Pretreatment of Extruded Corn Stover with Polyethylene Glycol to Enhance Enzymatic Hydrolysis: Optimization, Kinetics, and Mechanism of Action

Due to the high potential of the extrusion technique for pretreatment of lignocellulosic substrates, several attempts have been conducted in previous studies to further increase the subsequent sugar yield from extrusion pretreatment. Examples include application of chemicals along with extrusion, such as alkali-extrusion and ethylene glycol-extrusion, or before extrusion, such as hot water extraction. In this study, a new sequential technique has been developed for pretreatment of corn stover (CS), which utilizes an initial extrusion pretreatment (155?rpm screw speed and temperatures of 90°C, 180°C and 180°C corresponding to feed, barrel and die zones, respectively at a reaction time of 45?C90?s) followed by pretreatment with polyethylene glycol 6,000 (PEG). In order to fully characterize the response for sugar yield over the range of surfactant treatment conditions assessed, response surface methodology was used. Treatment temperature, incubation time and PEG concentration were varied between 45?C55°C, 1?C4?h, 0.15?C0.6?g PEG/g glucan, respectively. Statistical analysis was conducted by fitting the glucose and xylose yields to a quadratic polynomial model. PEG concentration and temperature were found to be the most significant factors in surfactant pretreatment. The optimum condition resulted in 25.4% and 10.3% increase in glucose and xylose yield, respectively. Using the combination of 10.8?FPU/g glucan of Ctec₂ and 0.3?g PEG/g glucan, the glucose yield of extruded CS reached 98%. A yield was 64% resulted from application of similar amounts of Ctec and Htec. Decreased adsorption of enzyme to the lignocellulosic substrate as well as increased enzyme activity and reaction velocity indicated by kinetic parameter evaluation and nitrogen combustion analysis suggested an increased solubilization of cellulase in the presence of PEG. We propose that a non-productive adsorption of enzymes occur during hydrolysis not only due to lignin but also due to crystalline cellulose. Comparison of enzyme adsorptions and increase in sugar yields between Avicel and CS suggests the presence of other potential mechanisms of action for PEG in addition to increase of enzyme solubilization.     

Search for seasonal rhythmicity of pineal melatonin production in rats under constant laboratory conditions: spectral chronobiological analysis, and relation to solar and geomagnetic variables

Earlier we reported that in a number of experiments pineal melatonin production in rats under constant laboratory conditions displayed seasonal rhythms but subsequently were not always able to confirm this. Since there was no indication under which conditions such rhythms may be present, we performed four consecutive identical experiments with untreated female Sprague-Dawley rats within the same animal room during 1997-2006. Nocturnal urine samples (19-23, 23-3, 3-7?h) were collected at monthly intervals over 494-658?d with 12 animals each in experiments I and II (1997-1999, 1999-2000), 30 animals in experiment III (2002-2004), and 15 in experiment IV (2005-2006). 6-Sulfatoxymelatonin (aMT6s) was measured by ELISA. The excreted aMT6s at each time interval as well as total nocturnal aMT6s-excretion (19-7?h) was submitted to standard statistical analyses as well as to a spectral chronobiological analysis to determine the period lengths of the components involved which was followed by processing with the single cosinor method. Seasonal rhythm components (circannual period length: 360 ± 60?d) were detected in experiment III (2002-2004) for the overall nocturnal excretion as well as for two sub-intervals (23-3 and 3-7?h) and in one night interval of experiment II (23-3?h). Multiple components with mostly short period lengths of around 100?d and some long ones of 500-650?d were found in the other experiments. Systematic MESOR and amplitude variations were observed during the experiments, being highest in experiment II (19-7?h, also 23-3?h and 3-7?h) and lowest in experiments I and IV. These results illustrate that seasonal melatonin rhythms are not a general phenomenon in female laboratory rats indicating an involvement of unknown environmental cues. As an extension of our earlier hypothesis regarding a seasonal Zeitgeber function of the horizontal intensity H of the geomagnetic field showing circannual variations, we assume further modulation by the 11-yrs' sunspot cycle which leads to geomagnetic disturbances and could facilitate seasonal aMT6s rhythmicity during specific years. (Author correspondence: christian.bartsch@uni-tuebingen.de ).     

Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis

Background  

Proline (Pro) accumulation is a widespread response of prokaryotic and eukaryotic cells subjected to osmotic stress or dehydration. When the cells are released from stress, Pro is degraded to glutamate by Pro-dehydrogenase (ProDH) and Pyrroline-5-carboxylate dehydrogenase (P5CDH), which are both mitochondrial enzymes in eukaryotes. While P5CDH is a single copy gene in Arabidopsis, two ProDH genes have been identified in the genome. Until now, only ProDH1 (At3g30775) had been functionally characterised.     

Targeted inhibition of sarcoplasmic reticulum CaMKII activity results in alterations of Ca2+ homeostasis and cardiac contractility

Transgenic (TG) mice expressing a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr17 and develop dilated myopathy when stressed by gestation and parturition (Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. J Biol Chem 278: 25063-25071, 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of sarcoplasmic reticulum (SR) CaMKII activity on the contractile characteristics and Ca2+ cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (+/-dP/dt) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild-type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40-47% decrease in the maximal rates of myocyte shortening and relengthening under both basal and Iso-stimulated conditions. Although twitch Ca2+ transient amplitudes were not significantly altered, the rate of twitch intracellular Ca2+ concentration decline was reduced by approximately 47% in TG myocytes, indicating decreased SR Ca2+ uptake function. Caffeine-induced Ca2+ transients indicated unaltered SR Ca2+ content and Na+/Ca2+ exchange function. Phosphorylation assays revealed an approximately 30% decrease in the phosphorylation of ryanodine receptor Ser2809. Iso stimulation increased the phosphorylation of both phospholamban Ser16 and the ryanodine receptor Ser2809 but not phospholamban Thr17 in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca2+ handling in TG hearts.     

Sodium-dependent and sodium-independent phosphate uptake by full-grown, prophase-arrested oocytes of Xenopus laevis before and after progesterone-induced maturation

The uptake of inorganic phosphate by full-grown, prophase-arrested oocytes occurs by two essentially independent transport systems, which accomplish the delivery of extracellular phosphate into two different cellular compartments. One transport system is sodium-dependent, the other is sodium-independent. Both systems can be inhibited by sulfhydryl reagents and the sodium-dependent system becomes inhibited during progesterone-induced oocyte maturation.     

Immunogold localization of a developmentally regulated,tapetal-specific, 15 kDa lily anther protein

Summary We have confirmed that the LLA-15 polypeptide ofLilium longiflorum is (a) tapetum specific with some expression possible in the adjacent middle layer cells and (b) relatively abundant as evidenced by the high density of gold particles localized to the tapetal cells. We have established that the protein is cytoplasmic and not associated with organelles, membranes, extracellular matrix or wall. We also report an amino acid composition of the molecule and a partial sequence which bears no resemblance to any protein yet described.Abbreviations BSA bovine serum albumin - PMSF phenyl methyl sulfonyl fluoride     

The structure and stability of trypsin-resistant segments from rabbit tropomyosin.

Tropomyosin was found to undergo only limited digestion by trypsin at 0 degrees C and the two segments that accumulated amounted to two-thirds of the original protein. They are referred to as segments A and B. These segments were not resistant to trypsin digestion at 20 degrees C and at the latter temperature no large fragments remained as judged by disc gel electrophoresis. Segments A and B were separated from each other on the basis of solubility differences and were found to have molecular weights of 24600 and 21900 respectively. Each of the segments appeared to retain about 70-75% of the helical conformation as judged by circular dichroism at 20 degrees C. However, the segments did not show any of the inhibitory activity of the parent tropomyosin molecule when mixed with troponin in the Mg2+-actomyosin ATPase system. Amino acid analysis showed that the portion of tropomyosin that was digested by trypsin (EC 3.4.21.4) had a lower content of the helix stabilizing residues Glu and Leu and a higher content of the helix-destabilizing residues Arg and Lys. These differences indicate that the digested portion should be less stable in the helical conformation than the two trypsin-resistant segments. End group determinations along with the results of the amino acid analysis indicated that segment A was probably derived from the central one-third of tropomyosin and segment B from the C-terminal one-third. By the process of elimination the N-terminal third appears to have been more liable region that was digested by trypsin. The segments A and B were shown to differ in their stability to denaturation by guanidine-HCl and elevated temperature. All of these observations indicate that tropomyosin is not a uniform structure and is composed of regions of different stability.