A kinetic model of growth and product formation of the anaerobic microorganism Thermoanaerobacter thermohydrosulfuricus

A model is developed to describe the growth, product formation and enzyme synthesis of the microorganism Thermoanaerobacter thermohydrosulfuricus. It differentiates between extracellular enzymatic starch degradation and the microorganism's constant specific growth rate, combined with the ingestion of maltose and maltotriose (the products of starch cleavage). The formation of growth dependent products like ethanol, acetate and the synthesis of the cell-associated enzyme, amylopullulanase, are formulated with constant yield coefficients. In contrast, both the non-growth dependent product lactate, and extracellular enzyme activity are linked to an intracellular substrate concentration. Most of the parameters used in the model are evaluated from batch culture or literature. The calculated results are compared with experimental data. Keywords: Continuous culture; Thermoanaerobacter thermohydrosulfuricus; Starch; Model     

Nervöse Überträgersubstanzen und ihr fermentativer Abbau

Zusammenfassung In sensiblen Nerven der Wirbeltiere kommen zwei Überträger substanzen vor, Dorsin in den dorsalen Rückenmarkswurzeln, Opticin im Nervus opticus und im Nervus stato-acusticus; von beiden ist es möglich, daß sie auch im Zentralnervensystem vorkommen. Beide sind im Bienentest durch Kreise nach der Seite des angestochenen Auges nachweisbar, im Test am denervierten Kaninchenohr wirkt Dorsin schon wenige Tage nach der Nervendurchschneidung gut, Opticin wirkt in den ersten 2–3 Wochen sehr schwach und erst nach der 4. Woche, evtl. nach einer zweiten Nervendurchschneidung, gut.Durch Kochen der Nerven in wäßriger Lösung erhält man Dorsin und Opticin in gebundener Form, durch Kochen in 75%igem Alkohol und Überführen in wäßrige Lösung in freier Form.Durchleiten von Sauerstoff durch Lösungen von Überträgersubstanzen zerstört Opticin rascher als Dorsin und jeweils die freie Form rascher als die gebundene. 5-Oxytryptamin, das im Bienentest nach der Seite des nicht angestochenen Auges wirkt, wird durch Sauerstoff in eine Substanz verwandelt, die im Bienentest nach der Seite des angestochenen Auges wirkt.Lösungen von Dorsin vertragen kurzes Kochen, Opticin wird in Lösung schon bei 60° C in mehreren Minuten zerstört, wobei freies Opticin empfindlicher ist als gebundenes.Von den freien Überträgersubstanzen wird jede durch ein eigenes Ferment abgebaut. Die Mengen von Dorsinase, die Dorsin abbaut, in den dorsalen Wurzeln und von Opticinase, die Opticin abbaut, im Nervus opticus sind so, daß sie die Überträgersubstanzen unter vergleichbaren Bedingungen in ähnlichen Zeiten abbauen, wie Cholinesterase aus ventralen Wurzeln Acetylcholin abbaut.Gebundenes Dorsin der Wirbeltiere wird durch Pease gespalten, ein Ferment, das man erhält, wenn man eine stark verdünnte, nicht sterile Aufschwemmung aus zerriebenen dorsalen Wurzeln einen Tag lang bei 36° C inkubiert. Die sehr rasche Wirkung dieses Fermentes läßt sich auch mit dem Test am Meerschweinchen-Ileum an der Abnahme der P-Wirkung eines Extraktes aus dorsalen Wurzeln verfolgen.Gebundenes Opticin und andere gebundene Überträgersubstanzen der Wirbeltiere werden durch Dorsinase gespalten. Dorsinase führt diese Spaltung ähnlich rasch durch wie Pease die Spaltungen von gebundenem Dorsin und etwa 50mal so rasch wie den Abbau von freiem Dorsin.Gebundenes Acetylcholin ist als Überträgersubstanz vom Hornhautepithel auf die freien Nervenenden und von sekundären Sinneszellen auf die sensiblen Nerven anzunehmen.Bei der Nervendegeneration erfahren Opticin und Dorsin ähnliche Veränderungen wie Acetylcholin.Bei Mollusken sind als nervöse Überträgersubstanzen wenigstens Opticin, 5-Oxytryptamin und Acetylcholin anzunehmen, bei Arthropoden wenigstens Dorsin, Opticin, Acetylcholin, 5-Oxytryptamin und eine noch kaum untersuchte Substanz, deren fermentativer Abbau durch Strychnin gehemmt wird, bei Anneliden dieselben Substanzen mit Ausnahme von Dorsin.Die Krämpfe lassen sich durch die Hemmung des fermentativen Abbaues von Überträgersubstanzen durch die Krampfgifte erklären. Bei Mollusken und bei Arthropoden hemmen verschiedene Krampfgifte verschiedene Fermente und damit den Abbau verschiedener Überträgersubstanzen. Bei den Wirbeltieren ist die Hemmung der Dorsinase am wichtigsten. Die typischen Krampfgifte hemmen die Dorsinase in denselben gegenseitigen Verhältnissen, in denen sie Krämpfe auslösen. Die Hemmung der Dorsinase bedeutet eine Hemmung des Abbaues von freiem Dorsin und eine Hemmung der Spaltung anderer gebundener Überträgersubstanzen; damit dürfte auch die Wirkung sekundärer Sinneszellen auf die sensiblen Nerven gesteigert werden. Die bei den verschiedenen Krampfgiften verschieden starke zusätzliche Hemmung der Cholinesterase beeinflußt den Charakter der Krämpfe. Als Erklärung für den spezifischen Charakter der Strychninund Brucinkrämpfe bleibt noch die Blockierung der Hemmungen, die bei Wirbeltieren nur durch diese beiden Krampfgifte erfolgt, oder die Hemmung des fermentativen Abbaues von Crosslands Kleinhirnfaktor.Fräulein Ilse Silberbauer und Herrn Helmut Gübitz danken wir für ihre Mithilfe bei einem Teil der Versuche.Wir danken allen Tierärzten des Grazer Schlachthauses für ihr stets freundliches und verständnisvolles Entgegenkommen, welches sie uns bei dieser Arbeit und schon seit 1946 bei den im Literaturverzeichnis genannten Arbeiten von Hellauer und Umrath gezeigt haben.     

