Proceedings of the National Academy of Sciences, 2010
Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The effects of BoNT serot... more Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The effects of BoNT serotype A (BoNT/A) can last several months, whereas the effects of BoNT serotype E (BoNT/E), which shares the same synaptic target, synaptosomal-associated protein 25 (SNAP25), last only several weeks. The long-lasting effects or persistence of BoNT/A, although desirable for therapeutic applications, presents a challenge for medical treatment of BoNT intoxication. Although the mechanisms for BoNT toxicity are well known, little is known about the mechanisms that govern the persistence of the toxins. We show that the recombinant catalytic light chain (LC) of BoNT/E is ubiquitylated and rapidly degraded in cells. In contrast, BoNT/A LC is considerably more stable. Differential susceptibility of the catalytic LCs to ubiquitin-dependent proteolysis therefore might explain the differential persistence of BoNT serotypes. In this regard we show that TRAF2, a RING finger protein implicated in ubiqui...
A device is described which measures, on line, the bubble size distribution and volume fraction o... more A device is described which measures, on line, the bubble size distribution and volume fraction of gas in a liquid foam. There are four elements in the device: a flow channel, meniscus detection, velocity detection, and microcomputer data acquisition. The foam flows into a gently converging capillary to reorder the foam bubbles into alternating slugs of gas and liquid. The passage of the meniscus between a gas and liquid slug is detected by a matched light emitting diode/phototransistor pair. When gas is in the detection path, the detector signal is ``low,'' and when liquid is in the path, the signal is ``high.'' The volume fraction of gas is determined from the ratio of time the detector signal is low to the total elapsed time. A second detector pair is located a fixed distance further down the capillary. From the time shift between signals at the first detector and those at the second, the velocity of the foam in the capillary can be calculated. From the velocity, time that the detector signal is ``low,'' and known capillary geometry, the volume of a gas bubble can be determined. An IBM PC is used for rapid data acquisition and calculation. The device has been tested under a range of foam conditions to validate its performance.
Synaptosomal associated protein of 25 kDa (SNAP-25) is a cytoplasmic protein that participates in... more Synaptosomal associated protein of 25 kDa (SNAP-25) is a cytoplasmic protein that participates in the docking and fusion of synaptic vesicles with the nerve terminal in preparation for neurotransmitter release. SNAP-25 is also a substrate for three of the seven serotypes of botulinum neurotoxin (BoNT). Intoxication by BoNT/A, /C1 or /E results in weakness and paralysis of skeletal muscle due to cleavage of SNAP-25 (and syntaxin la in the case /C1) at discrete serotype-specific sites. To elucidate the role of SNAP-25 in muscle function in more detail, contractility and neuromuscular transmission were studied in a mutant mouse model termed coloboma. The coloboma mutation results from a contiguous deletion of 1-2 centiMorgans on chromosome 2, which includes the entire SNAP-25 locus and three other identified genes. Homozygotes do not survive beyond gestation day 6; heterozygotes (Cm/+) have a normal life-span but express reduced levels of SNAP-25 mRNA and protein in the brain. The consequences of the Cm/+ mutation on twitch and tetanic tension, quantal release of neurotransmitter and spinal motoneuron expression of SNAP-25 were examined in the present study. Contrary to expectations, Cm/+ mice exhibited no alteration in twitch tension and generated normal tetanic tension even at the highest frequency examined (800 Hz). Microelectrode recordings revealed that MEPP amplitude and frequency were both within control limits. The ventral spinal cord of Cm/+ mice showed no deficiency in SNAP-25 content and immunohistochemical examination of nerve terminals in Cm/+ mice disclosed that SNAP-25 levels and distribution were similar to those of control mice. It is concluded that spinal motor neurons up-regulate SNAP-25 to preserve vital neuromuscular function.
