ihrer Lebensweise und -umgebung sind effiziente Strategien zur Abwehr bedrohender Einflüsse essentiell für die Porifera. Eine dieser Strategien stellen die Apoptose in höheren Metazoen, sowie ein effizientes Immunsystem dar. Diese sichern sowohl das Überleben des Organismus als auch die Entfernung beschädigter, infizierter oder redundanter Zellen. Bei Untersuchungen der Porifera auf Moleküle, die an diesen Prozessen beteiligt sind, konnten in den letzten Jahren beachtliche Erfolge erzielt werden. So konnten das in der Apoptose involvierte Protein GCDD2 (proapoptotisch), die antiapoptotischen GCBHP1 und GCBHP2 Proteine (Wiens et al., 2001), sowie ein LPS induzierbarer TNF (Wiens et al., 2007) und zwei Caspasen (Wiens et al., 2003) in Schwämmen identifiziert werden. Um diese essentiellen Mechanismen besser verstehen zu können, sollte ein möglicher Tumor-Nekrose-Faktor-Rezeptor identifiziert werden. Hierzu wurde die SpongeBase Datenbank nach Proteinen mit Todesdomänen durchsucht und diese unter Anwendung von PCR- und Screening-Techniken in einer cDNA-Bank des marinen Schwammes S. domuncula komplettiert. Im Anschluss an ihre Sequenzierung wurde ein Klon ausgewählt, dessen Todesdomäne größte Homologie zu einem TNFR zeigte. Dieser Klon SD_TNFR-like (Suberites domuncula TNFR-homologes Protein) wurde anschließend diversen Sequenz- und Strukturanalysen unterzogen. Diese offenbarten die Existenz zweier funktional bedeutsamer Domänen (Ubiquitin-like und Todesdomäne). Vor allem die Todesdomäne impliziert eine Beteiligung des Proteins an apoptotischen Prozessen. Über einen „Yeast Two Hybrid Screen“ sollten Proteine identifiziert werden, welche mit dem Ausgangsprotein interagieren. Hierbei wurde ein Protein identifiziert, das Ähnlichkeit mit einem antimikrobiellen Peptid aufweist. Dieses Protein kann analog zu einer Gruppe von antimikrobiellen Peptiden, den α-helikalen kationischen Peptiden, in drei Teile gespalten werden. Das Signalpeptid sowie ein anionisches Propeptid werden abgespalten und es entsteht ein kationisches, antimykotisch wirksames Peptid. Beide Proteine sollten, sofern sie in die Abwehrreaktionen involviert sind, durch Inkubation mit mikrobiellen Strukturen vermehrt exprimiert werden. Eine Überprüfung der Transkription mittels Northern Blot Analysen bestätigte dies für das SD_TNFR-like nach Inkubation mit LPS und TNF- α sowie für SD_Brevinin-like nach Inkubation mit LPS, PAM und Hefe. Mit der Herstellung eines rekombinanten SD_TNFR-like-Proteins wurde die Immunisierung von Kaninchen und die folgende Gewinnung eines polyklonalen SD_TNFR-like-Antikörpers ermöglicht. Dieser gestattete den Nachweis der SD_TNFR-like -Expression mittels Western Blot-Analysen sowie die stressinduzierte erhöhte Expression mittels Dot Blot-Analysen auch auf Proteinebene. Um die Funktion des SD_TNFR-like Proteins zu charakterisierten, wurde ein Test mit RAW-Blue™-Zellen durchgeführt. Die Ergebnisse implizieren, dass das Protein Teil der Immunreaktion analog der der TLR- bzw. NLR- Reaktion ist. Auch die Interaktion mit einem antimikrobiellen Protein, welches für das Überleben des Organismus und die Bekämpfung der Mikroorganismen sorgt, deutet auf eine solche Beteiligung hin. Zusätzlich wird diese These durch ein Ergebnis der Strukturanalysen unterstützt, nämlich die Identifizierung einer TRAF2 Bindestelle. TRAF2 ist ein Adapterprotein der TNFR und aktiviert Überlebensfaktoren über den NF - B-Weg. Immunohistochemische Analysen zeigten, dass das SD_TNFR-like Protein im Organismus vor allem um die Bakteriozysten, um verschiedene Mikroorganismen und am Rand des Schwammes exprimiert wird, was ebenfalls für eine immunologische Funktionsweise spricht. Auch im restlichen Gewebe wird es kontinuierlich, auch ohne vorherige LPS Inkubation exprimiert. Diese Akkumulation zeigt deutlich, dass das Protein in einen Schutzmechanismus gegen äußere Bedrohungen involviert ist. Es scheint dabei direkt an den eindringenden Mikroorganismen zu wirken. Das SD_TNFR-like ist demnach ein potentieller Bestandteil der Immunantwort des Schwammes, welches Apoptose verhindern und Überlebensmechanismen aktivieren kann. Das SD_Brevinin-like Protein besitzt antimykotische Aktivität, wie in einem antimikrobiellen Test gezeigt werden konnte. Weiterhin scheint es für das SD_TNFR-like Protein als positiver bzw. negativer Regulator von Bedeutung zu sein, der eine Reaktion entweder beendet oder die Expression von Überlebensfaktoren verstärkt. Die in dieser Arbeit präsentierten Ergebnisse und Schlussfolgerungen demonstrieren somit die Identifizierung eines neuen Schwammproteins, welches eine Rolle in der Immunantwort spielt, sowie eines neuen antimikrobiellen Peptids, welches die Wirkung des TNFR-like moduliert. Es müssen jedoch noch weitere Funktionsanalysen folgen, um den Mechanismus des SD_TNFR-like Proteins und seine Regulation genauer charakterisieren zu können