Cell size at S phase initiation: an emergent property of the G1/S network

The eukaryotic cell cycle is the repeated sequence of events that enable the division of a cell into two daughter cells. It is divided into four phases: G₁, S, G₂, and M. Passage through the cell cycle is strictly regulated by a molecular interaction network, which involves the periodic synthesis and destruction of cyclins that bind and activate cyclin-dependent kinases that are present in nonlimiting amounts. Cyclin-dependent kinase inhibitors contribute to cell cycle control. Budding yeast is an established model organism for cell cycle studies, and several mathematical models have been proposed for its cell cycle. An area of major relevance in cell cycle control is the G₁ to S transition. In any given growth condition, it is characterized by the requirement of a specific, critical cell size, P_S, to enter S phase. The molecular basis of this control is still under discussion. The authors report a mathematical model of the G₁ to S network that newly takes into account nucleo/cytoplasmic localization, the role of the cyclin-dependent kinase Sic1 in facilitating nuclear import of its cognate Cdk1-Clb5, Whi5 control, and carbon source regulation of Sic1 and Sic1-containing complexes. The model was implemented by a set of ordinary differential equations that describe the temporal change of the concentration of the involved proteins and protein complexes. The model was tested by simulation in several genetic and nutritional setups and was found to be neatly consistent with experimental data. To estimate P_S, the authors developed a hybrid model including a probabilistic component for firing of DNA replication origins. Sensitivity analysis of P_S provides a novel relevant conclusion: P_S is an emergent property of the G₁ to S network that strongly depends on growth rate.     

The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.     

Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1

The interaction of amyloid aggregates with the cell plasma membrane is currently considered among the basic mechanisms of neuronal dysfunction in amyloid neurodegeneration. We used amyloid oligomers and fibrils grown from the yeast prion Sup35p, responsible for the specific prion trait [PSI(+)], to investigate how membrane lipids modulate fibril interaction with the membranes of cultured H-END cells and cytotoxicity. Sup35p shares no homology with endogenous mammalian polypeptide chains. Thus, the generic toxicity of amyloids and the molecular events underlying cell degeneration can be investigated without interference with analogous polypeptides encoded by the cell genome. Sup35 fibrils bound to the cell membrane without increasing its permeability to Ca(2+). Fibril binding resulted in structural reorganization and aggregation of membrane rafts, with GM1 clustering and alteration of its mobility. Sup35 fibril binding was affected by GM1 or its sialic acid moiety, but not by cholesterol membrane content, with complete inhibition after treatment with fumonisin B1 or neuraminidase. Finally, cell impairment resulted from caspase-8 activation after Fas receptor translocation on fibril binding to the plasma membrane. Our observations suggest that amyloid fibrils induce abnormal accumulation and overstabilization of raft domains in the cell membrane and provide a reasonable, although not unique, mechanistic and molecular explanation for fibril toxicity.     

