A cluster of amyotrophic lateral sclerosis in New Hampshire: a possible role for toxic cyanobacteria blooms.

PubMed

Caller, Tracie A; Doolin, James W; Haney, James F; Murby, Amanda J; West, Katherine G; Farrar, Hannah E; Ball, Andrea; Harris, Brent T; Stommel, Elijah W

2009-01-01

Cyanobacteria produce many neurotoxins including beta-methylamino-L-alanine (BMAA) that has been liked to amyotrophic lateral sclerosis (ALS) and neurodegenerative disease. A number of ALS cases have been diagnosed among residents of Enfield, NH, a town encompassing a lake with a history of cyanobacteria algal blooms. To investigate an association between toxic cyanobacterial blooms in New Hampshire and development of ALS, we reviewed records from our institution and other community databases to obtain demographic information on patients diagnosed with ALS within New England. We identified nine ALS patients who lived near Lake Mascoma in Enfield, NH, an incidence of sporadic ALS that is 10 to 25 times the expected incidence of 2/100,000/year. We suggest that the high incidence of ALS in this potential cluster could be directly related to chronic exposure to cyanobacterial neurotoxins such as BMAA. Possible routes of toxin exposure include inhalation of aerosolized toxins, consuming fish, or ingestion of lake water. Further investigation, including analysis of brain tissue for cyanobacterial toxins, will be helpful to test for an association between BMAA and ALS.

Presence of the Neurotoxin BMAA in Aquatic Ecosystems: What Do We Really Know?

PubMed Central

Faassen, Elisabeth J.

2014-01-01

The neurotoxin β-N-methylamino-l-alanine (BMAA) is suspected to play a role in the neurological diseases amyotrophic lateral sclerosis, Alzheimer’s disease, and Parkinson’s disease. BMAA production by cyanobacteria has been reported and contact with cyanobacteria infested waters or consumption of aquatic organisms are possible pathways to human exposure. However, there is little consensus regarding whether BMAA is present in cyanobacteria or not, and if so, at what concentrations. The aim of this review is to indicate the current state of knowledge on the presence of BMAA in aquatic ecosystems. Some studies have convincingly shown that BMAA can be present in aquatic samples at the µg/g dry weight level, which is around the detection limit of some equally credible studies in which no BMAA was detected. However, for the majority of the reviewed articles, it was unclear whether BMAA was correctly identified, either because inadequate analytical methods were used, or because poor reporting of analyses made it impossible to verify the results. Poor analysis, reporting and prolific errors have shaken the foundations of BMAA research. First steps towards estimation of human BMAA exposure are to develop and use selective, inter-laboratory validated methods and to correctly report the analytical work. PMID:24662480

Analysis of β-N-methylamino-L-alanine (BMAA) in spirulina-containing supplements by liquid chromatography-tandem mass spectrometry

PubMed Central

2014-01-01

Over the last decade the amino acid beta-N-methylamino-L-alanine (BMAA) has come under intense scrutiny. International laboratory and epidemiological research continues to support the hypothesis that environmental exposure to BMAA (e.g., through dietary practices, water supply) can promote the risk of various neurodegenerative diseases. A wide variety of cyanobacteria spp. have previously been reported to produce BMAA, with production levels dependent upon species, strain and environmental conditions. Since spirulina (Arthrospira spp.) is a member of the cyanobacteria phylum frequently consumed via dietary supplements, the presence of BMAA in such products may have public health implications. In the current work, we have analyzed ten spirulina-containing samples for the presence of BMAA; six pure spirulina samples from two separate raw materials suppliers, and four commercially-available multi-ingredient products containing 1.45 g of spirulina per 8.5 g serving. Because of controversy surrounding the measurement of BMAA, we have used two complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods: one based on reversed phase LC (RPLC) with derivatization and the other based on hydrophilic interaction LC (HILIC). Potential matrix effects were corrected for by internal standardization using a stable isotope labeled BMAA standard. BMAA was not detected at low limits of detection (80 ng/g dry weight) in any of these product samples. Although these results are reassuring, BMAA analyses should be conducted on a wider sample selection and, perhaps, as part of ongoing spirulina production quality control testing and specifications. PMID:25120905

Analysis of β-N-methylamino-L-alanine (BMAA) in spirulina-containing supplements by liquid chromatography-tandem mass spectrometry.

PubMed

McCarron, Pearse; Logan, Alan C; Giddings, Sabrina D; Quilliam, Michael A

2014-01-01

Over the last decade the amino acid beta-N-methylamino-L-alanine (BMAA) has come under intense scrutiny. International laboratory and epidemiological research continues to support the hypothesis that environmental exposure to BMAA (e.g., through dietary practices, water supply) can promote the risk of various neurodegenerative diseases. A wide variety of cyanobacteria spp. have previously been reported to produce BMAA, with production levels dependent upon species, strain and environmental conditions. Since spirulina (Arthrospira spp.) is a member of the cyanobacteria phylum frequently consumed via dietary supplements, the presence of BMAA in such products may have public health implications. In the current work, we have analyzed ten spirulina-containing samples for the presence of BMAA; six pure spirulina samples from two separate raw materials suppliers, and four commercially-available multi-ingredient products containing 1.45 g of spirulina per 8.5 g serving. Because of controversy surrounding the measurement of BMAA, we have used two complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods: one based on reversed phase LC (RPLC) with derivatization and the other based on hydrophilic interaction LC (HILIC). Potential matrix effects were corrected for by internal standardization using a stable isotope labeled BMAA standard. BMAA was not detected at low limits of detection (80 ng/g dry weight) in any of these product samples. Although these results are reassuring, BMAA analyses should be conducted on a wider sample selection and, perhaps, as part of ongoing spirulina production quality control testing and specifications.

A critical review of the postulated role of the non-essential amino acid, β-N-methylamino-L-alanine, in neurodegenerative disease in humans

EPA Science Inventory

The compound BMAA (β-N-methylamino-L-alanine) has been hypothesized to play a significant role in four serious neurological diseases in humans: Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) found on Guam, and ALS, parkinsonism, and dementia that occur glob...

Genotoxic and Cytotoxic Effects on the Immune Cells of the Freshwater Bivalve Dreissena polymorpha Exposed to the Environmental Neurotoxin BMAA

PubMed Central

Milliote, Nadia; Bonnard, Marc; Palos-Ladeiro, Mélissa; Rioult, Damien; Bonnard, Isabelle; Bastien, Fanny; Geffard, Alain

2018-01-01

The environmental neurotoxin β-N-Methylamino-l-alanine (BMAA) has been pointed out to be involved in human neurodegenerative diseases. This molecule is known to be bioaccumulated by bivalves. However, little data about its toxic effects on freshwater mussels is available, particularly on the hemolymphatic compartment and its hemocyte cells involved in various physiological processes such as immune defenses, digestion and excretion, tissue repair, and shell production. Here we exposed Dreissena polymorpha to dissolved BMAA, at the environmental concentration of 7.5 µg of /mussel/3 days, during 21 days followed by 14 days of depuration in clear water, with the objective of assessing the BMAA presence in the hemolymphatic compartment, as well as the impact of the hemocyte cells in terms of potential cytotoxicity, immunotoxicity, and genotoxiciy. Data showed that hemocytes were in contact with BMAA. The presence of BMAA in hemolymph did not induce significant effect on hemocytes phagocytosis activity. However, significant DNA damage on hemocytes occurred during the first week (days 3 and 8) of BMAA exposure, followed by an increase of hemocyte mortality after 2 weeks of exposure. Those effects might be an indirect consequence of the BMAA-induced oxidative stress in cells. However, DNA strand breaks and mortality did not persist during the entire exposure, despite the BMAA persistence in the hemolymph, suggesting potential induction of some DNA-repair mechanisms. PMID:29494483

BMAA extraction of cyanobacteria samples: which method to choose?

PubMed

Lage, Sandra; Burian, Alfred; Rasmussen, Ulla; Costa, Pedro Reis; Annadotter, Heléne; Godhe, Anna; Rydberg, Sara

2016-01-01

β-N-Methylamino-L-alanine (BMAA), a neurotoxin reportedly produced by cyanobacteria, diatoms and dinoflagellates, is proposed to be linked to the development of neurological diseases. BMAA has been found in aquatic and terrestrial ecosystems worldwide, both in its phytoplankton producers and in several invertebrate and vertebrate organisms that bioaccumulate it. LC-MS/MS is the most frequently used analytical technique in BMAA research due to its high selectivity, though consensus is lacking as to the best extraction method to apply. This study accordingly surveys the efficiency of three extraction methods regularly used in BMAA research to extract BMAA from cyanobacteria samples. The results obtained provide insights into possible reasons for the BMAA concentration discrepancies in previous publications. In addition and according to the method validation guidelines for analysing cyanotoxins, the TCA protein precipitation method, followed by AQC derivatization and LC-MS/MS analysis, is now validated for extracting protein-bound (after protein hydrolysis) and free BMAA from cyanobacteria matrix. BMAA biological variability was also tested through the extraction of diatom and cyanobacteria species, revealing a high variance in BMAA levels (0.0080-2.5797 μg g(-1) DW).

Transfer of a cyanobacterial neurotoxin within a temperate aquatic ecosystem suggests pathways for human exposure

PubMed Central

Jonasson, Sara; Eriksson, Johan; Berntzon, Lotta; Spáčil, Zdenĕk; Ilag, Leopold L.; Ronnevi, Lars-Olof; Rasmussen, Ulla; Bergman, Birgitta

2010-01-01

β-methylamino-L-alanine (BMAA), a neurotoxic nonprotein amino acid produced by most cyanobacteria, has been proposed to be the causative agent of devastating neurodegenerative diseases on the island of Guam in the Pacific Ocean. Because cyanobacteria are widespread globally, we hypothesized that BMAA might occur and bioaccumulate in other ecosystems. Here we demonstrate, based on a recently developed extraction and HPLC-MS/MS method and long-term monitoring of BMAA in cyanobacterial populations of a temperate aquatic ecosystem (Baltic Sea, 2007–2008), that BMAA is biosynthesized by cyanobacterial genera dominating the massive surface blooms of this water body. BMAA also was found at higher concentrations in organisms of higher trophic levels that directly or indirectly feed on cyanobacteria, such as zooplankton and various vertebrates (fish) and invertebrates (mussels, oysters). Pelagic and benthic fish species used for human consumption were included. The highest BMAA levels were detected in the muscle and brain of bottom-dwelling fishes. The discovery of regular biosynthesis of the neurotoxin BMAA in a large temperate aquatic ecosystem combined with its possible transfer and bioaccumulation within major food webs, some ending in human consumption, is alarming and requires attention. PMID:20439734

β-N-Methylamino-L-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling.

PubMed

Engskog, Mikael K R; Ersson, Lisa; Haglöf, Jakob; Arvidsson, Torbjörn; Pettersson, Curt; Brittebo, Eva

2017-05-01

β-Methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid that induces long-term cognitive deficits, as well as an increased neurodegeneration and intracellular fibril formation in the hippocampus of adult rodents following short-time neonatal exposure and in vervet monkey brain following long-term exposure. It has also been proposed to be involved in the etiology of neurodegenerative disease in humans. The aim of this study was to identify metabolic effects not related to excitotoxicity or oxidative stress in human neuroblastoma SH-SY5Y cells. The effects of BMAA (50, 250, 1000 µM) for 24 h on cells differentiated with retinoic acid were studied. Samples were analyzed using LC-MS and NMR spectroscopy to detect altered intracellular polar metabolites. The analysis performed, followed by multivariate pattern recognition techniques, revealed significant perturbations in protein biosynthesis, amino acid metabolism pathways and citrate cycle. Of specific interest were the BMAA-induced alterations in alanine, aspartate and glutamate metabolism and as well as alterations in various neurotransmitters/neuromodulators such as GABA and taurine. The results indicate that BMAA can interfere with metabolic pathways involved in neurotransmission in human neuroblastoma cells.

Detection of cyanotoxins, β-N-methylamino-L-alanine and microcystins, from a lake surrounded by cases of amyotrophic lateral sclerosis.

PubMed

Banack, Sandra Anne; Caller, Tracie; Henegan, Patricia; Haney, James; Murby, Amanda; Metcalf, James S; Powell, James; Cox, Paul Alan; Stommel, Elijah

2015-01-29

A cluster of amyotrophic lateral sclerosis (ALS) has been previously described to border Lake Mascoma in Enfield, NH, with an incidence of ALS approximating 25 times expected. We hypothesize a possible association with cyanobacterial blooms that can produce β-N-methylamino-L-alanine (BMAA), a neurotoxic amino acid implicated as a possible cause of ALS/PDC in Guam. Muscle, liver, and brain tissue samples from a Lake Mascoma carp, as well as filtered aerosol samples, were analyzed for microcystins (MC), free and protein-bound BMAA, and the BMAA isomers 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl)glycine (AEG). In carp brain, BMAA and DAB concentrations were 0.043 μg/g ± 0.02 SD and 0.01 μg/g ± 0.002 SD respectively. In carp liver and muscle, the BMAA concentrations were 1.28 μg/g and 1.27 μg/g respectively, and DAB was not detected. BMAA was detected in the air filters, as were the isomers DAB and AEG. These results demonstrate that a putative cause for ALS, BMAA, exists in an environment that has a documented cluster of ALS. Although cause and effect have not been demonstrated, our observations and measurements strengthen the association.

β-methylamino-L-alanine (BMAA) is not found in the brains of patients with confirmed Alzheimer’s disease

NASA Astrophysics Data System (ADS)

Meneely, Julie P.; Chevallier, Olivier P.; Graham, Stewart; Greer, Brett; Green, Brian D.; Elliott, Christopher T.

2016-11-01

Controversy surrounds the proposed hypothesis that exposure to β-methylamino-L-alanine (BMAA) could play a role in various neurodegenerative conditions including Alzheimer’s disease (AD). Here we present the results of the most comprehensive scientific study on BMAA detection ever undertaken on brain samples from patients pathologically confirmed to have suffered from AD, and those from healthy volunteers. Following the full validation of a highly accurate and sensitive mass spectrometric method, no trace of BMAA was detected in the diseased brain or in the control specimens. This contradicts the findings of other reports and calls into question the significance of this compound in neurodegenerative disease. We have attempted to explain the potential causes of misidentification of BMAA in these studies.

Nitrogen starvation of cyanobacteria results in the production of β-N-methylamino-L-alanine.

PubMed

Downing, S; Banack, S A; Metcalf, J S; Cox, P A; Downing, T G

2011-08-01

β-N-Methylamino-L-alanine, an unusual amino acid implicated in neurodegenerative disease, has been detected in cultures of nearly all genera of environmentally ubiquitous cyanobacteria tested. The compound is present within cyanobacterial cells in free and protein-associated forms, with large variations occurring in the concentration of these pools between species as well as within single strains. With a lack of knowledge and supporting data on the regulation of BMAA production and the role of this compound in cyanobacteria, the association between BMAA and cyanobacteria is still subject to debate. In this study we investigated the biosynthesis of BMAA in axenic non-diazotrophic cyanobacterial cultures using the stable isotope ¹⁵N. Nitrogen starvation of nutritionally replete cells resulted in an increase in free cellular ¹⁵N BMAA suggesting that BMAA may be the result of catabolism to provide nitrogen or that BMAA is synthesised to serve a functional role in the cell in response to nitrogen deprivation. The addition of NO₃⁻ and NH₄⁺ to the culture medium following starvation resulted in a decrease of free cellular BMAA without a corresponding increase in the protein-associated fraction. The use of ammonia as a nitrogen source resulted in a more rapid reduction of BMAA when compared to nitrate. This study provides the first data regarding the regulation of intracellular BMAA concentrations in cyanobacteria with results conclusively showing the production of ¹⁵N BMAA by an axenic cyanobacterial culture. Copyright © 2011 Elsevier Ltd. All rights reserved.

Neurotoxic flying foxes as dietary items for the Chamorro people, Marianas Islands.

PubMed

Banack, Sandra Anne; Murch, Susan J; Cox, Paul Alan

2006-06-15

Fanihi -- flying foxes (Pteropus mariannus mariannus, Pteropodidae) -- are a highly salient component of the traditional Chamorro diet. A neurotoxic, non-protein amino acid, beta-methylamino-l-alanine (BMAA) accumulates in flying foxes, which forage on the seeds of Cycas micronesica (Cycadaceae) in Guam's forests. BMAA occurs throughout flying fox tissues both as a free amino acid and in a protein-bound form. It is not destroyed by cooking. Protein-bound BMAA also remains in cycad flour which has been washed and prepared by the Chamorro people as tortillas, dumplings, and thickened soups. Other animals that forage on cycad seeds may also provide BMAA inputs into the traditional Chamorro diet.

Potential transfer of neurotoxic amino acid β-N-methylamino-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines.

PubMed

Andersson, Marie; Ersson, Lisa; Brandt, Ingvar; Bergström, Ulrika

2017-04-01

β-N-methylamino-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria, diatoms and dinoflagellates. BMAA has potential to biomagnify in a terrestrial food chain, and to bioaccumulate in fish and shellfish. We have reported that administration of [ 14 C]l-BMAA to lactating mice and rats results in a mother to off-spring transfer via the milk. A preferential enantiomer-specific uptake of [ 14 C]l-BMAA has also been demonstrated in differentiated murine mammary epithelium HC11 cells. These findings, together with neurotoxic effects of BMAA demonstrated both in vitro and in vivo, highlight the need to determine whether such transfer could also occur in humans. Here, we used four cell lines of human origin to examine and compare the transport of the two BMAA enantiomers in vitro. The uptake patterns of [ 14 C]l- and [ 14 C]d-BMAA in the human mammary MCF7 cell line were in agreement with the results in murine HC11 cells, suggesting a potential secretion of BMAA into human breast milk. The permeability coefficients for both [ 14 C]l- and [ 14 C]d-BMAA over monolayers of human intestinal Caco2 cells supported an efficient absorption from the human intestine. As a final step, transport experiments confirmed that [ 14 C]l-and [ 14 C]d-BMAA can be taken up by human SHSY5Y neuroblastoma cells and even more efficiently by human U343 glioblastoma cells. In competition experiments with various amino acids, the ASCT2 specific inhibitor benzylserine was the most effective inhibitor of [ 14 C]l-BMAA uptake tested here. Altogether, our results suggest that BMAA can be transferred from an exposed mother, via the milk, to the brain of the nursed infant. Copyright © 2017. Published by Elsevier Inc.

Optimization of the Determination Method for Dissolved Cyanobacterial Toxin BMAA in Natural Water.

PubMed

Yan, Boyin; Liu, Zhiquan; Huang, Rui; Xu, Yongpeng; Liu, Dongmei; Lin, Tsair-Fuh; Cui, Fuyi

2017-10-17

There is a serious dispute on the existence of β-N-methylamino-l-alanine (BMAA) in water, which is a neurotoxin that may cause amyotrophic lateral sclerosis/Parkinson's disease (ALS/PDC) and Alzheimer' disease. It is believed that a reliable and sensitive analytical method for the determination of BMAA is urgently required to resolve this dispute. In the present study, the solid phase extraction (SPE) procedure and the analytical method for dissolved BMAA in water were investigated and optimized. The results showed both derivatized and underivatized methods were qualified for the measurement of BMAA and its isomer in natural water, and the limit of detection and the precision of the two methods were comparable. Cartridge characteristics and SPE conditions could greatly affect the SPE performance, and the competition of natural organic matter is the primary factor causing the low recovery of BMAA, which was reduced from approximately 90% in pure water to 38.11% in natural water. The optimized SPE method for BMAA was a combination of rinsed SPE cartridges, controlled loading/elution rates and elution solution, evaporation at 55 °C, reconstitution of a solution mixture, and filtration by polyvinylidene fluoride membrane. This optimized method achieved > 88% recovery of BMAA in both algal solution and river water. The developed method can provide an efficient way to evaluate the actual concentration levels of BMAA in actual water environments and drinking water systems.

Assessing Environmental Exposure to β-N-Methylamino-L-Alanine (BMAA) in Complex Sample Matrices: a Comparison of the Three Most Popular LC-MS/MS Methods.

PubMed

Baker, Teesha C; Tymm, Fiona J M; Murch, Susan J

2018-01-01

β-N-Methylamino-L-alanine (BMAA) is a naturally occurring non-protein amino acid produced by cyanobacteria, accumulated through natural food webs, found in mammalian brain tissues. Recent evidence indicates an association between BMAA and neurological disease. The accurate detection and quantification of BMAA in food and environmental samples are critical to understanding BMAA metabolism and limiting human exposure. To date, there have been more than 78 reports on BMAA in cyanobacteria and human samples, but different methods give conflicting data and divergent interpretations in the literature. The current work was designed to determine whether orthogonal chromatography and mass spectrometry methods give consistent data interpretation from a single sample matrix using the three most common analytical methods. The methods were recreated as precisely as possible from the literature with optimization of the mass spectrometry parameters specific to the instrument. Four sample matrices, cyanobacteria, human brain, blue crab, and Spirulina, were analyzed as 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives, propyl chloroformate (PCF) derivatives separated by reverse phase chromatography, or underivatized extracts separated by HILIC chromatography. The three methods agreed on positive detection of BMAA in cyanobacteria and no detected BMAA in the sample of human brain matrix. Interpretation was less clear for a sample of blue crab which was strongly positive for BMAA by AQC and PCF but negative by HILIC and for four spirulina raw materials that were negative by PCF but positive by AQC and HILIC. Together, these data demonstrate that the methods gave different results and that the choices in interpretation of the methods determined whether BMAA was detected. Failure to detect BMAA cannot be considered proof of absence.

Potential transfer of neurotoxic amino acid β-N-methylamino-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersson, Marie

β-N-methylamino-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria, diatoms and dinoflagellates. BMAA has potential to biomagnify in a terrestrial food chain, and to bioaccumulate in fish and shellfish. We have reported that administration of [{sup 14}C]L-BMAA to lactating mice and rats results in a mother to off-spring transfer via the milk. A preferential enantiomer-specific uptake of [{sup 14}C]L-BMAA has also been demonstrated in differentiated murine mammary epithelium HC11 cells. These findings, together with neurotoxic effects of BMAA demonstrated both in vitro and in vivo, highlight the need to determine whether such transfer could also occur in humans. Here,more » we used four cell lines of human origin to examine and compare the transport of the two BMAA enantiomers in vitro. The uptake patterns of [{sup 14}C]L- and [{sup 14}C]D-BMAA in the human mammary MCF7 cell line were in agreement with the results in murine HC11 cells, suggesting a potential secretion of BMAA into human breast milk. The permeability coefficients for both [{sup 14}C]L- and [{sup 14}C]D-BMAA over monolayers of human intestinal Caco2 cells supported an efficient absorption from the human intestine. As a final step, transport experiments confirmed that [{sup 14}C]L-and [{sup 14}C]D-BMAA can be taken up by human SHSY5Y neuroblastoma cells and even more efficiently by human U343 glioblastoma cells. In competition experiments with various amino acids, the ASCT2 specific inhibitor benzylserine was the most effective inhibitor of [{sup 14}C]L-BMAA uptake tested here. Altogether, our results suggest that BMAA can be transferred from an exposed mother, via the milk, to the brain of the nursed infant. - Highlights: • Transport of BMAA in human intestinal, mammary and CNS cell lines was examined. • The transport of L-BMAA over intestinal cell monolayers was unidirectional. • Enantiomer-selective uptake of L-BMAA in breast, neuron and glia cells was evident. • Competition experiments indicate that L-BMAA uptake involved several transporters. • A potential for mother to infant transfer of BMAA is proposed.« less

Potential for dietary exposure to β-N-methylamino-L-alanine and microcystin from a freshwater system.

PubMed

Scott, Laura L; Downing, Simoné; Downing, Tim

2018-06-18

The suggested link between β-N-methylamino-L-alanine (BMAA) and the onset of neurodegenerative diseases and the detection of this cyanotoxin in aquatic organisms has prompted research into the potential human exposure risk associated with sourcing food items from eutrophied water bodies worldwide. The Hartbeespoort Dam reservoir in the North West province of South Africa has persistent cyanobacterial blooms and is used extensively by anglers, many of whom consume their catch. The commercial sale of fish species harvested from this reservoir as part of a recent biomanipulative remediation strategy may pose an additional hazard. BMAA and Microcystins (MC) were detected in fish sourced from this reservoir. BMAA levels of up to 1630 ng g -1 dry weight and MC concentrations of up to 29.44 ng g -1 dry weight were detected in fish sourced during an extensive bloom episode, with a clear correlation between the total amount of BMAA detected in the fish muscle tissue and their relative position in the Hartbeespoort Dam reservoir food web. Interestingly, fish sourced from this reservoir in winter when dense cyanobacterial blooms were lacking contained BMAA levels of up to 3055 ng g -1 dry weight. We also comment on the observed seasonal variations of BMAA levels in phytoplankton and fish sourced from this water body as well as the potential exposure risks associated with harvesting food items from this reservoir. Copyright © 2018 Elsevier Ltd. All rights reserved.

Amino acid neurotoxins in feathers of the Lesser Flamingo, Phoeniconaias minor.

PubMed

Metcalf, J S; Banack, S A; Kotut, K; Krienitz, L; Codd, G A

2013-01-01

The Lesser Flamingo (Phoeniconaias minor) is known to use cyanobacteria (primarily Arthrospira) as a major food source in the East African Rift Valley lakes. Periodically, mass mortalities have occurred, associated with the cyanobacterial toxins (cyanotoxins), microcystins and anatoxin-a. Deposition of these cyanotoxins into P. minor feathers has been shown to occur, consistent with the presence of cyanotoxins in the livers, stomach and faecal contents after dietary intake. As cyanobacteria have been shown to also produce the neurotoxins β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB), stored wing feathers, previously recovered from flamingos which had been exposed to microcystins and anatoxin-a and had subsequently died, were analysed for these neurotoxic amino acids. Trace amounts of BMAA were detected in extracts from Lake Nakuru flamingo feathers, with DAB also present at concentrations between 3.5 and 8.5 μg g(-1) dry weight in feathers from both lakes. Toxin recovery by solid-phase extraction of feather digests was tested with spiked deuterated BMAA and showed good recovery when analysed by LC-MS/MS (80-94%). This is the first report of these neurotoxic amino acids in birds. We discuss the origin and significance of DAB, alongside other cyanotoxins of dietary origin, in the feathers of the Lesser Flamingo. Copyright © 2012 Elsevier Ltd. All rights reserved.

Environmental modulation of microcystin and β-N-methylamino-L-alanine as a function of nitrogen availability.

PubMed

Scott, L L; Downing, S; Phelan, R R; Downing, T G

2014-09-01

The most significant modulators of the cyanotoxins microcystin and β-N-methylamino-L-alanine in laboratory cyanobacterial cultures are the concentration of growth-medium combined nitrogen and nitrogen uptake rate. The lack of field studies that support these observations led us to investigate the cellular content of these cyanotoxins in cyanobacterial bloom material isolated from a freshwater impoundment and to compare these to the combined nitrogen availability. We established that these toxins typically occur in an inverse relationship in nature and that their presence is mainly dependent on the environmental combined nitrogen concentration, with cellular microcystin present at exogenous combined nitrogen concentrations of 29 μM and higher and cellular BMAA correlating negatively with exogenous nitrogen at concentrations below 40 μM. Furthermore, opposing nutrient and light gradients that form in dense cyanobacterial blooms may result in both microcystin and BMAA being present at a single sampling site. Copyright © 2014 Elsevier Ltd. All rights reserved.

A critical review of the postulated role of the non-essential amino acid, β-N-methylamino-L-alanine, in neurodegenerative disease in humans

USGS Publications Warehouse

Chernoff, Neil; Hill, D. J.; Diggs, D. L.; Faison, B. D.; Francis, B. M.; Lang, J. R.; Larue, M. M.; Le, T.-T.; Loftin, Keith A.; Lugo, J. N.; Schmid, J. E.; Winnik, W. W.

2017-01-01

The compound BMAA (β-N-methylamino-L-alanine) has been postulated to play a significant role in four serious neurological human diseases: Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) found on Guam, and ALS, Parkinsonism, and dementia that occur globally. ALS/PDC with symptoms of all three diseases first came to the attention of the scientific community during and after World War II. It was initially associated with cycad flour used for food because BMAA is a product of symbiotic cycad root-dwelling cyanobacteria. Human consumption of flying foxes that fed on cycad seeds was later suggested as a source of BMAA on Guam and a cause of ALS/PDC. Subsequently, the hypothesis was expanded to include a causative role for BMAA in other neurodegenerative diseases including Alzheimer’s disease (AD) through exposures attributed to proximity to freshwaters and/or consumption of seafood due to its purported production by most species of cyanobacteria. The hypothesis that BMAA is the critical factor in the genesis of these neurodegenerative diseases received considerable attention in the medical, scientific, and public arenas. This review examines the history of ALS/PDC and the BMAA-human disease hypotheses; similarities and differences between ALS/PDC and the other diseases with similar symptomologies; the relationship of ALS/PDC to other similar diseases, studies of BMAA-mediated effects in lab animals, inconsistencies and data gaps in the hypothesis; and other compounds and agents that were suggested as the cause of ALS/PDC on Guam. The review concludes that the hypothesis of a causal BMAA neurodegenerative disease relationship is not supported by existing data.

Cyanobacterial Neurotoxin BMAA and Mercury in Sharks

PubMed Central

Hammerschlag, Neil; Davis, David A.; Mondo, Kiyo; Seely, Matthew S.; Murch, Susan J.; Glover, William Broc; Divoll, Timothy; Evers, David C.; Mash, Deborah C.

2016-01-01

Sharks have greater risk for bioaccumulation of marine toxins and mercury (Hg), because they are long-lived predators. Shark fins and cartilage also contain β-N-methylamino-l-alanine (BMAA), a ubiquitous cyanobacterial toxin linked to neurodegenerative diseases. Today, a significant number of shark species have found their way onto the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Many species of large sharks are threatened with extinction due in part to the growing high demand for shark fin soup and, to a lesser extent, for shark meat and cartilage products. Recent studies suggest that the consumption of shark parts may be a route to human exposure of marine toxins. Here, we investigated BMAA and Hg concentrations in fins and muscles sampled in ten species of sharks from the South Atlantic and Pacific Oceans. BMAA was detected in all shark species with only seven of the 55 samples analyzed testing below the limit of detection of the assay. Hg concentrations measured in fins and muscle samples from the 10 species ranged from 0.05 to 13.23 ng/mg. These analytical test results suggest restricting human consumption of shark meat and fins due to the high frequency and co-occurrence of two synergistic environmental neurotoxic compounds. PMID:27537913

Cyanobacterial Neurotoxin BMAA and Mercury in Sharks.

PubMed

Hammerschlag, Neil; Davis, David A; Mondo, Kiyo; Seely, Matthew S; Murch, Susan J; Glover, William Broc; Divoll, Timothy; Evers, David C; Mash, Deborah C

2016-08-16

Sharks have greater risk for bioaccumulation of marine toxins and mercury (Hg), because they are long-lived predators. Shark fins and cartilage also contain β-N-methylamino-l-alanine (BMAA), a ubiquitous cyanobacterial toxin linked to neurodegenerative diseases. Today, a significant number of shark species have found their way onto the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Many species of large sharks are threatened with extinction due in part to the growing high demand for shark fin soup and, to a lesser extent, for shark meat and cartilage products. Recent studies suggest that the consumption of shark parts may be a route to human exposure of marine toxins. Here, we investigated BMAA and Hg concentrations in fins and muscles sampled in ten species of sharks from the South Atlantic and Pacific Oceans. BMAA was detected in all shark species with only seven of the 55 samples analyzed testing below the limit of detection of the assay. Hg concentrations measured in fins and muscle samples from the 10 species ranged from 0.05 to 13.23 ng/mg. These analytical test results suggest restricting human consumption of shark meat and fins due to the high frequency and co-occurrence of two synergistic environmental neurotoxic compounds.

Diverse taxa of cyanobacteria produce β-N-methylamino-l-alanine, a neurotoxic amino acid

PubMed Central

Cox, Paul Alan; Banack, Sandra Anne; Murch, Susan J.; Rasmussen, Ulla; Tien, Georgia; Bidigare, Robert Richard; Metcalf, James S.; Morrison, Louise F.; Codd, Geoffrey A.; Bergman, Birgitta

2005-01-01

Cyanobacteria can generate molecules hazardous to human health, but production of the known cyanotoxins is taxonomically sporadic. For example, members of a few genera produce hepatotoxic microcystins, whereas production of hepatotoxic nodularins appears to be limited to a single genus. Production of known neurotoxins has also been considered phylogenetically unpredictable. We report here that a single neurotoxin, β-N-methylamino-l-alanine, may be produced by all known groups of cyanobacteria, including cyanobacterial symbionts and free-living cyanobacteria. The ubiquity of cyanobacteria in terrestrial, as well as freshwater, brackish, and marine environments, suggests a potential for wide-spread human exposure. PMID:15809446

Chemistry and Chemical Equilibrium Dynamics of BMAA and Its Carbamate Adducts

PubMed Central

Diaz-parga, Pedro

2018-01-01

Beta-N-methylamino-L-alanine (BMAA) has been demonstrated to contribute to the onset of the ALS/Parkinsonism-dementia complex (ALS/PDC) and is implicated in the progression of other neurodegenerative diseases. While the role of BMAA in these diseases is still debated, one of the suggested mechanisms involves the activation of excitatory glutamate receptors. In particular, the excitatory effects of BMAA are shown to be dependent on the presence of bicarbonate ions, which in turn forms carbamate adducts in physiological conditions. The formation of carbamate adducts from BMAA and bicarbonate is similar to the formation of carbamate adducts from non-proteinogenic amino acids. Structural, chemical, and biological information related to non-proteinogenic amino acids provide insight into the formation of and possible neurological action of BMAA. This article reviews the carbamate formation of BMAA in the presence of bicarbonate ions, with a particular focus on how the chemical equilibrium of BMAA carbamate adducts may affect the molecular mechanism of its function. Highlights of nuclear magnetic resonance (NMR)-based studies on the equilibrium process between free BMAA and its adducts are presented. The role of divalent metals on the equilibrium process is also explored. The formation and the equilibrium process of carbamate adducts of BMAA may answer questions on their neuroactive potency and provide strong motivation for further investigations into other toxic mechanisms. PMID:28921378

Chemistry and Chemical Equilibrium Dynamics of BMAA and Its Carbamate Adducts.

