In contrast to mammals, some fish and amphibians have retained the ability to regenerate complex body structures or organs, such as the limb, the tail, the eye lens or even parts of the heart. One major difference in the response to injury is the appearance of a mesenchymal growth zone or blastema in these regenerative species instead of the scarring seen in mammals. This blastema is thought to largely derive from the dedifferentiation of various functional cell types, such as skeletal muscle, skin and cartilage. In the case of multinucleated skeletal muscle fibres, cell cycle re-entry into S-phase as well as fragmentation into mononucleated progenitors is observed both in vitro and in vivo. In order to identify molecules that initiate dedifferentiation of cells at the wound site in amphibians we have established a cellular assay with a cultured newt myogenic cell line. Using this assay we have found a serum activity that stimulates cell cycle re-entry in differentiated multinucleated newt myotubes. The activity is present in serum of all mammalian species tested so far and, interestingly, thrombin proteolysis amplifies the activity from both serum and plasma. We think this serum factor provides a link between wounding and regeneration and its identification will be a key step in understanding the remarkable differences in wound healing between mammals and amphibians. In the course of this PhD thesis we have characterized the serum factor as a thermo-labile, pH- and proteinase K-sensitive, high molecular weight protein that is resistant to denaturing conditions such as SDS, urea or organic solvents. Surprisingly, under denaturing conditions the activity behaves as a low molecular weight protein that displays charge heterogeneity on isoelectric focusing. Using these characteristics of the serum factor we have performed a systematic investigation of commonly used protein chromatography modes and separation techniques to develop a successful purification procedure. After four column chromatography steps -- cation exchange, hydrophobic interaction, heparin affinity and size exclusion chromatography under denaturing conditions -- we have achieved a 2,000-fold purification starting from a commercially available Crude Bovine Thrombin preparation. This represents about 40,000-fold purification over bovine serum. Silver stained gels of the most purified fractions revealed ten major protein bands. In order to finally identify the cell cycle re-entry factor, we are currently analyzing the purification by quantitative mass spectrometry by correlating the abundance of tryptic peptides with activity in sequential fractions across a chromatography run.