Antioxidant, cytotoxic and hemolytic effects of sulfated galactans from edible red alga Hypnea musciformis

Polysaccharides, galactans, obtained from edible red seaweed Hypnea musciformis were characterized by molecular weight and infrared spectroscopy analysis and were evaluated for antioxidant activity in vitro and for their effects on cell viability. The main components were galactose and sulfate presenting low protein contamination. These sulfated galactans (F1.0) showed a polydisperse profile, and signs in infrared analysis were attributed to a sulfate ester S?=?O bond, the presence of a 3,6-anhydrogalactose C–O bond, nonsulfated β-d-galactose, and a C–O–SO₄ bond in galactose C4. The NMR analysis showed signals at about 95 and 92 attributed to anomeric carbon of 4-linked 3,6-anhydro-α-d-galactopyranose residue of κ-carrageenans and 4-linked 3,6-anhydro-α-d-galactopyranose2-sulfate of ι-carrageenans. Sulfated galactan F1.0 showed strong antioxidant activity under lipid peroxidation assay where F1.0 at 8 mg mL^?1 promoted 57.92% peroxidation inhibition and displayed the scavenging activity on hydroxyl radicals in a dose-dependent manner leading to 32.5% scavenging of these radicals when 5 mg mL^?1 of sulfated galactan F1.0 was used. The sulfated galactan fraction also exhibited strong inhibition on the H₂O₂-induced hemolysis model. Sulfated galactan F1.0 displayed low cytotoxic action in 3 T3 cells and moderate antitumoral action in HeLa cells. These results suggest that sulfated galactan F1.0 from H. musciformis has antioxidant potential, which is a great effect for a compound used as food and in the food industry.     

Association of Genetic Variants at 3q22 with Nephropathy in Patients with Type 1 Diabetes Mellitus

Diabetic nephropathy (DN) is the primary cause of morbidity and mortality in patients with type 1 diabetes mellitus (T1DM) and affects about 30% of these patients. We have previously localized a DN locus on chromosome 3q with suggestive linkage in Finnish individuals. Linkage to this region has also been reported earlier by several other groups. To fine map this locus, we conducted a multistage case-control association study in T1DM patients, comprising 1822 cases with nephropathy and 1874 T1DM patients free of nephropathy, from Finland, Iceland, and the British Isles. At the screening stage, we genotyped 3072 tag SNPs, spanning a 28 Mb region, in 234 patients and 215 controls from Finland. SNPs that met the significance threshold of p < 0.01 at this stage were followed up by a series of sample sets. A genetic variant, rs1866813, in the noncoding region at 3q22 was associated with increased risk of DN (overall p = 7.07 × 10⁻⁶, combined odds ratio [OR] of the allele = 1.33). The estimated genotypic ORs of this variant in all Finnish samples suggested a codominant effect, resulting in significant association, with a p value of 4.7 × 10⁻⁵ (OR = 1.38; 95% confidence interval = 1.18–1.62). Additionally, an 11 kb segment flanked by rs62408925 and rs1866813, two strongly correlated variants (r² = 0.95), contains three elements highly conserved across multiple species. Independent replication will clarify the role of the associated variants at 3q22 in influencing the risk of DN.     

Monte Carlo analysis of an ODE Model of the Sea Urchin Endomesoderm Network

Background  

Gene Regulatory Networks (GRNs) control the differentiation, specification and function of cells at the genomic level. The levels of interactions within large GRNs are of enormous depth and complexity. Details about many GRNs are emerging, but in most cases it is unknown to what extent they control a given process, i.e. the grade of completeness is uncertain. This uncertainty stems from limited experimental data, which is the main bottleneck for creating detailed dynamical models of cellular processes. Parameter estimation for each node is often infeasible for very large GRNs. We propose a method, based on random parameter estimations through Monte-Carlo simulations to measure completeness grades of GRNs.     

Noninvasive,In Vivo Assessment of Mouse Retinal Structure Using Optical Coherence Tomography

Background

Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration.

Methodology/Principal Findings

We achieved to adapt a commercial 3^rd generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber''s congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified.

Conclusions/Significance

We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.