PubMed

Diaz-Parga, Pedro; Goto, Joy J; Krishnan, V V

2018-01-01

Beta-N-methylamino-L-alanine (BMAA) has been demonstrated to contribute to the onset of the ALS/Parkinsonism-dementia complex (ALS/PDC) and is implicated in the progression of other neurodegenerative diseases. While the role of BMAA in these diseases is still debated, one of the suggested mechanisms involves the activation of excitatory glutamate receptors. In particular, the excitatory effects of BMAA are shown to be dependent on the presence of bicarbonate ions, which in turn forms carbamate adducts in physiological conditions. The formation of carbamate adducts from BMAA and bicarbonate is similar to the formation of carbamate adducts from non-proteinogenic amino acids. Structural, chemical, and biological information related to non-proteinogenic amino acids provide insight into the formation of and possible neurological action of BMAA. This article reviews the carbamate formation of BMAA in the presence of bicarbonate ions, with a particular focus on how the chemical equilibrium of BMAA carbamate adducts may affect the molecular mechanism of its function. Highlights of nuclear magnetic resonance (NMR)-based studies on the equilibrium process between free BMAA and its adducts are presented. The role of divalent metals on the equilibrium process is also explored. The formation and the equilibrium process of carbamate adducts of BMAA may answer questions on their neuroactive potency and provide strong motivation for further investigations into other toxic mechanisms.

BMAA Inhibits Nitrogen Fixation in the Cyanobacterium Nostoc sp. PCC 7120

PubMed Central

Berntzon, Lotta; Erasmie, Sven; Celepli, Narin; Eriksson, Johan; Rasmussen, Ulla; Bergman, Birgitta

2013-01-01

Cyanobacteria produce a range of secondary metabolites, one being the neurotoxic non-protein amino acid β-N-methylamino-L-alanine (BMAA), proposed to be a causative agent of human neurodegeneration. As for most cyanotoxins, the function of BMAA in cyanobacteria is unknown. Here, we examined the effects of BMAA on the physiology of the filamentous nitrogen-fixing cyanobacterium Nostoc sp. PCC 7120. Our data show that exogenously applied BMAA rapidly inhibits nitrogenase activity (acetylene reduction assay), even at micromolar concentrations, and that the inhibition was considerably more severe than that induced by combined nitrogen sources and most other amino acids. BMAA also caused growth arrest and massive cellular glycogen accumulation, as observed by electron microscopy. With nitrogen fixation being a process highly sensitive to oxygen species we propose that the BMAA effects found here may be related to the production of reactive oxygen species, as reported for other organisms. PMID:23966039

Searching for a link between the L-BMAA neurotoxin and amyotrophic lateral sclerosis: a study protocol of the French BMAALS programme

PubMed Central

Delzor, Aurélie; Couratier, Philippe; Boumédiène, Farid; Nicol, Marie; Druet-Cabanac, Michel; Paraf, François; Méjean, Annick; Ploux, Olivier; Leleu, Jean-Philippe; Brient, Luc; Lengronne, Marion; Pichon, Valérie; Combès, Audrey; El Abdellaoui, Saïda; Bonneterre, Vincent; Lagrange, Emmeline; Besson, Gérard; Bicout, Dominique J; Boutonnat, Jean; Camu, William; Pageot, Nicolas; Juntas-Morales, Raul; Rigau, Valérie; Masseret, Estelle; Abadie, Eric; Preux, Pierre-Marie; Marin, Benoît

2014-01-01

Introduction Amyotrophic lateral sclerosis (ALS) is the most common motor neurone disease. It occurs in two forms: (1) familial cases, for which several genes have been identified and (2) sporadic cases, for which various hypotheses have been formulated. Notably, the β-N-methylamino-L-alanine (L-BMAA) toxin has been postulated to be involved in the occurrence of sporadic ALS. The objective of the French BMAALS programme is to study the putative link between L-BMAA and ALS. Methods and analysis The programme covers the period from 1 January 2003 to 31 December 2011. Using multiple sources of ascertainment, all the incident ALS cases diagnosed during this period in the area under study (10 counties spread over three French regions) were collected. First, the standardised incidence ratio will be calculated for each municipality under concern. Then, by applying spatial clustering techniques, overincidence and underincidence zones of ALS will be sought. A case–control study, in the subpopulation living in the identified areas, will gather information about patients’ occupations, leisure activities and lifestyle habits in order to assess potential risk factors to which they are or have been exposed. Specimens of drinking water, food and biological material (brain tissue) will be examined to assess the presence of L-BMAA in the environment and tissues of ALS cases and controls. Ethics and dissemination The study has been reviewed and approved by the French ethical committee of the CPP SOOM IV (Comité de Protection des Personnes Sud-Ouest & Outre-Mer IV). The results will be published in peer-reviewed journals and presented at national and international conferences. PMID:25180055

Co-occurrence of the cyanotoxins BMAA, DABA and anatoxin-a in Nebraska reservoirs, fish, and aquatic plants.

PubMed

Al-Sammak, Maitham Ahmed; Hoagland, Kyle D; Cassada, David; Snow, Daniel D

2014-01-28

Several groups of microorganisms are capable of producing toxins in aquatic environments. Cyanobacteria are prevalent blue green algae in freshwater systems, and many species produce cyanotoxins which include a variety of chemical irritants, hepatotoxins and neurotoxins. Production and occurrence of potent neurotoxic cyanotoxins β-N-methylamino-L-alanine (BMAA), 2,4-diaminobutyric acid dihydrochloride (DABA), and anatoxin-a are especially critical with environmental implications to public and animal health. Biomagnification, though not well understood in aquatic systems, is potentially relevant to both human and animal health effects. Because little is known regarding their presence in fresh water, we investigated the occurrence and potential for bioaccumulation of cyanotoxins in several Nebraska reservoirs. Collection and analysis of 387 environmental and biological samples (water, fish, and aquatic plant) provided a snapshot of their occurrence. A sensitive detection method was developed using solid phase extraction (SPE) in combination with high pressure liquid chromatography-fluorescence detection (HPLC/FD) with confirmation by liquid chromatography-tandem mass spectrometry (LC/MS/MS). HPLC/FD detection limits ranged from 5 to 7 µg/L and LC/MS/MS detection limits were <0.5 µg/L, while detection limits for biological samples were in the range of 0.8-3.2 ng/g depending on the matrix. Based on these methods, measurable levels of these neurotoxic compounds were detected in approximately 25% of the samples, with detections of BMAA in about 18.1%, DABA in 17.1%, and anatoxin-a in 11.9%.

Co-occurrence of the Cyanotoxins BMAA, DABA and Anatoxin-a in Nebraska Reservoirs, Fish, and Aquatic Plants

PubMed Central

Al-Sammak, Maitham Ahmed; Hoagland, Kyle D.; Cassada, David; Snow, Daniel D.

2014-01-01

Several groups of microorganisms are capable of producing toxins in aquatic environments. Cyanobacteria are prevalent blue green algae in freshwater systems, and many species produce cyanotoxins which include a variety of chemical irritants, hepatotoxins and neurotoxins. Production and occurrence of potent neurotoxic cyanotoxins β-N-methylamino-l-alanine (BMAA), 2,4-diaminobutyric acid dihydrochloride (DABA), and anatoxin-a are especially critical with environmental implications to public and animal health. Biomagnification, though not well understood in aquatic systems, is potentially relevant to both human and animal health effects. Because little is known regarding their presence in fresh water, we investigated the occurrence and potential for bioaccumulation of cyanotoxins in several Nebraska reservoirs. Collection and analysis of 387 environmental and biological samples (water, fish, and aquatic plant) provided a snapshot of their occurrence. A sensitive detection method was developed using solid phase extraction (SPE) in combination with high pressure liquid chromatography-fluorescence detection (HPLC/FD) with confirmation by liquid chromatography-tandem mass spectrometry (LC/MS/MS). HPLC/FD detection limits ranged from 5 to 7 µg/L and LC/MS/MS detection limits were <0.5 µg/L, while detection limits for biological samples were in the range of 0.8–3.2 ng/g depending on the matrix. Based on these methods, measurable levels of these neurotoxic compounds were detected in approximately 25% of the samples, with detections of BMAA in about 18.1%, DABA in 17.1%, and anatoxin-a in 11.9%. PMID:24476710

Parkinson-dementia complex and development of a new stable isotope dilution assay for BMAA detection in tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Snyder, Laura R.; Cruz-Aguado, Reyniel; Sadilek, Martin

2009-10-15

{beta}-Methylamino-L-alanine (BMAA) has been proposed as a global contributor to neurodegenerative diseases, including Parkinson-dementia complex (PDC) of Guam and Alzheimer's disease (AD). The literature on the effects of BMAA is conflicting with some but not all in vitro data supporting a neurotoxic action, and experimental animal data failing to replicate the pattern of neurodegeneration of these human diseases, even at very high exposures. Recently, BMAA has been reported in human brain from individuals afflicted with PDC or AD. Some of the BMAA in human tissue reportedly is freely extractable (free) while some is protein-associated and liberated by techniques that hydrolyzemore » the peptide bond. The latter is especially intriguing since BMAA is a non-proteinogenic amino acid that has no known tRNA. We attempted to replicate these findings with techniques similar to those used by others; despite more than adequate sensitivity, we were unable to detect free BMAA. Recently, using a novel stable isotope dilution assay, we again were unable to detect free or protein-associated BMAA in human cerebrum. Here we review the development of our new assay for tissue detection of BMAA and show that we are able to detect free BMAA in liver but not cerebrum, nor do we detect any protein-associated BMAA in mice fed this amino acid. These studies demonstrate the importance of a sensitive and specific assay for tissue BMAA and seriously challenge the proposal that BMAA is accumulating in human brain.« less

Detection of the Cyanotoxins L-BMAA Uptake and Accumulation in Primary Neurons and Astrocytes.

PubMed

Tan, Vanessa X; Mazzocco, Claire; Varney, Bianca; Bodet, Dominique; Guillemin, Tristan A; Bessede, Alban; Guillemin, Gilles J

2018-01-01

We show for the first time that a newly developed polyclonal antibody (pAb) can specifically target the cyanotoxin β-methylamino-L-alanine (BMAA) and can be used to enable direct visualization of BMAA entry and accumulation in primary brain cells. We used this pAb to investigate the effect of acute and chronic accumulation, and toxicity of both BMAA and its natural isomer 2,4-diaminobutyric acid (DAB), separately or in combination, on primary cultures of rat neurons. We further present evidence that co-treatment with BMAA and DAB increased neuronal death, as measured by MAP2 fluorescence level, and appeared to reduce BMAA accumulation. DAB is likely to be acting synergistically with BMAA resulting in higher level of cellular toxicity. We also found that glial cells such as microglia and astrocytes are also able to directly uptake BMAA indicating that additional brain cell types are affected by BMAA-induced toxicity. Therefore, BMAA clearly acts at multiple cellular levels to possibly increase the risk of developing neurodegenerative diseases, including neuro- and gliotoxicity and synergetic exacerbation with other cyanotoxins.

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction.

PubMed

Faassen, Elisabeth J; Antoniou, Maria G; Beekman-Lukassen, Wendy; Blahova, Lucie; Chernova, Ekaterina; Christophoridis, Christophoros; Combes, Audrey; Edwards, Christine; Fastner, Jutta; Harmsen, Joop; Hiskia, Anastasia; Ilag, Leopold L; Kaloudis, Triantafyllos; Lopicic, Srdjan; Lürling, Miquel; Mazur-Marzec, Hanna; Meriluoto, Jussi; Porojan, Cristina; Viner-Mozzini, Yehudit; Zguna, Nadezda

2016-02-29

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D₃BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D₃BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Creating a Simian Model of Guam ALS/PDC Which Reflects Chamorro Lifetime BMAA Exposures.

PubMed

Banack, Sandra Anne; Cox, Paul Alan

2018-01-01

The theory that β-N-methylamino-L-alanine (BMAA), a cyanobacterial toxin, contaminates traditional food supplies of the Chamorro people of Guam is supported by the recent finding that chronic dietary exposure to L-BMAA in vervets (Chlorocebus sabaeus) triggers the formation of neurofibrillary tangles (NFT) and β-amyloid plaques in the brain. In the first experiment, we found that all four vervets receiving a 210 mg/kg dose for 140 days developed NFT and sparse amyloid deposits. In the second experiment, all eight vervets receiving a 210 mg/kg dose for 140 days developed NFT and amyloid deposits, as well as all eight vervets that received only 21 mg/kg. Based on dietary surveys of the Chamorro people, we estimated lifetime chronic BMAA exposure at a high and a low level: 1) adult male Chamorros eating two flying foxes per month plus one 30 g serving of cycad flour per week; and 2) adult male Chamorros eating one 30 g serving of cycad flour per day combined with the consumption of eight flying foxes per month. The resultant cumulative lifetime Chamorro exposures ranged from 1 to 41 g/kg and are comparable to the total lifetime vervet exposures in our experiments of 2 and 22 g/kg, respectively. Furthermore, measured protein-bound BMAA concentrations of vervets fed L-BMAA powder are comparable to measured protein-bound BMAA concentrations in postmortem brain tissues of Chamorros who died with ALS/PDC.

Consumption of fa cai Nostoc soup: a potential for BMAA exposure from Nostoc cyanobacteria in China?

PubMed

Roney, Britton R; Renhui, Li; Banack, Sandra Anne; Murch, Susan; Honegger, Rosmarie; Cox, Paul Alan

2009-01-01

Grown in arid regions of western China the cyanobacterium Nostoc flagelliforme--called fa cai in Mandarin and fat choy in Cantonese--is wild-harvested and used to make soup consumed during New Year's celebrations. High prices, up to $125 USD/kg, led to overharvesting in Inner Mongolia, Ningxia, Gansu, Qinghai, and Xinjiang. Degradation of arid ecosystems, desertification, and conflicts between Nostoc harvesters and Mongol herdsmen concerned the Chinese environmental authorities, leading to a government ban of Nostoc commerce. This ban stimulated increased marketing of a substitute made from starch. We analysed samples purchased throughout China as well as in Chinese markets in the United States and the United Kingdom. Some were counterfeits consisting of dyed starch noodles. A few samples from California contained Nostoc flagelliforme but were adulterated with starch noodles. Other samples, including those from the United Kingdom, consisted of pure Nostoc flagelliforme. A recent survey of markets in Cheng Du showed no real Nostoc flagelliforme to be marketed. Real and artificial fa cai differ in the presence of beta-N-methylamino-L-alanine (BMAA). Given its status as a high-priced luxury food, the government ban on collection and marketing, and the replacement of real fa cai with starch substitutes consumed only on special occasions, it is anticipated that dietary exposure to BMAA from fa cai will be reduced in the future in China.

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

PubMed Central

Faassen, Elisabeth J.; Antoniou, Maria G.; Beekman-Lukassen, Wendy; Blahova, Lucie; Chernova, Ekaterina; Christophoridis, Christophoros; Combes, Audrey; Edwards, Christine; Fastner, Jutta; Harmsen, Joop; Hiskia, Anastasia; Ilag, Leopold L.; Kaloudis, Triantafyllos; Lopicic, Srdjan; Lürling, Miquel; Mazur-Marzec, Hanna; Meriluoto, Jussi; Porojan, Cristina; Viner-Mozzini, Yehudit; Zguna, Nadezda

2016-01-01

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis. PMID:26938542

Cytotoxic Effects of Environmental Toxins on Human Glial Cells.

PubMed

D'Mello, Fiona; Braidy, Nady; Marçal, Helder; Guillemin, Gilles; Rossi, Fanny; Chinian, Mirielle; Laurent, Dominique; Teo, Charles; Neilan, Brett A

2017-02-01

Toxins produced by cyanobacteria and dinoflagellates have increasingly become a public health concern due to their degenerative effects on mammalian tissue and cells. In particular, emerging evidence has called attention to the neurodegenerative effects of the cyanobacterial toxin β-N-methylamino-L-alanine (BMAA). Other toxins such as the neurotoxins saxitoxin and ciguatoxin, as well as the hepatotoxic microcystin, have been previously shown to have a range of effects upon the nervous system. However, the capacity of these toxins to cause neurodegeneration in human cells has not, to our knowledge, been previously investigated. This study aimed to examine the cytotoxic effects of BMAA, microcystin-LR (MC-LR), saxitoxin (STX) and ciguatoxin (CTX-1B) on primary adult human astrocytes. We also demonstrated that α-lipoate attenuated MC-LR toxicity in primary astrocytes and characterised changes in gene expression which could potentially be caused by these toxins in primary astrocytes. Herein, we are the first to show that all of these toxins are capable of causing physiological changes consistent with neurodegeneration in glial cells, via oxidative stress and excitotoxicity, leading to a reduction in cell proliferation culminating in cell death. In addition, MC-LR toxicity was reduced significantly in astrocytes-treated α-lipoic acid. While there were no significant changes in gene expression, many of the probes that were altered were associated with neurodegenerative disease pathogenesis. Overall, this is important in advancing our current understanding of the mechanism of toxicity of MC-LR on human brain function in vitro, particularly in the context of neurodegeneration.

UPLC-ESI-MS/MS method for the quantitative measurement of aliphatic diamines, trimethylamine N-oxide, and β-methylamino-l-alanine in human urine.

PubMed

Bhandari, Deepak; Bowman, Brett A; Patel, Anish B; Chambers, David M; De Jesús, Víctor R; Blount, Benjamin C

2018-04-15

This work describes a quantitative high-throughput analytical method for the simultaneous measurement of small aliphatic nitrogenous biomarkers, i.e., 1,6-hexamethylenediamine (HDA), isophoronediamine (IPDA), β-methylamino-l-alanine (BMAA), and trimethylamine N-oxide (TMAO), in human urine. Urinary aliphatic diamines, HDA and IPDA, are potential biomarkers of environmental exposure to their corresponding diisocyanates. Urinary BMAA forms as a result of human exposure to blue-green algae contaminated food. And, TMAO is excreted in urine due to the consumption of carnitine- and choline-rich diets. These urinary biomarkers represent classes of small aliphatic nitrogen-containing compounds (N-compounds) that have a high aqueous solubility, low logP, and/or high basic pK a . Because of the highly polar characteristics, analysis of these compounds in complex sample matrices is often challenging. We report on the development of ion-pairing chemistry based ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method for the simultaneous measurement of these biomarkers in human urine. Chromatographic separation was optimized using heptafluorobutyric acid-(HFBA-) based mobile phase and a reversed-phase C18 column. All four analytes were baseline separated within 2.6 min with an overall run time of 5 min per sample injection. Sample preparation involved 4 h of acid hydrolysis followed by automated solid phase extraction (SPE) performed using strong cation exchange sorbent bed with 7 N ammonia solution in methanol as eluent. Limits of detection ranged from 0.05 ng/mL to 1.60 ng/mL. The inter-day and intra-day accuracy were within 10%, and reproducibility within 15%. The method is accurate, fast, and well-suited for biomonitoring studies within targeted groups, as well as larger population-based studies such as the U. S. National Health and Nutrition Examination Survey (NHANES). Published by Elsevier B.V.

Responses of the antioxidative and biotransformation enzymes in the aquatic fungus Mucor hiemalis exposed to cyanotoxins.

PubMed

Balsano, Evelyn; Esterhuizen-Londt, Maranda; Hoque, Enamul; Lima, Stephan Pflugmacher

2017-08-01

To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications. Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M. hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100 µg MC-LR l -1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100 µg l -1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-L-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected. M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

Transport of BMAA into Neurons and Astrocytes by System xc.

PubMed

Albano, Rebecca; Lobner, Doug

2018-01-01

The study of the mechanism of β-N-methylamino-L-alanine (BMAA) neurotoxicity originally focused on its effects at the N-methyl-D-aspartate (NMDA) receptor. In recent years, it has become clear that its mechanism of action is more complicated. First, there are certain cell types, such as motor neurons and cholinergic neurons, where the dominate mechanism of toxicity is through action at AMPA receptors. Second, even in cortical neurons where the primary mechanism of toxicity appears to be activation of NMDA receptors, there are other mechanisms involved. We found that along with NMDA receptors, activation of mGLuR5 receptors and effects on the cystine/glutamate antiporter (system x c -) were involved in the toxicity. The effects on system x c - are of particular interest. System x c - mediates the transport of cystine into the cell in exchange for releasing glutamate into the extracellular fluid. By releasing glutamate, system x c - can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and in this way may protect cells against oxidative stress. We have previously published that BMAA inhibits cystine uptake leading to GSH depletion and had indirect evidence that BMAA is transported into the cells by system x c -. We now present direct evidence that BMAA is transported into both astrocytes and neurons through system x c -. The fact that BMAA is transported by system x c - also provides a mechanism for BMAA to enter brain cells potentially leading to misincorporation into proteins and protein misfolding.

The magical and medicinal usage of Stangeria eriopus in South Africa.

PubMed

Osborne, R; Grove, A; Oh, P; Mabry, T J; Ng, J C; Seawright, A A

1994-07-08

The underground caudex of the cycad Stangeria eriopus is used extensively by several ethnic groups in South Africa, mainly as an ingredient in magical potions but also as an emetic. An assessment of two main outlets showed that 3410 plants were sold in the month of July 1992; continued usage of this material now threatens the remaining plant populations. A proximate analysis of the caudex material gives high carbohydrate content with only small percentages of fat, protein, fibre and ash. An unusually high content of sodium sulphate may explain the efficacy of Stangeria-containing preparations as an emetic. The phytosterols sitosterol and stigmasterol are present in a 4:1 ratio while the fatty acid component comprises palmitic, oleic, stearic and arachidic acids. Twelve amino acids were identified in the material, including the non-protein amino acids beta-alanine, gamma-aminobutyric acid and pyroglutamic acid. The candidate neurotoxin beta-N-methylamino-L-alanine could not be detected but cycasin is present at the levels of 0.17% and 0.21% in fresh and dry caudex material, respectively and appears to be accompanied by the related toxin, macrozamin.

Cyanotoxins at low doses induce apoptosis and inflammatory effects in murine brain cells: Potential implications for neurodegenerative diseases.

PubMed

Takser, Larissa; Benachour, Nora; Husk, Barry; Cabana, Hubert; Gris, Denis

2016-01-01

Cyanotoxins have been shown to be highly toxic for mammalian cells, including brain cells. However, little is known about their effect on inflammatory pathways. This study investigated whether mammalian brain and immune cells can be a target of certain cyanotoxins, at doses approximating those in the guideline levels for drinking water, either alone or in mixtures. We examined the effects on cellular viability, apoptosis and inflammation signalling of several toxins on murine macrophage-like RAW264.7, microglial BV-2 and neuroblastoma N2a cell lines. We tested cylindrospermopsin (CYN), microcystin-LR (MC-LR), and anatoxin-a (ATX-a), individually as well as their mixture. In addition, we studied the neurotoxins β- N -methylamino-l-alanine (BMAA) and its isomer 2,4-diaminobutyric acid (DAB), as well as the mixture of both. Cellular viability was determined by the MTT assay. Apoptosis induction was assessed by measuring the activation of caspases 3/7. Cell death and inflammation are the hallmarks of neurodegenerative diseases. Thus, our final step was to quantify the expression of a major proinflammatory cytokine TNF-α by ELISA. Our results show that CYN, MC-LR and ATX-a, but not BMAA and DAB, at low doses, especially when present in a mixture at threefold less concentrations than individual compounds are 3-15 times more potent at inducing apoptosis and inflammation. Our results suggest that common cyanotoxins at low doses have a potential to induce inflammation and apoptosis in immune and brain cells. Further research of the neuroinflammatory effects of these compounds in vivo is needed to improve safety limit levels for cyanotoxins in drinking water and food.

Paralytic Shellfish Toxins and Cyanotoxins in the Mediterranean: New Data from Sardinia and Sicily (Italy).

PubMed

Lugliè, Antonella; Giacobbe, Maria Grazia; Riccardi, Elena; Bruno, Milena; Pigozzi, Silvia; Mariani, Maria Antonietta; Satta, Cecilia Teodora; Stacca, Daniela; Bazzoni, Anna Maria; Caddeo, Tiziana; Farina, Pasqualina; Padedda, Bachisio Mario; Pulina, Silvia; Sechi, Nicola; Milandri, Anna

2017-11-16

Harmful algal blooms represent a severe issue worldwide. They affect ecosystem functions and related services and goods, with consequences on human health and socio-economic activities. This study reports new data on paralytic shellfish toxins (PSTs) from Sardinia and Sicily (Italy), the largest Mediterranean islands where toxic events, mainly caused by Alexandrium species (Dinophyceae), have been ascertained in mussel farms since the 2000s. The toxicity of the A. minutum, A. tamarense and A. pacificum strains, established from the isolation of vegetative cells and resting cysts, was determined by high performance liquid chromatography (HPLC). The analyses indicated the highest toxicity for A. pacificum strains (total PSTs up to 17.811 fmol cell-1). The PSTs were also assessed in a strain of A. tamarense. The results encourage further investigation to increase the knowledge of toxic species still debated in the Mediterranean. This study also reports new data on microcystins (MCs) and β-N-methylamino-L-alanine (BMAA) from a Sardinian artificial lake (Lake Bidighinzu). The presence of MCs and BMAA was assessed in natural samples and in cell cultures by enzyme-linked immunosorbent assay (ELISA). BMAA positives were found in all the analysed samples with a maximum of 17.84 µg L-1. The obtained results added further information on cyanotoxins in Mediterranean reservoirs, particularly BMAA, which have not yet been thoroughly investigated.

Paralytic Shellfish Toxins and Cyanotoxins in the Mediterranean: New Data from Sardinia and Sicily (Italy)

PubMed Central

Giacobbe, Maria Grazia; Riccardi, Elena; Bruno, Milena; Pigozzi, Silvia; Mariani, Maria Antonietta; Stacca, Daniela; Caddeo, Tiziana; Farina, Pasqualina; Padedda, Bachisio Mario; Pulina, Silvia; Sechi, Nicola; Milandri, Anna

2017-01-01

Harmful algal blooms represent a severe issue worldwide. They affect ecosystem functions and related services and goods, with consequences on human health and socio-economic activities. This study reports new data on paralytic shellfish toxins (PSTs) from Sardinia and Sicily (Italy), the largest Mediterranean islands where toxic events, mainly caused by Alexandrium species (Dinophyceae), have been ascertained in mussel farms since the 2000s. The toxicity of the A. minutum, A. tamarense and A. pacificum strains, established from the isolation of vegetative cells and resting cysts, was determined by high performance liquid chromatography (HPLC). The analyses indicated the highest toxicity for A. pacificum strains (total PSTs up to 17.811 fmol cell−1). The PSTs were also assessed in a strain of A. tamarense. The results encourage further investigation to increase the knowledge of toxic species still debated in the Mediterranean. This study also reports new data on microcystins (MCs) and β-N-methylamino-l-alanine (BMAA) from a Sardinian artificial lake (Lake Bidighinzu). The presence of MCs and BMAA was assessed in natural samples and in cell cultures by enzyme-linked immunosorbent assay (ELISA). BMAA positives were found in all the analysed samples with a maximum of 17.84 µg L−1. The obtained results added further information on cyanotoxins in Mediterranean reservoirs, particularly BMAA, which have not yet been thoroughly investigated. PMID:29144421

Methods for simultaneous detection of the cyanotoxins BMAA, DABA, and anatoxin-a in environmental samples.

PubMed

Al-Sammak, Maitham Ahmed; Hoagland, Kyle D; Snow, Daniel D; Cassada, David

2013-12-15

Blue-green algae, also known as cyanobacteria, can produce several different groups of toxins in the environment including hepatotoxins (microcystins), neurotoxic non-protein amino acids β-methylamino-l-alanine (BMAA), and 2,4-diaminobutyric (DABA), as well as the bicyclic amine alkaloid anatoxin-a. Few studies have addressed the methods necessary for an accurate determination of cyanotoxins in environmental samples, and none have been published that can detect these cyanotoxins together in a single sample. Cyanotoxins occur in a wide range of environmental samples including water, fish, and aquatic plant samples. Using polymeric cation exchange solid phase extraction (SPE) coupled with liquid chromatography and fluorescence detection (HPLC/FD), and liquid chromatography ion trap tandem mass spectrometry (LC/MS/MS), these compounds can for the first time be simultaneously quantified in a variety of environmental sample types. The extraction method for biological samples can distinguish bound and free cyanotoxins. Detection limits for water ranged from 5 to 7 μg/L using HPLC/FD, while detection limits for and LC/MS were in the range of 0.8-3.2 μg/L. Copyright © 2013 Elsevier Ltd. All rights reserved.

Is exposure to cyanobacteria an environmental risk factor for amyotrophic lateral sclerosis and other neurodegenerative diseases?

USGS Publications Warehouse

Bradley, Walter G.; Borenstein, Amy R.; Nelson, Lorene M.; Codd, Geoffrey A.; Rosen, Barry H.; Stommel, Elijah W.; Cox, Paul Alan

2013-01-01

There is a broad scientific consensus that amyotrophic lateral sclerosis (ALS) is caused by gene-environment interactions. Mutations in genes underlying familial ALS (fALS) have been discovered in only 5–10% of the total population of ALS patients. Relatively little attention has been paid to environmental and lifestyle factors that may trigger the cascade of motor neuron death leading to the syndrome of ALS, although exposure to chemicals including lead and pesticides, and to agricultural environments, smoking, certain sports, and trauma have all been identified with an increased risk of ALS. There is a need for research to quantify the relative roles of each of the identified risk factors for ALS. Recent evidence has strengthened the theory that chronic environmental exposure to the neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) produced by cyanobacteria may be an environmental risk factor for ALS. Here we describe methods that may be used to assess exposure to cyanobacteria, and hence potentially to BMAA, namely an epidemiologic questionnaire and direct and indirect methods for estimating the cyanobacterial load in ecosystems. Rigorous epidemiologic studies could determine the risks associated with exposure to cyanobacteria, and if combined with genetic analysis of ALS cases and controls could reveal etiologically important gene-environment interactions in genetically vulnerable individuals.

Determination of BMAA and three alkaloid cyanotoxins in lake water using dansyl chloride derivatization and high-resolution mass spectrometry.

PubMed

Roy-Lachapelle, Audrey; Solliec, Morgan; Sauvé, Sébastien

2015-07-01

A new analytical method was developed for the detection of alkaloid cyanotoxins in harmful algal blooms. The detection of the nonproteinogenic amino acid β-N-methylamino-L-alanine (BMAA) and two of its conformation isomers, 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl) glycine (AEG), as well as three alkaloid cyanotoxins, anatoxin-a (ANA-a), cylindrospermopsin (CYN), and saxitoxin (STX), is presented. The use of a chemical derivatization with dansyl chloride (DNS) allows easier separation with reversed phase liquid chromatography. Detection with high-resolution mass spectrometry (HRMS) with the Q-Exactive enables high selectivity with specific fragmentation as well as exact mass detection, reducing considerably the possibilities of isobaric interferences. Previous to analysis, a solid phase extraction (SPE) step is used for purification and preconcentration. After DNS derivatization, samples are submitted to ultra high-performance liquid chromatography coupled with heated electrospray ionisation and the Q-Exactive mass spectrometer (UHPLC-HESI-HRMS). With an internal calibration using isotopically-labeled DAB-D3, the method was validated with good linearity (R (2)  > 0.998), and method limits of detection and quantification (MLD and MLQ) for target compounds ranged from 0.007 to 0.01 μg L(-1) and from 0.02 to 0.04 μg L(-1), respectively. Accuracy and within-day/between-days variation coefficients were below 15%. SPE recovery values ranged between 86 and 103%, and matrix effects recovery values ranged between 75 and 96%. The developed analytical method was successfully validated with 12 different lakes samples, and concentrations were found ranging between 0.009 and 0.3 μg L(-1) except for STX which was not found in any sample.

Metal Dyshomeostasis and Inflammation in Alzheimer's and Parkinson's Diseases: Possible Impact of Environmental Exposures

PubMed Central

Myhre, Oddvar; Utkilen, Hans; Duale, Nur; Brunborg, Gunnar; Hofer, Tim

2013-01-01

A dysregulated metal homeostasis is associated with both Alzheimer's (AD) and Parkinson's (PD) diseases; AD patients have decreased cortex and elevated serum copper levels along with extracellular amyloid-beta plaques containing copper, iron, and zinc. For AD, a putative hepcidin-mediated lowering of cortex copper mechanism is suggested. An age-related mild chronic inflammation and/or elevated intracellular iron can trigger hepcidin production followed by its binding to ferroportin which is the only neuronal iron exporter, thereby subjecting it to lysosomal degradation. Subsequently raised neuronal iron levels can induce translation of the ferroportin assisting and copper binding amyloid precursor protein (APP); constitutive APP transmembrane passage lowers the copper pool which is important for many enzymes. Using in silico gene expression analyses, we here show significantly decreased expression of copper-dependent enzymes in AD brain and metallothioneins were upregulated in both diseases. Although few AD exposure risk factors are known, AD-related tauopathies can result from cyanobacterial microcystin and β-methylamino-L-alanine (BMAA) intake. Several environmental exposures may represent risk factors for PD; for this disease neurodegeneration is likely to involve mitochondrial dysfunction, microglial activation, and neuroinflammation. Administration of metal chelators and anti-inflammatory agents could affect disease outcomes. PMID:23710288

Characterizing lake water quality, cyanotoxins, and Amyotrophic Lateral Sclerosis (ALS).

NASA Astrophysics Data System (ADS)

Torbick, N.; Ziniti, B.; Stommel, E.; Linder, E.; Andrew, A.; Bradley, W.; Shi, X.

2016-12-01

Concern over toxins and public health threats resulting from Cyanobacterial Harmful Algal Blooms (CHABs) have gained attention as reoccurring and seasonal blooms persist in many waters. Concordantly, climate change has been suggested to increase the intensity, duration, and frequency of CHAB events. Humans may be exposed to the cyanotoxins produced by cyanobacteria via the food chain, drinking water, recreational use of waterbodies and by aerosolization. Exposure to the cyanobacterial neurotoxin, β-N-methylamino-L-alanine (BMAA) that has been found in the brains of ALS patients is a hypothesized mechanism. The goals of this research initiative are to investigate spatiotemporal relationships between inland lake water quality and ALS across northern New England (NNE). Multiscale satellite remote sensing was integrated with in situ lake and toxin sampling to provide robust spatiotemporal exposure risk metrics characterizing CHAB. Semi-analytical, shape, and empirical algorithms were bldned together tp generate spatiotemporal measures of chl-a and PC with R2 ranging from 0.65-0.92 using withheld samples. Postmortem aerosolization analysis found 85% of high risk patients to express phycobillin in lung tissue using fluroesence microscopy. To scal eup to the region we employed complementing spatial statistics and a Bayesian hierarchical framework to model relationships between lake risk metrics and ALS case location across NNE. The eco-epidemiolgical modeling results show that on average poorer water quality conditions and higher measures of cyanobacteria are associated with increased odds of belonging to a normalized ALS hot spots and risk of ALS. This has broad societal impacts as the frequency, duration, and magnitude of cyanobacterial harmful algal blooms are expanding and this work helps characterize lake ecosystem services and human health.