Einleitung Adipositas ist ein zunehmendes Problem bei Menschen und Haustieren, z.B. in Pferden. Ponyrassen sind dabei besonders prädisponiert, wobei die Gründe bisher nicht abschließend geklärt werden konnten. Humane Adipositas geht mit einer fettigen Infiltration der Leber einher, die sogenannte Non-Alcoholic Fatty Liver Disease, welche zu einer hepatozellulären Entzündung führt. Es ist bisher nicht bekannt, ob Adipositas in Equiden auch zu hepatischen Veränderungen führt. Menschliche Fettleibigkeit ist zusätzlich mit systemischer Entzündung und gesteigertem oxidativen Stress verbunden. Das führte zu intensiven Untersuchungen von anti-inflammatorischen und antioxidativen Faktoren (z.B. Vitamin A - Retinol und Vitamin E - α-Tocopherol) in der humanen Adipositas Forschung. Viele Studien konnten ein Absinken von Vitamin A und Vitamin E in fettleibigen Menschen feststellen. Ziele Die vorliegende Studie sollte den Einfluss von zunehmendem Körpergewicht (KG) in Ponys und Pferden auf mehrere Parameter untersuchen: (1) Serum Leberenzymaktivitäten und Serum Gallensäuren (GS), (2) Leberfettgehalt, (3) hepatische messenger Ribonukleinsäure (mRNA) Level von Entzündungsmarkern und Markern des Lipidmetabolismus und (4) Serum Konzentrationen von Retinol und α-Tocopherol. Zusätzlich sollten Ponys und Pferde im Verlauf dieser Studie verglichen werden, um eventuelle Gründe für die Rasseprädisposition der Ponys für metabolische Störungen zu identifizieren. Material und Methoden Zehn Shetland Ponys und 9 Warmblut Pferde, die initial nicht adipös waren, wurden über 2 Jahre mit 200% des Erhaltungsbedarfes für umsetzbare Energie gefüttert. Die Entwicklung des KG, des Body Condition Scores (BCS) und des Cresty Neck Scores (CNS) wurde wöchentlich erfasst. Während der Fütterungsphase wurde zu 6 Zeitpunkten (ZP) Blut für die Bestimmung von Serum Leberenzymaktivitäten (Alkaline Phosphatase (ALP), Aspartat Aminotransferase (AST), Glutamat Dehydrogenase (GLDH), Gamma-Glutamyl Transferase (GGT)) und Serum GS entnommen und zu 7 ZP wurde Blut für die Analyse von Serum Retinol und α-Tocopherol gewonnen. An 3 ZP wurde durch Laparotomie Lebergewebe in Vollnarkose entnommen. Die Leberbiopsien wurden histologisch auf ihren Fettgehalt untersucht und mittels quantitativer Echtzeit Polymerase-Kettenreaktion (RT-qPCR) wurden die mRNA Level von Entzündungsmarkern (Nuclear Factor-κB (NF-κB), Interleukin-1β (IL-1β), IL-6, Tumor Nekrose Faktor α (TNFα), Differenzierungsgruppe 68 (CD68), Chemerin) und Lipid Metabolismus Markern (Lipoprotein Lipase (LPL), Fettsäuren Bindungsprotein 1 (FABP1) bestimmt. Die Daten wurden mittels statistischem Software Programm ausgewertet (STATISTICA, version 12, StatSoft GmbH, Hamburg, Deutschland). Nach Prüfung auf Normalverteilung der Daten, wurden geeignete statistische Tests angewendet mit einem statistischen Signifikanzniveau bei P < 0,05. Die Tierschutzkommission des Bezirks Leipzig genehmigte das Projekt in Übereinstimmung mit deutschen Rechtsvorschriften (Nr. TVV 32/15). Ergebnisse Ponys und Pferde zeigten einen signifikanten Anstieg von KG (Mittelwert ± SD; Ponys: 29,9 ± 19,4%; Pferde: 17 ± 6,74%), BCS (Median (25./75. Perzentil); Ponys: 157% (115/349); Pferde: 142% (128/192)) und CNS (Median (25./75. Perzentil); Ponys: 165% (123/500); Pferde: 200% (160/225)) induziert durch die hyperkalorische Fütterung über 2 Jahre. Das ansteigende KG hat keine Steatosis in der Mehrheit der Equiden ausgelöst. Die mRNA Level von IL-6, TNFα, CD68 und IL-1β in der Leber wurden nicht beeinflusst. Die Leber mRNA Level von Chemerin sind signifikant angestiegen in Ponys (x-facher Anstieg: 1,89) und Pferden (x-facher Anstieg: 2,04). Signifikante Unterschiede zwischen den Rassen hinsichtlich der Serum GLDH Aktivitäten, Serum GS Konzentrationen und der hepatischen mRNA LPL Level konnten festgestellt werden. Die Serum α-Tocopherol Konzentrationen stiegen in Ponys und Pferden signifikant an und korrelierten positiv mit der Vitamin E Aufnahme. Die Serum Retinol Konzentrationen fluktuierten während der Studie, ohne mit der Aufnahme zu korrelieren. Schlussfolgerungen Frühe Fettleibigkeit in Equiden führt nicht zwangsläufig zu einer Steatose mit hepatozellulärer Entzündung. Gemäß der Hypothese zeigten Ponys und Pferde allerdings unterschiedliche hepatische Reaktionsmuster nach KG Zunahme. Das könnte die höhere Empfänglichkeit von Ponys für metabolische Erkrankungen erklären. Chemerin konnte als interessanter Marker für die equine Adipositas Forschung identifiziert werden. Serum Konzentrationen von Retinol und α-Tocopherol wurden durch die KG Zunahme nicht beeinflusst.

The structural and mechanical properties of a surface often play an integral part in the determination of the cell adhesion strength and design parameters for creating a biodegradable electrospun scaffold. Nanofibers composed of the globular proteins bovine serum albumin (BSA) and fibronectin were produced by electrospinning with the electrospun protein scaffold serving as an extracellular matrix to which adhesion interaction will exist with cells via cell surface integrin. This interaction is vital in regulation cell differentiation, growth and migration and cell adhesion. We will demonstrate the ability to manipulate ligand-receptor interaction, the properties of the electrospun fibers, control and the formation of focal adhesions sites in cells cultured on the fibers with the ultimate goal of developing a biomimetric scaffold to investigate how cell adhesion molecules modulate cell behavior in a 3-dimentional culture.

The potential use of polymeric, functionalized nanoparticles (NPs) as drug delivery vectors was explored. Covalent conjugation of albumin to the surface of NPs via maleimide chemistry proved problematic. However, microwave assisted synthesis of NPs was not only time efficient, but enabled the exploration of size control by changing the following parameters: temperature, microwave power, reaction time, initiator concentration, and percentage of monomer used. About 1.5 g of fluorescently-labeled, carboxylic acid-functionalized NPs (100 nm diameter) were synthesized for a total cost of less than $1. Future work will address further functionalization of the NPs for the coupling of albumin (or other targeted proteins), and tests for in vivo biodistribution.

