Fesselin, an intrinsically disordered smooth muscle protein, organizes and stabilizes actin-myosin and myosin

Fesselin is an intrinsically disordered protein that is known to bind a large variety of cytoskeletal proteins. The proteins fesselin is known to bind include: actin (Leinweber et al. 1999), [alpha]-actinin (Pham et al. 2006), calmodulin (Schroeter et al. 2004), filamin (Weins et al. 2001), and smooth myosin (Schroeter et al. 2005). The binding of fessilin to smooth myosin is of particular interest because unphosphorylated smooth muscle myosin filaments are unstable in the presence of ATP (Trybus et al. 1982, Ikebe et al. 1983, Suzuki et al. 1978). However, in smooth muscle cells unphosphorylated myosin filaments are maintained (Milton et al. 2011). Several proteins have been identified that stabilize myosin filaments and actin-myosin filament interactions. Our experiments show that fesselin may be one such protein. The organization of F-actin and myosin filaments by fesselin was observed by monitoring the rate of dissociation of actin-myosin by ATP in a stopped-flow device. Actin-myosin dissociation was measured by light scattering (a measure of particle size) and by pyrene-actin or acrylodan-tropomyosin fluorescence (a measure of myosin-actin bond breaking). The stopped-flow studies were further supported with electron microscopy analysis. These experiments showed that fesselin was able to tether actin and myosin filaments together without significantly impacting the rate of the actin-myosin bond breaking. The stabilization of myosin filaments by fesselin was tested using a similar method. First, stopped-flow rapid kinetics were used to measure the rate of ATP induced myosin filament break down. Next, electron microscopy was used to support the stopped-flow data and observe the effects of fesselin on myosin filament organization. Through the stopped-flow and electron microscopy experiments it was found that fesselin stabilizes myosin filaments. The electron microscopy experiments further revealed that fesselin enhanced myosin filament size and organized them into bundles. Previously published results showed that the interaction between fesselin and actin is regulated by calmodulin (Schroeter et al. 2004). The final set of experiments presented here examined the possibility that calmodulin regulates the interactions between fessilin and myosin. The calmodulin regulation of fesselin myosin interactions would greatly expand the role that fesselin has within smooth muscle by placing it within the calcium signaling pathway. The regulation of fesselin-myosin interactions by calmodulin was tested using pyrene labeled actin as well as N,N'-Dimethyl-N-(Iodoacetyl)-N'-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine labeled fesselin (IANBD-fesselin). These experiments showed that the interactions between fesselin and myosin are regulated by calmodulin. Overall, our results support that fesselin plays a critical role within smooth muscle to organize contractile elements.M.S

Quantitative Evaluation of E1 Endoglucanase Recovery from Tobacco Leaves Using the Vacuum Infiltration-Centrifugation Method

As a production platform for recombinant proteins, plant leaf tissue has many advantages, but commercialization of this technology has been hindered by high recovery and purification costs. Vacuum infiltration-centrifugation (VI-C) is a technique to obtain extracellularly-targeted products from the apoplast wash fluid (AWF). Because of its selective recovery of secreted proteins without homogenizing the whole tissue, VI-C can potentially reduce downstream production costs. Lab scale experiments were conducted to quantitatively evaluate the VI-C method and compared to homogenization techniques in terms of product purity, concentration, and other desirable characteristics. From agroinfiltrated Nicotiana benthamiana leaves, up to 81% of a truncated version of E1 endoglucanase from Acidothermus cellulolyticus was recovered with VI-C versus homogenate extraction, and average purity and concentration increases of 4.2-fold and 3.1-fold, respectively, were observed. Formulas were developed to predict recovery yields of secreted protein obtained by performing multiple rounds of VI-C on the same leaf tissue. From this, it was determined that three rounds of VI-C recovered 97% of the total active recombinant protein accessible to the VI-C procedure. The results suggest that AWF recovery is an efficient process that could reduce downstream processing steps and costs for plant-made recombinant proteins