Studies of Environmental Risk Factors in Amyotrophic Lateral Sclerosis (ALS) and a Phase I Clinical Trial of L-Serine.

PubMed

Bradley, Walter G; Miller, R X; Levine, T D; Stommel, E W; Cox, P A

2018-01-01

β-N-Methylamino-L-alanine (BMAA) has been linked to Guam ALS/PDC and shown to produce neurodegeneration in vitro and in vivo (Drosophila, mice, rats, primates). BMAA misincorporation into neuroproteins produces protein misfolding and is inhibited by L-serine. Case-control studies in Northern New England indicate that living near to water-bodies with cyanobacterial blooms increases the risk of developing amyotrophic lateral sclerosis (ALS). The distribution of addresses of ALS cases in New Hampshire, Vermont, and Florida was compared to that of controls. Areas of statistically significantly increased numbers of ALS cases were examined for sources of environmental toxins. A phase I trial of oral L-serine was performed in 20 ALS patients (0.5 to 15 g twice daily). Safety and tolerability were assessed by comparing the rate of deterioration with 430 matched placebo controls. The distribution of residential addresses of ALS cases in New England and Florida revealed many areas where the age- and gender-adjusted frequency of ALS was greater than expected (P < 0.01). GIS studies of these "hot spots" in relation to sources of environmental pollutants, like cyanobacterial blooms, Superfund and Brownfield sites, and landfills, are ongoing. In the phase I trial of L-serine, two patients withdrew from because of gastrointestinal side effects. Three patients died during the study, which was about the expected number. The ALSFRS-R in the L-serine-treated patients showed a dose-related decrease in the rate of progression (34% reduction in slope, P = 0.044). The non-random distribution of addresses of ALS patients suggests that residential exposure to environmental pollutants may play an important role in the etiology of ALS. L-Serine in doses up to 15 g twice daily appears to be safe in patients with ALS. Exploratory studies of efficacy suggested that L-serine might slow disease progression. A phase II trial is planned.

Metabolic solutions to the biosynthesis of some diaminomonocarboxylic acids in nature: Formation in cyanobacteria of the neurotoxins 3-N-methyl-2,3-diaminopropanoic acid (BMAA) and 2,4-diaminobutanoic acid (2,4-DAB).

PubMed

Nunn, Peter B; Codd, Geoffrey A

2017-12-01

The non-encoded diaminomonocarboxylic acids, 3-N-methyl-2,3-diaminopropanoic acid (syn: α-amino-β-methylaminopropionic acid, MeDAP; β-N-methylaminoalanine, BMAA) and 2,4-diaminobutanoic acid (2,4-DAB), are distributed widely in cyanobacterial species in free and bound forms. Both amino acids are neurotoxic in whole animal and cell-based bioassays. The biosynthetic pathway to 2,4-DAB is well documented in bacteria and in one higher plant species, but has not been confirmed in cyanobacteria. The biosynthetic pathway to BMAA is unknown. This review considers possible metabolic routes, by analogy with reactions used in other species, by which these amino acids might be biosynthesised by cyanobacteria, which are a widespread potential environmental source of these neurotoxins. Where possible, the gene expression that might be implicated in these biosyntheses is discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pyrylium Salts as Reactive Matrices for MALDI-MS Imaging of Biologically Active Primary Amines

NASA Astrophysics Data System (ADS)

Shariatgorji, Mohammadreza; Nilsson, Anna; Källback, Patrik; Karlsson, Oskar; Zhang, Xiaoqun; Svenningsson, Per; Andren, Per E.

2015-06-01

Many neuroactive substances, including endogenous biomolecules, environmental compounds, and pharmaceuticals possess primary amine functional groups. Among these are catecholamine neurotransmitters (e.g., dopamine), many substituted phenethylamines (e.g., amphetamine), as well as amino acids and neuropeptides. In most cases, mass spectrometric (ESI and MALDI) analyses of trace amounts of such compounds are challenging because of their poor ionization properties. We present a method for chemical derivatization of primary amines by reaction with pyrylium salts that facilitates their detection by MALDI-MS and enables the imaging of primary amines in brain tissue sections. A screen of pyrylium salts revealed that the 2,4-diphenyl-pyranylium ion efficiently derivatizes primary amines and can be used as a reactive MALDI-MS matrix that induces both derivatization and desorption. MALDI-MS imaging with such matrix was used to map the localization of dopamine and amphetamine in brain tissue sections and to quantitatively map the distribution of the neurotoxin β- N-methylamino-L-alanine.

Animal Models of Brain Maldevelopment Induced by Cycad Plant Genotoxins

PubMed Central

Kisby, Glen E.; Moore, Holly; Spencer, Peter S.

2014-01-01

Cycads are long-lived tropical and subtropical plants that contain azoxyglycosides (e.g., cycasin, macrozamin) and neurotoxic amino acids (notably β-N-methylamino-L-alanine L-BMAA), toxins that have been implicated in the etiology of a disappearing neurodegenerative disease, amyotrophic lateral sclerosis and parkinsonism-dementia complex that has been present in high incidence among three genetically distinct populations in the western Pacific. The neuropathology of amyotrophic lateral sclerosis/parkinsonism-dementia complex includes features suggestive of brain maldevelopment, an experimentally proven property of cycasin attributable to the genotoxic action of its aglycone methylazoxymethanol (MAM). This property of MAM has been exploited by neurobiologists as a tool to study perturbations of brain development. Depending on the neurodevelopmental stage, MAM can induce features in laboratory animals that model certain characteristics of epilepsy, schizophrenia, or ataxia. Studies in DNA repair-deficient mice show that MAM perturbs brain development through a DNA damage-mediated mechanism. The brain DNA lesions produced by systemic MAM appear to modulate the expression of genes that regulate neurodevelopment and contribute to neurodegeneration. Epigenetic changes (histone lysine methylation) have also been detected in the underdeveloped brain after MAM administration. The DNA damage and epigenetic changes produced by MAM and, perhaps by chemically related substances (e.g., nitrosamines, nitrosoureas, hydrazines), might be an important mechanism by which early-life exposure to genotoxicants can induce long-term brain dysfunction. PMID:24339036

Metabolism of designer drugs of abuse: an updated review.

PubMed

Meyer, Markus R; Maurer, Hans H

2010-06-01

This paper reviews the metabolism of new designer drugs of abuse that have emerged on the black market during the last years and is an update of a review published in 2005. The presented review contains data concerning the so-called 2C compounds (phenethylamine type) such as 4-bromo-2,5-dimethoxy-beta-phenethylamine (2C-B), 4-iodo-2,5-dimethoxy-beta-phenethylamine (2C-I), 2,5-dimethoxy-4-methyl-beta-phenethylamine (2C-D), 4-ethyl-2,5-dimethoxy-beta-phenethylamine (2C-E), 4-ethylthio-2,5-dimethoxy-beta-phenethylamine (2C-T-2), and 2,5-dimethoxy-4-propylthio-beta-phenethylamine (2C-T-7), beta-keto designer drugs such as 2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one (butylone, bk-MBDB), 2-ethylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (ethylone, bk-MDEA), 2-methylamino-1-(3,4-methylene notdioxy notphenyl)propan-1-one (methylone, bk-MDMA), and 2-methylamino-1-p-tolylpropane-1-one (mephedrone, 4-methyl-methcathinone), pyrrolidino notphenones such as 4-methyl-pyrrolidinobutyrophenone (MPBP) and alpha-pyrrolidinovalerophenone (PVP), phencyclidine-derived drugs such as N (1 phenylcyclohexyl) propanamine (PCPr), N-(1-phenylcyclohexyl)-2-ethoxyethanamine (PCEEA), N-(1-phenylcyclohexyl)-3-methoxypropanamine (PCMPA), and N-(1-phenylcyclohexyl)-2-methoxyethanamine (PCMEA), tryptamines such as 5-methoxy-N,N-diisopropyl nottryptamine (5-MeO-DIPT), and finally alpha-methylfentanyl (alpha-MF) and 3-methylfentanyl (3-MF). Papers have been considered and reviewed on the identification of in vivo or in vitro human or animal metabolites and the cytochrome P450 or monoamineoxidase isoenzyme-dependent metabolism.

The Effects of the Toxic Cyanobacterium Limnothrix (Strain AC0243) on Bufo marinus Larvae

PubMed Central

Daniels, Olivia; Fabbro, Larelle; Makiela, Sandrine

2014-01-01

Limnothrix (strain AC0243) is a cyanobacterium, which has only recently been identified as toxin producing. Under laboratory conditions, Bufo marinus larvae were exposed to 100,000 cells mL−1 of Limnothrix (strain AC0243) live cultures for seven days. Histological examinations were conducted post mortem and revealed damage to the notochord, eyes, brain, liver, kidney, pancreas, gastrointestinal tract, and heart. The histopathological results highlight the toxicological impact of this strain, particularly during developmental stages. Toxicological similarities to β-N-Methylamino-l-alanine are discussed. PMID:24662524

Structural and functional characterization of a novel homodimeric three-finger neurotoxin from the venom of Ophiophagus hannah (king cobra).

PubMed

Roy, Amrita; Zhou, Xingding; Chong, Ming Zhi; D'hoedt, Dieter; Foo, Chun Shin; Rajagopalan, Nandhakishore; Nirthanan, Selvanayagam; Bertrand, Daniel; Sivaraman, J; Kini, R Manjunatha

2010-03-12

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (alphabetagammadelta) and neuronal (alpha(7), alpha(3)beta(2), and alpha(4)beta(2)) nicotinic acetylcholine receptors (nAChRs) with highest affinity for alpha(7)-nAChRs. The high resolution (1.5 A) crystal structure revealed haditoxin to be a homodimer, like kappa-neurotoxins, which target neuronal alpha(3)beta(2)- and alpha(4)beta(2)-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain alpha-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain alpha-neurotoxins and kappa-neurotoxins notwithstanding, haditoxin exhibited unique blockade of alpha(7)-nAChRs (IC(50) 180 nm), which is recognized by neither short-chain alpha-neurotoxins nor kappa-neurotoxins. This is the first report of a dimeric short-chain alpha-neurotoxin interacting with neuronal alpha(7)-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.

Grazing livestock are exposed to terrestrial cyanobacteria.

PubMed

McGorum, Bruce C; Pirie, R Scott; Glendinning, Laura; McLachlan, Gerry; Metcalf, James S; Banack, Sandra A; Cox, Paul A; Codd, Geoffrey A

2015-02-25

While toxins from aquatic cyanobacteria are a well-recognised cause of disease in birds and animals, exposure of grazing livestock to terrestrial cyanobacteria has not been described. This study identified terrestrial cyanobacteria, predominantly Phormidium spp., in the biofilm of plants from most livestock fields investigated. Lower numbers of other cyanobacteria, microalgae and fungi were present on many plants. Cyanobacterial 16S rDNA, predominantly from Phormidium spp., was detected in all samples tested, including 6 plant washings, 1 soil sample and ileal contents from 2 grazing horses. Further work was performed to test the hypothesis that ingestion of cyanotoxins contributes to the pathogenesis of some currently unexplained diseases of grazing horses, including equine grass sickness (EGS), equine motor neuron disease (EMND) and hepatopathy. Phormidium population density was significantly higher on EGS fields than on control fields. The cyanobacterial neurotoxic amino acid 2,4-diaminobutyric acid (DAB) was detected in plant washings from EGS fields, but worst case scenario estimations suggested the dose would be insufficient to cause disease. Neither DAB nor the cyanobacterial neurotoxins β-N-methylamino-L-alanine and N-(2-aminoethyl) glycine were detected in neural tissue from 6 EGS horses, 2 EMND horses and 7 control horses. Phormidium was present in low numbers on plants where horses had unexplained hepatopathy. This study did not yield evidence linking known cyanotoxins with disease in grazing horses. However, further study is warranted to identify and quantify toxins produced by cyanobacteria on livestock fields, and determine whether, under appropriate conditions, known or unknown cyanotoxins contribute to currently unexplained diseases in grazing livestock.

Amyotrophic lateral sclerosis and environmental factors

PubMed Central

Bozzoni, Virginia; Pansarasa, Orietta; Diamanti, Luca; Nosari, Guido; Cereda, Cristina; Ceroni, Mauro

2016-01-01

Summary Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that affects central and peripheral motor neuron cells. Its etiology is unknown, although a relationship between genetic background and environmental factors may play a major role in triggering the neurodegeneration. In this review, we analyze the role of environmental factors in ALS: heavy metals, electromagnetic fields and electric shocks, pesticides, β-N-methylamino-L-alanine, physical activity and the controversial role of sports. The literature on the single issues is analyzed in an attempt to clarify, as clearly as possible, whether each risk factor significantly contributes to the disease pathogenesis. After summarizing conflicting observations and data, the authors provide a final synthetic statement. PMID:27027889

Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity.

PubMed Central

Gojković, Z; Sandrini, M P; Piskur, J

2001-01-01

beta-Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamyl-beta-alanine as the sole nitrogen source and exhibits diminished beta-alanine synthase activity was used to clone analogous genes from different eukaryotes. Putative PYD3 sequences from the yeast S. kluyveri, the slime mold Dictyostelium discoideum, and the fruit fly Drosophila melanogaster complemented the pyd3 defect. When the S. kluyveri PYD3 gene was expressed in S. cerevisiae, which has no pyrimidine catabolic pathway, it enabled growth on N-carbamyl-beta-alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta-alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial N-carbamyl amidohydrolases. All three beta-alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N-carbamyl-beta-alanine, but not by uracil. This work establishes S. kluyveri as a model organism for studying pyrimidine degradation and beta-alanine production in eukaryotes. PMID:11454750

Detection of Cyanotoxins in Algae Dietary Supplements

PubMed Central

Roy-Lachapelle, Audrey; Solliec, Morgan; Bouchard, Maryse F.; Sauvé, Sébastien

2017-01-01

Algae dietary supplements are marketed worldwide as natural health products. Although their proprieties have been claimed as beneficial to improve overall health, there have been several previous reports of contamination by cyanotoxins. These products generally contain non-toxic cyanobacteria, but the methods of cultivation in natural waters without appropriate quality controls allow contamination by toxin producer species present in the natural environment. In this study, we investigated the presence of total microcystins, seven individual microcystins (RR, YR, LR, LA, LY, LW, LF), anatoxin-a, dihydroanatoxin-a, epoxyanatoxin-a, cylindrospermopsin, saxitoxin, and β-methylamino-l-alanine in 18 different commercially available products containing Spirulina or Aphanizomenon flos-aquae. Total microcystins analysis was accomplished using a Lemieux oxidation and a chemical derivatization using dansyl chloride was needed for the simultaneous analysis of cylindrospermopsin, saxitoxin, and β-methylamino-l-alanine. Moreover, the use of laser diode thermal desorption (LDTD) and ultra-high performance liquid chromatography (UHPLC) both coupled to high resolution mass spectrometry (HRMS) enabled high performance detection and quantitation. Out of the 18 products analyzed, 8 contained some cyanotoxins at levels exceeding the tolerable daily intake values. The presence of cyanotoxins in these algal dietary supplements reinforces the need for a better quality control as well as consumer’s awareness on the potential risks associated with the consumption of these supplements. PMID:28245621

Detection of Cyanotoxins in Algae Dietary Supplements.

PubMed

Roy-Lachapelle, Audrey; Solliec, Morgan; Bouchard, Maryse F; Sauvé, Sébastien

2017-02-25

Algae dietary supplements are marketed worldwide as natural health products. Although their proprieties have been claimed as beneficial to improve overall health, there have been several previous reports of contamination by cyanotoxins. These products generally contain non-toxic cyanobacteria, but the methods of cultivation in natural waters without appropriate quality controls allow contamination by toxin producer species present in the natural environment. In this study, we investigated the presence of total microcystins, seven individual microcystins (RR, YR, LR, LA, LY, LW, LF), anatoxin-a, dihydroanatoxin-a, epoxyanatoxin-a, cylindrospermopsin, saxitoxin, and β-methylamino-l-alanine in 18 different commercially available products containing Spirulina or Aphanizomenon flos-aquae . Total microcystins analysis was accomplished using a Lemieux oxidation and a chemical derivatization using dansyl chloride was needed for the simultaneous analysis of cylindrospermopsin, saxitoxin, and β-methylamino-l-alanine. Moreover, the use of laser diode thermal desorption (LDTD) and ultra-high performance liquid chromatography (UHPLC) both coupled to high resolution mass spectrometry (HRMS) enabled high performance detection and quantitation. Out of the 18 products analyzed, 8 contained some cyanotoxins at levels exceeding the tolerable daily intake values. The presence of cyanotoxins in these algal dietary supplements reinforces the need for a better quality control as well as consumer's awareness on the potential risks associated with the consumption of these supplements.

L-beta-ODAP alters mitochondrial Ca2+ handling as an early event in excitotoxicity.

PubMed

Van Moorhem, Marijke; Decrock, Elke; Coussee, Evelyne; Faes, Liesbeth; De Vuyst, Elke; Vranckx, Katleen; De Bock, Marijke; Wang, Nan; D'Herde, Katharina; Lambein, Fernand; Callewaert, Geert; Leybaert, Luc

2010-03-01

The neurotoxin beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (L-beta-ODAP) is an L-glutamate analogue at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors in neurons and therefore acts as an excitotoxic substance. Chronic exposure to L-beta-ODAP present in Lathyrus sativus L. (L. sativus) seeds is proposed as the cause of the neurodegenerative disease neurolathyrism, but the mechanism of its action has not been conclusively identified. A key factor in excitotoxic neuronal cell death is a disturbance of the intracellular Ca2+ homeostasis, including changes in the capacity of intracellular Ca2+ stores like the endoplasmic reticulum (ER) or mitochondria. In this study, aequorin and other Ca2+ indicators were used in N2a neuroblastoma cells to investigate alterations of cellular Ca2+ handling after 24 h exposure to L-beta-ODAP. Our data demonstrate increased mitochondrial Ca2+ loading and hyperpolarization of the mitochondrial membrane potential (Psi(m)), which was specific for L-beta-ODAP and not observed with L-glutamate. We conclude that L-beta-ODAP disturbs the ER-mitochondrial Ca2+ signaling axis and thereby renders the cells more vulnerable to its excitotoxic effects that ultimately will lead to cell death. 2010 Elsevier Ltd. All rights reserved.

Extraterrestrial Amino Acids in Orgueil and Ivuna: Tracing the Parent Body of CI Type Carbonaceous Chondrites

NASA Technical Reports Server (NTRS)

Meyer, Michael (Technical Monitor); Ehrenfreund, Pascale; Glavin, Daniel P.; Bota, Oliver; Cooper, George; Bada, Jeffrey

2001-01-01

Amino acid analyses using HPLC of pristine interior pieces of the CI carbonaceous chondrites Orgueil and Ivuna have found that beta-alanine, glycine, and gamma-amino-n-butyric acid (ABA) are the most abundant amino acids in these two meteorites, with concentrations ranging from approx. 600 to 2,000 parts per billion (ppb). Other alpha-amino acids such as alanine, alpha-ABA, alpha-aminoisobutyric acid (AIB), and isovaline are present only in trace amounts (less than 200 ppb). Carbon isotopic measurements of beta-alanine and glycine and the presence of racemic (D/L 1) alanine and beta-ABA in Orgueil suggest that these amino acids are extraterrestrial in origin. In comparison to the CM carbonaceous chondrites Murchison and Murray, the amino acid composition of the CIs is strikingly distinct, suggesting that these meteorites came from a different type of parent body, possibly an extinct comet, than did the CM carbonaceous chondrites.

CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

PubMed

Tsukamoto, Kentaro; Ozeki, Chikako; Kohda, Tomoko; Tsuji, Takao

2015-01-01

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

Role of beta-alanine supplementation on muscle carnosine and exercise performance.

PubMed

Artioli, Guilherme Giannini; Gualano, Bruno; Smith, Abbie; Stout, Jeffrey; Lancha, Antonio Herbert

2010-06-01

In this narrative review, we present and discuss the current knowledge available on carnosine and beta-alanine metabolism as well as the effects of beta-alanine supplementation on exercise performance. Intramuscular acidosis has been attributed to be one of the main causes of fatigue during intense exercise. Carnosine has been shown to play a significant role in muscle pH regulation. Carnosine is synthesized in skeletal muscle from the amino acids l-histidine and beta-alanine. The rate-limiting factor of carnosine synthesis is beta-alanine availability. Supplementation with beta-alanine has been shown to increase muscle carnosine content and therefore total muscle buffer capacity, with the potential to elicit improvements in physical performance during high-intensity exercise. Studies on beta-alanine supplementation and exercise performance have demonstrated improvements in performance during multiple bouts of high-intensity exercise and in single bouts of exercise lasting more than 60 s. Similarly, beta-alanine supplementation has been shown to delay the onset of neuromuscular fatigue. Although beta-alanine does not improve maximal strength or VO2max, some aspects of endurance performance, such as anaerobic threshold and time to exhaustion, can be enhanced. Symptoms of paresthesia may be observed if a single dose higher than 800 mg is ingested. The symptoms, however, are transient and related to the increase in plasma concentration. They can be prevented by using controlled release capsules and smaller dosing strategies. No important side effect was related to the use of this amino acid so far. In conclusion, beta-alanine supplementation seems to be a safe nutritional strategy capable of improving high-intensity anaerobic performance.

Structure of aureobasidin A.

PubMed

Ikai, K; Takesako, K; Shiomi, K; Moriguchi, M; Umeda, Y; Yamamoto, J; Kato, I; Naganawa, H

1991-09-01

Aureobasidin A, a new antifungal antibiotic, was isolated from the culture medium of Aureobasidium pullulans R106. Aureobasidin A was a cyclic depsipeptide consisting of eight alpha-amino acid units and one hydroxy acid unit. The structures of the units were found by acid hydrolysis of the antibiotic to be 2(R)-hydroxy-3(R)-methylpentanoic acid, beta-hydroxy-N-methyl-L-valine, N-methyl-L-valine, L-proline, allo-L-isoleucine, N-methyl-L-phenylalanine, L-leucine, and L-phenyl-alanine. The sequence of the units was identified by NMR and FAB-MS of the products from the alkaline hydrolysis of aureobasidin A.

Entropic stabilization of isolated beta-sheets.

PubMed

Dugourd, Philippe; Antoine, Rodolphe; Breaux, Gary; Broyer, Michel; Jarrold, Martin F

2005-04-06

Temperature-dependent electric deflection measurements have been performed for a series of unsolvated alanine-based peptides (Ac-WA(n)-NH(2), where Ac = acetyl, W = tryptophan, A = alanine, and n = 3, 5, 10, 13, and 15). The measurements are interpreted using Monte Carlo simulations performed with a parallel tempering algorithm. Despite alanine's high helix propensity in solution, the results suggest that unsolvated Ac-WA(n)-NH(2) peptides with n > 10 adopt beta-sheet conformations at room temperature. Previous studies have shown that protonated alanine-based peptides adopt helical or globular conformations in the gas phase, depending on the location of the charge. Thus, the charge more than anything else controls the structure.

Effects of beta-alanine supplementation and high-intensity interval training on endurance performance and body composition in men; a double-blind trial.

PubMed

Smith, Abbie E; Walter, Ashley A; Graef, Jennifer L; Kendall, Kristina L; Moon, Jordan R; Lockwood, Christopher M; Fukuda, David H; Beck, Travis W; Cramer, Joel T; Stout, Jeffrey R

2009-02-11

Intermittent bouts of high-intensity exercise result in diminished stores of energy substrates, followed by an accumulation of metabolites, promoting chronic physiological adaptations. In addition, beta-alanine has been accepted has an effective physiological hydrogen ion (H+) buffer. Concurrent high-intensity interval training (HIIT) and beta-alanine supplementation may result in greater adaptations than HIIT alone. The purpose of the current study was to evaluate the effects of combining beta-alanine supplementation with high-intensity interval training (HIIT) on endurance performance and aerobic metabolism in recreationally active college-aged men. Forty-six men (Age: 22.2 +/- 2.7 yrs; Ht: 178.1 +/- 7.4 cm; Wt: 78.7 +/- 11.9; VO2peak: 3.3 +/- 0.59 l.min-1) were assessed for peak O2 utilization (VO2peak), time to fatigue (VO2TTE), ventilatory threshold (VT), and total work done at 110% of pre-training VO2peak (TWD). In a double-blind fashion, all subjects were randomly assigned into one either a placebo (PL - 16.5 g dextrose powder per packet; n = 18) or beta-alanine (BA - 1.5 g beta-alanine plus 15 g dextrose powder per packet; n = 18) group. All subjects supplemented four times per day (total of 6 g/day) for the first 21-days, followed by two times per day (3 g/day) for the subsequent 21 days, and engaged in a total of six weeks of HIIT training consisting of 5-6 bouts of a 2:1 minute cycling work to rest ratio. Significant improvements in VO2peak, VO2TTE, and TWD after three weeks of training were displayed (p < 0.05). Increases in VO2peak, VO2TTE, TWD and lean body mass were only significant for the BA group after the second three weeks of training. The use of HIIT to induce significant aerobic improvements is effective and efficient. Chronic BA supplementation may further enhance HIIT, improving endurance performance and lean body mass.

Kinetic and crystallographic studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

PubMed Central

Emanuele, J. J.; Jin, H.; Jacobson, B. L.; Chang, C. Y.; Einspahr, H. M.; Villafranca, J. J.

1996-01-01

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studies included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, kcat was equal to 980 +/- 40 min-1, while K(m) values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 microM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/K(m) for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine. PMID:8976565

[Model antigens and their significance for occupational dermatology].

PubMed

Schwartze, G; Lübbe, D; Wozniak, K D

1989-07-01

By means of epicutaneous tests we studied the contact hypersensitivity to DNP-amino acids in individuals sensitized to dinitrochlorobenzene (DNCB). We found a high incidence of positive skin responses to DNP-glycine, di-DNP-L-cystine, and DNP-L-alpha-alanine, but only in some cases DNP-beta-alanine induced skin reactivity. The results are discussed both in connection with the influence of varying molecular sizes and chemical structure on the immunological reactivity and the possibility to develop a beta-alanine containing protective ointment against protein-reactive haptens.

Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli.

PubMed

Liger, D; Masson, A; Blanot, D; van Heijenoort, J; Parquet, C

1995-05-15

The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli was over-produced in strains harbouring recombinant plasmids bearing the murC gene under the control of the lac or trc promoter. Plasmid pAM1005, in which the promoter and ribosome-binding site region of murC were removed and in which the gene was directly under the control of promoter trc, led to a 2000-fold amplification of the L-alanine-adding activity after induction by isopropyl-thio-beta-D-galactopyranoside. The murC gene product was visualized as a 50-kDa protein accounting for approximately 50% of the cell protein. A two-step purification led to 1 g of a homogeneous protein from an 18-1 culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the murC gene. The presence of 2-mercaptoethanol and glycerol was essential for the stability of the enzyme. The Km values for UDP-N-acetylmuramic acid, L-alanine and ATP/Mg2+ were estimated at 100, 20 and 450 microM, respectively. Under the optimal in vitro conditions a turnover number of 928 min-1 was calculated and a copy number/cell of 600 could be roughly estimated. The specificity of the enzyme for its substrates was investigated with various analogues. The enzyme also catalysed the reverse reaction.

A review of cyanobacteria and cyanotoxins removal/inactivation in drinking water treatment.

PubMed

Westrick, Judy A; Szlag, David C; Southwell, Benjamin J; Sinclair, James

2010-07-01

This review focuses on the efficiency of different water treatment processes for the removal of cyanotoxins from potable water. Although several investigators have studied full-scale drinking water processes to determine the efficiency of cyanotoxin inactivation, many of the studies were based on ancillary practice. In this context, "ancillary practice" refers to the removal or inactivation of cyanotoxins by standard daily operational procedures and without a contingency operational plan utilizing specific treatment barriers. In this review, "auxiliary practice" refers to the implementation of inactivation/removal treatment barriers or operational changes explicitly designed to minimize risk from toxin-forming algae and their toxins to make potable water. Furthermore, the best drinking water treatment practices are based on extension of the multibarrier approach to remove cyanotoxins from water. Cyanotoxins are considered natural contaminants that occur worldwide and specific classes of cyanotoxins have shown regional prevalence. For example, freshwaters in the Americas often show high concentrations of microcystin, anatoxin-a, and cylindrospermopsin, whereas Australian water sources often show high concentrations of microcystin, cylindrospermopsin, and saxitoxins. Other less frequently reported cyanotoxins include lyngbyatoxin A, debromoaplysiatoxin, and beta-N-methylamino-L-alanine. This review focuses on the commonly used unit processes and treatment trains to reduce the toxicity of four classes of cyanotoxins: the microcystins, cylindrospermopsin, anatoxin-a, and saxitoxins. The goal of this review is to inform the reader of how each unit process participates in a treatment train and how an auxiliary multibarrier approach to water treatment can provide safer water for the consumer.

beta-Alanine elevates dopamine levels in the rat nucleus accumbens: antagonism by strychnine.

PubMed

Ericson, Mia; Clarke, Rhona B C; Chau, PeiPei; Adermark, Louise; Söderpalm, Bo

2010-04-01

Glycine receptors (GlyRs) in the nucleus accumbens (nAc) have recently been suggested to be involved in the reinforcing and dopamine-elevating properties of ethanol via a neuronal circuitry involving the VTA. Apart from ethanol, both glycine and taurine have the ability to modulate dopamine output via GlyRs in the same brain region. In the present study, we wanted to explore whether yet another endogenous ligand for the GlyR, beta-alanine, had similar effects. To this end, we monitored dopamine in the nAc by means of in vivo microdialysis and found that local perfusion of beta-alanine increased dopamine output. In line with previous observations investigating ethanol, glycine and taurine, the competitive GlyR antagonist strychnine completely blocked the dopamine elevation. The present results suggest that beta-alanine has the ability to modulate dopamine levels in the nAc via strychnine-sensitive GlyRs, and are consistent with previous studies suggesting the importance of this receptor for modulating dopamine output.

L-alanine-glyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate beta-lyase activity: a contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes?

PubMed Central

Cooper, Arthur J L; Krasnikov, Boris F; Okuno, Etsuo; Jeitner, Thomas M

2003-01-01

Several halogenated alkenes are metabolized in part to cysteine S-conjugates, which are mitochondrial toxicants of kidney and, to a lesser extent, other organs. Toxicity is due to cysteine S-conjugate beta-lyases, which convert the cysteine S-conjugate into pyruvate, ammonia and a reactive sulphur-containing fragment. A section of the human population is exposed to halogenated alkenes. To understand the health effects of such exposure, it is important to identify cysteine S-conjugate beta-lyases that contribute to mitochondrial damage. Mitochondrial aspartate aminotransferase [Cooper, Bruschi, Iriarte and Martinez-Carrion (2002) Biochem. J. 368, 253-261] and mitochondrial branched-chain aminotransferase [Cooper, Bruschi, Conway and Hutson (2003) Biochem. Pharmacol. 65, 181-192] exhibit beta-lyase activity toward S -(1,2-dichlorovinyl)-L-cysteine (the cysteine S-conjugate of trichloroethylene) and S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene). Turnover leads to eventual inactivation of these enzymes. Here we report that mitochondrial L-alanine-glyoxylate aminotransferase II, which, in the rat, is most active in kidney, catalyses cysteine S-conjugate beta-lyase reactions with S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(benzothiazolyl-L-cysteine); turnover leads to inactivation. Previous workers showed that the reactive-sulphur-containing fragment released from S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and S -(1,2-dichlorovinyl)-L-cysteine is toxic by acting as a thioacylating agent - particularly of lysine residues in nearby proteins. Toxicity, however, may also involve 'self-inactivation' of key enzymes. The present findings suggest that alanine-glyoxylate aminotransferase II may be an important factor in the well-established targeting of rat kidney mitochondria by toxic halogenated cysteine S-conjugates. Previous reports suggest that alanine-glyoxylate aminotransferase II is absent in some humans, but present in others. Alanine-glyoxylate aminotransferase II may contribute to the bioactivation (toxification) of halogenated cysteine S-conjugates in a subset of individuals exposed to halogenated alkenes. PMID:12859250

Forced swimming and imipramine modify plasma and brain amino acid concentrations in mice.

PubMed

Murakami, Tatsuro; Yamane, Haruka; Tomonaga, Shozo; Furuse, Mitsuhiro

2009-01-05

The relationships between monoamine metabolism and forced swimming or antidepressants have been well studied, however information is lacking regarding amino acid metabolism under these conditions. Therefore, the aim of the present study was to investigate the effects of forced swimming and imipramine on amino acid concentrations in plasma, the cerebral cortex and the hypothalamus in mice. Forced swimming caused cerebral cortex concentrations of L-glutamine, L-alanine, and taurine to be increased, while imipramine treatment caused decreased concentrations of L-glutamate, L-alanine, L-tyrosine, L-methionine, and L-ornithine. In the hypothalamus, forced swimming decreased the concentration of L-serine while imipramine treatment caused increased concentration of beta-alanine. Forced swimming caused increased plasma concentration of taurine, while concentrations of L-serine, L-asparagine, L-glutamine and beta-alanine were decreased. Imipramine treatment caused increased plasma concentration of all amino acid, except for L-aspartate and taurine. In conclusion, forced swimming and imipramine treatment modify central and peripheral amino acid metabolism. These results may aid in the identification of amino acids that have antidepressant-like effects, or may help to refine the dosages of antidepressant drugs.

Aspects of cuticular sclerotization in the locust, Scistocerca gregaria, and the beetle, Tenebrio molitor.