Nerve-evoked contractions were studied in vitro in phrenic nerve–hemidiaphragm preparations from ... more Nerve-evoked contractions were studied in vitro in phrenic nerve–hemidiaphragm preparations from strain 129X1 acetylcholinesterase knockout (AChE−/−) mice and their wild-type littermates (AChE+/+). The AChE−/− mice fail to express AChE but have normal levels of butyrylcholinesterase (BChE) and can survive into adulthood. Twitch tensions elicited in diaphragms of AChE−/− mice by single supramaximal stimuli had larger amplitudes and slower rise and decay times than did those in wild-type animals. In AChE−/− preparations, repetitive stimulation at frequencies of 20 and 50 Hz and at 200 and 400 Hz produced decremental muscle tensions; however, stimulation at 70 and 100 Hz resulted in little or no loss of tension during trains. Muscles from AChE+/+ mice maintained tension at all frequencies examined but exhibited tetanic fade after exposure to the selective AChE inhibitor 1,5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW 284C51). The ability of diaphragm muscles from AChE−/− mice to maintain tension at 70 and 100 Hz suggests a partial compensation for impairment of acetylcholine (ACh) hydrolysis. Three mechanisms—including a reliance on BChE activity for termination of ACh action, downregulation of nicotinic acetylcholine receptors (nAChRs), and morphological remodeling of the endplate region—were identified. Studies of neuromuscular transmission in this model system provide an excellent opportunity to evaluate the role of AChE without complications arising from use of inhibitors. Muscle Nerve 30: 317–327, 2004
Primary dissociated fetal mouse spinal cord cultures were used to study the mechanisms underlying... more Primary dissociated fetal mouse spinal cord cultures were used to study the mechanisms underlying the differences in persistence of botulinum neurotoxin A (BoNT/A) and botulinum neurotoxin/E (BoNT/E) activities. Spinal cord cultures were exposed to BoNT/A (0.4 pM) for 2^3 days, which converted approximately half of the SNAP-25 to an altered form lacking the final nine C-terminal residues. The distribution of toxindamaged to control SNAP-25 remained relatively unchanged for up to 80 days thereafter. Application of a high concentration of BoNT/E (250 pM) either 25 or 60 days following initial intoxication with BoNT/A converted both normal and BoNT/ A-truncated SNAP-25 into a single population lacking the final 26 C-terminal residues. Excess BoNT/E was removed by washout, and recovery of intact SNAP-25 was monitored by Western blot analysis. The BoNT/E-truncated species gradually diminished during the ensuing 18 days, accompanied by the reappearance of both normal and BoNT/A-truncated SNAP-25. Return of BoNT/A-truncated SNAP-25 was observed in spite of the absence of BoNT/A in the culture medium during all but the first 3 days of exposure. These results indicate that proteolytic activity associated with the BoNT/A light chain persists inside cells for s 11 weeks, while recovery from BoNT/E is complete in 6 3 weeks. This longer duration of enzymatic activity appears to account for the persistence of serotype A action.
A targeted delivery vehicle (DV) was developed for intracellular transport of emerging botulinum ... more A targeted delivery vehicle (DV) was developed for intracellular transport of emerging botulinum neurotoxin (BoNT) antagonists. The DV consisted of the isolated heavy chain (HC) of BoNT/A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The HC served to target BoNT-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells. To determine the ability of this chimeric glycoprotein to enter neurons, dextran and HC were labeled independently with the fluorescent dyes Oregon green 488 and Cy3, respectively. Internalization of DV was monitored in primary cortical cells using laser confocal microscopy. Incubation of cells for 24 h with DV resulted in discrete punctate labeling of both soma and processes. The Cy3 and Oregon green 488 signals were generally co-localized, suggesting that the complex remained in the same intracellular compartment during the initial 24 h. The DVassociated fluorescence was reduced progressively by co-application of increasing concentrations of unlabeled BoNT/A holotoxin. The results suggest that the BoNT/A HC is able to mediate internalization of a coupled dextran, even though the latter bears no resemblance to the BoNT/A light chain (LC). The HC of BoNT/A thus offers promise as a selective carrier to deliver BoNT antagonists to the nerve terminal cytoplasm for inhibiting the proteolytic activity of internalized BoNT/A LC. ß
Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release by clea... more Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release by cleaving the 206-amino-acid SNARE protein, SNAP-25. For each BoNT serotype, cleavage of SNAP-25 results in the loss of intact protein, the production of an N-terminal truncated protein, and the generation of a small C-terminal peptide. Peptides that mimic the C-terminal fragments of SNAP-25 following BoNT/A or BoNT/E cleavage were shown to depress transmitter release in bovine chromaffin cells and in Aplysia buccal ganglion cells. Similarly, the N-terminal–truncated SNAP-25 resulting from BoNT/A or BoNT/E cleavage has been found to inhibit transmitter exocytosis in various systems. With one exception, however, the inhibitory action of truncated SNAP-25 has not been demonstrated at a well-defined cholinergic synapse. The goal of the current study was to determine the level of inhibition of neurotransmitter release by N-terminal BoNT/A- or BoNT/E-truncated SNAP-25 in two different neuronal systems: cholinergically coupled Aplysia neurons and rat hippocampal cell cultures. Both truncated SNAP-25 products inhibited depolarization-dependent glutamate release from hippocampal cultures and depressed synaptic transmission in Aplysia buccal ganglion cells. These results suggest that truncated SNAP-25 can compete with endogenous SNAP-25 for binding with other SNARE proteins involved in transmitter release, thus inhibiting neurotransmitter exocytosis.