This project was undertaken to establish whether the Onderstepoort Biological Products Fowl Typhoid (OBPft) vaccine registered as an injectable vaccine was effective and safe when administered orally to commercial layers. Its efficacy and duration of protection were compared to the intramuscular injectable route. Commercial brown layer hens were used as they were found to be highly susceptible to Salmonella gallinarum infections. In the safety trial birds were euthanased at timed intervals spanning 4-weeks post vaccination. Necropsies were performed and samples were taken and tested. No clinical signs or mortalities could be attributed to the OBPft vaccine. No active shedding of the vaccine strain could be detected. Slight pathological changes were noted with both routes of vaccination; however these changes were transient, returning to normal within the observation period. The injected group showed a better serological response with the serum agglutination test than the orally vaccinated groups. In the duration of protection trial the two routes of vaccination were compared, the birds were challenged at three 8-week intervals post vaccination. All the unvaccinated birds died. The protection offered to the vaccinated groups was good when birds were challenged 8 and 16-weeks after vaccination. However, this dipped steeply by the challenge 24-weeks post vaccination. Statistically (ANOVA, p<0.05) it was found that there was no significant difference between the protection offered by either the oral or injected route of vaccination with the OBPft vaccine. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2006. / Production Animal Studies / unrestricted

BACKGROUND: With the proportion of the world’s elderly population continuing to increase dramatically, tremendous amounts of research have focused on detecting the earliest signs of cognitive impairment before the onset of dementia. The pathophysiology of dementia is complex and recent genetic and biomarker studies have identified new biological pathways that might modify the risk of dementia. One potential risk factor, altered serum phosphorus levels, has been studied with respect to its potential impact on human cognition. The association between serum phosphorus and cognition needs further investigation using a population that is free of CKD. OBJECTIVE: I used data from the Framingham Offspring Study (FOS) cohort to investigate the cross-sectional association between measured serum phosphorus within the normal range and cognitive performance in women and men. METHODS: Participants from the FOS who attended the ninth examination cycle were included in this analysis (N=1253). The Wechsler Memory Scale: Logical Memory – Immediate and Delayed recall (LM-IR and LM-DR) and Visual reproduction – Immediate and Delayed recall (VR-IR and VR-DR) tests were used to assess verbal and visual memory, respectively. Times for the Trailmaking Test – Parts A and B along with the difference between the two tests (B-A) were used to assess attention, psychomotor speed, and executive functioning. Participants were categorized according to levels of fasting serum phosphorus as follows: Low Phosphorus - 2.0-<3.1 mg/dL for men and 2.6-<3.6 mg/dL for women, Moderate Phosphorus - 3.1-<3.6 mg/dL for men and 3.6-<3.9 mg/dL for women, High Phosphorus - 3.6-<5.2 mg/dL for men and 3.9-<5.3 mg/dL for women. Mean cognitive scores were compared across categories using a least squares general fit linear model. Multivariable logistic regression models were used to investigate the association between higher serum phosphorus levels and odds of cognitive impairment within each cognitive test. RESULTS: We did not find any statistically significant differences in mean scores on the Logical Memory, Visual Reproduction, and Trailmaking – Part A tests among the categories of fasting serum phosphorus. There was no association between higher serum phosphorus levels and odds of cognitive impairment on any of the verbal and visual memory tests. In men, higher serum phosphorus levels were associated with poorer performance on the Trailmaking Test – Part B (High phosphorus: -0.50 ± 0.04, Moderate phosphorus: -0.40 ± 0.03, Low phosphorus : -0.33 ± 0.04; P-trend: <0.002) and the difference in log-transformed times between the Trailmaking Test – Part B and A tests (High phosphorus: -1.16 ± 0.02, Moderate phosphorus: -1.10 ± 0.02, Low phosphorus: -1.07 ± 0.02; P-trend: <0.004). Higher serum phosphorus levels were associated with an 80% greater odds of having a cognitively impaired score on Trailmaking Test – Part B-A in men (OR: 1.81, 1.11-2.94), and this association was strengthened when adjusting for additional confounding variables (OR: 2.02, 1.15-3.54). There was no such association in women. Using a cubic spline regression analysis, we treated serum phosphorus as a continuous variable and observed a positive linear association between phosphorus and total time or Trailmaking – Part B-A in men. In particular, the odds of cognitive impairment increased at levels of phosphorus above 3.5 mg/dL. CONCLUSIONS: This study demonstrated that higher serum phosphorus levels were associated with poorer performance on the Trailmaking Test – Part B, and in times represented by the Trailmaking Test – Part B-A time in men. We also observed that higher fasting serum phosphorus levels as a continuous variable were associated with increased odds of cognitive impairment on Trailmaking Test – Part B-A in men. We found no association between higher serum phosphorus levels and lower verbal and visual memory scores or increased odds of cognitive impairment on those scores. Since fewer women had cognitive impairment on these test, statistical power was limited for some of these analyses. Future studies are necessary to examine the mechanistic pathways by which serum phosphorus could impact cognition and whether these effects are independent of cardiovascular disease.