PubMed

Andersen, Svend Olav; Roepstorff, Peter

2007-03-01

The number of reactive amino groups in cuticular proteins decreases during the early period of insect cuticular sclerotization, presumably due to reaction with oxidation products of N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD). We have quantitated the decrease in cuticular N-terminal amino groups and lysine epsilon-amino groups during the first 24h of sclerotization in adult locusts, Schistocerca gregaria, and in larval and adult beetles, Tenebrio molitor, as well as the increase in beta-alanine amino groups in Tenebrio cuticle. The results indicate that nearly all glycine N-terminal groups and a significant part of the epsilon-amino groups from lysine residues are involved in the sclerotization process in both locusts and Tenebrio. A pronounced increase in the amount of free beta-alanine amino groups was observed in cuticle from adult Tenebrio and to a lesser extent also in Tenebrio larval cuticle, but from locust cuticle no beta-alanine was obtained. Hydrolysis of sclerotized cuticles from locusts and Tenebrio by dilute hydrochloric acid released a large number of compounds containing amino acids linked to catecholic moieties. Products have been identified which contain histidine residues linked via their imidazole group to the beta-position of various catechols, such as dopamine, 3,4-dihydroxyphenyl-ethanol (DOPET), and 3,4-dihydroxyphenyl-acetaldehyde (DOPALD), and a ketocatecholic compound has also been identified composed of lysine linked via its epsilon-amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone. Some of the hydrolysis products have previously been obtained from sclerotized pupal cuticle of Manduca sexta [Xu, R., Huang, X., Hopkins, T.L., Kramer, K.J., 1997. Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle. Insect Biochemistry and Molecular Biology 27, 101-108; Kerwin, J.L., Turecek, F., Xu, R., Kramer, K.J., Hopkins, T.L., Gatlin, C.L., Yates, J.R., 1999. Mass spectrometric analysis of catechol-histidine adducts from insect cuticle. Analytical Biochemistry 268, 229-237; Kramer, K.J., Kanost, M.R., Hopkins, T.L., Jiang, H., Zhu, Y.C., Xu, R., Kerwin, J.L., Turecek, F., 2001. Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron 57, 385-392], but the lysine-dihydroxyacetophenone compound and the histidine-DOPALD adduct have not been reported before. It is suggested that the compounds are derived from NADA and NBAD residues which were incorporated into the cuticle during sclerotization, and that the lysine-dihydroxyacetophenone as well as the DOPET and DOPALD containing adducts are degradation products derived from cross-links between the cuticular proteins, whereas the dopamine-containing adducts are derived from a non-crosslinking reaction product.

Spatial and thematic distribution of research on cyanotoxins.

PubMed

Merel, Sylvain; Villarín, María C; Chung, Khrystyne; Snyder, Shane

2013-12-15

Cyanobacteria in surface water are well known for their ability to form toxic blooms responsible for animal mortality and human poisoning. Accompanying major progress in science and technology, the state of knowledge of cyanotoxins has dramatically increased over the last two decades. The bibliometric approach applied in this study shows the evolution of research and identifies major gaps to be filled by future work. Although the publication rate has gradually increased from one hundred to three hundred articles per year since the 1990s, half of the literature available focuses on microcystins and another quarter on saxitoxins. Other cyanotoxins such as beta-N-methylamino-l-alanine or cylindrospermopsin remain vastly disregarded. Moreover, most of the publications deal with toxicity and ecology while other research areas, such as environmental and public health, require additional investigation. The analysis of the literature highlights the main journals for the communication of knowledge on cyanotoxins but also reveals that 90% of the research is originated from only ten countries. These countries are also those with the highest H-index and average number of citation per article. Nonetheless, the ranking of these countries is significantly altered when the amount of publications is normalized based on the population, the number of universities, the national gross domestic product or the government revenue. However, the lower amount of publications from Eastern Europe, Africa and South America could also reflect the lack of monitoring campaigns in these regions. This lack could potentially lead to the underestimation of the prevalence of toxic cyanobacterial blooms and the diversity of toxins worldwide. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification of risk factors associated with onset and progression of amyotrophic lateral sclerosis using systematic review and meta-analysis.

PubMed

Wang, Ming-Dong; Little, Julian; Gomes, James; Cashman, Neil R; Krewski, Daniel

2017-07-01

Although amyotrophic lateral sclerosis (ALS) was identified as a neurological condition 150 years ago, risk factors related to the onset and progression of ALS remain largely unknown. Monogenic mutations in over 30 genes are associated with about 10% of ALS cases. The age at onset of ALS and disease types has been found to influence ALS progression. The present study was designed to identify additional putative risk factors associated with the onset and progression of ALS using systematic review and meta-analysis of observational studies. Risk factors that may be associated with ALS include: 1) genetic mutations, including the intermediate CAG repeat expansion in ATXN2; 2) previous exposure to heavy metals such as lead and mercury; 3) previous exposure to organic chemicals, such as pesticides and solvents; 4) history of electric shock; 5) history of physical trauma/injury (including head trauma/injury); 6) smoking (a weak risk factor for ALS in women); and 6) other risk factors, such as participating in professional sports, lower body mass index, lower educational attainment, or occupations requiring repetitive/strenuous work, military service, exposure to Beta-N-methylamino-l-alanin and viral infections. Risk factors that may be associated with ALS progression rate include: 1) nutritional status, including vitamin D deficiency; 2) comorbidities; 3) ethnicity and genetic factors; 4) lack of supportive care; and 4) smoking. The extent to which these associations may be causal is discussed, with further research recommended to strengthen the evidence on which determinations of causality may be based. Copyright © 2016. Published by Elsevier B.V.

Effects of Plyometric Training and Beta-Alanine Supplementation on Maximal-Intensity Exercise and Endurance in Female Soccer Players

PubMed Central

Rosas, Fabián; Ramírez-Campillo, Rodrigo; Martínez, Cristian; Cañas-Jamet, Rodrigo; McCrudden, Emma; Meylan, Cesar; Moran, Jason; Nakamura, Fábio Y.; Pereira, Lucas A.; Loturco, Irineu; Diaz, Daniela; Izquierdo, Mikel

2017-01-01

Abstract Plyometric training and beta-alanine supplementation are common among soccer players, although its combined use had never been tested. Therefore, a randomized, double-blind, placebo-controlled trial was conducted to compare the effects of a plyometric training program, with or without beta-alanine supplementation, on maximal-intensity and endurance performance in female soccer players during an in-season training period. Athletes (23.7 ± 2.4 years) were assigned to either a plyometric training group receiving a placebo (PLACEBO, n = 8), a plyometric training group receiving beta-alanine supplementation (BA, n = 8), or a control group receiving placebo without following a plyometric training program (CONTROL, n = 9). Athletes were evaluated for single and repeated jumps and sprints, endurance, and change-of-direction speed performance before and after the intervention. Both plyometric training groups improved in explosive jumping (ES = 0.27 to 1.0), sprinting (ES = 0.31 to 0.78), repeated sprinting (ES = 0.39 to 0.91), 60 s repeated jumping (ES = 0.32 to 0.45), endurance (ES = 0.35 to 0.37), and change-of-direction speed performance (ES = 0.36 to 0.58), whereas no significant changes were observed for the CONTROL group. Nevertheless, compared to the CONTROL group, only the BA group showed greater improvements in endurance, repeated sprinting and repeated jumping performances. It was concluded that beta-alanine supplementation during plyometric training may add further adaptive changes related to endurance, repeated sprinting and jumping ability. PMID:28828081

Effects of Plyometric Training and Beta-Alanine Supplementation on Maximal-Intensity Exercise and Endurance in Female Soccer Players.

PubMed

Rosas, Fabián; Ramírez-Campillo, Rodrigo; Martínez, Cristian; Caniuqueo, Alexis; Cañas-Jamet, Rodrigo; McCrudden, Emma; Meylan, Cesar; Moran, Jason; Nakamura, Fábio Y; Pereira, Lucas A; Loturco, Irineu; Diaz, Daniela; Izquierdo, Mikel

2017-09-01

Plyometric training and beta-alanine supplementation are common among soccer players, although its combined use had never been tested. Therefore, a randomized, double-blind, placebo-controlled trial was conducted to compare the effects of a plyometric training program, with or without beta-alanine supplementation, on maximal-intensity and endurance performance in female soccer players during an in-season training period. Athletes (23.7 ± 2.4 years) were assigned to either a plyometric training group receiving a placebo (PLACEBO, n = 8), a plyometric training group receiving beta-alanine supplementation (BA, n = 8), or a control group receiving placebo without following a plyometric training program (CONTROL, n = 9). Athletes were evaluated for single and repeated jumps and sprints, endurance, and change-of-direction speed performance before and after the intervention. Both plyometric training groups improved in explosive jumping (ES = 0.27 to 1.0), sprinting (ES = 0.31 to 0.78), repeated sprinting (ES = 0.39 to 0.91), 60 s repeated jumping (ES = 0.32 to 0.45), endurance (ES = 0.35 to 0.37), and change-of-direction speed performance (ES = 0.36 to 0.58), whereas no significant changes were observed for the CONTROL group. Nevertheless, compared to the CONTROL group, only the BA group showed greater improvements in endurance, repeated sprinting and repeated jumping performances. It was concluded that beta-alanine supplementation during plyometric training may add further adaptive changes related to endurance, repeated sprinting and jumping ability.

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice.

PubMed

Bozue, Joel A; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K; Toothman, Ronald G; Dankmeyer, Jennifer L; Klimko, Christopher P; Wilhelmsen, Catherine L; Raymond, Jolynn W; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

2016-01-01

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice

PubMed Central

Bozue, Joel A.; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K.; Toothman, Ronald G.; Dankmeyer, Jennifer L.; Klimko, Christopher P.; Wilhelmsen, Catherine L.; Raymond, Jolynn W.; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

2016-01-01

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders. PMID:26955620

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

PubMed

Xu, Shuhua; Soroka, Carol J; Sun, An-Qiang; Backos, Donald S; Mennone, Albert; Suchy, Frederick J; Boyer, James L

2016-01-01

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

Isolation and characterization at cholinergic nicotinic receptors of a neurotoxin from the venom of the Acanthophis sp. Seram death adder.

PubMed

Wickramaratna, Janith C; Fry, Bryan G; Loiacono, Richard E; Aguilar, Marie-Isabel; Alewood, Paul F; Hodgson, Wayne C

2004-07-15

The present study describes the isolation of the first neurotoxin (acantoxin IVa) from Acanthophis sp. Seram death adder venom and an examination of its activity at nicotinic acetylcholine receptor (nAChR) subtypes. Acantoxin IVa (MW 6815; 0.1-1.0 microM) caused concentration-dependent inhibition of indirect twitches (0.1 Hz, 0.2 ms, supramaximal V) and inhibited contractile responses to exogenous nicotinic agonists in the chick biventer cervicis nerve-muscle, confirming that this toxin is a postsynaptic neurotoxin. Acantoxin IVa (1-10 nM) caused pseudo-irreversible antagonism at skeletal muscle nAChR with an estimated pA2 of 8.36+/-0.17. Acantoxin IVa was approximately two-fold less potent than the long-chain (Type II) neurotoxin, alpha-bungarotoxin. With a pKi value of 4.48, acantoxin IVa was approximately 25,000 times less potent than alpha-bungarotoxin at alpha7-type neuronal nAChR. However, in contrast to alpha-bungarotoxin, acantoxin IVa completely inhibited specific [3H]-methyllycaconitine (MLA) binding in rat hippocampus homogenate. Acantoxin IVa had no activity at ganglionic nAChR, alpha4beta2 subtype neuronal nAChR or cytisine-resistant [3H]-epibatidine binding sites. While long-chain neurotoxin resistant [3H]-MLA binding in hippocampus homogenate requires further investigation, we have shown that a short-chain (Type I) neurotoxin is capable of fully inhibiting specific [3H]-MLA binding.

Cyanobacteria and cyanotoxins are present in drinking water impoundments and groundwater wells in desert environments.

PubMed

Chatziefthimiou, Aspassia D; Metcalf, James S; Glover, W Broc; Banack, Sandra A; Dargham, Soha R; Richer, Renee A

2016-05-01

Desert environments and drylands experience a drastic scarcity of water resources. To alleviate dependence on freshwater for drinking water needs, countries have invested in infrastructure development of desalination plants. Collectively, the countries of the Arabian Gulf produce 45% of the world's desalinated water, which is stored in dams, mega-reservoirs and secondary house water tanks to secure drinking water beyond daily needs. Improper storage practices of drinking water in impoundments concomitant with increased temperatures and light penetration may promote the growth of cyanobacteria and accumulation of cyanotoxins. To shed light on this previously unexplored research area in desert environments, we examined drinking and irrigation water of urban and rural environments to determine whether cyanobacteria and cyanotoxins are present, and what are the storage and transportation practices as well as the environmental parameters that best predict their presence. Cyanobacteria were present in 80% of the urban and 33% of the rural water impoundments. Neurotoxins BMAA, DAB and anatoxin-a(S) were not detected in any of the water samples, although they have been found to accumulate in the desert soils, which suggests a bioaccumulation potential if they are leached into the aquifer. A toxic BMAA isomer, AEG, was found in 91.7% of rural but none of the urban water samples and correlated with water-truck transportation, light exposure and chloride ions. The hepatotoxic cyanotoxin microcystin-LR was present in the majority of all sampled impoundments, surpassing the WHO provisional guideline of 1 μg/l in 30% of the urban water tanks. Finally, we discuss possible management strategies to improve storage and transportation practices in order to minimize exposure to cyanobacteria and cyanotoxins, and actions to promote sustainable use of limited water resources. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of beta-alanine supplementation on the onset of blood lactate accumulation (OBLA) during treadmill running: Pre/post 2 treatment experimental design.

PubMed

Jordan, Thomas; Lukaszuk, Judith; Misic, Mark; Umoren, Josephine

2010-05-19

beta-Alanine (betaA) has been shown to improve performance during cycling. This study was the first to examine the effects of betaA supplementation on the onset of blood lactate accumulation (OBLA) during incremental treadmill running. Seventeen recreationally-active men (mean +/- SE 24.9 +/- 4.7 yrs, 180.6 +/- 8.9 cm, 79.25 +/- 9.0 kg) participated in this randomized, double-blind, placebo-controlled pre/post test 2-treatment experimental design. Subjects participated in two incremental treadmill tests before and after 28 days of supplementation with either betaA (6.0 g.d-1)(betaA, n = 8) or an equivalent dose of Maltodextrin as the Placebo (PL, n = 9). Heart rate, percent heart rate maximum (%HRmax), %VO2max@OBLA (4.0 mmol.L-1 blood lactate concentration) and VO2max (L.min-1) were determined for each treadmill test. Friedman test was used to determine within group differences; and Mann-Whitney was used to determine between group differences for pre and post values (p < 0.05). The betaA group experienced a significant rightward shift in HR@OBLA beats.min-1 (p < 0.01) pre/post (161.6 +/- 19.2 to 173.6 +/- 9.9) but remained unchanged in the PL group (166.8 +/- 15.8 to 169.6 +/- 16.1). The %HRmax@OBLA increased (p < 0.05) pre/post in the betaA group (83.0% +/- 9.7 to 88.6% +/- 3.7) versus no change in the PL group (86.3 +/- % 4.8 to 87.9% +/- 7.2). The %VO2max@OBLA increased (p < 0.05) in the betaA group pre/post (69.1 +/- 11.0 to 75.6 +/- 10.7) but remained unchanged in the PL group (73.3 +/- 7.3 to 74.3 +/- 7.3). VO2max (L.min-1) decreased (p < 0.01) in the betaA group pre/post (4.57 +/- 0.8 to 4.31 +/- 0.8) versus no change in the PL group (4.04 +/- 0.7 to 4.18 +/- 0.8). Body mass kg increased (p < 0.05) in the betaA group pre/post (77.9 +/- 9.0 to 78.3 +/- 9.3) while the PL group was unchanged (80.6 +/- 9.1 to 80.4 +/- 9.0). betaA supplementation for 28 days enhanced sub-maximal endurance performance by delaying OBLA. However, betaA supplemented individuals had a reduced aerobic capacity as evidenced by the decrease in VO2max values post supplementation.

The lathyrus toxin, {beta}-N-oxalyl-L-{alpha},{beta}-diaminopropionic acid (ODAP), and homocysteic acid sensitize CA1 pyramidal neurons to cystine and L-2-amino-6-phosphonohexanoic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chase, L.A.; Peterson, N.L.; Koerner, J.F.

2007-02-15

A brief exposure of hippocampal slices to L-quisqualic acid (QUIS) sensitizes CA1 pyramidal neurons 30- to 250-fold to depolarization by certain excitatory amino acids analogues, e.g., L-2-amino-6-phosphonohexanoic acid (L-AP6), and by the endogenous compound, L-cystine. This phenomenon has been termed QUIS sensitization. A mechanism similar to that previously described for QUIS neurotoxicity has been proposed to describe QUIS sensitization. Specifically, QUIS has been shown to be sequestered into GABAergic interneurons by the System x{sub c} {sup -} and subsequently released by heteroexchange with cystine or L-AP6, resulting in activation of non-NMDA receptors. We now report two additional neurotoxins, the Lathyrusmore » excitotoxin, {beta}-N-oxalyl-L-{alpha},{beta}-diaminopropionic acid (ODAP), and the endogenous compound, L-homocysteic acid (HCA), sensitize CA1 hippocampal neurons > 50-fold to L-AP6 and > 10-fold to cystine in a manner similar to QUIS. While the cystine- or L-AP6-mediated depolarization can be inhibited by the non-NMDA receptor antagonist CNQX in ODAP- or QUIS-sensitized slices, the NMDA antagonist D-AP5 inhibits depolarization by cystine or L-AP6 in HCA-sensitized slices. Thus, HCA is the first identified NMDA agonist that induces phosphonate or cystine sensitization. Like QUIS sensitization, the sensitization evoked by either ODAP or HCA can be reversed by a subsequent exposure to 2 mM {alpha}-aminoadipic acid. Finally, we have demonstrated that there is a correlation between the potency of inducers for triggering phosphonate or cystine sensitivity and their affinities for System x{sub c} {sup -} and either the non-NMDA or NMDA receptor. Thus, the results of this study support our previous model of QUIS sensitization and have important implications for the mechanisms of neurotoxicity, neurolathyrism and hyperhomocystinemia.« less

Transplastomic expression of bacterial L-aspartate-alpha-decarboxylase enhances photosynthesis and biomass production in response to high temperature stress.

PubMed

Fouad, W M; Altpeter, F

2009-10-01

Metabolic engineering for beta-alanine over-production in plants is expected to enhance environmental stress tolerance. The Escherichia coli L-aspartate-alpha-decarboxylase (AspDC) encoded by the panD gene, catalyzes the decarboxylation of L-aspartate to generate beta-alanine and carbon dioxide. The constitutive E. coli panD expression cassette was co-introduced with the constitutive, selectable aadA expression cassette into the chloroplast genome of tobacco via biolistic gene transfer and homologous recombination. Site specific integration of the E. coli panD expression cassette into the chloroplast genome and generation of homotransplastomic plants were confirmed by PCR and Southern blot analysis, respectively, following plant regeneration and germination of seedlings on selective media. PanD expression was verified by assays based on transcript detection and in vitro enzyme activity. The AspDC activities in transplastomic plants expressing panD were drastically increased by high-temperature stress. beta-Alanine accumulated in transplastomic plants at levels four times higher than in wildtype plants. Analysis of chlorophyll fluorescence on plants subjected to severe heat stress at 45 degrees C under light verified that photosystem II (PSII) in transgenic plants had higher thermotolerance than in wildtype plants. The CO(2) assimilation of transplastomic plants expressing panD was more tolerant to high temperature stress than that of wildtype plants, resulting in the production of 30-40% more above ground biomass than wildtype control. The results presented indicate that chloroplast engineering of the beta-alanine pathway by over-expression of the E. coli panD enhances thermotolerance of photosynthesis and biomass production following high temperature stress.

Ruthenium(II) arene complexes with chelating chloroquine analogue ligands: Synthesis, characterization and in vitro antimalarial activity†

PubMed Central

Glans, Lotta; Ehnbom, Andreas; de Kock, Carmen; Martínez, Alberto; Estrada, Jesús; Smith, Peter J.; Haukka, Matti; Sánchez-Delgado, Roberto A.; Nordlander, Ebbe

2012-01-01

Three new ruthenium complexes with bidentate chloroquine analogue ligands, [Ru(η6-cym)(L1)Cl]Cl (1, cym = p-cymene, L1 = N-(2-((pyridin-2-yl)methylamino)ethyl)-7-chloroquinolin-4-amine), [Ru(η6-cym)(L2)Cl]Cl (2, L2 = N-(2-((1-methyl-1H-imidazol-2-yl)methylamino)ethyl)-7-chloroquinolin-4-amine) and [Ru(η6-cym)(L3)Cl] (3, L3 = N-(2-((2-hydroxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine) have been synthesized and characterized. In addition, the X-ray crystal structure of 2 is reported. The antimalarial activity of complexes 1–3 and ligands L1, L2 and L3, as well as the compound N-(2-(bis((pyridin-2-yl)methyl)amino)ethyl)-7-chloroquinolin-4-amine (L4), against chloroquine sensitive and chloroquine resistant Plasmodium falciparum malaria strains was evaluated. While 1 and 2 are less active than the corresponding ligands, 3 exhibits high antimalarial activity. The chloroquine analogue L2 also shows good activity against both the choloroquine sensitive and the chloroquine resistant strains. Heme aggregation inhibition activity (HAIA) at an aqueous buffer/n-octanol interface (HAIR50) and lipophilicity (D, as measured by water/n-octanol distribution coefficients) have been measured for all ligands and metal complexes. A direct correlation between the D and HAIR50 properties cannot be made because of the relative structural diversity of the complexes, but it may be noted that these properties are enhanced upon complexation of the inactive ligand L3 to ruthenium, to give a metal complex (3) with promising antimalarial activity. PMID:22249579

Comparison of the UDP-N-Acetylmuramate:l-Alanine Ligase Enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

PubMed Central

Mahapatra, Sebabrata; Crick, Dean C.; Brennan, Patrick J.

2000-01-01

In the peptidoglycan of Mycobacterium leprae, l-alanine of the side chain is replaced by glycine. When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:l-alanine ligase) of M. leprae showed Km and Vmax for l-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine. PMID:11073931

Genome Sequence Alterations Detected upon Passage of Burkholderia mallei ATCC 23344 in Culture and in Mammalian Hosts

DTIC Science & Technology

2006-09-05

NA NA no yes BMAA1128 ABC Transporter 3 GGGAAACGCGAAAC 6 5 yes no BMAA1873 Hypothetical protein 4 No (-C) NA NA no yes BMAA1868 Aconitate hydratase 5...no yes BMAA1868 Aconitate hydratase 3 GTGCTGTC 21 22 no yes BMAA0375 Transcriptional regulator Human Blood 1 TTGGCGC 111 109 no no BMAA1866 Conserved...NA NA no yes BMAA1128 ABC transporter 5 No (-C) NA NA no yes BMAA1868 Aconitate hydratase NA: Not applicable.Page 3 of 11 (page number not for

Dose- and Glucose-Dependent Effects of Amino Acids on Insulin Secretion from Isolated Mouse Islets and Clonal INS-1E Beta-Cells

PubMed Central

Liu, Zhenping; Jeppesen, Per B.; Gregersen, Søren; Chen, Xiaoping; Hermansen, Kjeld

2008-01-01

BACKGROUND: The influence of glucose and fatty acids on beta-cell function is well established whereas little is known about the role of amino acids (AAs). METHODS: Islets isolated from NMRI mice were incubated overnight. After preincubation, isolated islets as well as clonal INS-1E beta-cells were incubated for 60 min in a modified Krebs Ringer buffer containing glucose and AAs. RESULTS: At 16.7 mmol/l (mM) glucose, L-arginine, L-lysine, L-alanine, L-proline, L-leucine, and L-glutamine potentiated glucose-stimulated insulin secretion dose-dependently, while DL-homocysteine inhibited insulin secretion. Maximal insulin stimulation was obtained at 20 mM L-proline, L-lysine, L-alanine, L-arginine (islets: 2.5 to 6.7 fold increase; INS-1E cells: 1.6 to 2.2 fold increase). L-glutamine and L-leucine only increased glucose-stimulated (16.7 mM) insulin secretion (INS-1E cells: 1.5 and 1.3 fold, respectively) at an AA concentration of 20 mM. Homocysteine inhibited insulin secretion both at 5.6 mM and 16.7 mM glucose. At glucose levels ranging from 1.1 to 25 mM, the equimolar concentration of 10 mM, L-proline, L-lysine, L-arginine increased insulin secretion from mouse islets and INS-1E cells at all glucose levels applied, with a maximal effect obtained at 25 mM glucose. At a concentration of 10 mM, L-arginine and L-lysine had the highest insulinotropic potency among the AAs investigated. CONCLUSION: L-arginine, L-lysine, L-alanine, L-proline, L-leucine and L-glutamine acutely stimulate insulin secretion from mouse islets and INS-1E cells in a dose- and glucose-dependent manner, whereas DL-homocysteine inhibits insulin release. PMID:19290384

The Amino Acid Composition of the Sutter's Mill Carbonaceous Chondrite

NASA Technical Reports Server (NTRS)

Glavin, D. P.; Burton, A. S.; Elsila, J. E.; Dworkin, J. P.; Yin, Q. Z.; Cooper, G.; Jenniskens, P.

2012-01-01

In contrast to the Murchison meteorite which had a complex distribution of amino acids with a total C2 to Cs amino acid abundance of approx.14,000 parts-per-billion (ppb) [2], the Sutters Mill meteorite was found to be highly depleted in amino acids. Much lower abundances (approx.30 to 180 ppb) of glycine, beta-alanine, L-alanine and L-serine were detected in SM2 above procedural blank levels indicating that this meteorite sample experienced only minimal terrestrial amino acid contamination after its fall to Earth. Carbon isotope measurements will be necessary to establish the origin of glycine and beta-alanine in SM2. Other non-protein amino acids that are rare on Earth, yet commonly found in other CM meteorites such as aaminoisobutyric acid (alpha-AIB) and isovaline, were not identified in SM2. However, traces of beta-AIB (approx.1 ppb) were detected in SM2 and could be" extraterrestrial in origin. The low abundances of amino acids in the Sutter's Mill meteorite is consistent with mineralogical evidence that at least some parts of the Sutter's Mill meteorite parent body experienced extensive aqueous and/or thermal alteration.

Acquisition and Assimilation of Nitrogen as Peptide-Bound and D-Enantiomers of Amino Acids by Wheat

PubMed Central

Hill, Paul W.; Quilliam, Richard S.; DeLuca, Thomas H.; Farrar, John; Farrell, Mark; Roberts, Paula; Newsham, Kevin K.; Hopkins, David W.; Bardgett, Richard D.; Jones, David L.

2011-01-01

Nitrogen is a key regulator of primary productivity in many terrestrial ecosystems. Historically, only inorganic N (NH4 + and NO3 -) and L-amino acids have been considered to be important to the N nutrition of terrestrial plants. However, amino acids are also present in soil as small peptides and in D-enantiomeric form. We compared the uptake and assimilation of N as free amino acid and short homopeptide in both L- and D-enantiomeric forms. Sterile roots of wheat (Triticum aestivum L.) plants were exposed to solutions containing either 14C-labelled L-alanine, D-alanine, L-trialanine or D-trialanine at a concentration likely to be found in soil solution (10 µM). Over 5 h, plants took up L-alanine, D-alanine and L-trialanine at rates of 0.9±0.3, 0.3±0.06 and 0.3±0.04 µmol g−1 root DW h−1, respectively. The rate of N uptake as L-trialanine was the same as that as L-alanine. Plants lost ca.60% of amino acid C taken up in respiration, regardless of the enantiomeric form, but more (ca.80%) of the L-trialanine C than amino acid C was respired. When supplied in solutions of mixed N form, N uptake as D-alanine was ca.5-fold faster than as NO3 -, but slower than as L-alanine, L-trialanine and NH4 +. Plants showed a limited capacity to take up D-trialanine (0.04±0.03 µmol g−1 root DW h−1), but did not appear to be able to metabolise it. We conclude that wheat is able to utilise L-peptide and D-amino acid N at rates comparable to those of N forms of acknowledged importance, namely L-amino acids and inorganic N. This is true even when solutes are supplied at realistic soil concentrations and when other forms of N are available. We suggest that it may be necessary to reconsider which forms of soil N are important in the terrestrial N cycle. PMID:21541281

The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II.

PubMed

Brunt, Jason; Carter, Andrew T; Pye, Hannah V; Peck, Michael W

2018-05-04

Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca 2+ -DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II.

International Traffic in Arms Regulations (ITAR), (22 CFR 120-130)

DTIC Science & Technology

1996-06-01

Butadienenitrileoxide (BNO); (6) Catocene, N-butyl- ferrocene and other ferrocene derivatives; (7) 3-nitraza-l,5 pentane diisocyanate; (8) Bis(2,2...Tris-l-(2-methyl)aziridinyl phosphine oxide (MAPO), bis(2-methyl aziridinyl) 2-(2-hydroxypropanoxy) propylamino phosphine oxide (BOBBA 8), and...other MAPO derivatives; (22) Bis(2-methyl aziridinyl) methylamino phosphine oxide (methyl BAPO); (23) Organo-metallic coupling agents, specifi

Algal Blooms and Cyanotoxins in Jordan Lake, North Carolina.

PubMed

Wiltsie, Daniel; Schnetzer, Astrid; Green, Jason; Vander Borgh, Mark; Fensin, Elizabeth

2018-02-24

The eutrophication of waterways has led to a rise in cyanobacterial, harmful algal blooms (CyanoHABs) worldwide. The deterioration of water quality due to excess algal biomass in lakes has been well documented (e.g., water clarity, hypoxic conditions), but health risks associated with cyanotoxins remain largely unexplored in the absence of toxin information. This study is the first to document the presence of dissolved microcystin, anatoxin-a, cylindrospermopsin, and β- N -methylamino-l-alanine in Jordan Lake, a major drinking water reservoir in North Carolina. Saxitoxin presence was not confirmed. Multiple toxins were detected at 86% of the tested sites and during 44% of the sampling events between 2014 and 2016. Although concentrations were low, continued exposure of organisms to multiple toxins raises some concerns. A combination of discrete sampling and in-situ tracking (Solid Phase Adsorption Toxin Tracking [SPATT]) revealed that microcystin and anatoxin were the most pervasive year-round. Between 2011 and 2016, summer and fall blooms were dominated by the same cyanobacterial genera, all of which are suggested producers of single or multiple cyanotoxins. The study's findings provide further evidence of the ubiquitous nature of cyanotoxins, and the challenges involved in linking CyanoHAB dynamics to specific environmental forcing factors are discussed.

Beta-Alanine Supplementation Improves Throwing Velocities in Repeated Sprint Ability and 200-m Swimming Performance in Young Water Polo Players.

PubMed

Claus, Gabriel Machado; Redkva, Paulo Eduardo; Brisola, Gabriel Mota Pinheiro; Malta, Elvis Sousa; de Araujo Bonetti de Poli, Rodrigo; Miyagi, Willian Eiji; Zagatto, Alessandro Moura

2017-05-01

The purpose of this study was to investigate the effects of beta-alanine supplementation on specific tests for water polo. Fifteen young water polo players (16 ± 2 years) underwent a 200-m swimming performance, repeated-sprint ability test (RSA) with free throw (shooting), and 30-s maximal tethered eggbeater kicks. Participants were randomly allocated into two groups (placebo × beta-alanine) and supplemented with 6.4g∙day -1 of beta-alanine or a placebo for six weeks. The mean and total RSA times, the magnitude based inference analysis showed a likely beneficial effect for beta-alanine supplementation (both). The ball velocity measured in the throwing performance after each sprint in the RSA presented a very like beneficial inference in the beta-alanine group for mean (96.4%) and percentage decrement of ball velocity (92.5%, likely beneficial). Furthermore, the percentage change for mean ball velocity was different between groups (beta-alanine=+2.5% and placebo=-3.5%; p = .034). In the 30-s maximal tethered eggbeater kicks the placebo group presented decreased peak force, mean force, and fatigue index, while the beta-alanine group maintained performance in mean force (44.1%, possibly beneficial), only presenting decreases in peak force. The 200-m swimming performance showed a possibly beneficial effect (68.7%). Six weeks of beta-alanine supplementation was effective for improving ball velocity shooting in the RSA, maintaining performance in the 30-s test, and providing possibly beneficial effects in the 200-m swimming performance.

Oligopeptides and copeptides of homochiral sequence, via beta-sheets, from mixtures of racemic alpha-amino acids, in a one-pot reaction in water; relevance to biochirogenesis.

PubMed

Illos, Roni A; Bisogno, Fabricio R; Clodic, Gilles; Bolbach, Gerard; Weissbuch, Isabelle; Lahav, Meir

2008-07-09

As part of our studies on the biochirogenesis of peptides of homochiral sequence during early evolution, the formation of oligopeptides composed of 14-24 residues of the same handedness in the polymerization of dl-leucine (Leu), dl-phenylalanine (Phe), and dl-valine (Val) in aqueous solutions, by activation with N, N'-carbonyldiimidazole and then initiation with a primary amine, in a one-pot reaction, was demonstrated by MALDI-TOF MS using deuterium enantio-labeled alpha-amino acids. The formation of long isotactic peptides is rationalized by the following steps occurring in tandem: (i) creation of a library of short diasteroisomeric oligopeptides containing isotactic peptides in excess in comparison to a binomial kinetics, as a result of an asymmetric induction exerted by the N-terminal residue of a given handedness; (ii) precipitation of the less soluble racemic isotactic penta- and hexapeptides in the form of beta-sheets that are delineated by homochiral rims; (iii) regio-enantiospecific chain elongation occurring heterogeneously at the beta-sheets/solution interface. Polymerization of l-Leu with l-isoleucine (Ile) or l-Phe with l- (1) N-Me-histidine yielded mixtures of copeptides containing both residues. In contrast, in the polymerization of the corresponding mixtures of l- + d-alpha-amino acids, the long oligopeptides were composed mainly from oligo- l-Leu and oligo- d-Ile in the first system and oligo- d-Phe in the second. Furthermore, in the polymerization of mixtures of hydrophobic racemic alpha-amino acids dl-Leu, dl-Val, and dl-Phe and with added racemic dl-alanine and dl-tyrosine, copeptides of homochiral sequences are most dominantly represented. Possible routes for a spontaneous "mirror-symmetry breaking" process of the racemic mixtures of homochiral peptides are presented.

Blood harmane is correlated with cerebellar metabolism in essential tremor: a pilot study.