Microalgae have the potential to revolutionize biotechnology in a number of areas including nutri... more Microalgae have the potential to revolutionize biotechnology in a number of areas including nutrition, aquaculture, pharmaceuticals, and biofuels. Although algae have been commercially cultivated for over 50 years, metabolic engineering now seems necessary in order to achieve their full processing capabilities. Recently, the development of a number of transgenic algal strains boasting recombinant protein expression, engineered photosynthesis, and enhanced metabolism encourage the prospects of designer microalgae. Given the vast contributions that these solar-powered, carbon dioxide-sequestering organisms can provide to current global markets and the environment, an intensified focus on microalgal biotechnology is warranted. Ongoing advances in cultivation techniques coupled with genetic manipulation of crucial metabolic networks will further promote microalgae as an attractive platform for the production of numerous highvalue compounds.
A RTI C LE S The Bax protein is widely known as a pro-apoptotic Bcl-2 family member that when ove... more A RTI C LE S The Bax protein is widely known as a pro-apoptotic Bcl-2 family member that when overexpressed can trigger apoptosis in multiple cell types and is important for the developmental cell death of neurons 1,2 . However, Bax was found here to be a potent inhibitor of neuronal cell death in mice infected with Sindbis virus. Newborn mice, which are highly susceptible to a fatal infection with neurotropic Sindbis virus, were significantly protected from neuronal apoptosis and fatal disease when infected with a recombinant Sindbis virus encoding Bax. Deletion of the N terminus of Bax, which mimics cleaved Bax, converted Bax into a pro-apoptotic factor in vivo. As mice mature during the first week after birth, they acquire resistance to a fatal Sindbis virus infection 3,4 . However, Bax-deficient mice remained very sensitive to fatal disease compared with their control littermates, indicating that endogenous Bax functions as a survival factor and contributes to age-dependent resistance to Sindbis virus-induced mortality. The protective effects of Bax were reproduced in cultured hippocampal neurons but not in cultured dorsal root ganglia neurons. These findings indicate that cell-specific factors determine the anti-apoptotic versus pro-apoptotic function of Bax.
Nannochloropsis oculata CCMP 525, Dunaliella salina FACHB 435, and Chlorella sorokiniana CCTCC M2... more Nannochloropsis oculata CCMP 525, Dunaliella salina FACHB 435, and Chlorella sorokiniana CCTCC M209220 were compared in mixotrophic and photoautotrophic cultures in terms of growth rate, protein, and lipid content. Growth improved in glucose, and the biomass productivities of N. oculata, D. salina, and C. sorokiniana were found to be 1.4-, 2.2- and 4.2-fold that observed photoautotrophically. However, biomass and lipid production decreased at the highest glucose concentrations. Meanwhile, the content of protein and lipid were significantly augmented for mixotrophic conditions at least for some species. C. sorokiniana was found to be well suited for lipid production based on its high biomass production rate and lipid content reaching 51% during mixotrophy. Expression levels of accD (heteromeric acetyl-CoA carboxylase beta subunit), acc1 (homomeric acetyl-CoA carboxylase), rbcL (ribulose 1, 5-bisphosphate carboxylase/oxygenase large subunit) genes in C. sorokiniana were studied by real-time PCR. Increased expression levels of accD reflect the increased lipid content in stationary phase of mixotrophic growth, but expression of the acc1 gene remains low, suggesting that this gene may not be critical to lipid accumulation. Additionally, reduction of expression of the rbcL gene during mixotrophy indicated that utilization of glucose was found to reduce the role of this gene and photosynthesis.