A Study of Alternative Catalysts and Analysis Methods for Biodiesel Production

This project aims to develop a cost efficient process for biodiesel production and can be divided in three main components: 1) production of biodiesel from a variety of fuel stocks using liquid morpholine as catalyst; 2) production of biodiesel using a homogeneous phase transfer catalyst; and 3) development of a method for using Infrared Spectroscopy (IR) to determine the extent of conversion of oil to biodiesel. The production of biodiesel from various fuel stocks in the presence of methanol using liquid morpholine as catalyst reduces the problems related to purification of the biodiesel since morpholine can be recovered by distillation. Furthermore the use of two homogeneous phase transfer catalyst, tetramethylammonium hydroxide (TMAH) and choline hydroxide (CH), was evaluated. The advantage of using these catalysts is that it allows for a better separation between the fuel and glycerin, thus additionally simplifying the purification procedure. Finally, this project endeavored to develop a way to use FT-IR to determine the purity of biodiesel samples obtained since FT-IR is faster and more readily available than the standard method of gas chromatographic analysis. For educational applications, a calibration curve was created by comparing data on the purity of biodiesel samples obtained from the GC-FID analysis to the ratio of the absorbances at 1197 cm-1 to 1166 cm-1 from the FT-IR spectrum. For field application, a similar method was developed using a portable IR spectrometer. The data collected gave a good linear fit for % purity of the samples versus absorbance ratio

Quantitative evaluation of E1 endoglucanase recovery from tobacco leaves using the vacuum infiltration-centrifugation method.

As a production platform for recombinant proteins, plant leaf tissue has many advantages, but commercialization of this technology has been hindered by high recovery and purification costs. Vacuum infiltration-centrifugation (VI-C) is a technique to obtain extracellularly-targeted products from the apoplast wash fluid (AWF). Because of its selective recovery of secreted proteins without homogenizing the whole tissue, VI-C can potentially reduce downstream production costs. Lab scale experiments were conducted to quantitatively evaluate the VI-C method and compared to homogenization techniques in terms of product purity, concentration, and other desirable characteristics. From agroinfiltrated Nicotiana benthamiana leaves, up to 81% of a truncated version of E1 endoglucanase from Acidothermus cellulolyticus was recovered with VI-C versus homogenate extraction, and average purity and concentration increases of 4.2-fold and 3.1-fold, respectively, were observed. Formulas were developed to predict recovery yields of secreted protein obtained by performing multiple rounds of VI-C on the same leaf tissue. From this, it was determined that three rounds of VI-C recovered 97% of the total active recombinant protein accessible to the VI-C procedure. The results suggest that AWF recovery is an efficient process that could reduce downstream processing steps and costs for plant-made recombinant proteins

Quantitative Evaluation of E1 Endoglucanase Recovery from Tobacco Leaves Using the Vacuum Infiltration-Centrifugation Method

As a production platform for recombinant proteins, plant leaf tissue has many advantages, but commercialization of this technology has been hindered by high recovery and purification costs. Vacuum infiltration-centrifugation (VI-C) is a technique to obtain extracellularly-targeted products from the apoplast wash fluid (AWF). Because of its selective recovery of secreted proteins without homogenizing the whole tissue, VI-C can potentially reduce downstream production costs. Lab scale experiments were conducted to quantitatively evaluate the VI-C method and compared to homogenization techniques in terms of product purity, concentration, and other desirable characteristics. From agroinfiltrated Nicotiana benthamiana leaves, up to 81% of a truncated version of E1 endoglucanase from Acidothermus cellulolyticus was recovered with VI-C versus homogenate extraction, and average purity and concentration increases of 4.2-fold and 3.1-fold, respectively, were observed. Formulas were developed to predict recovery yields of secreted protein obtained by performing multiple rounds of VI-C on the same leaf tissue. From this, it was determined that three rounds of VI-C recovered 97% of the total active recombinant protein accessible to the VI-C procedure. The results suggest that AWF recovery is an efficient process that could reduce downstream processing steps and costs for plant-made recombinant proteins