PubMed

Louis, Elan D; Zheng, Wei; Mao, Xiangling; Shungu, Dikoma C

2007-08-07

On proton magnetic resonance spectroscopic imaging ((1)H MRSI), there is a decrease in cerebellar N-acetylaspartate/total creatine (NAA/tCr) in essential tremor (ET), signifying cerebellar neuronal dysfunction or degeneration. Harmane, which is present in the human diet, is a potent tremor-producing neurotoxin. Blood harmane concentrations seem to be elevated in ET. To assess in patients with ET whether blood harmane concentration is correlated with cerebellar NAA/tCR, a neuroimaging measure of neuronal dysfunction or degeneration. Twelve patients with ET underwent (1)H MRSI. The major neuroanatomic structure of interest was the cerebellar cortex. Secondary regions were the central cerebellar white matter, cerebellar vermis, thalamus, and basal ganglia. Blood concentrations of harmane and another neurotoxin, lead, were also assessed. Mean +/- SD cerebellar NAA/tCR was 1.52 +/- 0.41. In a linear regression model that adjusted for age and gender, log blood harmane concentration was a predictor of cerebellar NAA/tCR (beta = -0.41, p = 0.009); every 1 g(-10)/mL unit increase in log blood harmane concentration was associated with a 0.41 unit decrease in cerebellar NAA/tCR. The association between blood harmane concentration and brain NAA/tCR only occurred in the cerebellar cortex; it was not observed in secondary brain regions of interest. Furthermore, the association was specific to harmane and not another neurotoxin, lead. This study provides additional support for the emerging link between harmane, a neurotoxin, and ET. Further studies are warranted to address whether cerebellar harmane concentrations are associated with cerebellar pathology in postmortem studies of the ET brain.

Photoactivatable Rhodamine Spiroamides and Diazoketones Decorated with "Universal Hydrophilizer" or Hydroxyl Groups.

PubMed

Roubinet, Benoit; Bischoff, Matthias; Nizamov, Shamil; Yan, Sergey; Geisler, Claudia; Stoldt, Stefan; Mitronova, Gyuzel Y; Belov, Vladimir N; Bossi, Mariano L; Hell, Stefan W

2018-05-11

Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor superresolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-L-alanyl - beta-alanine dipeptide (a "universal hydrophilizer") or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The superresolution images of tubulin and keratin filaments in fixed and living cells exemplify the performance of "blinking" spiroamides derived from N,N,N',N'-tetramethyl rhodamine.

Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum

USDA-ARS?s Scientific Manuscript database

Aspartate 1-decarboxylase (ADC) and dopa decarboxylase (DDC) provide b–alanine and dopamine used in insect cuticle tanning. Beta-alanine is conjugated with dopamine to yield N-b-alanyldopamine (NBAD), a substrate for the phenoloxidase laccase that catalyzes the synthesis of cuticle protein cross-li...

Biosynthesis and accumulation of osmoprotective compounds by halophytic plants of the genus Limonium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanson, A.D.; Rathinasabapathi, B.; Gage, D.A.

1991-05-01

Analyses of quaternary ammonium compounds in leaf and root tissues of halophytic Limonium spp. using fast atom bombardment mass spectrometry revealed that only 3 out of 21 spp. accumulated glycine betaine, the common angiosperm osmolyte. The 18 other spp. accumulated {beta}-alanine betaine instead. However, all the Limonium spp. studied accumulated choline-O-sulfate and their leaf disks metabolized ({sup 14}C) choline to choline-O-sulfate. Only the glycine betaine accumulators oxidized ({sup 14}C) choline to glycine betaine and only {beta}-alanine betaine accumulators converted {beta}-({sup 14}C)alanine to {beta}-alanine betaine. When {beta}-alanine betaine and glycine betaine accumulators were salinized with NaCl, the levels of their respectivemore » betaines and of choline sulfate were closely correlated with solute potential. Glycine betaine accumulators had less choline-O-sulfate than {beta}-alanine betaine accumulators and increasing the SO{sub 4}{sup 2}/Cl ratio in the medium increased choline-O-sulfate and caused a matching decrease in glycine betaine. Thus, it appears that {beta}-alanine betaine has replaced glycine betaine in most members of this genus, eliminating a possible competition between glycine betaine and choline-O-sulfate for choline.« less

Formation of [b3 - 1 + cat]+ ions from metal-cationized tetrapeptides containing beta-alanine, gamma-aminobutyric acid or epsilon-aminocaproic acid residues.

PubMed

Osburn, Sandra M; Ochola, Sila O; Talaty, Erach R; Van Stipdonk, Michael J

2008-11-01

The presence and position of a single beta-alanine (betaA), gamma-aminobutyric acid (gammaABu) or epsilon-aminocaproic acid (Cap) residue has been shown to have a significant influence on the formation of b(n)+ and y(n)+ product ions from a series of model, protonated peptides. In this study, we examined the effect of the same residues on the formation of analogous [b3 - 1 + cat]+ products from metal (Li+, Na+ and Ag+)-cationized peptides. The larger amino acids suppress formation of b3+ from protonated peptides with general sequence AAXG (where X = beta-alanine, gamma-aminobutyric acid or epsilon-aminocaproic acid), presumably because of the prohibitive effect of larger cyclic intermediates in the 'oxazolone' pathway. However, abundant [b3 - 1 + cat]+ products are generated from metal-cationized versions of AAXG. Using a group of deuterium-labeled and exchanged peptides, we found that formation of [b3 - 1 + cat]+ involves transfer of either amide or alpha-carbon position H atoms, and the tendency to transfer the atom from the alpha-carbon position increases with the size of the amino acid in position X. To account for the transfer of the H atom, a mechanism involving formation of a ketene product as [b3 - 1 + cat]+ is proposed.

Centers for Oceans and Human Health: a unified approach to the challenge of harmful algal blooms

PubMed Central

Erdner, Deana L; Dyble, Julianne; Parsons, Michael L; Stevens, Richard C; Hubbard, Katherine A; Wrabel, Michele L; Moore, Stephanie K; Lefebvre, Kathi A; Anderson, Donald M; Bienfang, Paul; Bidigare, Robert R; Parker, Micaela S; Moeller, Peter; Brand, Larry E; Trainer, Vera L

2008-01-01

Background Harmful algal blooms (HABs) are one focus of the national research initiatives on Oceans and Human Health (OHH) at NIEHS, NOAA and NSF. All of the OHH Centers, from the east coast to Hawaii, include one or more research projects devoted to studying HAB problems and their relationship to human health. The research shares common goals for understanding, monitoring and predicting HAB events to protect and improve human health: understanding the basic biology of the organisms; identifying how chemistry, hydrography and genetic diversity influence blooms; developing analytical methods and sensors for cells and toxins; understanding health effects of toxin exposure; and developing conceptual, empirical and numerical models of bloom dynamics. Results In the past several years, there has been significant progress toward all of the common goals. Several studies have elucidated the effects of environmental conditions and genetic heterogeneity on bloom dynamics. New methods have been developed or implemented for the detection of HAB cells and toxins, including genetic assays for Pseudo-nitzschia and Microcystis, and a biosensor for domoic acid. There have been advances in predictive models of blooms, most notably for the toxic dinoflagellates Alexandrium and Karenia. Other work is focused on the future, studying the ways in which climate change may affect HAB incidence, and assessing the threat from emerging HABs and toxins, such as the cyanobacterial neurotoxin β-N-methylamino-L-alanine. Conclusion Along the way, many challenges have been encountered that are common to the OHH Centers and also echo those of the wider HAB community. Long-term field data and basic biological information are needed to develop accurate models. Sensor development is hindered by the lack of simple and rapid assays for algal cells and especially toxins. It is also critical to adequately understand the human health effects of HAB toxins. Currently, we understand best the effects of acute toxicity, but almost nothing is known about the effects of chronic, subacute toxin exposure. The OHH initiatives have brought scientists together to work collectively on HAB issues, within and across regions. The successes that have been achieved highlight the value of collaboration and cooperation across disciplines, if we are to continue to advance our understanding of HABs and their relationship to human health. PMID:19025673

Involvement of tyrosine residues, N-terminal amino acids, and beta-alanine in insect cuticular sclerotization.

PubMed

Andersen, Svend Olav

2007-09-01

During sclerotization of insect cuticle the acyldopamines, N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD), are oxidatively incorporated into the cuticular matrix, thereby hardening and stabilizing the material by forming crosslinks between the proteins in the cuticular matrix and by forming polymers filling the intermolecular spaces in the cuticle. Sclerotized cuticle from the locust, Schistocerca gregaria, and the beetle, Tenebrio molitor, was hydrolyzed in dilute hydrochloric acid, and from the hydrolysates some components presumably degradation products of cuticular crosslinks were isolated. In two of the components, the sidechain of 3,4-dihydroxyacetophenone was linked to the amino groups of glycine and beta-alanine, respectively, and in the third component to the phenolic group of tyrosine. These three compounds, glycino-dihydroxyacetophenone, beta-alanino-dihydroxyacetophenone, and O-tyrosino-dihydroxyacetophenone, as well as the previously reported compound, lysino-dihydroxyacetophenone [Andersen, S.O., Roepstorff, P., 2007. Aspects of cuticular sclerotization in the locust, Schistocerca gregaria, and the beetle, Tenebrio molitor. Insect Biochem. Mol. Biol. 37, 223-234], are suggested to be degradation products of cuticular crosslinks, in which amino acid residues formed linkages to both the alpha- and beta-positions of the sidechain of acyldopamines.

40 CFR 721.9582 - Certain perfluoroalkyl sulfonates.

Code of Federal Regulations, 2012 CFR

2012-07-01

...-0319306979-40-8 Poly(oxy-1,2-ethanediyl), .alpha.-[2-(methylamino)ethyl]-.omega.-[(1,1,3,3-tetramethylbutyl...]amino]ethyl]-.omega.-hydroxy- 38850-52-1 1-Propanaminium, 3-[(carboxymethyl)[(1,1,2,2,3,3,4,4,5,5,6,6,6...-heptadecafluorooctyl)sulfonyl]methylamino]carbonyl]-.omega.-butoxy- 52166-82-2 1-Propanaminium, N,N,N-trimethyl-3-[[(1...

40 CFR 721.9582 - Certain perfluoroalkyl sulfonates.

Code of Federal Regulations, 2013 CFR

2013-07-01

...-0319306979-40-8 Poly(oxy-1,2-ethanediyl), .alpha.-[2-(methylamino)ethyl]-.omega.-[(1,1,3,3-tetramethylbutyl...]amino]ethyl]-.omega.-hydroxy- 38850-52-1 1-Propanaminium, 3-[(carboxymethyl)[(1,1,2,2,3,3,4,4,5,5,6,6,6...-heptadecafluorooctyl)sulfonyl]methylamino]carbonyl]-.omega.-butoxy- 52166-82-2 1-Propanaminium, N,N,N-trimethyl-3-[[(1...

40 CFR 721.9582 - Certain perfluoroalkyl sulfonates.

Code of Federal Regulations, 2011 CFR

2011-07-01

...-0319306979-40-8 Poly(oxy-1,2-ethanediyl), .alpha.-[2-(methylamino)ethyl]-.omega.-[(1,1,3,3-tetramethylbutyl...]amino]ethyl]-.omega.-hydroxy- 38850-52-1 1-Propanaminium, 3-[(carboxymethyl)[(1,1,2,2,3,3,4,4,5,5,6,6,6...-heptadecafluorooctyl)sulfonyl]methylamino]carbonyl]-.omega.-butoxy- 52166-82-2 1-Propanaminium, N,N,N-trimethyl-3-[[(1...

40 CFR 721.9582 - Certain perfluoroalkyl sulfonates.

Code of Federal Regulations, 2010 CFR

2010-07-01

...-0319306979-40-8 Poly(oxy-1,2-ethanediyl), .alpha.-[2-(methylamino)ethyl]-.omega.-[(1,1,3,3-tetramethylbutyl...]amino]ethyl]-.omega.-hydroxy- 38850-52-1 1-Propanaminium, 3-[(carboxymethyl)[(1,1,2,2,3,3,4,4,5,5,6,6,6...-heptadecafluorooctyl)sulfonyl]methylamino]carbonyl]-.omega.-butoxy- 52166-82-2 1-Propanaminium, N,N,N-trimethyl-3-[[(1...

Compound-specific nitrogen isotope analysis of D-alanine, L-alanine, and valine: application of diastereomer separation to delta15N and microbial peptidoglycan studies.

PubMed

Takano, Yoshinori; Chikaraishi, Yoshito; Ogawa, Nanako O; Kitazato, Hiroshi; Ohkouchi, Naohiko

2009-01-01

We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (R)-(-)-2-butanol or (S)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of D- and L-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus, Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., delta15N ( per thousand vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 +/- 0.5 per thousand, n = 4) and L-alanine (21.3 +/- 0.8 per thousand, n = 4) and in Bacillus subtilis for D-alanine (6.2 +/- 0.2 per thousand, n = 3) and L-alanine (8.2 +/- 0.4 per thousand, n = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in 15N-depleted D-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers.

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification.

PubMed

Osipov, Alexey V; Kasheverov, Igor E; Makarova, Yana V; Starkov, Vladislav G; Vorontsova, Olga V; Ziganshin, Rustam Kh; Andreeva, Tatyana V; Serebryakova, Marina V; Benoit, Audrey; Hogg, Ronald C; Bertrand, Daniel; Tsetlin, Victor I; Utkin, Yuri N

2008-05-23

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

A serendipitous discovery of antifreeze protein-specific activity in C-linked antifreeze glycoprotein analogs.

PubMed

Eniade, Adewale; Purushotham, Madhusudhan; Ben, Robert N; Wang, J B; Horwath, Kathleen

2003-01-01

Structurally diverse carbon-linked (C-linked) analogs of antifreeze glycoprotein (AFGP) have been prepared via linear or convergent solid phase synthesis. These analogs range in molecular weight from approx 1.5-4.1 KDa and do not possess the beta-D-galactose-1,3-alpha-D-N-acetylgalactosamine carbohydrate moiety or the L-threonine-L-alanine-L-alanine polypeptide backbone native to the AFGP wild-type. Despite these dramatic structural modifications, the 2.7-KDa and 4.1-KDa analogs possess antifreeze protein-specific activity as determined by recrystallization-inhibition (RI) and thermal hysteresis (TH) assays. These analogs are weaker than the wild-type in their activity, but nanoliter osmometry indicates that these compounds are binding to ice and affecting a localized freezing point depression. This is the first example of a C-linked AFGP analog that possesses TH and RI activity and suggests that the rational design and synthesis of chemically and biologically stable AFGP analogs is a feasible and worthwhile endeavor. Given the low degree of TH activity, these compounds may prove useful for the protection of cells during freezing and thawing cycles.

Effect of various alanine analogues on the L-alanine-adding enzyme from Escherichia coli.

PubMed

Liger, D; Blanot, D; van Heijenoort, J

1991-05-01

An extract from Escherichia coli containing the L-alanine-adding enzyme with a high specific activity was prepared. Several compounds structurally related to L-alanine were tested as inhibitors of this activity. Intact amino and carboxyl groups were necessary for an interaction with the enzyme. Certain halogenated (haloalanines) or unsaturated (L-vinylglycine, L-propargylglycine, 3-cyano-L-alanine) amino acids were good inhibitors. Radioactive glycine, serine and 1-aminoethylphosphonic acid were tested as substrates. Whereas glycine or L-serine gave rise to the formation of the corresponding nucleotide product, no synthesis of UDP-N-acetylmuramyl-L-1-aminoethylphosphonic acid could be detected.

Regulation of endothelial nitric oxide synthase: involvement of protein kinase G 1 beta, serine 116 phosphorylation and lipid structures.

PubMed

John, Theresa A; Ibe, Basil O; Raj, J Usha

2008-02-01

1. Endothelial nitric oxide synthase (NOS3) is important for vascular homeostasis. The role of protein kinase G (PKG) in regulation of NOS3 activity was studied in primary cultures of newborn lamb lung microvascular endothelial cells (LMVEC). 2. We determined the presence of PKG in fetal and neonatal LMVEC as well as subcellular localization of PKG isoforms in the neonatal cells by fluorescence immunohistochemistry. We used diaminofluorescein (DAF) fluorophore to measure nitric oxide (NO) production from neonatal LMVEC. We confirmed that NO measured was from constitutive NOS3 by inhibiting it with NOS inhibitors. 3. To identify a role for PKG in basal NO production, we measured NO release from LMVEC cells using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM; 0.5-0.8 micromol/L) with and without prior stimulation with the PKG activator 8-bromo-cGMP (8-Br-cGMP; 0.3 and 3 micromol/L) or prior PKG inhibition with beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (BPC; 0.3 and 3 micromol/L). With the same drugs, we determined the role of PKG on cellular expression of NOS3 and serine 116 phosphorylated NOS (pSer116-NOS) by qualitative and quantitative immunofluorescence assays, as well as western blotting. 4. Because PKG 1 beta was distributed throughout the cytosol in a punctate expression, we used 2 mmol/L cyclodextrin, a cholesterol extractor, to determine a role for lipid vesicles in PKG regulation of NO production. 5. Protein kinase G 1 beta gave a punctate appearance, indicating its presence in intracellular vesicles. Nitric oxide production decreased by approximately 20% with 300 nmol/L and 3 micromol/L 8-Br cGMP (P < 0.05) and increased by 20.8 +/- 3.7% with 3 micromol/L BPC (P < 0.001), indicating that both stimulated and basal PKG activity has inhibitory effects on basal NOS3 function. Nitric oxide synthase immunofluorescence and immunoblot expression were decreased and pSer116-NOS immunofluorescence was increased by 800 nmol/L 8-Br-cGMP and 170 micromol/L (Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1, 2-diolate (DETANONOate). The effect of cyclodextrin indicated that cholesterol extraction interfered with PKG inhibition of NOS. Further examination of pSer116-NOS by immunohistochemistry showed it abundant in the endoplasmic reticulum and colocalized with PKG 1 beta, especially in nuclear vesicles. 6. We conclude that endothelial PKG is involved in endogenous regulation of basal NOS3 activity with the involvement of lipid structures, the endoplasmic reticulum and the nucleus. Protein kinase G 1 beta is colocalized with pSer116-NOS, indicating that PKG action may involve serine 116 phosphorylation on NOS.

Enzymatic preparation of. cap alpha. - and. beta. -deuterated or tritiated amino acids with l-methionine. gamma. -lyase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Esaki, N.; Sawada, S.; Tanaka, H.

L-Methionine ..gamma..-lyase catalyzes the exchange of ..cap alpha..- and ..beta..-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium or tritium of solvents. The rate of ..cap alpha..-hydrogen exchange with deuterium was about 40 times faster than that of the elimination reactions. The deuterium and tritium were exchanged also with the ..cap alpha..- and ..beta..-hydrogens of the straight-chain amino acids which do not undergo the elimination: L-alanine, L-..cap alpha..-aminobutyrate, L-norvaline, and L-norleucine. No exchange occurs for the D-isomers, acidic L-amino acids, basic L-amino acids, and branched-chain L-amino acids, although ..cap alpha..-hydrogen of glycine, L-trypotophan, and L-phenylalanine is exchanged slowly. These enzymatic hydrogen-exchange reactionsmore » facilitate specific labeling of the L-amino acids with deuterium and tritium.« less

The effects of beta alanine plus creatine administration on performance during repeated bouts of supramaximal exercise in sedentary men.

PubMed

Okudan, N; Belviranli, M; Pepe, H; Gökbel, H

2015-11-01

The aim of this study was to investigate the effects of beta alanine and/or creatine supplementation on performance during repeated bouts of supramaximal exercise in sedentary men. Forty-four untrained healthy men (aged 20-22 years, weight: 68-72 kg, height: 174-178 cm) participated in the present study. After performing the Wingate Test (WAnT) for three times in the baseline exercise session, the subjects were assigned to one of four treatment groups randomly: 1) placebo (P; 10 g maltodextrose); 2) creatine (Cr; 5 g creatine plus 5 g maltodextrose); 3) beta-alanine (β-ALA; 1,6 g beta alanine plus 8,4 g maltodextrose); and 4) beta-alanine plus creatine (β-ALA+Cr; 1,6 g beta alanine plus 5 g creatine plus 3,4 g maltodextrose). Participants were given the supplements orally twice a day for 22 consecutive days, then four times a day for the following 6 days. After 28 days, the second exercise session was applied during which peak power (PP) and mean power (MP) were measured and fatigue index (FI) was calculated. PP and MP decreased and FI increased in all groups during exercise before and after the treatment. During the postsupplementation session PP2 and PP3 increased in creatine supplemented group (from 642.7±148.6 to 825.1±205.2 in PP2 and from 522.9±117.5 to 683.0±148.0 in PP3, respectively). However, MP increased in β-ALA+Cr during the postsupplementation compared to presupplementation in all exercise sessions (from 586.2±55.4 to 620.6±49.6 in MP1, from 418.1±37.2 to 478.3±30.3 in MP2 and from 362.0±41.3 to 399.1±3 in MP3, respectively). FI did not change with beta alanine and beta alanine plus creatine supplementation during the postsupplementation exercise session. Beta-alanine and beta alanine plus creatine supplementations have strong performance enhancing effect by increasing mean power and delaying fatigue Index during the repeated WAnT.

Inhibition of cystathionine-gamma-lyase leads to loss of glutathione and aggravation of mitochondrial dysfunction mediated by excitatory amino acid in the CNS.

PubMed

Diwakar, Latha; Ravindranath, Vijayalakshmi

2007-01-01

Oxidative stress has been implicated in the pathogenesis and progression of neurodegenerative disorders and antioxidants potentially have a major role in neuroprotection. Optimum levels of glutathione (gamma-glutamylcysteinyl glycine), an endogenous thiol antioxidant are required for the maintenance of the redox status of cells. Cystathionine gamma-lyase is the rate-limiting enzyme for the synthesis of cysteine from methionine and availability of cysteine is a critical factor in glutathione synthesis. In the present study, we have examined the role of cystathionine gamma-lyase in maintaining the redox homeostasis in brain, particularly with reference to mitochondrial function since the complex I of the electron transport chain is sensitive to redox perturbation. Inhibition of cystathionine gamma-lyase by l-propargylglycine caused loss of glutathione and decrease in complex I activity in the brain although the enzyme activity in mouse brain was 1% of the corresponding hepatic activity. We then examined the effect of this inhibition on the neurotoxicity mediated by the excitatory amino acid, l-beta-oxalyl amino-l-alanine, which is the causative factor of a type of motor neuron disease, neurolathyrism. l-beta-Oxalyl amino-l-alanine toxicity was exacerbated by l-propargylglycine measured as loss of complex I activity indicating the importance of cystathionine gamma-lyase in maintaining glutathione levels and in turn the mitochondrial function during excitotoxicity. Oxidative stress generated by l-beta-oxalyl amino-l-alanine itself inhibited cystathionine gamma-lyase, which could be prevented by prior treatment with thiol antioxidant. Thus, cystathionine gamma-lyase itself is susceptible to inactivation by oxidative stress and this can potentially exacerbate oxidant-induced damage. Cystathionine gamma-lyase is present in neuronal cells in human brain and its activity is several-fold higher compared to mouse brain. It could potentially play an important role in maintaining glutathione and protein thiol homeostasis in brain and hence afford neuroprotection.

Synthesis and characterization of poly(L-alanine)-block-poly(ethylene glycol) monomethyl ether as amphiphilic biodegradable co-polymers.

PubMed

Zhang, Guolin; Ma, Jianbiao; Li, Yanhong; Wang, Yinong

2003-01-01

Di-block co-polymers of poly(L-alanine) with poly(ethylene glycol) monomethyl ether (MPEG) were synthesized as amphiphilic biodegradable co-polymers. The ring-opening polymerization of N-carboxy-L-alanine anhydride (NCA) in dichloromethane was initiated by amino-terminated poly(ethylene glycol) monomethyl ether (MPEG-NH2, M(n) = 2000) to afford poly(L-alanine)-block-MPEG. The weight ratio of two blocks in the co-polymers could be altered by adjusting the feeding ratio of NCA to MPEG-NH2. Their chemical structures were characterized on the basis of infrared spectrometry and nuclear magnetic resonance. According to circular dichroism measurement, the poly(L-alanine) chain on the co-polymers in an aqueous medium had a alpha-helix conformation. Two melting points from MPEG block and poly(L-alanine), respectively, could be observed in differential scanning calorimetry curves of the co-polymers, suggesting that a micro-domain phase separation appeared in their bulky states. The co-polymers could take up some water and the capacity was dependent on the ratio of poly(L-alanine) block to MPEG. Such co-polymers might be useful in drug-delivery systems and other biomedical applications.

N-Methylamino Pyrimidyl Amides (MAPA): Highly Reactive, Electronically-Activated Amides in Catalytic N-C(O) Cleavage.

PubMed

Meng, Guangrong; Lalancette, Roger; Szostak, Roman; Szostak, Michal

2017-09-01

Despite recent progress in catalytic cross-coupling technologies, the direct activation of N-alkyl-N-aryl amides has been a challenging transformation. Here, we report the first Suzuki cross-coupling of N-methylamino pyrimidyl amides (MAPA) enabled by the controlled n N → π Ar conjugation and the resulting remodeling of the partial double bond character of the amide bond. The new mode of amide activation is suitable for generating acyl-metal intermediates from unactivated primary and secondary amides.

VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion.

PubMed Central

Regazzi, R; Wollheim, C B; Lang, J; Theler, J M; Rossetto, O; Montecucco, C; Sadoul, K; Weller, U; Palmer, M; Thorens, B

1995-01-01

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta-cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP-2 and cellubrevin, but not VAMP-1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta-cells contain secretory granules that store and secrete insulin as well as synaptic-like microvesicles carrying gamma-aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP-2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP-2 with insulin-containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta-cells. Pretreatment of streptolysin-O permeabilized insulin-secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP-2 and cellubrevin and abolished Ca(2+)-induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S-stimulated insulin secretion. Taken together, our results show that pancreatic beta-cells express VAMP-2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)-mediated insulin secretion. Images PMID:7796801

Study of the overproduced uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli.

PubMed

Liger, D; Masson, A; Blanot, D; van Heijenoort, J; Parquet, C

1996-01-01

The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli is responsible for the addition of the first amino acid of the peptide moiety in the assembly of the monomer unit of peptidoglycan. It catalyzes the formation of the amide bond between UDP-N-acetylmuramic acid (UDP-MurNAc) and L-alanine. The UDP-MurNAc-L-alanine ligase was overproduced 2000-fold in a strain harboring a recombinant plasmid (pAM1005) with the murC gene under the control of the inducible promoter trc. The murC gene product appears as a 50-kDa protein accounting for ca. 50% of total cell proteins. A two-step purification led to 1 g of a homogeneous protein from an 8-liter culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the gene. The stability of the enzymatic activity is strictly dependent on the presence of 2-mercaptoethanol. The K(m) values for substrates UDP-N-acetylmuramic acid, L-alanine, and ATP were estimated; 100, 20, and 450 microM, respectively. The specificity of the enzyme for its substrates was investigated with various analogues. Preliminary experiments attempting to elucidate the enzymatic mechanism were consistent with the formation of an acylphosphate intermediate.

Algal Blooms and Cyanotoxins in Jordan Lake, North Carolina

PubMed Central

Wiltsie, Daniel; Schnetzer, Astrid; Green, Jason; Vander Borgh, Mark; Fensin, Elizabeth

2018-01-01

The eutrophication of waterways has led to a rise in cyanobacterial, harmful algal blooms (CyanoHABs) worldwide. The deterioration of water quality due to excess algal biomass in lakes has been well documented (e.g., water clarity, hypoxic conditions), but health risks associated with cyanotoxins remain largely unexplored in the absence of toxin information. This study is the first to document the presence of dissolved microcystin, anatoxin-a, cylindrospermopsin, and β-N-methylamino-l-alanine in Jordan Lake, a major drinking water reservoir in North Carolina. Saxitoxin presence was not confirmed. Multiple toxins were detected at 86% of the tested sites and during 44% of the sampling events between 2014 and 2016. Although concentrations were low, continued exposure of organisms to multiple toxins raises some concerns. A combination of discrete sampling and in-situ tracking (Solid Phase Adsorption Toxin Tracking [SPATT]) revealed that microcystin and anatoxin were the most pervasive year-round. Between 2011 and 2016, summer and fall blooms were dominated by the same cyanobacterial genera, all of which are suggested producers of single or multiple cyanotoxins. The study’s findings provide further evidence of the ubiquitous nature of cyanotoxins, and the challenges involved in linking CyanoHAB dynamics to specific environmental forcing factors are discussed. PMID:29495289

Staphylococcus aureus MurC participates in L-alanine recognition via histidine 343, a conserved motif in the shallow hydrophobic pocket.

PubMed

Kurokawa, Kenji; Nishida, Satoshi; Ishibashi, Mihoko; Mizumura, Hikaru; Ueno, Kohji; Yutsudo, Takashi; Maki, Hideki; Murakami, Kazuhisa; Sekimizu, Kazuhisa

2008-03-01

UDP-N-acetylmuramic acid:L-alanine ligase that is encoded by the murC gene, is indispensable for bacterial peptidoglycan biosynthesis and an important target for the development of antibacterial agents. Structure of MurC ligase with substrates has been described, however, little validation via studying the effects of mutations on the structure of MurC has been performed. In this study, we carried out a functional in vitro and in vivo characterization of Staphylococcus aureus MurCH343Y protein that has a temperature-sensitive mutation of a conserved residue in the predicted shallow hydrophobic pocket that holds a short L-alanine side chain. Purified H343Y and wild-type MurC had K(m) values for L-alanine of 3.2 and 0.44 mM, respectively, whereas there was no significant difference in their K(m) values for ATP and UDP-N-acetylmuramic acid, suggesting the specific alteration of L-alanine recognition in MurCH343Y protein. In a synthetic medium that excluded L-alanine, S. aureus murCH343Y mutant cells showed an allele-specific slow growth phenotype that was suppressed by addition of L-alanine. These results suggest that His343 of S. aureus MurC is essential for high-affinity binding to L-alanine both in vitro and in vivo and provide experimental evidence supporting the structural information of MurC ligase.

Biochemistry of snake venom neurotoxins and their application to the study of synapse. [Neurotoxins isolated from venom of the Formosan banded krait

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanley, M.R.

1978-11-01

The crude venom of the Formosan banded krait, Bungarus multicinctus, was separated into eleven lethal protein fractions. Nine fractions were purified to final homogeneous toxins, designated ..cap alpha..-bungarotoxin, ..beta..-bungarotoxin, and toxins 7, 8, 9A, 11, 12, 13, and 14. Three of the toxins, ..cap alpha..-bungarotoxin, 7, and 8, were identified as post-synaptic curarimimetic neurotoxins. The remaining toxins were identified as pre-synaptic neurotoxins. ..cap alpha..-Bungarotoxin, toxin 7, and toxin 8 are all highly stable basic polypeptides of approx. 8000 daltons molecular weight. The pre-synaptic toxins fell into two structural groups: toxin 9A and 14 which were single basic chains of approx.more » 14,000 daltons, and ..beta..-bungarotoxin, and toxins 11 thru 13 which were composed of two chains of approx. 8000 and approx. 13,000 daltons covalently linked by disulfides. All the pre-synaptic neurotoxins were shown to have intrinsic calcium-dependent phospholipase A activities. Under certain conditions, intact synaptic membranes were hydrolyzed more rapidly than protein-free extracted synaptic-lipid liposomes which, in turn, were hydrolyzed more rapidly than any other tested liposomes. It was speculated that cell-surface arrays of phosphatidyl serine/glycolipids created high affinity target sites for ..beta..-bungarotoxin. Single-chain toxins were found to be qualitatively different from the two-chain toxins in their ability to block the functioning of acetylcholine receptors, and were quantitatively different in their enzymatic and membrane disruptive activities. ..beta..-Bungarotoxin was shown to be an extremely potent neuronal lesioning agent. There was no apparent selectivity for cholinergic over non-cholinergic neurons, nor for nerve terminals over cell bodies. It was suggested that ..beta..-bungarotoxin can be considered a useful new histological tool, which may exhibit some regional selectivity.« less

Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system.

PubMed

Hodgson, A J; Penke, B; Erdei, A; Chubb, I W; Somogyi, P

1985-03-01

Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.

H NMR studies of substrate hydrogen exchange reactions catalyzed by L-methionine gamma-lyase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Esaki, N.; Nakayama, T.; Sawada, S.

Hydrogen exchange reactions of various L-amino acids catalyzed by L-methionine gamma-lyase (EC 4.4.1.11) have been studied. The enzyme catalyzes the rapid exchange of the alpha- and beta-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium from the solvent. The rate of alpha-hydrogen exchange was about 40 times faster than that of the enzymatic elimination reaction of the sulfur-containing amino acids. The enzyme also catalyzes the exchange reaction of alpha- and beta-hydrogens of the straight-chain L-amino acids which are not susceptible to elimination. The exchange rates of the alpha-hydrogen and the total beta-hydrogens of L-alanine and L-alpha-aminobutyrate with deuterium followed first-order kinetics. Formore » L-norvaline, L-norleucine, S-methyl-L-cysteine, and L-methionine, the rate of alpha-hydrogen exchange followed first-order kinetics, but the rate of total beta-hydrogen exchange decreased due to a primary isotope effect at the alpha-position. L-Phenylalanine and L-tryptophan slowly underwent alpha-hydrogen exchange. The pro-R hydrogen of glycine was deuterated stereospecifically.« less

Anaerobic degradation of amino acids generated from the hydrolysis of sewage sludge.

PubMed

Park, Junghoon; Park, Seyong; Kim, Moonil

2014-01-01

The anaerobic degradation of each amino acid that could be generated through the hydrolysis of sewage sludge was evaluated. Stickland reaction as an intermediate reaction between two kinds of amino acids was restricted in order to evaluate each amino acid. Changes in the chemical oxygen demand (COD), T-N, NH4(+)-N, biogas, and CH4 were analysed for the anaerobic digestion process. The initial nitrogen concentration of all amino acids is adjusted as 1000 mg/L. The degradation rate of the amino acids was determined based on the ammonia form of nitrogen, which is generated by the deamination of amino acids. Among all amino acids, such as alpha-alanine, beta-alanine, lysine, arginine, glycine, histidine, cysteine, methionine, and leucine, deamination rates of cysteine, leucine, and methionine were just 61.55%, 54.59%, and 46.61%, respectively, and they had low removal rates of organic matter and showed very low methane production rates of 13.55, 71.04, and 80.77 mL CH4/g CODin, respectively. Especially for cysteine, the methane content was maintained at approximately 7% during the experiment. If wastewater contains high levels of cysteine, leucine, and methionine and Stickland reaction is not prepared, these amino acids may reduce the efficiency of the anaerobic digestion.

Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nuemket, Nipawan; Tanaka, Yoshikazu; Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810

2011-07-29

Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinummore » neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.« less

Cellular and Physiological Effects of Dietary Supplementation with β-Hydroxy-β-Methylbutyrate (HMB) and β-Alanine in Late Middle-Aged Mice.

PubMed

Vallejo, Julian; Spence, Madoka; Cheng, An-Lin; Brotto, Leticia; Edens, Neile K; Garvey, Sean M; Brotto, Marco

2016-01-01

There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C2C12 myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid β-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C2C12 myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C2C12 myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an ex vivo muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with β-alanine, or diet with HMB and β-alanine. In β-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus β-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm2, P = 0.021). Also in EDL muscle, β-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and β-alanine supplementation on skeletal muscle function in aging mice.

Cellular and Physiological Effects of Dietary Supplementation with β-Hydroxy-β-Methylbutyrate (HMB) and β-Alanine in Late Middle-Aged Mice

PubMed Central

Vallejo, Julian; Spence, Madoka; Cheng, An-Lin; Brotto, Leticia; Edens, Neile K.; Garvey, Sean M.; Brotto, Marco

2016-01-01

There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C2C12 myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid β-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C2C12 myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C2C12 myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an ex vivo muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with β-alanine, or diet with HMB and β-alanine. In β-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus β-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm2, P = 0.021). Also in EDL muscle, β-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and β-alanine supplementation on skeletal muscle function in aging mice. PMID:26953693

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ngo, Sam; Guo, Zhefeng, E-mail: zhefeng@ucla.edu

Highlights: Black-Right-Pointing-Pointer A{beta} oligomers are neurotoxins and likely the causing agents for Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}42 fusion protein form globular oligomers. Black-Right-Pointing-Pointer A{beta}42 fusion protein oligomers contain SDS-resistant tetramers and hexamers. Black-Right-Pointing-Pointer Cysteine substitutions at residues 31, 32, 34, 39-41 disrupt A{beta}42 oligomerization. -- Abstract: Deposition of amyloid fibrils consisting of amyloid {beta} (A{beta}) protein as senile plaques in the brain is a pathological hallmark of Alzheimer's disease. However, a growing body of evidence shows that soluble A{beta} oligomers correlate better with dementia than fibrils, suggesting that A{beta} oligomers may be the primary toxic species. The structure and oligomerization mechanismmore » of these A{beta} oligomers are crucial for developing effective therapeutics. Here we investigated the oligomerization of A{beta}42 in the context of a fusion protein containing GroES and ubiquitin fused to the N-terminus of A{beta} sequence. The presence of fusion protein partners, in combination with a denaturing buffer containing 8 M urea at pH 10, is unfavorable for A{beta}42 aggregation, thus allowing only the most stable structures to be observed. Transmission electron microscopy showed that A{beta}42 fusion protein formed globular oligomers, which bound weakly to thioflavin T and Congo red. SDS-PAGE shows that A{beta}42 fusion protein formed SDS-resistant hexamers and tetramers. In contrast, A{beta}40 fusion protein remained as monomers on SDS gel, suggesting that the oligomerization of A{beta}42 fusion protein is not due to the fusion protein partners. Cysteine scanning mutagenesis at 22 residue positions further revealed that single cysteine substitutions of the C-terminal hydrophobic residues (I31, I32, L34, V39, V40, and I41) led to disruption of hexamer and tetramer formation, suggesting that hydrophobic interactions between these residues are most critical for A{beta}42 oligomerization.« less

Does α-Amino-β-methylaminopropionic Acid (BMAA) Play a Role in Neurodegeneration?

PubMed Central

Chiu, Alexander S.; Gehringer, Michelle M.; Welch, Jeffrey H.; Neilan, Brett A.

2011-01-01

The association of α-amino-β-methylaminopropionic acid (BMAA) with elevated incidence of amyotrophic lateral sclerosis/Parkinson’s disease complex (ALS/PDC) was first identified on the island of Guam. BMAA has been shown to be produced across the cyanobacterial order and its detection has been reported in a variety of aquatic and terrestrial environments worldwide, suggesting that it is ubiquitous. Various in vivo studies on rats, mice, chicks and monkeys have shown that it can cause neurodegenerative symptoms such as ataxia and convulsions. Zebrafish research has also shown disruption to neural development after BMAA exposure. In vitro studies on mice, rats and leeches have shown that BMAA acts predominantly on motor neurons. Observed increases in the generation of reactive oxygen species (ROS) and Ca2+ influx, coupled with disruption to mitochondrial activity and general neuronal death, indicate that the main mode of activity is via excitotoxic mechanisms. The current review pertaining to the neurotoxicity of BMAA clearly demonstrates its ability to adversely affect neural tissues, and implicates it as a potentially significant compound in the aetiology of neurodegenerative disease. When considering the potential adverse health effects upon exposure to this compound, further research to better understand the modes of toxicity of BMAA and the environmental exposure limits is essential. PMID:22016712

Staphylococcus aureus Peptidoglycan Stem Packing by Rotational-Echo Double Resonance NMR Spectroscopy

PubMed Central

Kim, Sung Joon; Singh, Manmilan; Preobrazhenskaya, Maria; Schaefer, Jacob

2013-01-01

Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with D-[1-13C]alanine and L-[15N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between 13C and 15N of labeled amino acids incorporated in the peptidoglycan, and from those labels to 19F of a glycopeptide drug specifically bound to the peptidoglycan. The observed 13C-15N average distance of 4.1 to 4.4 Å between D- and L-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus. PMID:23617832

Influence of proline upon the folding and geometry of the WALP19 transmembrane peptide.

PubMed

Thomas, Rachel; Vostrikov, Vitaly V; Greathouse, Denise V; Koeppe, Roger E

2009-12-22

The orientations, geometries, and lipid interactions of designed transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because the amino acid proline will introduce a known discontinuity into an alpha helix, we have sought to measure the extent of helix kinking caused by a single proline within the isolated TM helical domain of WALP19. For this purpose, we synthesized acetyl-GWWLALALAP(10)ALALALWWA-ethanolamide and included pairs of deuterated alanines by using 60-100% Fmoc-l-Ala-d(4) at selected sequence positions. Solid-state deuterium ((2)H) magnetic resonance spectra from oriented, hydrated samples (1/40, peptide/lipid; using several lipids) reveal signals from many of the alanine backbone C(alpha) deuterons as well as the alanine side-chain C(beta) methyl groups, whereas signals from C(alpha) deuterons generally have not been observed for similar peptides without proline. It is conceivable that altered peptide dynamics may be responsible for the apparent "unmasking" of the backbone resonances in the presence of the proline. Data analysis using the geometric analysis of labeled alanines (GALA) method reveals that the peptide helix is distorted due to the presence of the proline. To provide additional data points for evaluating the segmental tilt angles of the two halves of the peptide, we substituted selected leucines with l-Ala-d(4). Using this approach, we were able to deduce that the apparent average tilt of the C-terminal increases from approximately 4 degrees to approximately 12 degrees when Pro(10) is introduced. The segment N-terminal to proline is more complex and possibly is more dynamically flexible; Leu to Ala mutations within the N-terminal segment alter the average orientations of alanines in both segments. Nevertheless, in DOPC, we could estimate an apparent kink angle of approximately 19 degrees . Together, the results suggest that the central proline influences not only the geometry but also the dynamics of the membrane-spanning peptide. The results make up an important basis for understanding the functional role of proline in several families of membrane proteins.

New Poly(amide-imide)/Nanocomposites Reinforced Silicate Nanoparticles Based on N-pyromellitimido-L-phenyl Alanine Containing Ether Moieties

NASA Astrophysics Data System (ADS)

Faghihi, Khalil; Shabanian, Meisam; Dadfar, Ehsan

2012-02-01

A series of Poly(amide-imide)/montmorillonite nanocomposites containing N-pyromellitimido-L-phenyl alanine moiety in the main chain were synthesized by a convenient solution intercalation technique. Poly(amide-imide) (PAI) 5 as a source of polymer matrix was synthesized by the direct polycondensation reaction of N-pyromellitimido-L-phenyl alanine 3 with 4,4'-diamino diphenyl ether 4 in the presence of triphenyl phosphite (TPP), CaCl2, pyridine and N-methyl-2-pyrrolidone (NMP). The resulting nanocomposite films were characterized by Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). The results showed that organo-modified clay was dispersed homogeneously in PAI matrix. TGA indicated an enhancement of thermal stability of new nanocomposites compared with the pure polymer.

Structure-activity relationships of N-beta-phenylpropionyl-L-tyrosine and its derivatives on the inhibition of an identifiable giant neurone of an African giant snail (Achatina fulica Férussac).

PubMed Central

Ariyoshi, Y.; Takeuchi, H.

1982-01-01

1 Inhibitory effects of N-beta-phenylpropionyl-L-tyrosine, N-beta-phenylpropionyl-L-tryptophan and their derivatives on an identifiable giant neurone, TAN (tonically autoactive neurone) of an African giant snail (Achatina fulica Férussac) were examined in an attempt to elucidate which structural features are necessary to produce the effect. 2 Of the compounds examined, N-beta-cyclohexylpropionyl-L-tyrosine showed the strongest effect. Its critical concentration (c.c.) was 3 X 10(-8)-10(-7)M, about ten times lower than that of N-beta-phenylpropionyl-L-tyrosine (c.c., 3 X 10(-7)-10(-6)M). N-beta-cyclohexylpropionyl-L-tryptophan (c.c., 10(-6)M) had an effect almost similar to that of N-beta-phenylpropionyl-L-tryptophan (c.c., 10(-6)M). 3 N-beta-Phenylpropionyl-N-methyl-L-tyrosine had no effect at a high concentration. 4 Effects of N-beta-phenylpropionyl-L-tyrosine amide (c.c., 3 X 10(-7)-10(-6)M) and N-beta-phenylpropionyl-L-tryptophan amide (c.c., 10(-6)M) were very similar to those of N-beta-phenylpropionyl-L-tyrosine and N-beta-phenylpropionyl-L-tryptophan respectively. 5 N-beta-Phenylpropionyl-p-amino-L-phenylalanine (c.c., 3 X 10(-5)-10(-4)M) and N-beta-phenylpropionyl-p-chloro-L-phenylalanine (c.c., 10(-4)M) had only a weak effect. 6 It is proposed that the structural features producing the effect are as follows: the active compound has a phenyl or a cyclohexyl group (hydrophobic binding group), after a suitable distance a peptide bond (proton donor and proton acceptor), adjacently a carbonyl group (proton acceptor), and a phenolic hydroxyl or an indolyl imino group (proton donor) in the molecule. PMID:7150871

The hybrid modulatory/pattern generating N1L interneuron in the buccal feeding system of Lymnaea is cholinergic.

PubMed

Vehovszky, A; Elliott, C J

1995-01-01

This study examines neurotransmission between identified buccal interneurons in the feeding system of the snail Lymnaea stagnalis. We compare the pharmacology of the individual synaptic connections from a hybrid modulatory/pattern generating interneuron (N1L) to a pattern generating interneuron (N1M) with that from a modulatory interneuron (SO) to the same follower cell (N1M). The pharmacological properties of the N1L to N1M and the SO to N1M connections closely resemble each other. Both interneurons produce fast cholinergic EPSPs as judged by the blocking effects of cholinergic antagonists hexamethonium, d-tubocurarine and the cholinergic neurotoxin AF-64A. A slower, more complex but non-cholinergic component of the synaptic response is also present after stimulating either the presynaptic N1L or SO interneurons. This second component of the postsynaptic response is not dopaminergic, on the basis of its persistence in the presence of dopaminergic antagonists ergometrine and fluphenazine and the dopaminergic neurotoxin MPP+. We conclude that, although there has been an evolutionary divergence in function, the modulatory SO and the hybrid modulatory/pattern generating N1L are pharmacologically similar. Neither of them contributes directly to dopaminergic modulation of the feeding activity. These neurons also resemble the N1M protraction phase pattern generating neurons which are cholinergic (Elliott and Kemenes, 1992).

Design and synthesis of novel N-benzylidenesulfonohydrazide inhibitors of MurC and MurD as potential antibacterial agents.

PubMed

Frlan, Rok; Kovac, Andreja; Blanot, Didier; Gobec, Stanislav; Pecar, Slavko; Obreza, Ales

2008-01-11

A series of novel N-benzylidenesulfonohydrazide compounds were designed and synthesized as inhibitors of UDP-N-acetylmuramic acid: L-alanine ligase (MurC) and UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase (MurD) from E. coli, involved in the biosynthesis of bacterial cell-walls. Some compounds possessed inhibitory activity against both enzymes with IC(50) values as low as 30 microM. In addition, a new, one-pot synthesis of amidobenzaldehydes is reported.

X-ray diffraction study of nanocrystalline and amorphous structure within major and minor ampullate dragline spider silks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sampath, Sujatha; Isdebski, Thomas; Jenkins, Janelle E.

Synchrotron X-ray micro-diffraction experiments were carried out on Nephila clavipes (NC) and Argiope aurantia (AA) major (MA) and minor ampullate (MiA) fibers that make up dragline spider silk. The diffraction patterns show a semi-crystalline structure with {beta}-poly(L-alanine) nanocrystallites embedded in a partially oriented amorphous matrix. A superlattice reflection 'S' diffraction ring is observed, which corresponds to a crystalline component larger in size and is poorly oriented, when compared to the {beta}-poly(L-alanine) nanocrystallites that are commonly observed in dragline spider silks. Crystallite size, crystallinity and orientation about the fiber axis have been determined from the wide-angle X-ray diffraction (WAXD) patterns. Inmore » both NC and AA, the MiA silks are found to be more highly crystalline, when compared with the corresponding MA silks. Detailed analysis on the amorphous matrix shows considerable differences in the degree of order of the oriented amorphous component between the different silks studied and may play a crucial role in determining the mechanical properties of the silks.« less

Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

PubMed

Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

2007-10-01

The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

Dietary BMAA exposure in an amyotrophic lateral sclerosis cluster from southern France.

PubMed

Masseret, Estelle; Banack, Sandra; Boumédiène, Farid; Abadie, Eric; Brient, Luc; Pernet, Fabrice; Juntas-Morales, Raoul; Pageot, Nicolas; Metcalf, James; Cox, Paul; Camu, William

2013-01-01

Dietary exposure to the cyanotoxin BMAA is suspected to be the cause of amyotrophic lateral sclerosis in the Western Pacific Islands. In Europe and North America, this toxin has been identified in the marine environment of amyotrophic lateral sclerosis clusters but, to date, only few dietary exposures have been described. We aimed at identifying cluster(s) of amyotrophic lateral sclerosis in the Hérault district, a coastal district from Southern France, and to search, in the identified area(s), for the existence of a potential dietary source of BMAA. A spatio-temporal cluster analysis was performed in the district, considering all incident amyotrophic lateral sclerosis cases identified from 1994 to 2009 by our expert center. We investigated the cluster area with serial collections of oysters and mussels that were subsequently analyzed blind for BMAA concentrations. We found one significant amyotrophic lateral sclerosis cluster (p = 0.0024), surrounding the Thau lagoon, the most important area of shellfish production and consumption along the French Mediterranean coast. BMAA was identified in mussels (1.8 µg/g to 6.0 µg/g) and oysters (0.6 µg/g to 1.6 µg/g). The highest concentrations of BMAA were measured during summer when the highest picocyanobacteria abundances were recorded. While it is not possible to ascertain a direct link between shellfish consumption and the existence of this ALS cluster, these results add new data to the potential association of BMAA with sporadic amyotrophic lateral sclerosis, one of the most severe neurodegenerative disorder.

Effects of Beta-Alanine Supplementation on Brain Homocarnosine/Carnosine Signal and Cognitive Function: An Exploratory Study

PubMed Central

Hobson, Ruth M; Artioli, Guilherme G.; Otaduy, Maria C.; Roschel, Hamilton; Robertson, Jacques; Martin, Daniel; S. Painelli, Vitor; Harris, Roger C.; Gualano, Bruno

2015-01-01

Objectives Two independent studies were conducted to examine the effects of 28 d of beta-alanine supplementation at 6.4 g d-1 on brain homocarnosine/carnosine signal in omnivores and vegetarians (Study 1) and on cognitive function before and after exercise in trained cyclists (Study 2). Methods In Study 1, seven healthy vegetarians (3 women and 4 men) and seven age- and sex-matched omnivores undertook a brain 1H-MRS exam at baseline and after beta-alanine supplementation. In study 2, nineteen trained male cyclists completed four 20-Km cycling time trials (two pre supplementation and two post supplementation), with a battery of cognitive function tests (Stroop test, Sternberg paradigm, Rapid Visual Information Processing task) being performed before and after exercise on each occasion. Results In Study 1, there were no within-group effects of beta-alanine supplementation on brain homocarnosine/carnosine signal in either vegetarians (p = 0.99) or omnivores (p = 0.27); nor was there any effect when data from both groups were pooled (p = 0.19). Similarly, there was no group by time interaction for brain homocarnosine/carnosine signal (p = 0.27). In study 2, exercise improved cognitive function across all tests (P<0.05), although there was no effect (P>0.05) of beta-alanine supplementation on response times or accuracy for the Stroop test, Sternberg paradigm or RVIP task at rest or after exercise. Conclusion 28 d of beta-alanine supplementation at 6.4g d-1 appeared not to influence brain homocarnosine/carnosine signal in either omnivores or vegetarians; nor did it influence cognitive function before or after exercise in trained cyclists. PMID:25875297

Effects of beta-alanine supplementation on brain homocarnosine/carnosine signal and cognitive function: an exploratory study.

PubMed

Solis, Marina Yazigi; Cooper, Simon; Hobson, Ruth M; Artioli, Guilherme G; Otaduy, Maria C; Roschel, Hamilton; Robertson, Jacques; Martin, Daniel; S Painelli, Vitor; Harris, Roger C; Gualano, Bruno; Sale, Craig

2015-01-01

Two independent studies were conducted to examine the effects of 28 d of beta-alanine supplementation at 6.4 g d(-1) on brain homocarnosine/carnosine signal in omnivores and vegetarians (Study 1) and on cognitive function before and after exercise in trained cyclists (Study 2). In Study 1, seven healthy vegetarians (3 women and 4 men) and seven age- and sex-matched omnivores undertook a brain 1H-MRS exam at baseline and after beta-alanine supplementation. In study 2, nineteen trained male cyclists completed four 20-Km cycling time trials (two pre supplementation and two post supplementation), with a battery of cognitive function tests (Stroop test, Sternberg paradigm, Rapid Visual Information Processing task) being performed before and after exercise on each occasion. In Study 1, there were no within-group effects of beta-alanine supplementation on brain homocarnosine/carnosine signal in either vegetarians (p = 0.99) or omnivores (p = 0.27); nor was there any effect when data from both groups were pooled (p = 0.19). Similarly, there was no group by time interaction for brain homocarnosine/carnosine signal (p = 0.27). In study 2, exercise improved cognitive function across all tests (P < 0.05), although there was no effect (P>0.05) of beta-alanine supplementation on response times or accuracy for the Stroop test, Sternberg paradigm or RVIP task at rest or after exercise. 28 d of beta-alanine supplementation at 6.4 g d(-1) appeared not to influence brain homocarnosine/carnosine signal in either omnivores or vegetarians; nor did it influence cognitive function before or after exercise in trained cyclists.

Short-duration beta-alanine supplementation increases training volume and reduces subjective feelings of fatigue in college football players.

PubMed

Hoffman, Jay R; Ratamess, Nicholas A; Faigenbaum, Avery D; Ross, Ryan; Kang, Jie; Stout, Jeffrey R; Wise, John A

2008-01-01

The purpose of this study was to examine the effect of 30 days of beta-alanine supplementation in collegiate football players on anaerobic performance measures. Subjects were randomly divided into a supplement (beta-alanine group [BA], 4.5 g x d(-1) of beta-alanine) or placebo (placebo group [P], 4.5 g x d(-1) of maltodextrin) group. Supplementation began 3 weeks before preseason football training camp and continued for an additional 9 days during camp. Performance measures included a 60-second Wingate anaerobic power test and 3 line drills (200-yd shuttle runs with a 2-minute rest between sprints) assessed on day 1 of training camp. Training logs recorded resistance training volumes, and subjects completed questionnaires on subjective feelings of soreness, fatigue, and practice intensity. No difference was seen in fatigue rate in the line drill, but a trend (P = .07) was observed for a lower fatigue rate for BA compared with P during the Wingate anaerobic power test. A significantly higher training volume was seen for BA in the bench press exercise, and a trend (P = .09) for a greater training volume was seen for all resistance exercise sessions. In addition, subjective feelings of fatigue were significantly lower for BA than P. In conclusion, despite a trend toward lower fatigue rates during 60 seconds of maximal exercise, 3 weeks of beta-alanine supplementation did not result in significant improvements in fatigue rates during high-intensity anaerobic exercise. However, higher training volumes and lower subjective feelings of fatigue in BA indicated that as duration of supplementation continued, the efficacy of beta-alanine supplementation in highly trained athletes became apparent.

Dipeptide transport and hydrolysis in isolated loops of rat small intestine: effects of stereospecificity.

PubMed Central

Lister, N; Sykes, A P; Bailey, P D; Boyd, C A; Bronk, J R

1995-01-01

1. Isolated jejunal loops of rat small intestine were perfused by a single pass of bicarbonate Krebs-Ringer solution containing either D- or L-phenylalanine or one of eight dipeptides formed from D- or L-alanine plus D- or L-phenylalanine. 2. At 0.5 mM L-phenylalanyl-L-alanine increased serosal phenylalanine appearance to forty times the control rate giving a value similar to that found with 0.5 mM free L-phenylalanine. No serosal dipeptide could be detected. 3. Perfusions with the two mixed dipeptides with N-terminal D-amino acids (D-alanyl-L-phenylalanine and D-phenylalanyl-L-alanine) gave rise to the appearance of intact dipeptides in the serosal secretions although there were substantial differences in their rates of absorption and subsequent hydrolysis. 4. L-Alanyl-D-phenylalanine was absorbed from the lumen three to five times as fast as L-phenylalanyl-D-alanine. At 1 mM L-alanyl-D-phenylalanine transferred D-phenylalanine across the epithelial layer at more than seven times the rate found with the same concentration of the free D-amino acid. 5. Perfusions with D-alanyl-D-phenylalanine or D-phenylalanyl-D-alanine showed that these two dipeptides are poor substrates for both transport and hydrolysis by the rat small intestine. 6. Analysis of mucosal tissue extracts after perfusion with the two mixed dipeptides with N-terminal D-amino acids revealed that both dipeptides were accumulated within the mucosa and suggested that exit across the basolateral membrane was rate limiting for transepithelial dipeptide transport. Images Figure 5 PMID:7602518

Molecular and chiral analyses of some protein amino acid derivatives in the Murchison and Murray meteorite

NASA Astrophysics Data System (ADS)

Pizzarello, Sandra; Cooper, George W.

2001-07-01

The varied organic suite extracted from the Murchison meteorite contains several amino acids that are common to the biosphere. Some of these have been found to be non-racemic, but the indigenous nature of their L-enantiomeric excesses has been subject to debate in view of possible terrestrial contamination. We have investigated two amino acids of common terrestrial and meteoritic occurrence, alanine and glutamic acid, and assessed their indigenous enantiomeric ratios in the Murchison and Murray meteorites through the ratios of some of their derivatives. Analyzed were: N-acetyl alanine, ??imino propioacetic acid, N-acetyl glutamic acid and pyroglutamic acid. Both alanine derivatives were found to be racemic, while those of glutamic acid showed L-enantiomeric excesses varying from 16% to 47.2% for pyroglutamic acid, and from 8.6% to 41% for N-acetyl glutamic acid. The ?13C was determined for the two enantiomers of Murchison pyroglutamic acid both before and after acid hydrolysis of the lactam to glutamic acid. The values of +27.7 (D-pyro), +10.0 (L-pyro), +32.2 (D-glu) and +14.6 (L-glu) were obtained. The racemic nature of alanine derivatives strongly suggests that alanine itself, as indigenous to the meteorite, is racemic. The explanation of the L-enantiomeric excesses found for glutamic acid derivatives is less direct; however, the variability of the enantiomeric ratios for these compounds and the distinctly lower ?13C values determined for pyroglutamic L-enantiomer point to a terrestrial contamination, possibly dating to the time of fall.

Characterization of the stereochemical selectivity of beta-hairpin formation by molecular dynamics simulations.

PubMed

Soto, Patricia; Zangi, Ronen

2005-01-27

The stability of secondary structure motifs found in proteins is influenced by the choice of the configuration of the chiral centers present in the amino acid residues (i.e., D vs L). Experimental studies showed that the structural properties of the tetrapeptide (L)V(L)P(L)A(L)L (all-L) are drastically altered upon mutating the L-proline and the L-alanine by their d-enantiomers [J. Am. Chem. Soc. 1996, 118, 6975]. The all-L diastereomer is unstructured, experiencing little or no beta-hairpin formation, while the (L)V(D)P(D)A(L)L peptide exhibits a substantial population of beta-hairpin conformation. In this study, we perform molecular dynamics simulations to investigate the folding propensity of these two model peptides. The results confirm the experimental findings, namely, that the presence of d-amino acids in the loop region strongly induces beta-hairpin formation (a population increase from about 1.5% to 50% is observed). The major factor determining the different behavior is found to be the large difference in energy between the two diastereomers, approximately 22 kJ/mol, when they adopt a beta-hairpin structure. The higher energy observed for the all-L peptide is a consequence of none-ideal hydrogen bond formation and of steric repulsions. The results suggest that selective incorporation of D-amino acids in proteins can be used to enhance certain secondary structure elements. The kinetic behavior of the folding process observed in the simulations is also investigated. We find that the decay rate of the folded structure fits to a biexponential function, suggesting that the folding/unfolding process of a beta-hairpin is governed by two different mechanisms.

Dietary BMAA Exposure in an Amyotrophic Lateral Sclerosis Cluster from Southern France

PubMed Central

Masseret, Estelle; Banack, Sandra; Boumédiène, Farid; Abadie, Eric; Brient, Luc; Pernet, Fabrice; Juntas-Morales, Raoul; Pageot, Nicolas; Metcalf, James; Cox, Paul; Camu, William

2013-01-01

Background Dietary exposure to the cyanotoxin BMAA is suspected to be the cause of amyotrophic lateral sclerosis in the Western Pacific Islands. In Europe and North America, this toxin has been identified in the marine environment of amyotrophic lateral sclerosis clusters but, to date, only few dietary exposures have been described. Objectives We aimed at identifying cluster(s) of amyotrophic lateral sclerosis in the Hérault district, a coastal district from Southern France, and to search, in the identified area(s), for the existence of a potential dietary source of BMAA. Methods A spatio-temporal cluster analysis was performed in the district, considering all incident amyotrophic lateral sclerosis cases identified from 1994 to 2009 by our expert center. We investigated the cluster area with serial collections of oysters and mussels that were subsequently analyzed blind for BMAA concentrations. Results We found one significant amyotrophic lateral sclerosis cluster (p = 0.0024), surrounding the Thau lagoon, the most important area of shellfish production and consumption along the French Mediterranean coast. BMAA was identified in mussels (1.8 µg/g to 6.0 µg/g) and oysters (0.6 µg/g to 1.6 µg/g). The highest concentrations of BMAA were measured during summer when the highest picocyanobacteria abundances were recorded. Conclusions While it is not possible to ascertain a direct link between shellfish consumption and the existence of this ALS cluster, these results add new data to the potential association of BMAA with sporadic amyotrophic lateral sclerosis, one of the most severe neurodegenerative disorder. PMID:24349504

Spectral investigations of amino acid picrates.

PubMed

Briget Mary, M; Sasirekha, V; Ramakrishnan, V

2006-10-01

FTIR and laser Raman spectra of beta-alanine beta-alaninium picrate and dl-phenylalanine dl-phenylalaninium picrate crystals of space group P1 (C(i)) have been me in the 4000-50 cm(-1) range, at room temperature. The former crystal consists of beta-alanine beta-alaninium and the later dl-phenylalanine dl-phenylalaninium as cations. The presence of both carbonyl (CO) and carboxylate COO(-) groups in these crystals is the evidence for the existence of the zwitterion and the protonated forms. Factor group analysis has been made and the numbers of vibrational modes have been calculated. The tentative assignments of the observed bands are given. Fermi resonance has also been observed in one of the crystal beta-alanine beta-alaninium picrate. The picrate group forms the anion in both crystals and the characteristic bands nu(as)NO(2), nu(s)NO(2), and nu(phen)C-O stretching are observed in the spectra. These suggest that the picrate ion is unaffected by the presence of the cations.

Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:l-Alanine Ligase (MurC) from Haemophilus influenzae

PubMed Central

Mol, Clifford D.; Brooun, Alexei; Dougan, Douglas R.; Hilgers, Mark T.; Tari, Leslie W.; Wijnands, Robert A.; Knuth, Mark W.; McRee, Duncan E.; Swanson, Ronald V.

2003-01-01

UDP-N-acetylmuramic acid:l-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg2+ and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-l-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn2+ have been determined to 1.85- and 1.7-Å resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the γ-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates. PMID:12837790

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae.

PubMed

Mol, Clifford D; Brooun, Alexei; Dougan, Douglas R; Hilgers, Mark T; Tari, Leslie W; Wijnands, Robert A; Knuth, Mark W; McRee, Duncan E; Swanson, Ronald V

2003-07-01

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.

The Origin of Amino Acids in Lunar Regolith Samples

NASA Technical Reports Server (NTRS)

Cook, Jamie E.; Callahan, Michael P.; Dworkin, Jason P.; Glavin, Daniel P.; McLain, Hannah L.; Noble, Sarah K.; Gibson, Everett K., Jr.

2016-01-01

We analyzed the amino acid content of seven lunar regolith samples returned by the Apollo 16 and Apollo 17 missions and stored under NASA curation since collection using ultrahigh-performance liquid chromatography with fluorescence detection and time-of-flight mass spectrometry. Consistent with results from initial analyses shortly after collection in the 1970s, we observed amino acids at low concentrations in all of the curated samples, ranging from 0.2 parts-per-billion (ppb) to 42.7 ppb in hot-water extracts and 14.5 ppb to 651.1 ppb in 6M HCl acid-vapor-hydrolyzed, hot-water extracts. Amino acids identified in the Apollo soil extracts include glycine, D- and L-alanine, D- and L-aspartic acid, D- and L-glutamic acid, D- and L-serine, L-threonine, and L-valine, all of which had previously been detected in lunar samples, as well as several compounds not previously identified in lunar regoliths: -aminoisobutyric acid (AIB), D-and L-amino-n-butyric acid (-ABA), DL-amino-n-butyric acid, -amino-n-butyric acid, -alanine, and -amino-n-caproic acid. We observed an excess of the L enantiomer in most of the detected proteinogenic amino acids, but racemic alanine and racemic -ABA were present in some samples.

Hydrophobic interaction between the SH2 domain and the kinase domain is required for the activation of Csk.

PubMed

Mikkola, Esa T; Gahmberg, Carl G

2010-06-18

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the beta3-alphaC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the beta3-alphaC loop. The mutation of the beta3-alphaC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the beta3-alphaC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli.

PubMed

Ihara, Kohei; Sato, Kazuki; Hori, Hatsuhiro; Makino, Yumiko; Shigenobu, Shuji; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

2017-04-01

The alaE gene in Escherichia coli encodes an l-alanine exporter that catalyzes the active export of l-alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of l-alanyl-l-alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene. A promoter less β-galactosidase gene was fused to an alaE upstream region (240 nucleotides). Cells that were lacZ-deficient and harbored this reporter plasmid showed significant induction of β-galactosidase activity (approximately 17-fold) in the presence of 6 mM l-alanine, l-leucine, and Ala-Ala. However, a reporter plasmid possessing a smaller alaE upstream region (180 nucleotides) yielded transformants with strikingly low enzyme activity under the same conditions. In contrast, lrp-deficient cells showed almost no β-galactosidase induction, indicating that Lrp positively regulates alaE expression. We next performed an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay using purified hexahistidine-tagged Lrp (Lrp-His). Consequently, we found that Lrp-His binds to the alaE upstream region spanning nucleotide -161 to -83 with a physiologically relevant affinity (apparent K D , 288.7 ± 83.8 nM). Furthermore, the binding affinity of Lrp-His toward its cis-element was increased by l-alanine and l-leucine, but not by Ala-Ala and d-alanine. Based on these results, we concluded that the gene expression of the alaE is regulated by Lrp in response to intracellular levels of l-alanine, which eventually leads to intracellular homeostasis of l-alanine concentrations. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Amino acids in the Martian meteorite Nakhla

NASA Technical Reports Server (NTRS)

Glavin, D. P.; Bada, J. L.; Brinton, K. L.; McDonald, G. D.

1999-01-01

A suite of protein and nonprotein amino acids were detected with high-performance liquid chromatography in the water- and acid-soluble components of an interior fragment of the Martian meteorite Nakhla, which fell in Egypt in 1911. Aspartic and glutamic acids, glycine, alanine, beta-alanine, and gamma-amino-n-butyric acid (gamma-ABA) were the most abundant amino acids detected and were found primarily in the 6 M HCl-hydrolyzed, hot water extract. The concentrations ranged from 20 to 330 parts per billion of bulk meteorite. The amino acid distribution in Nakhla, including the D/L ratios (values range from <0.1 to 0.5), is similar to what is found in bacterially degraded organic matter. The amino acids in Nakhla appear to be derived from terrestrial organic matter that infiltrated the meteorite soon after its fall to Earth, although it is possible that some of the amino acids are endogenous to the meteorite. The rapid amino acid contamination of Martian meteorites after direct exposure to the terrestrial environment has important implications for Mars sample-return missions and the curation of the samples from the time of their delivery to Earth.

Amino acids in the Martian meteorite Nakhla.