The cell density is an inherent constraint in commercial mammalian cell cultures. Here, we descri... more The cell density is an inherent constraint in commercial mammalian cell cultures. Here, we describe a cell engineering strategy utilizing the overexpression of the E2F-1 cell cycle transcription factor in CHO DG44 cells that produce a monoclonal antibody in serum-free, suspension culture. Stable pools and cell lines expressing E2F-1 were isolated that attained viable cell densities 20% higher than control cell lines and continued proliferation for an additional day in batch culture. There were no significant changes in antibody production, apoptosis, and cell cycle compared to control cells, nor were the growth effects evident in fed-batch conditions. Overall, E2F-1 overexpression postponed entry into stationary phase in mammalian cells, but perhaps novel E2F-1 variants or combination cell cycle engineering strategies will be necessary to realize significant growth benefits in commercial applications.
We investigate the effect of mitochondrial localization and the Bcl-x L C-terminal transmembrane ... more We investigate the effect of mitochondrial localization and the Bcl-x L C-terminal transmembrane (TM) domain on mitochondrial morphology and subcellular light scattering. CSM 14.1 cell lines stably expressed yellow fluorescent protein (YFP), YFP-Bcl-x L, YFP-Bcl-x L -DTM, containing the remainder of Bcl-x L after deletion of the last 21 amino acids corresponding to the TM domain, or YFP-TM, consisting of YFP fused at its C-terminal to the last 21 amino acids of Bcl-x L . YFP-Bcl-x L and YFP-TM localized to the mitochondria. Their expression decreased the intensity ratio of wide-to-narrow angle forward scatter by subcellular organelles, and correlated with an increase in the proportion of mitochondria with an expanded matrix having greatly reduced intracristal spaces as observed by electron microscopy. Cells expressing YFP-TM also exhibited significant autophagy. In contrast, YFP-Bcl-x L -DTM was diffusely distributed in the cells, and its expression did not alter light scattering or mitochondrial morphology compared with parental cells. Expression of YFP-Bcl-x L or YFP-Bcl-x L -DTM provided significant resistance to staurosporine-induced apoptosis. Surprisingly however, YFP-TM expression also conferred a moderate level of cell death resistance in response to staurosporine. Taken together, our results suggest the existence of a secondary Bcl-x L function that is mediated by the transmembrane domain, alters mitochondrial morphology, and is distinct from BH3 domain sequestration.
Biodiesel CO 2 sequestration Midwest Biorefinery a b s t r a c t Microalgae present some advantag... more Biodiesel CO 2 sequestration Midwest Biorefinery a b s t r a c t Microalgae present some advantageous qualities for reducing carbon dioxide (CO 2 ) emissions from ethanol biorefineries. As photosynthetic organisms, microalgae utilize sunlight and CO 2 to generate biomass. By integrating large-scale microalgal cultivation with ethanol biorefineries, CO 2 sequestration can be coupled with the growth of algae, which can then be used as feedstock for biodiesel production. In this case study, a 50-mgy ethanol biorefinery in Iowa was evaluated as a candidate for this process. Theoretical projections for the amount of land needed to grow algae in raceway ponds and the oil yields of this operation were based on the amount of CO 2 from the ethanol plant. A practical algal productivity of 20 g m À2 d À1 would require over 2,000 acres of ponds for complete CO 2 abatement, but with an aggressive productivity of 40e60 g m À2 d À1 , a significant portion of the CO 2 could be consumed using less than 1,000 acres. Due to the cold temperatures in Iowa, a greenhouse covering and a method to recover waste heat from the biorefinery were devised. While an algal strain, such as Chlorella vulgaris, would be able to withstand some temperature fluctuations, it was concluded that this process is limited by the amount of available heat, which could maintain only 41 acres at 73 F. Additional heating requirements result in a cost of 10e40 USD per gallon of algal oil, which is prohibitively expensive for biodiesel production, but could be profitable with the incorporation of high-value algal coproducts. ª A v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / b i o m b i o e b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 3 8 6 5 e3 8 7 6 0961-9534/$ e see front matter ª
Proceedings of the National Academy of Sciences, 2010
Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The effects of BoNT serot... more Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The effects of BoNT serotype A (BoNT/A) can last several months, whereas the effects of BoNT serotype E (BoNT/E), which shares the same synaptic target, synaptosomal-associated protein 25 (SNAP25), last only several weeks. The long-lasting effects or persistence of BoNT/A, although desirable for therapeutic applications, presents a challenge for medical treatment of BoNT intoxication. Although the mechanisms for BoNT toxicity are well known, little is known about the mechanisms that govern the persistence of the toxins. We show that the recombinant catalytic light chain (LC) of BoNT/E is ubiquitylated and rapidly degraded in cells. In contrast, BoNT/A LC is considerably more stable. Differential susceptibility of the catalytic LCs to ubiquitin-dependent proteolysis therefore might explain the differential persistence of BoNT serotypes. In this regard we show that TRAF2, a RING finger protein implicated in ubiqui...