The low-molecular weight proteome is of great interest due to the vast number of smaller proteins and peptides present within it, some of which are likely to be disease-related and may provide insights into disease mechanisms or even prediction and diagnosis. Serum is often the most preferred specimen for disease prediction or diagnosis, as it can be obtained less-invasively and its collection is fairly routine in a clinical setting. Although serum samples are one of the most preferred specimens for disease studies, additional disease information may also be obtained using other specimens, such as tissue. The study of tissue specimens can be extremely valuable in disease studies as it likely contains the highest concentration of potential markers for tissue related diseases. Therefore, despite the difficulty of sample collection, as compared to serum specimens, tissue specimens can be invaluable in studying tissue-related diseases. Tissue proteomics approaches for studying the low-molecular weight proteome of tissue have been infrequently described in the literature, which suggests the need to develop new methods or improve upon current proteomics approaches to better study this subset of the proteome in tissue. In this dissertation, I first report on a low-molecular weight tissue proteomics approach that we have developed based on a previously reported serum approach. As reported here, we conclude that this tissue proteomics approach cannot only distinguish the low-molecular weight proteome of different tissue types, but it also appears to be within the reproducibility range of other currently used proteomic approaches. Additionally, we also report on the use of a serum proteomics approach for biomarker discovery and identification in preeclampsia and endometriosis. These studies reveal many candidate biomarkers for preeclampsia prediction and endometriosis diagnosis. In the preeclampsia study we discovered several candidate biomarkers early in pregnancies (12-14 weeks gestation) that were able to predict preeclampsia later in pregnancy with statistical significance. In the endometriosis study, several candidate biomarkers were discovered that were statistically significant in diagnosing endometriosis. Further, for both preeclampsia and endometriosis, combinations of 3 or 4 biomarkers yielded an improved sensitivity and specificity in disease prediction and diagnosis with combined area under the curves of greater than 0.8. Thus, the low-molecular weight proteome of both tissue and serum could potentially provide important information regarding disease physiology, prediction, and diagnosis that may supplement our current understanding of these processes.

No description available.

Background Promoters are key players in gene regulation. They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG http://corg.molgen.mpg.de, a framework for studying upstream regions including untranslated exons (5' UTR). Description The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences. As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server http://tomcat.molgen.mpg.de:8080/das. The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set. Conclusion The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites).

C-reactive protein (CRP) is an evolutionarily conserved protein. From arthropods to humans, CRP has been found in every organism where the presence of CRP has been sought. Human CRP is a pentamer made up of five identical subunits which binds to phosphocholine (PCh) in a Ca2+-dependent manner. In various species, we define a protein as CRP if it has any two of the following three characteristics: First, it is a cyclic oligomer of almost identical subunits of molecular weight 20–30 kDa. Second, it binds to PCh in a Ca2+-dependent manner. Third, it exhibits immunological cross-reactivity with human CRP. In the arthropod horseshoe crab, CRP is a constitutively expressed protein, while in humans, CRP is an acute phase plasma protein and a component of the acute phase response. As the nature of CRP gene expression evolved from a constitutively expressed protein in arthropods to an acute phase protein in humans, the definition of CRP became distinctive. In humans, CRP can be distinguished from other homologous proteins such as serum amyloid P, but this is not the case for most other vertebrates and invertebrates. Literature indicates that the binding ability of CRP to PCh is less relevant than its binding to other ligands. Human CRP displays structure-based ligand-binding specificities, but it is not known if that is true for invertebrate CRP. During evolution, changes in the intrachain disulfide and interchain disulfide bonds and changes in the glycosylation status of CRP may be responsible for different structure-function relationships of CRP in various species. More studies of invertebrate CRP are needed to understand the reasons behind such evolution of CRP. Also, CRP evolved as a component of and along with the development of the immune system. It is important to understand the biology of ancient CRP molecules because the knowledge could be useful for immunodeficient individuals.