PubMed

Glavin, D P; Bada, J L; Brinton, K L; McDonald, G D

1999-08-03

A suite of protein and nonprotein amino acids were detected with high-performance liquid chromatography in the water- and acid-soluble components of an interior fragment of the Martian meteorite Nakhla, which fell in Egypt in 1911. Aspartic and glutamic acids, glycine, alanine, beta-alanine, and gamma-amino-n-butyric acid (gamma-ABA) were the most abundant amino acids detected and were found primarily in the 6 M HCl-hydrolyzed, hot water extract. The concentrations ranged from 20 to 330 parts per billion of bulk meteorite. The amino acid distribution in Nakhla, including the D/L ratios (values range from <0.1 to 0.5), is similar to what is found in bacterially degraded organic matter. The amino acids in Nakhla appear to be derived from terrestrial organic matter that infiltrated the meteorite soon after its fall to Earth, although it is possible that some of the amino acids are endogenous to the meteorite. The rapid amino acid contamination of Martian meteorites after direct exposure to the terrestrial environment has important implications for Mars sample-return missions and the curation of the samples from the time of their delivery to Earth.

Ability of L-canavanine to support nitrogen metabolism in the jack bean, Canavalia ensiformis (L. ) DC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosenthal, G.A.; Berge, M.A.; Ozinskas, A.J.

The ability of L-canavanine, a nonprotein amino acid of certain leguminous plants, to support the nitrogen metabolism of jack bean, Canavalia ensiformis (Leguminosae), was assessed by administration of L-(guanidino-N{sup 3}-{sup 15}N)arginine, L-(guanidinooxy-N{sup 3}-{sup 15}N)canavanine, or L-(guanidinooxy-N{sup 1}-{sup 15}N)canavanine into the cotyledons of 9-day-old plants. A strikingly similar pattern of {sup 15}N assimilation into de novo synthesized amino and imino acids resulted from feeding L-(guanidino-N{sup 3}-{sup 15}N)arginine and L-(guanidinooxy-N{sup 3}-{sup 15}N)canavanine. Glutamic acid plus glutamine and alanine were the most heavily labeled of the detected compounds. Some transfer of {sup 15}N from L-(gluanidino-N{sup 3}-{sup 15}N)arginine to canavanine was noted. This maymore » occur by a transamidination reaction between L-canaline and L-arginine. L-(guanidinooxy-N{sup 1}-{sup 15}N)Canavanine also supported amino and imino acid biosynthesis in this plant, but much more alanine and less glutamic acid and glutamine were labeled. These experiments provide substantive experimental evidence for the long-reputed hypothesis that canavanine functions as a nitrogen-storing metabolite.« less

Neutron inelastic scattering by amino acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thaper, C.L.; Sinha, S.K.; Dasannacharya, B.A.

Inelastic neutron scattering experiments on normal, N-deuterated glycine, normal and N-deuterated alanine, L-valine, L-tyrosine and, L-phenylalanine at 100 K, are reported. Coupling of the external modes to different hydrogens is discussed.

Beta-alanine/alpha-ketoglutarate aminotransferase for 3-hydroxypropionic acid production

DOEpatents

Jessen, Holly Jean [Chanhassen, MN; Liao, Hans H [Eden Prairie, MN; Gort, Steven John [Apple Valley, MN; Selifonova, Olga V [Plymouth, MN

2011-10-04

The present disclosure provides novel beta-alanine/alpha ketoglutarate aminotransferase nucleic acid and protein sequences having increased biological activity. Also provided are cells containing such enzymes, as well as methods of their use, for example to produce malonyl semialdehyde and downstream products thereof, such as 3-hydroxypropionic acid and derivatives thereof.

Beta-alanine/alpha-ketoglutarate aminotransferase for 3-hydroxypropionic acid production

DOEpatents

Jessen, Holly Jean; Liao, Hans H; Gort, Steven John; Selifonova, Olga V

2014-11-18

The present disclosure provides novel beta-alanine/alpha ketoglutarate aminotransferase nucleic acid and protein sequences having increased biological activity. Also provided are cells containing such enzymes, as well as methods of their use, for example to produce malonyl semialdehyde and downstream products thereof, such as 3-hydroxypropionic acid and derivatives thereof.

Determination of the carbon, hydrogen and nitrogen contents of alanine and their uncertainties using the certified reference material L-alanine (NMIJ CRM 6011-a).

PubMed

Itoh, Nobuyasu; Sato, Ayako; Yamazaki, Taichi; Numata, Masahiko; Takatsu, Akiko

2013-01-01

The carbon, hydrogen, and nitrogen (CHN) contents of alanine and their uncertainties were estimated using a CHN analyzer and the certified reference material (CRM) L-alanine. The CHN contents and their uncertainties, as measured using the single-point calibration method, were 40.36 ± 0.20% for C, 7.86 ± 0.13% for H, and 15.66 ± 0.09% for N; the results obtained using the bracket calibration method were also comparable. The method described in this study is reasonable, convenient, and meets the general requirement of having uncertainties ≤ 0.4%.

9-Methyl-beta-carboline has restorative effects in an animal model of Parkinson's disease.

PubMed

Wernicke, Catrin; Hellmann, Julian; Zieba, Barbara; Kuter, Katarzyna; Ossowska, Krystyna; Frenzel, Monika; Dencher, Norbert A; Rommelspacher, Hans

2010-01-01

In a previous study, a primary culture of midbrain cells was exposed to 9-methyl-beta-carboline for 48 h, which caused an increase in the number of tyrosine hydroxylase-positive cells. Quantitative RT-PCR revealed increased transcription of genes participating in the maturation of dopaminergic neurons. These in vitro findings prompted us to investigate the restorative actions of 9-methyl-beta-carboline in vivo. The compound was delivered for 14 days into the left cerebral ventricle of rats pretreated with the neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP+) for 28 days applying a dose which lowered dopamine by approximately 50%. Interestingly, 9-methyl-beta-carboline reversed the dopamine-lowering effect of the neurotoxin in the left striatum. Stereological counts of tyrosine hydroxylase-immunoreactive cells in the substantia nigra revealed that the neurotoxin caused a decrease in the number of those cells. However, when treated subsequently with 9-methyl-beta-carboline, the number reached normal values. In search of an explanation for the restorative activity, we analyzed the complexes that compose the respiratory chain in striatal mitochondria by 2-dimension gel electrophoresis followed by MALDI-TOF peptide mass fingerprinting.We found no changes in the overall composition of the complexes. However, the activity of complex I was increased by approximately 80% in mitochondria from rats treated with MPP+ and 9-methyl-beta-carboline compared to MPP+ and saline and to sham-operated rats, as determined by measurements of nicotinamide adenine dinucleotide dehydrogenase activity. Microarray technology and single RT-PCR revealed the induction of neurotrophins: brain-derived neurotrophic factor, conserved dopamine neurotrophic factor, cerebellin 1 precursor protein, and ciliary neurotrophic factor. Selected western blots yielded consistent results. The findings demonstrate restorative effects of 9-methyl-beta-carboline in an animal model of Parkinson's disease that improve the effectiveness of the respiratory chain and promote the transcription and expression of neurotrophin-related genes.

Mode of Action of Membrane Perturbing Agents: Snake Venom Cardiotoxins and Phospholipases A

DTIC Science & Technology

1989-07-15

PLAz neurotoxins. Experimental Methods: Materials. Vencm from 1 nAjA atra, CTX from Naja n9ja kaouthia venom (Lots 125F-4007), bee venom PLAz ( Apis ... mellifera ), melittin, B-bungarotoxin, Tris base, Hepes (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), Mes (4- morpholineethanesulfonic acid), bovine

State of knowledge and concerns on cyanobacterial blooms and cyanotoxins.

PubMed

Merel, Sylvain; Walker, David; Chicana, Ruth; Snyder, Shane; Baurès, Estelle; Thomas, Olivier

2013-09-01

Cyanobacteria are ubiquitous microorganisms considered as important contributors to the formation of Earth's atmosphere and nitrogen fixation. However, they are also frequently associated with toxic blooms. Indeed, the wide range of hepatotoxins, neurotoxins and dermatotoxins synthesized by these bacteria is a growing environmental and public health concern. This paper provides a state of the art on the occurrence and management of harmful cyanobacterial blooms in surface and drinking water, including economic impacts and research needs. Cyanobacterial blooms usually occur according to a combination of environmental factors e.g., nutrient concentration, water temperature, light intensity, salinity, water movement, stagnation and residence time, as well as several other variables. These environmental variables, in turn, have promoted the evolution and biosynthesis of strain-specific, gene-controlled metabolites (cyanotoxins) that are often harmful to aquatic and terrestrial life, including humans. Cyanotoxins are primarily produced intracellularly during the exponential growth phase. Release of toxins into water can occur during cell death or senescence but can also be due to evolutionary-derived or environmentally-mediated circumstances such as allelopathy or relatively sudden nutrient limitation. Consequently, when cyanobacterial blooms occur in drinking water resources, treatment has to remove both cyanobacteria (avoiding cell lysis and subsequent toxin release) and aqueous cyanotoxins previously released. Cells are usually removed with limited lysis by physical processes such as clarification or membrane filtration. However, aqueous toxins are usually removed by both physical retention, through adsorption on activated carbon or reverse osmosis, and chemical oxidation, through ozonation or chlorination. While the efficient oxidation of the more common cyanotoxins (microcystin, cylindrospermopsin, anatoxin and saxitoxin) has been extensively reported, the chemical and toxicological characterization of their by-products requires further investigation. In addition, future research should also investigate the removal of poorly considered cyanotoxins (β-methylamino-alanine, lyngbyatoxin or aplysiatoxin) as well as the economic impact of blooms. © 2013 Elsevier Ltd. All rights reserved.

Short-chain consensus alpha-neurotoxin: a synthetic 60-mer peptide with generic traits and enhanced immunogenic properties.

PubMed

de la Rosa, Guillermo; Corrales-García, Ligia L; Rodriguez-Ruiz, Ximena; López-Vera, Estuardo; Corzo, Gerardo

2018-07-01

The three-fingered toxin family and more precisely short-chain α-neurotoxins (also known as Type I α-neurotoxins) are crucial in defining the elapid envenomation process, but paradoxically, they are barely neutralized by current elapid snake antivenoms. This work has been focused on the primary structural identity among Type I neurotoxins in order to create a consensus short-chain α-neurotoxin with conserved characteristics. A multiple sequence alignment considering the twelve most toxic short-chain α-neurotoxins reported from the venoms of the elapid genera Acanthophis, Oxyuranus, Walterinnesia, Naja, Dendroaspis and Micrurus led us to propose a short-chain consensus α-neurotoxin, here named ScNtx. The synthetic ScNtx gene was de novo constructed and cloned into the expression vector pQE30 containing a 6His-Tag and an FXa proteolytic cleavage region. Escherichia coli Origami cells transfected with the pQE30/ScNtx vector expressed the recombinant consensus neurotoxin in a soluble form with a yield of 1.5 mg/L of culture medium. The 60-amino acid residue ScNtx contains canonical structural motifs similar to α-neurotoxins from African elapids and its LD 50 of 3.8 µg/mice is similar to the most toxic short-chain α-neurotoxins reported from elapid venoms. Furthermore, ScNtx was also able to antagonize muscular, but not neuronal, nicotinic acetylcholine receptors (nAChR). Rabbits immunized with ScNtx were able to immune-recognize short-chain α-neurotoxins within whole elapid venoms. Type I neurotoxins are difficult to isolate and purify from natural sources; therefore, the heterologous expression of molecules such ScNtx, bearing crucial motifs and key amino acids, is a step forward to create common immunogens for developing cost-effective antivenoms with a wider spectrum of efficacy, quality and strong therapeutic value.

Metabolomics approach to understand mechanisms of ß-N-Oxalyl-L-a,ß-diaminopropionic Acid (ß-ODAP) biosynthesis in grass pea (Lathyrus sativus L.)

USDA-ARS?s Scientific Manuscript database

Grass pea (Lathyrus sativus L.) is an important food crop for human health and food security, however its seed contains high levels of the neurotoxin ß-ODAP. Previous work demonstrated that ß-ODAP content changes during early developmental stages of grass pea. Further, the regulation and mechanisms ...

Precursors of Androctonus australis Scorpion Neurotoxins. Structures of Precursors, Processing Outcomes, and Expression of a Functional Recombinant Toxin II

DTIC Science & Technology

1989-05-24

29. Proudfoot, N. J., and Brownlee, G. G. (1976) NatUlgL263, 211-214 30. Detima, M. E ., Martin, M. F., Diniz , C. R., and Rochat, H. (1986) Biochem...Neurotoxins 7 12. PERSONAL AUTHOR(S) Drs. Pierre E . Bougis, Herve Rochat and Leonard A. Smith 13a. TYPE OF REPORT 13b. TIME COVERED 114. DATE OF REPORT...PRECURSORS, PROCESSING OUTCOMES, AND EXPRESSION OF A FUNCTIONAL RECOMBINANT TOXIN II Pierre E . Bougis, Herve Rochat1 and Leonard A. Smith from the

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klucevsek, K.; Daley, J.; Darshan, M.S.

We have investigated the nuclear import strategies of high-risk HPV18 L2 minor capsid protein. HPV18 L2 interacts with Kap {alpha}{sub 2} adapter, and Kap {beta}{sub 2} and Kap {beta}{sub 3} nuclear import receptors. Moreover, binding of RanGTP to either Kap {beta}{sub 2} or Kap {beta}{sub 3} inhibits their interaction with L2, suggesting that these Kap {beta}/L2 complexes are import competent. Mapping studies show that HPV18 L2 contains two NLSs: in the N-terminus (nNLS) and in the C-terminus (cNLS), both of which can independently mediate nuclear import. Both nNLS and cNLS form a complex with Kap {alpha}{sub 2}{beta}{sub 1} heterodimer andmore » mediate nuclear import via a classical pathway. The nNLS is also essential for the interaction of HPV18 L2 with Kap {beta}{sub 2} and Kap {beta}{sub 3}. Interestingly, both nNLS and cNLS interact with the viral DNA and this DNA binding occurs without nucleotide sequence specificity. Together, the data suggest that HPV18 L2 can interact via its NLSs with several Kaps and the viral DNA and may enter the nucleus via multiple import pathways mediated by Kap {alpha}{sub 2}{beta}{sub 1} heterodimers, Kap {beta}{sub 2} and Kap {beta}{sub 3}.« less

Prominent Human Health Impacts from Several Marine Microbes: History, Ecology, and Public Health Implications

PubMed Central

Bienfang, P. K.; DeFelice, S. V.; Laws, E. A.; Brand, L. E.; Bidigare, R. R.; Christensen, S.; Trapido-Rosenthal, H.; Hemscheidt, T. K.; McGillicuddy, D. J.; Anderson, D. M.; Solo-Gabriele, H. M.; Boehm, A. B.; Backer, L. C.

2011-01-01

This paper overviews several examples of important public health impacts by marine microbes and directs readers to the extensive literature germane to these maladies. These examples include three types of dinoflagellates (Gambierdiscus spp., Karenia brevis, and Alexandrium fundyense), BMAA-producing cyanobacteria, and infectious microbes. The dinoflagellates are responsible for ciguatera fish poisoning, neurotoxic shellfish poisoning, and paralytic shellfish poisoning, respectively, that have plagued coastal populations over time. Research interest on the potential for marine cyanobacteria to contribute BMAA into human food supplies has been derived by BMAA's discovery in cycad seeds and subsequent implication as the putative cause of amyotrophic lateral sclerosis/parkinsonism dementia complex among the Chamorro people of Guam. Recent UPLC/MS analyses indicate that recent reports that BMAA is prolifically distributed among marine cyanobacteria at high concentrations may be due to analyte misidentification in the analytical protocols being applied for BMAA. Common infectious microbes (including enterovirus, norovirus, Salmonella, Campylobacter, Shigella, Staphylococcus aureus, Cryptosporidium, and Giardia) cause gastrointestinal and skin-related illness. These microbes can be introduced from external human and animal sources, or they can be indigenous to the marine environment. PMID:20976073

The GABA uptake inhibitor beta-alanine reduces pilocarpine-induced tremor and increases extracellular GABA in substantia nigra pars reticulata as measured by microdialysis.

PubMed

Ishiwari, Keita; Mingote, Susana; Correa, Merce; Trevitt, Jennifer T; Carlson, Brian B; Salamone, John D

2004-12-30

Substantia nigra pars reticulata (SNr) is a major output nucleus of the basal ganglia that receives GABAergic projections from neostriatum and globus pallidus. Previous research has shown that local pharmacological manipulations of GABA in SNr can influence tremulous jaw movements in rats. Tremulous jaw movements are defined as rapid vertical deflections of the lower jaw that resemble chewing but are not directed at a particular stimulus, and evidence indicates that these movements share many characteristics with parkinsonian tremor in humans. In order to investigate the role of GABA in motor functions related to tremor, the present study tested the GABA uptake blocker beta-alanine for its ability to reduce pilocarpine-induced tremulous jaw movements. In a parallel experiment, the effect of an active dose of beta-alanine on dialysate levels of GABA in SNr was assessed using microdialysis methods. GABA levels in dialysis samples were measured using high performance liquid chromatography with electrochemical detection. beta-Alanine (250-500 mg/kg) significantly reduced tremulous jaw movements induced by pilocarpine (4.0 mg/kg). Moreover, systemic administration of beta-alanine at a dose that reduced tremulous jaw movements (500 mg/kg) resulted in a substantial increase in extracellular levels of GABA in SNr compared to the pre-injection baseline. Thus, the present results are consistent with the hypothesis that GABAergic tone in SNr plays a role in the regulation of tremulous jaw movements. This research may lead to a better understanding of how parkinsonian symptoms are modulated by SNr GABA mechanisms.

Oxygenated N-Acyl Alanine Methyl Esters (NAMEs) from the Marine Bacterium Roseovarius tolerans EL-164.

PubMed

Bruns, Hilke; Herrmann, Jennifer; Müller, Rolf; Wang, Hui; Wagner Döbler, Irene; Schulz, Stefan

2018-01-26

The marine bacterium Roseovarius tolerans EL-164 (Rhodobacteraceae) can produce unique N-acylalanine methyl esters (NAMEs) besides strucutrally related N-acylhomoserine lactones (AHLs), bacterial signaling compounds widespread in the Rhodobacteraceae. The structures of two unprecedented NAMEs carrying a rare terminally oxidized acyl chain are reported here. The compounds (Z)-N-16-hydroxyhexadec-9-enoyl-l-alanine methyl ester (Z9-16-OH-C16:1-NAME, 3) and (Z)-N-15-carboxypentadec-9-enoyl-l-alanine methyl ester (16COOH-C16:1-NAME, 4) were isolated, and the structures were determined by NMR and MS experiments. Both compounds were synthesized to prove assignments and to test their biological activity. Finally, non-natural, structurally related Z9-3-OH-C16:1-NAME (18) was synthesized to investigate the mass spectroscopy of structurally related NAMEs. Compound 3 showed moderate antibacterial activity against microorganisms such as Bacillus, Streptococcus, Micrococcus, or Mucor strains. In contrast to AHLs, quorum-sensing or quorum-quenching activity was not observed.

d-Alanine Oxidase from Escherichia coli: Localization and Induction by l-Alanine

PubMed Central

Raunio, R. P.; Jenkins, W. T.

1973-01-01

Dialyzed membranes of Escherichia coli prepared by an ethylenediaminetetraacetic acid-lysozyme method catalyze the oxidation of both l-alanine and d-alanine. The specific activities for the oxidations of both d-alanine and l-alanine are increased fivefold when the cells are grown in the presence of either l-alanine or dl-alanine, but are increased only slightly when grown in the presence of d-alanine. In the dl-alanine-induced system, the specific activities for the oxidations of some other d-amino acids are also raised. dl-alanine also induces two other alanine catabolizing enzymes, alanine dehydrogenase and alanine-glutamate aminotransferase which are found in the “soluble” fraction of lysozyme-treated cells. The oxidations of both l-alanine and d-alanine were associated with the membranes of induced cells. After the membranes were disintegrated by sonic treatment, both l-alanine and d-alanine oxidation catalysts sedimented in a sucrose density gradient together with d-lactate and l-lactate dehydrogenases, apparently as a single multienzyme complex. PMID:4146872

Efficient Enzymatic Preparation of (13) N-Labelled Amino Acids: Towards Multipurpose Synthetic Systems.

PubMed

da Silva, Eunice S; Gómez-Vallejo, Vanessa; Baz, Zuriñe; Llop, Jordi; López-Gallego, Fernando

2016-09-12

Nitrogen-13 can be efficiently produced in biomedical cyclotrons in different chemical forms, and its stable isotopes are present in the majority of biologically active molecules. Hence, it may constitute a convenient alternative to Fluorine-18 and Carbon-11 for the preparation of positron-emitter-labelled radiotracers; however, its short half-life demands for the development of simple, fast, and efficient synthetic processes. Herein, we report the one-pot, enzymatic and non-carrier-added synthesis of the (13) N-labelled amino acids l-[(13) N]alanine, [(13) N]glycine, and l-[(13) N]serine by using l-alanine dehydrogenase from Bacillus subtilis, an enzyme that catalyses the reductive amination of α-keto acids by using nicotinamide adenine dinucleotide (NADH) as the redox cofactor and ammonia as the amine source. The integration of both l-alanine dehydrogenase and formate dehydrogenase from Candida boidinii in the same reaction vessel to facilitate the in situ regeneration of NADH during the radiochemical synthesis of the amino acids allowed a 50-fold decrease in the concentration of the cofactor without compromising reaction yields. After optimization of the experimental conditions, radiochemical yields were sufficient to carry out in vivo imaging studies in small rodents. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Oligosaccharide composition of the neurotoxin responsive Na/sup +/ channel and the requirement of sialic acid for activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Negishi, M.; Shaw, G.W.; Glick, M.C.

1986-05-01

The neurotoxin responsive Na/sup +/ channel was purified to homogeneity in an 18% yield from a clonal cell line of mouse neuroblastoma, N-18, metabolically labeled with L-(/sup 3/H)fucose. The Na/sup +/ channel, a glycoprotein, M/sub r/=200,000 (gradient 7-14% PAGE) was digested with Pronase and the glycopeptides were characterized by serial lectin affinity chromatography. greater than 90% of the oligosaccharides contained sialic acid and 18% were biantennary, 39% were triantennary and 30% tetraantennary. The glycoprotein was reconstituted into artificial phospholipid vesicles and /sup 86/Rb flux was stimulated (65%) by 200 ..mu..M veratridine and 1.2 ..mu..g of scorpion venom and was inhibitedmore » (95%) by 5 ..mu..M tetrodotoxin. The requirement of sialic acid for Na/sup +/ channel activity was demonstrated since neuraminidase (0.01 U) treatment of the reconstituted glycoprotein eliminated the response of /sup 86/Rb flux to the stimulating neurotoxins. In other experiments, treatment of N-18 cells with 10 ..mu..M swainsonine, an inhibitor of glycoprotein processing, altered the oligosaccharide composition of the Na/sup +/ channel. When the abnormally glycosylated Na/sup +/ channel was reconstituted into artificial phospholipid vesicles, /sup 86/Rb flux in response to neurotoxins was impaired. Thus, glycosylation of the polypeptide with oligosaccharides of specific composition and structure is essential for expression of the biological activity of the neurotoxin responsive Na/sup +/ channel.« less

Identification and Partial Characterization of a Novel UDP-N-Acetylenolpyruvoylglucosamine Reductase/UDP-N-Acetylmuramate:l-Alanine Ligase Fusion Enzyme from Verrucomicrobium spinosum DSM 4136(T).

PubMed

Naqvi, Kubra F; Patin, Delphine; Wheatley, Matthew S; Savka, Michael A; Dobson, Renwick C J; Gan, Han Ming; Barreteau, Hélène; Blanot, Didier; Mengin-Lecreulx, Dominique; Hudson, André O

2016-01-01

The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/C Vs ) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44-46°C. Its apparent K m values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum.

Identification and Partial Characterization of a Novel UDP-N-Acetylenolpyruvoylglucosamine Reductase/UDP-N-Acetylmuramate:l-Alanine Ligase Fusion Enzyme from Verrucomicrobium spinosum DSM 4136T

PubMed Central

Naqvi, Kubra F.; Patin, Delphine; Wheatley, Matthew S.; Savka, Michael A.; Dobson, Renwick C. J.; Gan, Han Ming; Barreteau, Hélène; Blanot, Didier; Mengin-Lecreulx, Dominique; Hudson, André O.

2016-01-01

The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/CVs) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44–46°C. Its apparent Km values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum. PMID:27047475

Risk factors for amyotrophic lateral sclerosis

PubMed Central

Ingre, Caroline; Roos, Per M; Piehl, Fredrik; Kamel, Freya; Fang, Fang

2015-01-01

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. It is typically fatal within 2–5 years of symptom onset. The incidence of ALS is largely uniform across most parts of the world, but an increasing ALS incidence during the last decades has been suggested. Although recent genetic studies have substantially improved our understanding of the causes of ALS, especially familial ALS, an important role of non-genetic factors in ALS is recognized and needs further study. In this review, we briefly discuss several major genetic contributors to ALS identified to date, followed by a more focused discussion on the most commonly examined non-genetic risk factors for ALS. We first review factors related to lifestyle choices, including smoking, intake of antioxidants, physical fitness, body mass index, and physical exercise, followed by factors related to occupational and environmental exposures, including electromagnetic fields, metals, pesticides, β-methylamino-L-alanine, and viral infection. Potential links between ALS and other medical conditions, including head trauma, metabolic diseases, cancer, and inflammatory diseases, are also discussed. Finally, we outline several future directions aiming to more efficiently examine the role of non-genetic risk factors in ALS. PMID:25709501

A new comprehensive technique of catheterisation, blood sampling, sample preparation and sample analysis by means of high-pressure liquid chromatography for pharmacokinetic studies with estradiol-linked nitrosoureas and their metabolites.

PubMed

Betsch, B; Berger, M R; Spiegelhalder, B

1990-09-01

Estradiol-linked nitrosoureas are offering new perspectives in the antineoplastic chemotherapy of estradiol-receptor positive mammary carcinomas. In such a molecule estradiol has the function of a carrier which brings about a specific accumulation of the anticancer drug in estradiol-receptor containing tumor cells. However, there is only little knowledge about the pharmacokinetic behavior of this new group of anticancer agents. For that reason a new comprehensive technique of catheterisation, blood sampling, sample preparation and sample analysis with high-pressure liquid chromatography (HPLC) for preclinical pharmacokinetic studies with estradiol-linked nitrosoureas and their metabolites has been developed. N-(2-Chloroethyl)-N-nitroso-carbamoyl-L-alanine-estradiol-17-ester (CNC-alanine-estradiol-17-ester) and N-(2-chloroethyl)-N-nitroso-carbamoyl-L-alanine (CNC-alanine) were used as test compounds. The drugs were tested in female Sprague-Dawley rats with chemically induced mammary carcinomas. The laboratory animals were supplied with two catheters prior to the pharmacokinetic experiments. The blood samples were drawn from the vena cava catheter after the drug had been applied through a vena jugularis catheter. The compounds were extracted from plasma with C18 silicagel reversed phase cartridges. The clean-up technique delivered clear samples only slightly contaminated with the biological matrix. The recovery from plasma was 75 +/- 5% for the hormone-linked CNC-alanine-estradiol-17-ester and 70 +/- 5% for the unlinked CNC-alanine. The analysis was carried out by means of HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel type of murein transglycosylase in Escherichia coli.

PubMed Central

Höltje, J V; Mirelman, D; Sharon, N; Schwarz, U

1975-01-01

The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed. PMID:357

The effects of aromatic amino acid derivatives on the excitability of an identifiable giant neurone of the African giant snail (Achatina fulica Férussac).

PubMed Central

Takeuchi, H.; Tamura, H.

1980-01-01

1 The effects of derivatives of aromatic amino acids on the excitability of an identifiable giant neurone (TAN, tonically autoactive neurone) of the African giant snail (Achatina fulica Férussac) were examined. 2 The following substances had marked inhibitory effects on TAN using bath application: N-beta-phenylpropionyl-L-Tyr and N-beta-phenylpropionyl-L-Trp (critical concentration, 3 x 10(-7) M), N-beta-phenylpropionyl-L-Phe, N-cinnamoyl-DL-Trp and N-phenoxyacetyl-L-Trp (critical concentration, 10(-5) to 3 x 10(-5) M). However, N-beta-phenylpropionyl-D-Tyr and N-beta-phenylpropionyl tyramine had no effect. 3 Microdrop (150 micrometers in diameter) application of N-beta-phenylpropionyl-L-Tyr or N-beta-phenylpropionyl-l-trp containing about 100 pg resulted in marked inhibitory effects on TAN. The effect was observed in Ca2+-free, Mg2+-rich (24 mM) solution. Substitution of Cl- by acetate did not alter the response. This indicates that the two substances act directly on the TAN membrane and not via synaptic influences, and that the inhibition produced by the two substances is not due to the permeability increase of the TAN membrane to Cl-. PMID:7378654

Responses of single facial taste fibers in the channel catfish, Ictalurus punctatus, to amino acids.

PubMed

Kohbara, J; Michel, W; Caprio, J

1992-10-01

1. Amino acids and nucleotides stimulate taste receptors of teleosts. In this report, responses to these compounds of 105 facial taste fibers (79 fully characterized) that innervate maxillary barbel taste buds of the channel catfish (Ictalurus punctatus) were analyzed. 2. The fully characterized facial taste fibers that responded to amino acids (n = 68) were generally poorly responsive to nucleotides and related substances (NRS), whereas the fibers responsive to NRS (n = 11) were poorly responsive to amino acids. Spike discharge of the amino acid-responsive fibers to the most potent amino acid stimulus tested per fiber increased 44-fold from a mean spontaneous activity of 2.1 +/- 3.5 to 92.1 +/- 42.4 (SD) spikes/3 s. Spike activity of the NRS-responsive fibers to NRS increased 11.5-fold from a mean spontaneous activity of 3.4 +/- 5.9 to 39.1 +/- 27.4 spikes/3 s. There was no significant difference between the spontaneous rates, but stimulus evoked spike rates for the amino acid-responsive fibers were significantly greater (P < 0.05; Mann-Whitney test) than those for the NRS-responsive fibers. 3. Hierarchical cluster analysis based on the 3-s response time identified three major groups of neurons. The identified clusters comprised neurons that were highly responsive to either L-alanine (i.e., Ala cluster; n = 39), L-arginine (i.e., Arg cluster; n = 29), or NRS (NRS cluster; n = 11). Fibers comprising the Arg cluster were more narrowly tuned than those within the Ala cluster. This report further characterizes the responses to amino acids of the individual facial taste fibers comprising the Ala and Arg clusters. 4. Subclusters were evident within both of the amino acid-responsive clusters. The Arg cluster was divisible into two subclusters dependent on the response to 1 mM L-proline. Twelve neurons that were significantly (P < 0.05; Mann-Whitney test) more responsive to L-proline than the remaining 17 neurons within the Arg cluster formed the Arg/Pro subcluster; these latter 17 neurons comprised the Arg subcluster. However, there was no significant difference (Mann-Whitney test) in the response to L-arginine between fibers within either subcluster across four different response times analyzed. Fibers within the Ala cluster were generally poorly responsive to L-proline. Four alanine subclusters were suggested on the basis of their relative responses to L-alanine, D-alanine, L-arginine, and the NRS; however, of the 39 fibers comprising the alanine cluster, two alanine subclusters comprised only two fibers each, and the third subcluster consisted of four fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

A Structural and Mutagenic Blueprint for Molecular Recognition of Strychnine and d-Tubocurarine by Different Cys-Loop Receptors

PubMed Central

Kuzmin, Dmitry; van Elk, René; Krijnen, Liz; Yakel, Jerrel L.; Tsetlin, Victor; Smit, August B.; Ulens, Chris

2011-01-01

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT3R. PMID:21468359

A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.

PubMed

Brams, Marijke; Pandya, Anshul; Kuzmin, Dmitry; van Elk, René; Krijnen, Liz; Yakel, Jerrel L; Tsetlin, Victor; Smit, August B; Ulens, Chris

2011-03-01

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT(3)R.

Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA2 neurotoxin from the venom of Daboia russelli siamensis.

PubMed

Maung-Maung-Thwin; Gopalakrishnakone, P; Yuen, R; Tan, C H

1996-02-01

Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations, while charybdotoxin was the only K+ channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K+ nor Ca2+ channels are associated with DbTx binding sites.

Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

PubMed

Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

2011-08-19

The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT.

Interest of fluorine-19 nuclear magnetic resonance spectroscopy in the detection, identification and quantification of metabolites of anticancer and antifungal fluoropyrimidine drugs in human biofluids.

PubMed

Martino, Robert; Gilard, Véronique; Desmoulin, Franck; Malet-Martino, Myriam

2006-01-01

The metabolism of fluorouracil and fluorocytosine, two 5-fluoropyrimidine drugs in clinical use, was investigated. (19)F nuclear magnetic resonance (NMR) spectroscopy was used as an analytical technique for the detection, identification and quantification of fluorinated metabolites of these drugs in intact human biofluids as well as fluorinated degradation compounds of fluorouracil in commercial vials. (19)F NMR provides a highly specific tool for the detection and absolute quantification, in a single run, of all the fluorinated species, including unexpected substances, present in biofluids of patients treated with fluorouracil or fluorocytosine. Besides the parent drug and the already known fluorinated metabolites, nine new metabolites were identified for the first time with (19)F NMR in human biofluids. Six of them can only be observed with this technique: fluoride ion, N-carboxy-alpha-fluoro-beta-alanine, alpha-fluoro-beta-alanine conjugate with deoxycholic acid, 2-fluoro-3-hydroxypropanoic acid, fluoroacetic acid, O(2)-beta-glucuronide of fluorocytosine. (19)F NMR studies of biological fluids of patients treated with anticancer fluorouracil or antifungal fluorocytosine have furthered the understanding of their catabolic pathways.

Fluorine nuclear magnetic resonance spectroscopy of human biofluids in the field of metabolic studies of anticancer and antifungal fluoropyrimidine drugs.