A device is described which measures, on line, the bubble size distribution and volume fraction o... more A device is described which measures, on line, the bubble size distribution and volume fraction of gas in a liquid foam. There are four elements in the device: a flow channel, meniscus detection, velocity detection, and microcomputer data acquisition. The foam flows into a gently converging capillary to reorder the foam bubbles into alternating slugs of gas and liquid. The passage of the meniscus between a gas and liquid slug is detected by a matched light emitting diode/phototransistor pair. When gas is in the detection path, the detector signal is ``low,'' and when liquid is in the path, the signal is ``high.'' The volume fraction of gas is determined from the ratio of time the detector signal is low to the total elapsed time. A second detector pair is located a fixed distance further down the capillary. From the time shift between signals at the first detector and those at the second, the velocity of the foam in the capillary can be calculated. From the velocity, time that the detector signal is ``low,'' and known capillary geometry, the volume of a gas bubble can be determined. An IBM PC is used for rapid data acquisition and calculation. The device has been tested under a range of foam conditions to validate its performance.
Synaptosomal associated protein of 25 kDa (SNAP-25) is a cytoplasmic protein that participates in... more Synaptosomal associated protein of 25 kDa (SNAP-25) is a cytoplasmic protein that participates in the docking and fusion of synaptic vesicles with the nerve terminal in preparation for neurotransmitter release. SNAP-25 is also a substrate for three of the seven serotypes of botulinum neurotoxin (BoNT). Intoxication by BoNT/A, /C1 or /E results in weakness and paralysis of skeletal muscle due to cleavage of SNAP-25 (and syntaxin la in the case /C1) at discrete serotype-specific sites. To elucidate the role of SNAP-25 in muscle function in more detail, contractility and neuromuscular transmission were studied in a mutant mouse model termed coloboma. The coloboma mutation results from a contiguous deletion of 1-2 centiMorgans on chromosome 2, which includes the entire SNAP-25 locus and three other identified genes. Homozygotes do not survive beyond gestation day 6; heterozygotes (Cm/+) have a normal life-span but express reduced levels of SNAP-25 mRNA and protein in the brain. The consequences of the Cm/+ mutation on twitch and tetanic tension, quantal release of neurotransmitter and spinal motoneuron expression of SNAP-25 were examined in the present study. Contrary to expectations, Cm/+ mice exhibited no alteration in twitch tension and generated normal tetanic tension even at the highest frequency examined (800 Hz). Microelectrode recordings revealed that MEPP amplitude and frequency were both within control limits. The ventral spinal cord of Cm/+ mice showed no deficiency in SNAP-25 content and immunohistochemical examination of nerve terminals in Cm/+ mice disclosed that SNAP-25 levels and distribution were similar to those of control mice. It is concluded that spinal motor neurons up-regulate SNAP-25 to preserve vital neuromuscular function.