Synthesis of Biodiesel Using Liquid Morpholine as a Homogeneous Basic Catalyst

The described technique can be used to produce biodiesel from pure canola oil, waste vegetable oil, or animal fat. The method described in the project uses excess of methanol and liquid morpholine as a catalyst. With this method the biodiesel is produced without soapy water and in less corrosive reaction conditions. The excess methanol can be recovered by distillation and recovery of morpholine while not currently very successful (52%) can be improved with a more powerful vacuum in a vacuum distillation. This method uses a new catalyst that reduces the problems in the purification of biodiesel and glycerol. The described method uses simple liquid liquid extraction to separate the biodiesel produced from the glycerol. Once extracted purification involves filtrtion and evaporation. The final part of the method is testing using GC-MS, 1H NMR, and 13C NMR for the presence of biodiesel

Fesselin, an intrinsically disordered smooth muscle protein, organizes and stabilizes actin-myosin and myosin

Fesselin is an intrinsically disordered protein that is known to bind a large variety of cytoskeletal proteins. The proteins fesselin is known to bind include: actin (Leinweber et al. 1999), [alpha]-actinin (Pham et al. 2006), calmodulin (Schroeter et al. 2004), filamin (Weins et al. 2001), and smooth myosin (Schroeter et al. 2005). The binding of fessilin to smooth myosin is of particular interest because unphosphorylated smooth muscle myosin filaments are unstable in the presence of ATP (Trybus et al. 1982, Ikebe et al. 1983, Suzuki et al. 1978). However, in smooth muscle cells unphosphorylated myosin filaments are maintained (Milton et al. 2011). Several proteins have been identified that stabilize myosin filaments and actin-myosin filament interactions. Our experiments show that fesselin may be one such protein. The organization of F-actin and myosin filaments by fesselin was observed by monitoring the rate of dissociation of actin-myosin by ATP in a stopped-flow device. Actin-myosin dissociation was measured by light scattering (a measure of particle size) and by pyrene-actin or acrylodan-tropomyosin fluorescence (a measure of myosin-actin bond breaking). The stopped-flow studies were further supported with electron microscopy analysis. These experiments showed that fesselin was able to tether actin and myosin filaments together without significantly impacting the rate of the actin-myosin bond breaking. The stabilization of myosin filaments by fesselin was tested using a similar method. First, stopped-flow rapid kinetics were used to measure the rate of ATP induced myosin filament break down. Next, electron microscopy was used to support the stopped-flow data and observe the effects of fesselin on myosin filament organization. Through the stopped-flow and electron microscopy experiments it was found that fesselin stabilizes myosin filaments. The electron microscopy experiments further revealed that fesselin enhanced myosin filament size and organized them into bundles. Previously published results showed that the interaction between fesselin and actin is regulated by calmodulin (Schroeter et al. 2004). The final set of experiments presented here examined the possibility that calmodulin regulates the interactions between fessilin and myosin. The calmodulin regulation of fesselin myosin interactions would greatly expand the role that fesselin has within smooth muscle by placing it within the calcium signaling pathway. The regulation of fesselin-myosin interactions by calmodulin was tested using pyrene labeled actin as well as N,N'-Dimethyl-N-(Iodoacetyl)-N'-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine labeled fesselin (IANBD-fesselin). These experiments showed that the interactions between fesselin and myosin are regulated by calmodulin. Overall, our results support that fesselin plays a critical role within smooth muscle to organize contractile elements

Calcium Circadian Rhythmicity in the Suprachiasmatic Nucleus: Cell Autonomy and Network Modulation.

Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca2+ ([Ca2+]i), and neuronal firing rate. [Ca2+]i oscillates in SCN neurons even in the absence of neuronal firing. To determine the causal relationship between circadian clock gene expression and [Ca2+]i rhythms in the SCN, as well as the SCN neuronal network dependence of [Ca2+]i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca2+ indicator, into SCN neurons from PER2::LUC knock-in reporter mice. Then, PER2 and [Ca2+]i were imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca2+]i both exhibited cell autonomous circadian rhythms, but [Ca2+]i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model in which clock gene rhythms drive [Ca2+]i rhythms. As predicted by the model, PER2 and [Ca2+]i rhythms were both stronger in SCN slices than in dispersed cells and were weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca2+]i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca2+]i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca2+]i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network