The stress response protein Gls24 is induced by copper and interacts with the CopZ copper chaperone of Enterococcus hirae

Intracellular copper routing in Enterococcus hirae is accomplished by the CopZ copper chaperone. Under copper stress, CopZ donates Cu(+) to the CopY repressor, thereby releasing its bound zinc and abolishing repressor-DNA interaction. This in turn induces the expression of the cop operon, which encodes CopY and CopZ, in addition to two copper ATPases, CopA and CopB. To gain further insight into the function of CopZ, the yeast two-hybrid system was used to screen for proteins interacting with the copper chaperone. This led to the identification of Gls24, a member of a family of stress response proteins. Gls24 is part of an operon containing eight genes. The operon was induced by a range of stress conditions, but most notably by copper. Gls24 was overexpressed and purified, and was shown by surface plasmon resonance analysis to also interact with CopZ in vitro. Circular dichroism measurements revealed that Gls24 is partially unstructured. The current findings establish a novel link between Gls24 and copper homeostasis.

Acute Regulation of Renal Na+/H+ Exchanger NHE3 by Dopamine: Role of Protein Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56 delta (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, co-immunoprecipitation, blot overlay and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small t antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition. Key words: Natriuresis, Sodium transport, Signal transduction.

Plexiform hybrid granular cell tumor/perineurioma: a novel variant of benign peripheral nerve sheath tumor with divergent differentiation

The descriptive term hybrid peripheral nerve sheath tumor refers to any neoplasm of the neurilemmal apparatus composed of more than one pathologically defined tumoral equivalent derived from its constituent cells. Within this uncommon nosological category, participation of granular cell tumor - a neoplasm of modified Schwann cells - has been reported only exceptionally. We describe a hitherto not documented variant composed of an organoid mixture of granular cell tumor and perineurioma with plexiform growth. A solitary subcutaneous nodule of 1.5 cm diameter was excised from the right ring finger of a 19-year-old female with no antecedents of neurofibromatosis or relevant trauma. Histology revealed a monotonous, yet cytologically dimorphic proliferation of classical granular cells intermingled with flattened, inconspicuous perineurial cells. Immunohistochemical double labeling detected expression of S100 protein in the former and of EMA and GLUT-1 in the latter. While the respective staining patterns for S100 protein and EMA or GLUT-1 tended to be mutually exclusive, a minority of cells exhibited transitional granular cell/perineurial immunophenotype. Electron microscopy permitted direct visualization of a plethora of lysosomes in the granular cell moiety, and of pinocytotic vesicles and tight junctions in perineurial cells. Intratumoral axons were not detected. Expanding intraneurally, the lesion showed discrete encapsulation by the local perineurium, and resulted in plexiform growth. The MIB-1 labeling index averaged 1%. We interpret our findings as supporting evidence for the dual cell lineage to have arisen through metaplasia, with the tumor's dynamics probably having been driven by the granular cell component.

NETWORK TOPOLOGY IN HUMAN PROTEIN INTERACTION DATA PREDICTS FUNCTIONAL ASSOCIATION

High-throughput assays, such as yeast two-hybrid system, have generated a huge amount of protein-protein interaction (PPI) data in the past decade. This tremendously increases the need for developing reliable methods to systematically and automatically suggest protein functions and relationships between them. With the available PPI data, it is now possible to study the functions and relationships in the context of a large-scale network. To data, several network-based schemes have been provided to effectively annotate protein functions on a large scale. However, due to those inherent noises in high-throughput data generation, new methods and algorithms should be developed to increase the reliability of functional annotations. Previous work in a yeast PPI network (Samanta and Liang, 2003) has shown that the local connection topology, particularly for two proteins sharing an unusually large number of neighbors, can predict functional associations between proteins, and hence suggest their functions. One advantage of the work is that their algorithm is not sensitive to noises (false positives) in high-throughput PPI data. In this study, we improved their prediction scheme by developing a new algorithm and new methods which we applied on a human PPI network to make a genome-wide functional inference. We used the new algorithm to measure and reduce the influence of hub proteins on detecting functionally associated proteins. We used the annotations of the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) as independent and unbiased benchmarks to evaluate our algorithms and methods within the human PPI network. We showed that, compared with the previous work from Samanta and Liang, our algorithm and methods developed in this study improved the overall quality of functional inferences for human proteins. By applying the algorithms to the human PPI network, we obtained 4,233 significant functional associations among 1,754 proteins. Further comparisons of their KEGG and GO annotations allowed us to assign 466 KEGG pathway annotations to 274 proteins and 123 GO annotations to 114 proteins with estimated false discovery rates of <21% for KEGG and <30% for GO. We clustered 1,729 proteins by their functional associations and made pathway analysis to identify several subclusters that are highly enriched in certain signaling pathways. Particularly, we performed a detailed analysis on a subcluster enriched in the transforming growth factor β signaling pathway (P<10-50) which is important in cell proliferation and tumorigenesis. Analysis of another four subclusters also suggested potential new players in six signaling pathways worthy of further experimental investigations. Our study gives clear insight into the common neighbor-based prediction scheme and provides a reliable method for large-scale functional annotations in this post-genomic era.