Nerve-evoked contractions were studied in vitro in phrenic nerve–hemidiaphragm preparations from ... more Nerve-evoked contractions were studied in vitro in phrenic nerve–hemidiaphragm preparations from strain 129X1 acetylcholinesterase knockout (AChE−/−) mice and their wild-type littermates (AChE+/+). The AChE−/− mice fail to express AChE but have normal levels of butyrylcholinesterase (BChE) and can survive into adulthood. Twitch tensions elicited in diaphragms of AChE−/− mice by single supramaximal stimuli had larger amplitudes and slower rise and decay times than did those in wild-type animals. In AChE−/− preparations, repetitive stimulation at frequencies of 20 and 50 Hz and at 200 and 400 Hz produced decremental muscle tensions; however, stimulation at 70 and 100 Hz resulted in little or no loss of tension during trains. Muscles from AChE+/+ mice maintained tension at all frequencies examined but exhibited tetanic fade after exposure to the selective AChE inhibitor 1,5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW 284C51). The ability of diaphragm muscles from AChE−/− mice to maintain tension at 70 and 100 Hz suggests a partial compensation for impairment of acetylcholine (ACh) hydrolysis. Three mechanisms—including a reliance on BChE activity for termination of ACh action, downregulation of nicotinic acetylcholine receptors (nAChRs), and morphological remodeling of the endplate region—were identified. Studies of neuromuscular transmission in this model system provide an excellent opportunity to evaluate the role of AChE without complications arising from use of inhibitors. Muscle Nerve 30: 317–327, 2004
Primary dissociated fetal mouse spinal cord cultures were used to study the mechanisms underlying... more Primary dissociated fetal mouse spinal cord cultures were used to study the mechanisms underlying the differences in persistence of botulinum neurotoxin A (BoNT/A) and botulinum neurotoxin/E (BoNT/E) activities. Spinal cord cultures were exposed to BoNT/A (0.4 pM) for 2^3 days, which converted approximately half of the SNAP-25 to an altered form lacking the final nine C-terminal residues. The distribution of toxindamaged to control SNAP-25 remained relatively unchanged for up to 80 days thereafter. Application of a high concentration of BoNT/E (250 pM) either 25 or 60 days following initial intoxication with BoNT/A converted both normal and BoNT/ A-truncated SNAP-25 into a single population lacking the final 26 C-terminal residues. Excess BoNT/E was removed by washout, and recovery of intact SNAP-25 was monitored by Western blot analysis. The BoNT/E-truncated species gradually diminished during the ensuing 18 days, accompanied by the reappearance of both normal and BoNT/A-truncated SNAP-25. Return of BoNT/A-truncated SNAP-25 was observed in spite of the absence of BoNT/A in the culture medium during all but the first 3 days of exposure. These results indicate that proteolytic activity associated with the BoNT/A light chain persists inside cells for s 11 weeks, while recovery from BoNT/E is complete in 6 3 weeks. This longer duration of enzymatic activity appears to account for the persistence of serotype A action.
A targeted delivery vehicle (DV) was developed for intracellular transport of emerging botulinum ... more A targeted delivery vehicle (DV) was developed for intracellular transport of emerging botulinum neurotoxin (BoNT) antagonists. The DV consisted of the isolated heavy chain (HC) of BoNT/A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The HC served to target BoNT-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells. To determine the ability of this chimeric glycoprotein to enter neurons, dextran and HC were labeled independently with the fluorescent dyes Oregon green 488 and Cy3, respectively. Internalization of DV was monitored in primary cortical cells using laser confocal microscopy. Incubation of cells for 24 h with DV resulted in discrete punctate labeling of both soma and processes. The Cy3 and Oregon green 488 signals were generally co-localized, suggesting that the complex remained in the same intracellular compartment during the initial 24 h. The DVassociated fluorescence was reduced progressively by co-application of increasing concentrations of unlabeled BoNT/A holotoxin. The results suggest that the BoNT/A HC is able to mediate internalization of a coupled dextran, even though the latter bears no resemblance to the BoNT/A light chain (LC). The HC of BoNT/A thus offers promise as a selective carrier to deliver BoNT antagonists to the nerve terminal cytoplasm for inhibiting the proteolytic activity of internalized BoNT/A LC. ß
Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release by clea... more Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release by cleaving the 206-amino-acid SNARE protein, SNAP-25. For each BoNT serotype, cleavage of SNAP-25 results in the loss of intact protein, the production of an N-terminal truncated protein, and the generation of a small C-terminal peptide. Peptides that mimic the C-terminal fragments of SNAP-25 following BoNT/A or BoNT/E cleavage were shown to depress transmitter release in bovine chromaffin cells and in Aplysia buccal ganglion cells. Similarly, the N-terminal–truncated SNAP-25 resulting from BoNT/A or BoNT/E cleavage has been found to inhibit transmitter exocytosis in various systems. With one exception, however, the inhibitory action of truncated SNAP-25 has not been demonstrated at a well-defined cholinergic synapse. The goal of the current study was to determine the level of inhibition of neurotransmitter release by N-terminal BoNT/A- or BoNT/E-truncated SNAP-25 in two different neuronal systems: cholinergically coupled Aplysia neurons and rat hippocampal cell cultures. Both truncated SNAP-25 products inhibited depolarization-dependent glutamate release from hippocampal cultures and depressed synaptic transmission in Aplysia buccal ganglion cells. These results suggest that truncated SNAP-25 can compete with endogenous SNAP-25 for binding with other SNARE proteins involved in transmitter release, thus inhibiting neurotransmitter exocytosis.
