Production of cellulases by Trichoderma reesei QM 9414 in fed-batch and continuous-flow culture with cell recycle

The scope in improving enzyme productivities from the cellulose fermentation process is examined in laboratory-scale fermentors. The maximum productivity (30 IU/liter hr) is attained in a continuous-culture process with cell recycle using modified medium containing 0.5% cellulose. Optimum dilution rate and recycle ratio are determined as 0.025 hr-1 and 1.2, respectively, for the process. The system is analyzed and steady-state equations for predicting enzyme protein concentrations in the fermentor are developed. In fed-batch cultures, slow addition of cellulose at high concentrations can improve enzyme productivity by as much as 33% over a batch process. The scope and results of using modified medium for cellulase production are also presented.     

Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.     

Photosynthesis and water-use characteristics in Indian mangroves

Photosynthesis and water efflux were measured in different PAR and stomatal conductance in members of Avicenniaceae and Rhizophoraceae. Trend of leaf temperature with irradiance and its effect on photosynthesis were also estimated. In most of the studied species, photosynthesis and stomatal conductance followed similar trends with increase in irradiance. The rate of net photosynthesis and stomatal conductance were higher in members of Avicenniaceae than in Rhizophoraceae. In Avicenniaceae, the optimum PAR for maximum photosynthesis ranged between 1340–1685 (μmol m^-2s^-1, which was also higher than that of Rhizophoraceae (840-1557 μmol m^-2s^-1). Almost in all the studied taxa, transpiration and stomatal conductance followed similar trends and reached the maximal peaks at the same PAR value. The range of breakeven leaf temperature was almost the same in both the families (34-36°C in Avicenniaceae and 33.5-36.3°C in Rhizophoraceae), beyond which assimilation rate declined.     

Molecular characterization of gilthead sea bream (Sparus aurata) lipoprotein lipase. Transcriptional regulation by season and nutritional condition in skeletal muscle and fat storage tissues

Lipoprotein lipase (LPL) of gilthead sea bream (Sparus aurata) was cloned and sequenced using a RT-PCR approach completed by 3' and 5'RACE assays. The nucleotide sequence covered 1669 bp with an open reading frame of 525 amino acids, including a putative signal peptide of 23 amino acids long. Sequence alignment and phylogenetic analysis revealed a high degree of conservation among most fish and higher vertebrates, retaining the consensus sequence the polypeptide "lid", the catalytic triad and eight cysteine residues at the N-terminal region. A tissue-specific regulation of LPL was also found on the basis of changes in season and nutritional condition as a result of different dietary protein sources. First, the expression of LPL in mesenteric adipose tissue was several times higher than in liver and skeletal muscle. Secondly, the spring up-regulation of LPL expression in the mesenteric adipose tissue was coincident with a pronounced increase of whole body fat content. Thirdly, the highest expression of LPL in the skeletal muscle was found in summer, which may serve to cover the increased energy demands for muscle growth and protein accretion. Further, in fish fed plant-protein-based diets, hepatic LPL expression was up-regulated whereas an opposite trend was found in the mesenteric adipose tissue, which may contribute to drive dietary lipids towards liver fat storage. Finally, it is of interest that changes in circulating triglyceride (TG) levels support the key role of LPL in the clearance of TG-rich lipoproteins. This study is the first report in fish of a co-regulated expression of LPL in oxidative and fat storage tissues under different physiological conditions.     

Fibril formation from cellulose by a novel protein from Trichoderma reesei: A non-hydrolytic cellulolytic component?

A low molecular weight protein, named fibril-forming protein (FFP), was isolated from the culture supernatant of Avicel-grown Trichoderma reesei. The protein was purified to homogeneity and it exhibited a molecular weight of 11,400Da. Low amounts of this protein caused apparently non-hydrolytic disruption of filter paper, releasing fibrils without any detectable release of reducing sugars. It displayed no hydrolytic activity on carboxymethylcellulose (CMC), p-nitrophenyl--d-glucoside (pNPG) or 4-methylumbelliferyl cellobioside. The pH optimum of the protein was between 4 and 5. The temperature optimum was 40°C and the computed activation energy (E_a) for the filter paper disruption process was 4.18kcal/mol, suggesting disruption of non-covalent bonds. It had no immunological cross reactivity with reported cellulase components of T. reesei.     

Experimental studies on some trophic relationships in juvenile pike, Esox lucius L.

A postprandial increase in ammonia nitrogen excretion and oxygen consumption rates was observed in juvenile pike fed a natural diet or an artificial dry diet. Specific growth rate of natural diet fed pike (2.4%) was lower than that of pike fed the artificial diet (3.1%). Fifty per cent of ingesta was evacuated within 5–6 h in pike of 25 mg body weight and 9–10 h in those weighing 150mg. Daily nitrogen excretion rates were related to body weight. Respiratory quotient and energy retention efficiency were affected by the nature of the diet ingested by pike. Parameters of the energy balance (losses, retention, increment due to feeding) were related to energy intake.     

Hepatic glucokinase and glucose-6-phosphatase responses to dietary glucose and starch in gilthead sea bream (Sparus aurata) juveniles reared at two temperatures.