Microalgae have the potential to revolutionize biotechnology in a number of areas including nutri... more Microalgae have the potential to revolutionize biotechnology in a number of areas including nutrition, aquaculture, pharmaceuticals, and biofuels. Although algae have been commercially cultivated for over 50 years, metabolic engineering now seems necessary in order to achieve their full processing capabilities. Recently, the development of a number of transgenic algal strains boasting recombinant protein expression, engineered photosynthesis, and enhanced metabolism encourage the prospects of designer microalgae. Given the vast contributions that these solar-powered, carbon dioxide-sequestering organisms can provide to current global markets and the environment, an intensified focus on microalgal biotechnology is warranted. Ongoing advances in cultivation techniques coupled with genetic manipulation of crucial metabolic networks will further promote microalgae as an attractive platform for the production of numerous highvalue compounds.
A RTI C LE S The Bax protein is widely known as a pro-apoptotic Bcl-2 family member that when ove... more A RTI C LE S The Bax protein is widely known as a pro-apoptotic Bcl-2 family member that when overexpressed can trigger apoptosis in multiple cell types and is important for the developmental cell death of neurons 1,2 . However, Bax was found here to be a potent inhibitor of neuronal cell death in mice infected with Sindbis virus. Newborn mice, which are highly susceptible to a fatal infection with neurotropic Sindbis virus, were significantly protected from neuronal apoptosis and fatal disease when infected with a recombinant Sindbis virus encoding Bax. Deletion of the N terminus of Bax, which mimics cleaved Bax, converted Bax into a pro-apoptotic factor in vivo. As mice mature during the first week after birth, they acquire resistance to a fatal Sindbis virus infection 3,4 . However, Bax-deficient mice remained very sensitive to fatal disease compared with their control littermates, indicating that endogenous Bax functions as a survival factor and contributes to age-dependent resistance to Sindbis virus-induced mortality. The protective effects of Bax were reproduced in cultured hippocampal neurons but not in cultured dorsal root ganglia neurons. These findings indicate that cell-specific factors determine the anti-apoptotic versus pro-apoptotic function of Bax.
Nannochloropsis oculata CCMP 525, Dunaliella salina FACHB 435, and Chlorella sorokiniana CCTCC M2... more Nannochloropsis oculata CCMP 525, Dunaliella salina FACHB 435, and Chlorella sorokiniana CCTCC M209220 were compared in mixotrophic and photoautotrophic cultures in terms of growth rate, protein, and lipid content. Growth improved in glucose, and the biomass productivities of N. oculata, D. salina, and C. sorokiniana were found to be 1.4-, 2.2- and 4.2-fold that observed photoautotrophically. However, biomass and lipid production decreased at the highest glucose concentrations. Meanwhile, the content of protein and lipid were significantly augmented for mixotrophic conditions at least for some species. C. sorokiniana was found to be well suited for lipid production based on its high biomass production rate and lipid content reaching 51% during mixotrophy. Expression levels of accD (heteromeric acetyl-CoA carboxylase beta subunit), acc1 (homomeric acetyl-CoA carboxylase), rbcL (ribulose 1, 5-bisphosphate carboxylase/oxygenase large subunit) genes in C. sorokiniana were studied by real-time PCR. Increased expression levels of accD reflect the increased lipid content in stationary phase of mixotrophic growth, but expression of the acc1 gene remains low, suggesting that this gene may not be critical to lipid accumulation. Additionally, reduction of expression of the rbcL gene during mixotrophy indicated that utilization of glucose was found to reduce the role of this gene and photosynthesis.