The effects of carbohydrate sources/complexity and rearing temperature on hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) activities and gene expression were studied in gilthead sea bream juveniles. Two isonitrogenous (50% crude protein) and isolipidic (19% crude lipids) diets were formulated to contain 20% waxy maize starch or 20% glucose. Triplicate groups of fish (63.5 g initial body weight) were fed each diet to near satiation during four weeks at 18 degrees C or 25 degrees C. Growth, feed intake, feed efficiency and protein efficiency ratio, were higher at the higher water temperature. At each water temperatures fish growth and feed efficiency were higher with the glucose diet. Plasma glucose levels were not influenced by water temperature but were higher in fish fed the glucose diet. Hepatosomatic index and liver glycogen were higher at the lower water temperature and within each water temperature in fish fed the glucose diet. No effect of water temperature on enzymes activities was observed, except for hexokinase and GK which were higher at 25 degrees C. Hepatic hexokinase and pyruvate kinase activities were not influenced by diet composition, whereas glucose-6-phosphate dehydrogenase activity was higher in fish fed the glucose diet. Higher GK activity was observed in fish fed the glucose diet. GK gene expression was higher at 25 degrees C in fish fed the waxy maize starch diet while in fish fed the glucose diet, no temperature effect on GK gene expression was observed. Hepatic G6Pase activities and gene expression were neither influenced by dietary carbohydrates nor water temperature. Overall, our data suggest that in gilthead sea bream juveniles hepatocytes dietary carbohydrate source and temperature affect more intensively GK, the enzyme responsible for the first step of glucose uptake, than G6Pase the enzyme involved in the last step of glucose hepatic release.     

A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical

The mechanism of the antiulcer effect of omeprazole was studied placing emphasis on its role to block oxidative damage and apoptosis during ulceration. Dose-response studies on gastroprotection in stress and indomethacin-induced ulcer and inhibition of pylorus ligation-induced acid secretion indicate that omeprazole significantly blocks gastric lesions at lower dose (2.5 mg/kg) without inhibiting acid secretion, suggesting an independent mechanism for its antiulcer effect. Time course studies on gastroprotection and acid reduction also indicate that omeprazole almost completely blocks lesions at 1 h when acid inhibition is partial. The severity of lesions correlates well with the increased level of endogenous hydroxyl radical (*OH), which when scavenged by dimethyl sulfoxide causes around 90% reduction of the lesions, indicating that *OH plays a major role in gastric damage. Omeprazole blocks stress-induced increased generation of *OH and associated lipid peroxidation and protein oxidation, indicating that its antioxidant role plays a major part in preventing oxidative damage. Omeprazole also prevents stress-induced DNA fragmentation, suggesting its antiapoptotic role to block cell death during ulceration. The oxidative damage of DNA by *OH generated in vitro is also protected by omeprazole or its analogue, lansoprazole. Lansoprazole when incubated in a *OH-generating system scavenges *OH to produce four oxidation products of which the major one in mass spectroscopy shows a molecular ion peak at m/z 385, which is 16 mass units higher than that of lansoprazole (m/z 369). The product shows no additional aromatic proton signal for aromatic hydroxylation in (1)H NMR. The product absorbing at 278 nm shows no alkaline shift for phenols, thereby excluding the formation of hydroxylansoprazole. The product is assigned to lansoprazole sulfone formed by the addition of one oxygen atom at the sulfur center following attack by the *OH. Thus, omeprazole plays a significant role in gastroprotection by acting as a potent antioxidant and antiapoptotic molecule.     

A three-dimensional multiscale model for the prediction of thrombus growth under flow with single-platelet resolution

Modeling thrombus growth in pathological flows allows evaluation of risk under patient-specific pharmacological, hematological, and hemodynamical conditions. We have developed a 3D multiscale framework for the prediction of thrombus growth under flow on a spatially resolved surface presenting collagen and tissue factor (TF). The multiscale framework is composed of four coupled modules: a Neural Network (NN) that accounts for platelet signaling, a Lattice Kinetic Monte Carlo (LKMC) simulation for tracking platelet positions, a Finite Volume Method (FVM) simulator for solving convection-diffusion-reaction equations describing agonist release and transport, and a Lattice Boltzmann (LB) flow solver for computing the blood flow field over the growing thrombus. A reduced model of the coagulation cascade was embedded into the framework to account for TF-driven thrombin production. The 3D model was first tested against in vitro microfluidics experiments of whole blood perfusion with various antiplatelet agents targeting COX-1, P₂Y₁, or the IP receptor. The model was able to accurately capture the evolution and morphology of the growing thrombus. Certain problems of 2D models for thrombus growth (artifactual dendritic growth) were naturally avoided with realistic trajectories of platelets in 3D flow. The generalizability of the 3D multiscale solver enabled simulations of important clinical situations, such as cylindrical blood vessels and acute flow narrowing (stenosis). Enhanced platelet-platelet bonding at pathologically high shear rates (e.g., von Willebrand factor unfolding) was required for accurately describing thrombus growth in stenotic flows. Overall, the approach allows consideration of patient-specific platelet signaling and vascular geometry for the prediction of thrombotic episodes.