The cell density is an inherent constraint in commercial mammalian cell cultures. Here, we descri... more The cell density is an inherent constraint in commercial mammalian cell cultures. Here, we describe a cell engineering strategy utilizing the overexpression of the E2F-1 cell cycle transcription factor in CHO DG44 cells that produce a monoclonal antibody in serum-free, suspension culture. Stable pools and cell lines expressing E2F-1 were isolated that attained viable cell densities 20% higher than control cell lines and continued proliferation for an additional day in batch culture. There were no significant changes in antibody production, apoptosis, and cell cycle compared to control cells, nor were the growth effects evident in fed-batch conditions. Overall, E2F-1 overexpression postponed entry into stationary phase in mammalian cells, but perhaps novel E2F-1 variants or combination cell cycle engineering strategies will be necessary to realize significant growth benefits in commercial applications.
We investigate the effect of mitochondrial localization and the Bcl-x L C-terminal transmembrane ... more We investigate the effect of mitochondrial localization and the Bcl-x L C-terminal transmembrane (TM) domain on mitochondrial morphology and subcellular light scattering. CSM 14.1 cell lines stably expressed yellow fluorescent protein (YFP), YFP-Bcl-x L, YFP-Bcl-x L -DTM, containing the remainder of Bcl-x L after deletion of the last 21 amino acids corresponding to the TM domain, or YFP-TM, consisting of YFP fused at its C-terminal to the last 21 amino acids of Bcl-x L . YFP-Bcl-x L and YFP-TM localized to the mitochondria. Their expression decreased the intensity ratio of wide-to-narrow angle forward scatter by subcellular organelles, and correlated with an increase in the proportion of mitochondria with an expanded matrix having greatly reduced intracristal spaces as observed by electron microscopy. Cells expressing YFP-TM also exhibited significant autophagy. In contrast, YFP-Bcl-x L -DTM was diffusely distributed in the cells, and its expression did not alter light scattering or mitochondrial morphology compared with parental cells. Expression of YFP-Bcl-x L or YFP-Bcl-x L -DTM provided significant resistance to staurosporine-induced apoptosis. Surprisingly however, YFP-TM expression also conferred a moderate level of cell death resistance in response to staurosporine. Taken together, our results suggest the existence of a secondary Bcl-x L function that is mediated by the transmembrane domain, alters mitochondrial morphology, and is distinct from BH3 domain sequestration.
Biodiesel CO 2 sequestration Midwest Biorefinery a b s t r a c t Microalgae present some advantag... more Biodiesel CO 2 sequestration Midwest Biorefinery a b s t r a c t Microalgae present some advantageous qualities for reducing carbon dioxide (CO 2 ) emissions from ethanol biorefineries. As photosynthetic organisms, microalgae utilize sunlight and CO 2 to generate biomass. By integrating large-scale microalgal cultivation with ethanol biorefineries, CO 2 sequestration can be coupled with the growth of algae, which can then be used as feedstock for biodiesel production. In this case study, a 50-mgy ethanol biorefinery in Iowa was evaluated as a candidate for this process. Theoretical projections for the amount of land needed to grow algae in raceway ponds and the oil yields of this operation were based on the amount of CO 2 from the ethanol plant. A practical algal productivity of 20 g m À2 d À1 would require over 2,000 acres of ponds for complete CO 2 abatement, but with an aggressive productivity of 40e60 g m À2 d À1 , a significant portion of the CO 2 could be consumed using less than 1,000 acres. Due to the cold temperatures in Iowa, a greenhouse covering and a method to recover waste heat from the biorefinery were devised. While an algal strain, such as Chlorella vulgaris, would be able to withstand some temperature fluctuations, it was concluded that this process is limited by the amount of available heat, which could maintain only 41 acres at 73 F. Additional heating requirements result in a cost of 10e40 USD per gallon of algal oil, which is prohibitively expensive for biodiesel production, but could be profitable with the incorporation of high-value algal coproducts. ª A v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / b i o m b i o e b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 3 8 6 5 e3 8 7 6 0961-9534/$ e see front matter ª
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