PubMed

Malet-Martino, Myriam; Gilard, Véronique; Desmoulin, Franck; Martino, Robert

2006-04-01

Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy provides a highly specific tool for the detection, identification and quantification of fluorine-containing drugs and their metabolites in biofluids. The value and difficulties encountered in investigations on drug metabolism are first discussed. Then the metabolism of three fluoropyrimidines in clinical use, 5-fluorouracil, 5-fluorocytosine and capecitabine are reported. Besides the parent drug and the already known fluorinated metabolites, 12 new metabolites were identified for the first time with 19F NMR in human biofluids. Nine of them can only be observed with this technique: fluoride ion, N-carboxy-alpha-fluoro-beta-alanine, alpha-fluoro-beta-alanine conjugate with deoxycholic acid, 2-fluoro-3-hydroxypropanoic acid, fluoroacetic acid, O2-beta-glucuronide of fluorocytosine, fluoroacetaldehyde hydrate and its adduct with urea, fluoromalonic acid semi-aldehyde adducts with urea. This emphasizes the high analytical potential of 19F NMR for the furtherance in the understanding of fluoropyrimidine catabolic pathways. 19F NMR should also play a role in the therapeutic monitoring of FU and its prodrugs in specific groups of patients, e.g. hemodialyzed patients or patients with deficiency in FU catabolic enzymes.

Mechanism of d-Cycloserine Action: Transport Systems for d-Alanine, d-Cycloserine, l-Alanine, and Glycine1

PubMed Central

Wargel, Robert J.; Shadur, Craig A.; Neuhaus, Francis C.

1970-01-01

The accumulation of d-alanine, l-alanine, glycine, and d-cycloserine in Escherichia coli was found to be mediated by at least two transport systems. The systems for d-alanine and glycine are related, and are separate from that involved in the accumulation of l-alanine. d-Cycloserine appears to be primarily transported by the d-alanine-glycine system. The accumulation of d-alanine, glycine, and d-cycloserine was characterized by two line segments in the Lineweaver-Burk analysis, whereas the accumulation of l-alanine was characterized by a single line segment. d-Cycloserine was an effective inhibitor of glycine and d-alanine accumulation, and l-cycloserine was an effective inhibitor of l-alanine transport. The systems were further differentiated by effects of azide, enhancement under various growth conditions, and additional inhibitor studies. Since the primary access of d-cycloserine in E. coli is via the d-alanine-glycine system, glycine might be expected to be a better antagonist of d-cycloserine inhibition than l-alanine. Glycine and d-alanine at 10−5m antagonized the effect of d-cycloserine in E. coli, whereas this concentration of l-alanine had no effect. PMID:4919992

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides of DL-alanine and N-(tert-butoxycarbonyl)-DL-alanine

NASA Technical Reports Server (NTRS)

Profy, A. T.; Usher, D. A.

1984-01-01

The aminoacylation of diinosine monophosphate was studied experimentally. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-DL-alanine, a 40 percent enantiomeric excess of the isomer was incorporated at the 2' site and the positions of equilibrium for the reversible 2'-3' migration reaction differed for the D and L enantiomers. The reactivity of the nucleoside hydroxyl groups was found to decrease on the order 2'(3') less than internal 2' and less than 5', and the extent of the reaction was affected by the concentration of the imidazole buffer. Reaction of IpI with imidazolide of unprotected DL-alanine, by contrast, led to an excess of the D isomer at the internal 2' site. Finally, reaction with the N-carboxy anhydride of DL-alanine occurred without stereoselection. These results are found to be relevant to the study of the evolution of optical chemical activity and the origin of genetically directed protein synthesis.

Energy metabolism and metabolomics response of Pacific white shrimp Litopenaeus vannamei to sulfide toxicity.

PubMed

Li, Tongyu; Li, Erchao; Suo, Yantong; Xu, Zhixin; Jia, Yongyi; Qin, Jian G; Chen, Liqiao; Gu, Zhimin

2017-02-01

The toxicity and poisoning mechanisms of sulfide were studied in Litopenaeus vannamei from the perspective of energy metabolism and metabolomics. The lethal concentrations of sulfide in L. vannamei (LC50) at 24h, 48h, 72h, and 96h were determined. Sulfide at a concentration of 0, 1/10 (425.5μg/L), and 1/5 (851μg/L) of the LC 50 at 96h was used to test the metabolic responses of L. vannamei for 21days. The chronic exposure of shrimp to a higher sulfide concentration of 851μg/L decreased shrimp survival but did not affect weight gain or the hepatopancreas index. The glycogen content in the hepatopancreas and muscle and the activity of hepatopancreas cytochrome C oxidase of the shrimp exposed to all sulfide concentrations were significantly lower, and the serum glucose and lactic acid levels and lactic acid dehydrogenase activity were significantly lower than those in the control. Metabolomics assays showed that shrimp exposed to sulfide had lower amounts of serum pyruvic acid, succinic acid, glycine, alanine, and proline in the 425.5μg/L group and phosphate, succinic acid, beta-alanine, serine, and l-histidine in the 851μg/L group than in the control. Chronic sulfide exposure could disturb protein synthesis in shrimp but enhance gluconeogenesis and substrate absorption for ATP synthesis and tricarboxylic acid cycles to provide extra energy to cope with sulfide stress. Chronic sulfide exposure could adversely affect the health status of L. vannamei, as indicated by the high amounts of serum n-ethylmaleamic acid, pyroglutamic acid, aspartic acid and phenylalanine relative to the control. This study indicates that chronic exposure of shrimp to sulfide can decrease health and lower survival through functional changes in gluconeogenesis, protein synthesis and energy metabolism. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis and separation of residues important for the chemoattractant and antimicrobial activities of beta-defensin 3.

PubMed

Taylor, Karen; Clarke, David J; McCullough, Bryan; Chin, Wutharath; Seo, Emily; Yang, De; Oppenheim, Joost; Uhrin, Dusan; Govan, John R W; Campopiano, Dominic J; MacMillan, Derek; Barran, Perdita; Dorin, Julia R

2008-03-14

beta-Defensins are important in mammalian immunity displaying both antimicrobial and chemoattractant activities. Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, here we show that HBD3 (human beta-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant. Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. These findings are replicated by the murine ortholog Defb14. We restore the chemoattractant activity of Defb14 and HBD3 by introduction of a single cysteine in the fifth position (Cys V) of the beta-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position (Cys I) is inactive. Moreover, a range of overlapping linear fragments of Defb14 do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys V. Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of beta-defensins can be separated, and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design.

Clostridium botulinum serotype D neurotoxin and toxin complex bind to bovine aortic endothelial cells via sialic acid.

PubMed

Yoneyama, Tohru; Miyata, Keita; Chikai, Tomoyuki; Mikami, Akifumi; Suzuki, Tomonori; Hasegawa, Kimiko; Ikeda, Toshihiko; Watanabe, Toshihiro; Ohyama, Tohru; Niwa, Koichi

2008-12-01

Botulinum neurotoxin (BoNT) is produced as a large toxin complex (L-TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). The binding properties of BoNT to neurons and L-TC to intestinal epithelial cells are well documented, while those to other tissues are largely unknown. Here, to obtain novel insights into the pathogenesis of foodborne botulism, we examine whether botulinum toxins bind to vascular endothelial cells. BoNT and 750 kDa L-TC (a complex of BoNT, NTNHA and HAs) of Clostridium botulinum serotype D were incubated with bovine aortic endothelial cells (BAECs), and binding to the cells was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Both BoNT and L-TC bound to BAECs, with L-TC showing stronger binding. Binding of BoNT and L-TC to BAECs was significantly inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, galactose, lactose or N-acetyl galactosamine did not significantly inhibit toxin binding to the cells. This is the first report demonstrating that BoNT and L-TC bind to BAECs via sialic acid, and this mechanism may be important in the trafficking pathway of BoNT in foodborne botulism.

Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

PubMed

He, Ying-Ying; Lee, Wei-Hui; Zhang, Yun

2004-09-01

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice.

Influence of the amino acid moiety on deconjugation of bile acid amidates by cholylglycine hydrolase or human fecal cultures.

PubMed

Huijghebaert, S M; Hofmann, A F

1986-07-01

The influence of the chemical structure of the amino acid (or amino acid analogue) moiety of a number of synthetic cholyl amidates on deconjugation by cholylglycine hydrolase from Clostridium perfringens was studied in vitro at pH 5.4. Conjugates with alkyl homologues of glycine were hydrolyzed more slowly as the number of methylene units increased (cholylglycine greater than cholyl-beta-alanine greater than cholyl-gamma-aminobutyrate). In contrast, for conjugates with the alkyl homologues of taurine, cholylaminopropane sulfonate was hydrolyzed slightly faster than cholyltaurine, whereas cholylaminomethane sulfonate was hydrolyzed much more slowly. When glycine was replaced by other neutral alpha-amino acids, rates of hydrolysis decreased with increasing steric hindrance near the amide bond (cholyl-L-alpha-alanine much much greater than cholyl-L-leucine much greater than cholyl-L-valine greater than cholyl-L-tyrosine much greater than cholyl-D-valine). Conjugation with acidic or basic amino acids also greatly reduced the rates of hydrolysis, as cholyl-L-aspartate, cholyl-L-cysteate, cholyl-L-lysine, and cholyl-L-histidine were all hydrolyzed at a rate less than one-tenth that of cholylglycine. Methyl esterification of the carboxylic group of the amino acid moiety reduced the hydrolysis, but such substrates (cholylglycine methyl ester and cholyl-beta-alanine methyl ester) were completely hydrolyzed after overnight incubation with excess of enzyme. In contrast, cholyl-cholamine was not hydrolyzed at all, suggesting that a negative charge at the end of the side chain is required for optimal hydrolysis. Despite the lack of specificity for the amino acid moiety, a bile salt moiety was required, as the cholylglycine hydrolase did not display general carboxypeptidase activity for other non-bile acid substrates containing a terminal amide bond: hippuryl-L-phenylalanine and hippuryl-L-arginine, as well as oleyltaurine and oleylglycine, were not hydrolyzed. Fecal bacterial cultures from healthy volunteers also hydrolyzed cholyl-L-valine and cholyl-D-valine more slowly than cholylglycine, suggesting that cholylglycine hydrolase from Clostridium perfringens has a substrate specificity similar to that of the deconjugating enzymes of the fecal flora. The results indicate that modification of the position of the amide bond, introduction of steric hindrance near the amide bond, or loss of a negative charge on the terminal group of the amino acid moiety of the bile acid conjugate greatly reduces the rate of bacterial deconjugation in vitro when compared to that of the naturally occurring glycine and taurine conjugates.

The synthesis and biological evaluation of new DNA-directed alkylating agents, phenyl N-mustard-4-anilinoquinoline conjugates containing a urea linker.

PubMed

Marvania, Bhavin; Kakadiya, Rajesh; Christian, Wilson; Chen, Tai-Lin; Wu, Ming-Hsi; Suman, Sharda; Tala, Kiran; Lee, Te-Chang; Shah, Anamik; Su, Tsann-Long

2014-08-18

We synthesized a series of phenyl N-mustard-4-anilinoquinoline conjugates to study their antitumorigenic effects. These agents were prepared by the condensation of 4-[N,N-bis(2-chloroethyl)amino]phenyl isocyanate with 6-amino-4-methylamino or 4-anilinoquinolines. The structure-activity relationship (SAR) studies revealed that the C2-methylquinoline derivatives (18a-o) were generally more cytotoxic than the C2-phenylquinoline conjugates (23a-d) in inhibiting the cell growth of various human tumor cell lines in vitro. However, the methylamino or aniline substituents at C4 of quinoline did not influence the cytotoxic effects. The title conjugates were capable of inducing DNA cross-linking and promoting cell-cycle arrest at the G2/M phase. This study demonstrates that phenyl N-mustard-4-anilinoquinoline conjugates are generally more potent than phenyl N-mustard-4-anilinoquinazoline conjugates against the cell growth of various tumor cell-lines. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Structures of apo IRF-3 and IRF-7 DNA binding domains: effect of loop L1 on DNA binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Ioannes, Pablo; Escalante, Carlos R.; Aggarwal, Aneel K.

2013-11-20

Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-{beta} (IFN-{beta}) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-{beta} promoter elements revealed flexibility in the loops (L1-L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures ofmore » IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding.« less

Specific amino acid residues in the beta sliding clamp establish a DNA polymerase usage hierarchy in Escherichia coli.

PubMed

Sutton, Mark D; Duzen, Jill M

2006-03-07

Escherichia coli dnaN159 strains encode a mutant form of the beta sliding clamp (beta159), causing them to display altered DNA polymerase (pol) usage. In order to better understand mechanisms of pol selection/switching in E. coli, we have further characterized pol usage in the dnaN159 strain. The dnaN159 allele contains two amino acid substitutions: G66E (glycine-66 to glutamic acid) and G174A (glycine-174 to alanine). Our results indicated that the G174A substitution impaired interaction of the beta clamp with the alpha catalytic subunit of pol III. In light of this finding, we designed two additional dnaN alleles. One of these dnaN alleles contained a G174A substitution (beta-G174A), while the other contained D173A, G174A and H175A substitutions (beta-173-175). Examination of strains bearing these different dnaN alleles indicated that each conferred a distinct UV sensitive phenotype that was dependent upon a unique combination of Delta polB (pol II), Delta dinB (pol IV) and/or Delta umuDC (pol V) alleles. Taken together, these findings indicate that mutations in the beta clamp differentially affect the functions of these three pols, and suggest that pol II, pol IV and pol V are capable of influencing each others' abilities to gain access to the replication fork. These findings are discussed in terms of a model whereby amino acid residues in the vicinity of those mutated in beta159 (G66 and G174) help to define a DNA polymerase usage hierarchy in E. coli following UV irradiation.

Islet immunity and beta cell reserve of indigenous Black South Africans with ketoacidosis at initial diagnosis of diabetes.

PubMed

Ekpebegh, Chukwuma; Longo-Mbenza, Benjamin; Blanco-Blanco, Ernesto

2013-01-01

Islet immunity and beta cell reserve status were utilized to classify persons with ketoacidosis as the initial manifestation of diabetes. The clinical features of the various diabetes classes were also characterized. Prospective cross sectional study. Nelson Mandela Academic Hospital, Mthatha, Eastern Cape Province, South Africa. Indigenous Black South Africans with ketoacidosis as the initial manifestation of diabetes. Islet immunity and beta cell reserve were respectively assessed using serum anti-glutamic acid decarboxylase 65 (GAD) antibody and serum C-peptide after 1 mg of intravenous glucagon. Serum anti-GAD 65 antibody > or = 5 units/L and < 5 units/L, respectively defined anti-GAD 65 positive (A+) and negative (A-). Replete (beta+) and deplete (beta-) beta cell reserve were serum C-peptide after glucagon injection of > or = 0.5 ng/mL and < 0.5 ng/mL, respectively. The proportions of patients with A+beta-, A+beta+, A-beta- and A-beta+ and their clinical characteristics were determined. Of the 38 males and 33 females who participated in the study, patients were categorized in various classes: A-beta+, 46.5% (n=33/ 71); A-beta-, 26.8% (n=19/71); A+beta-, 22.5% (n=16/71); and A+beta+, 4.2% (n=3/71). The ages of the various classes were: 41.8 +/- 13.8 years for A-beta+ (n=33); 36.5 +/- 14.6 years for A-beta- (n=19); and 20.6 +/- 7.1 years for the combination of A+beta- with A+beta+ (n=19) (P<.0001, P<.0001 for the combination of A+beta- and A+beta+ vs A-beta+, P=.001 for the combination of A+beta- and A+beta+ vs A-beta-and P=.2 for A-beta- vs A-beta+. The clinical features of type 2 diabetes were most prevalent in A-beta+ class while the A+beta- and A+beta+ groups had the clinical profile of type 1A diabetes. Most of the indigenous Black South African patients with ketoacidosis as the initial manifestation of diabetes had islet immunity, beta cell reserve status and clinical profiles of type 2 diabetes.

Purification and characterization of a lectin from the white shrimp Litopenaeus setiferus (Crustacea decapoda) hemolymph.

PubMed

Alpuche, Juan; Pereyra, Ali; Agundis, Concepción; Rosas, Carlos; Pascual, Cristina; Slomianny, Marie-Christine; Vázquez, Lorena; Zenteno, Edgar

2005-06-20

A 291-kDa lectin (LsL) was purified from the hemolymph of the white shrimp Litopenaeus setiferus by affinity chromatography on glutaraldehyde-fixed stroma from rabbit erythrocytes. LsL is a heterotetramer of two 80-kDa and two 52-kDa subunits, with no covalently-liked carbohydrate, and mainly composed by aspartic and glutamic acids, glycine and alanine, with relatively lower methionine and cysteine contents. Edman degradation indicated that the NH2-terminal of the 80-kDa subunit is composed DASNAQKQHDVNFLL, whereas the NH2-terminal of the 52-kDa subunit is blocked. The peptide mass fingerprint of LsL was predicted from tryptic peptides from each subunit by MALDI-TOF, and revealed that each subunit showed 23 and 22%, respectively, homology with the hemocyanin precursor from Litopenaeus vannamei. Circular dichroism analysis revealed beta sheet and alpha helix contents of 52.7 and 6.1%, respectively. LsL agglutinate at higher titers guinea pig, murine, and rabbit erythrocytes its activity is divalent cation-dependent. N-acetylated sugars, such as GlcNAc, GalNAc, and NeuAc, were the most effective inhibitors of the LsL hemagglutinating activity. Sialylated O-glycosylated proteins, such as bovine submaxillary gland mucin, human IgA, and fetuin, showed stronger inhibitory activity than sialylated N-glycosylated proteins, such as human orosomucoid, IgG, transferrin, and lactoferrin. Desialylation of erythrocytes or inhibitory glycoproteins abolished their capacity to bind LsL, confirming the relevance of sialic acid in LsL-ligand interactions.

Cyanogenesis - a general phenomenon in the lepidoptera

DOE Office of Scientific and Technical Information (OSTI.GOV)

Witthohn, K.; Naumann, C.M.

1987-08-01

There are two different pathways known to be used for the detoxification of hydrocyanic acid in insects, viz., rhodanese and ..beta..-cyano-L-alanine synthase. The authors consider the latter to be indicative for cyanogenesis, while rhodanese might, in general, play a more important role in sulfur transfer for protein synthesis. This paper reports on the distribution of ..beta..-cyano-L-alanine (BCA) in the Lepidoptera. First reports of cyanogenesis are presented for the following families: Papilionidae, Pieridae, Lycaenidae, Hesperiidae, Lymantriidae, Arctiidae, Notodontidae, Megalopygidae, Limacodidae, Cymatophoridae, Noctuidae, Geometridae, and Yponomeutidae. New and old records for three other families, the Nymphalidae, Zygaenidae, and Heterogynidae, are included tomore » complete the present state of knowledge. Special emphasis has been laid on the Nymphalidae, where BCA has been detected in eight subfamilies. Taxonomic, geographic, and seasonal variation has been found in a number of cases. In all cases observed so far, the source of cyanogenesis in the Lepidoptera is most probably the cyanoglucosides linamarin and lotaustralin, although cyanogenesis based on mustard oil glucosides and cyclopentenoid glucosides might occur as well. BCA has been found in both cryptic and aposematic species, including taxa such as the Pieridae, Danainae, Ithomiinae, and Arctiidae, where the defensive biology is believed to be linked with other compounds, like mustard oil glucosides, cardenolides, or pyrrolizidinie alkaloids. The ecological interaction and significance of such secondary compounds is not yet understood.« less

Echinococcus granulosus: specificity of amino acid transport systems in protoscoleces.

PubMed

Jeffs, S A; Arme, C

1987-08-01

Protoscoleces of Echinococcus granulosus absorb the L-amino acids proline, methionine, leucine, alanine, serine, phenylalanine, lysine and glutamic acid by a combination of mediated transport and diffusion. All eight amino acids were accumulated against a concentration gradient. Comparison of Kt and Vmax values suggests that a low affinity for a particular compound is compensated for by a relatively larger number of transport sites for that compound. Four systems serve for the transport of the eight substrates studied: 2 for neutral (EgN1, EgN2) and 1 each for acidic (EgA) and basic (EgB) amino acids. All eight amino acids are incorporated into protein to varying degrees and substantial portions of absorbed L-alanine and L-methionine are metabolized into other compounds.

Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, T.; Weintraub, B.D.

1985-04-01

The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing (/sup 14/C)alanine and (/sup 3/H) glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, (/supmore » 14/C)alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. (/sup 3/H)Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function.« less

N-terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled-coil region of cartilage oligomeric matrix protein.

PubMed

Gunasekar, Susheel K; Asnani, Mukta; Limbad, Chandani; Haghpanah, Jennifer S; Hom, Wendy; Barra, Hanna; Nanda, Soumya; Lu, Min; Montclare, Jin Kim

2009-09-15

The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.

Enhanced amine and amino acid analysis using Pacific Blue and the Mars Organic Analyzer microchip capillary electrophoresis system.

PubMed

Chiesl, Thomas N; Chu, Wai K; Stockton, Amanda M; Amashukeli, Xenia; Grunthaner, Frank; Mathies, Richard A

2009-04-01

The fluorescent amine reactive probe Pacific Blue succinimidyl ester (PB) is used for the detection of trace amounts of amines and amino acids by microchip capillary electrophoresis on the Mars Organic Analyzer (MOA). The spectral and chemical properties of PB provide a 200-fold increase in sensitivity and improved resolution compared to fluorescamine derivatization. With the use of cross injection and PB labeling, the MOA detected amino acids at concentrations as low as 75 pM (sub-parts-per-trillion). Micellar electrokinetic chromatography (MEKC) which separates PB-labeled amino acids by their hydrophobicity is also demonstrated. The optimized MEKC conditions (45 mM CHAPSO, pH 6 at 5 degrees C) effectively separated amines and 25 amino acids with enantiomeric resolution of alanine, serine, and citrulline. Samples from the Yungay Hills region in the Atacama Desert, Chile, and from the Murchison meteorite are successfully analyzed using both techniques, and amino acids are found in the parts-per-billion range. Abiotic amino acids such as beta-alanine and epsilon-aminocaprioc acid are detected along with several neutral and acidic amino acids in the Murchison sample. The Atacama Desert sample is found to contain homochiral L-alanine and L-serine indicating the presence of extant or recently extinct life.

Titration of Alanine Monitored by NMR Spectroscopy: A Biochemistry Laboratory Experiment

ERIC Educational Resources Information Center

Waller, Francis J.; And Others

1977-01-01

The experiment described here involves simultaneous monitoring of pH and NMR chemical shifts during an aqueous titration of alpha- and beta-alanine. This experiment is designed for use in an undergraduate biochemistry course. (MR)

Synthesis and evaluation of N-alkyl-beta-D-glucosylamines on the growth of two wood fungi, Coriolus versicolor and Poria placenta.

PubMed

Muhizi, Théoneste; Coma, Véronique; Grelier, Stéphane

2008-09-22

Various glucosylamines were synthesized from glucose and different alkyl amine compounds. These amino compounds are beta-D-glucopyranosylamine (GPA), N-ethyl-beta-D-glucopyranosylamine (EtGPA), N-butyl-beta-D-glucopyranosylamine (BuGPA), N-hexyl-beta-D-glucopyranosylamine (HeGPA), N-octyl-beta-D-glucopyranosylamine (OcGPA), N-dodecyl-beta-D-glucopyranosylamine (DoGPA), N-(2-hydroxyethyl)-beta-D-glucopyranosylamine (HEtGPA) and N,N-di(2-hydroxyethyl)-beta-D-glucopyranosylamine (DHEtGPA). They were tested for their antifungal activity against the growth of Coriolus versicolor and Poria placenta. An improvement of the antifungal activity with the increase of alkyl chain length was observed. DoGPA exhibited the best antifungal activity against both strains. It completely inhibited the fungal growth at 0.01x10(-3)molmL(-1) and 0.0075x10(-3)molmL(-1) for C. versicolor and P. placenta, respectively. For other glucosylamines higher concentrations were needed for complete inhibition of fungi.

Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases

PubMed Central

Meziane-Cherif, Djalal; Stogios, Peter J.; Evdokimova, Elena; Egorova, Olga

2015-01-01

ABSTRACT Vancomycin resistance in Gram-positive bacteria results from the replacement of the d-alanyl–d-alanine target of peptidoglycan precursors with d-alanyl–d-lactate or d-alanyl–d-serine (d-Ala-d-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of d-Ala-d-Ser-terminating precursors by converting l-Ser to d-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in l-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5′-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the l-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against l-Ser versus l-Ala implied that this enzyme relies on its membrane-bound domain for l-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria. PMID:26265719

Structural and functional adaptation of vancomycin resistance VanT serine racemases

DOE PAGES

Meziane-Cherif, Djalal; Stogios, Peter J.; Evdokimova, Elena; ...

2015-08-11

Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl–D-alanine target of peptidoglycan precursors with D-alanyl–D-lactate or D-alanyl–D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanT G from VanG-type resistant Enterococcus faecalis BM4518more » was determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanT G and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn 696 which are responsible for theL-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanT G against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of D-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria.« less

Physical Characterization of Clostridium Botulinum Neurotoxin Genes

DTIC Science & Technology

1993-10-01

Acid Res. 14, 7809-78 12. 10 Fujii, N., Kimura, K., Murakami, T ., Indoh, I., Yashiki, T ., Tsuzuki, K., Yokosawa , N. & Oguma, K. (1990) Microbiol...from Clostridium spp. whose genomic DNA is of a high A+ T content (greater than 70% A+ T ), exhibit an extremely strong discrimination against all...BoNT/E, 4-12 Sathyamoorthy T T et al. (1985) H E L I H S L H G LE2 YES 5’-CACGAACTTATACATTCTCTACATGG-3’ 861-886 BoNT/E, 212-220 Wernars & T T A AT

Obtaining molecular and structural information from 13C-14N systems with 13C FIREMAT experiments.

PubMed

Strohmeier, Mark; Alderman, D W; Grant, David M

2002-04-01

The effect of dipolar coupling to 14N on 13C FIREMAT (five pi replicated magic angle turning) experiments is investigated. A method is developed for fitting the 13C FIREMAT FID employing the full theory to extract the 13C-14N dipolar and 13C chemical shift tensor information. The analysis requires prior knowledge of the electric field gradient (EFG) tensor at the 14N nucleus. In order to validate the method the analysis is done for the amino acids alpha-glycine, gamma-glycine, l-alanine, l-asparagine, and l-histidine on FIREMAT FIDs recorded at 13C frequencies of 50 and 100 MHz. The dipolar and chemical shift data obtained with this analysis are in very good agreement with the previous single-crystal 13C NMR results and neutron diffraction data on alpha-glycine, l-alanine, and l-asparagine. The values for gamma-glycine and l-histidine obtained with this new method are reported for the first time. The uncertainties in the EFG tensor on the resultant 13C chemical shift and dipolar tensor values are assessed. (c) 2002 Elsevier Science (USA).

The activity of hydrolases of larval stages of Anisakis simplex (Nematoda).

PubMed

Lopieńska-Biernat, Elzbieta; Zółtowska, Krystyna; Rokicki, Jerzy

2004-01-01

Activity of hydrolases during the third and fourth larval stage of Anisakis simplex was identified by applying the API ZYM test method. In A. simplex larvae the activity of phosphatases was high, particularly that of acid phosphatase (40 nmol/mg(-1)). Among esterases lack of activity of lipase (C14) is worth noticing while the activity of esterases (C4) and (C8) was high. The activity of those later two enzymes was higher in L3 larvae than in L4 larvae. The highest activity in the subclass of glucosidases was recorded for beta-fucosidase and N-acetyl-beta-glucosaminidase. A higher activity in L3 larvae than in L4 larvae was recorded for: beta-glucuronidase and N-acetyl-beta-glucosaminidase (2-fold) and beta-fucosidase (3-fold). Differently the activity of beta-galactosidase and beta-glucosidase was higher in L4 larvae than in L3 larvae. The tests did not show activity of alpha-galactosidase, beta-glucosidase and alpha-mannosidase on both larval forms.

Functional Characterization of Alanine Racemase from Schizosaccharomyces pombe: a Eucaryotic Counterpart to Bacterial Alanine Racemase

PubMed Central

Uo, Takuma; Yoshimura, Tohru; Tanaka, Naotaka; Takegawa, Kaoru; Esaki, Nobuyoshi

2001-01-01

Schizosaccharomyces pombe has an open reading frame, which we named alr1+, encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1+ gene in Escherichia coli and purified the gene product (Alr1p), with an Mr of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent Km and Vmax values as follows: for l-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for d-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but l-serine and l-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of l-alanine, respectively. S. pombe uses d-alanine as a sole nitrogen source, but deletion of the alr1+ gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for l-alanine coupled with racemization plays a major role in the catabolism of d-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses l-alanine but not d-alanine as a sole nitrogen source. Moreover, d-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1+ gene enabled S. cerevisiae to grow efficiently on d-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of d-alanine. PMID:11244061

Structure-activity relationships of C6-uridine derivatives targeting plasmodia orotidine monophosphate decarboxylase.

PubMed

Bello, Angelica M; Poduch, Ewa; Liu, Yan; Wei, Lianhu; Crandall, Ian; Wang, Xiaoyang; Dyanand, Christopher; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2008-02-14

Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on human health, especially due to multidrug resistance. In Plasmodia, orotidine 5'-monophosphate decarboxylase (ODCase) is an essential enzyme for the de novo synthesis of uridine 5'-monophosphate. Impairing ODCase in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure-activity relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6- N-methylamino, and 6- N, N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial ODCase. 6-Azidouridine 5'-monophosphate is a potent covalent inhibitor of P. falciparum ODCase. 6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6- N-Methylamino and 6- N, N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.

The effect of altered 5-hydroxytryptamine levels on beta-endorphin

NASA Technical Reports Server (NTRS)

Soliman, Karam F. A.; Mash, Deborah C.; Walker, Charles A.

1986-01-01

The purpose of the present study was to examine the effect of altering the concentration of 5-hydroxytryptamine (5-HT) on beta-endorphin (beta-Ep) content in the hypothalamus, thalamus, and periaqueductal gray (PAG)-rostral pons regions of the rat brain. The selective 5-HT reuptake inhibitor, fluoxetine (10 mg/kg), significantly lowered beta-Ep content in the hypothalamus and the PAG. Parachlorophenylalanine, which inhibits 5-HT synthesis, significantly elevated beta-Ep in all brain parts studied. Intracisternal injections of the neurotoxin 5-prime, 7-prime-dihydroxytryptamine with desmethylimipramine pretreatment significantly increased beta-Ep content in the hypothalamus and the PAG. In adrenalectomized rats, fluoxetine significantly decreased beta-Ep levels in the hypothalamus and increased the levels in the PAG. The results indicate that 5-HT may modulate the levels of brain beta-Ep.

Synthesis and structural characterization of lithium, sodium and potassium complexes supported by a tridentate amino-bisphenolate ligand

NASA Astrophysics Data System (ADS)

Durango-García, Clara J.; Rufino-Felipe, Ernesto; López-Cardoso, Marcela; Muñoz-Hernández, Miguel-Ángel; Montiel-Palma, Virginia

2018-07-01

Reactions of methylamino-N,N-bis(2-methylene-4,6-di-tert-butylphenol) (1) with one or two equivalents of bulk Li, Na or K metals in THF or DMSO render mono or dialkali metal complexes depending on the stoichiometric ratio of the reactants. The metal-methylamino-N-(2-methylene-4,6-tert-butylphenol)sbnd N-(2-methylene-4,6-tert-butylphenolate) complexes, 2Li, 2Na and 2K, are generated upon the substitution of a single phenol hydrogen of 1. In the solid state, complex 2Na is a dimer due to the establishment of two symmetric hydrogen bonds between two adjacent molecules. The Na center also engages into the formation of a ten-membered metallacycle ring with a butterfly-like structure. Due to dimerization, an intermolecular six-membered core is formed involving two sodium and four oxygen atoms. The weakly coordinated nitrogen atom from the ligand is nearly perpendicular to the hexagonal core. The dimetal-methylamino-N,N‧-bis(2-methylene-4,6-di-tert-butylphenolate) complexes, 3Li, 3Na and 3K result from metal substitution of the two phenol hydrogens from ligand 1. The SC-XRD structures of 3Li and 3Na are discreet, each incorporating two metal atoms in different coordination environments. Ten-membered rings with boat-boat conformations are also observed as are rhombic central M2O2 cores. The molecular structure of 3K in DMSO shows a higher degree of aggregation. It effectively comprises four K atoms, two ligand backbones and seven solvent molecules forming a central four-membered K2O2 ring perpendicular to an eight-membered structure formed also by K and O atoms spanning over the two ligand moieties.

The origin of amino acids in lunar regolith samples

NASA Astrophysics Data System (ADS)

Elsila, Jamie E.; Callahan, Michael P.; Dworkin, Jason P.; Glavin, Daniel P.; McLain, Hannah L.; Noble, Sarah K.; Gibson, Everett K.

2016-01-01

We analyzed the amino acid content of seven lunar regolith samples returned by the Apollo 16 and Apollo 17 missions and stored under NASA curation since collection using ultrahigh-performance liquid chromatography with fluorescence detection and time-of-flight mass spectrometry. Consistent with results from initial analyses shortly after collection in the 1970s, we observed amino acids at low concentrations in all of the curated samples, ranging from 0.2 parts-per-billion (ppb) to 42.7 ppb in hot-water extracts and 14.5-651.1 ppb in 6 M HCl acid-vapor-hydrolyzed, hot-water extracts. Amino acids identified in the Apollo soil extracts include glycine, D- and L-alanine, D- and L-aspartic acid, D- and L-glutamic acid, D- and L-serine, L-threonine, and L-valine, all of which had previously been detected in lunar samples, as well as several compounds not previously identified in lunar regoliths: α-aminoisobutyric acid (AIB), D- and L-β-amino-n-butyric acid (β-ABA), DL-α-amino-n-butyric acid, γ-amino-n-butyric acid, β-alanine, and ε-amino-n-caproic acid. We observed an excess of the L enantiomer in most of the detected proteinogenic amino acids, but racemic alanine and racemic β-ABA were present in some samples. We also examined seven samples from Apollo 15, 16, and 17 that had been previously allocated to a non-curation laboratory, as well as two samples of terrestrial dunite from studies of lunar module engine exhaust that had been stored in the same laboratory. The amino acid content of these samples suggested that contamination had occurred during non-curatorial storage. We measured the compound-specific carbon isotopic ratios of glycine, β-alanine, and L-alanine in Apollo regolith sample 70011 and found values of -21‰ to -33‰. These values are consistent with those seen in terrestrial biology and, together with the enantiomeric compositions of the proteinogenic amino acids, suggest that terrestrial biological contamination is a primary source of the amino acids in these samples. However, the presence of the non-proteinogenic amino acids such as AIB and β-ABA suggests the possibility of some contribution from exogenous sources. We did not observe a correlation of amino acid content with proximity to the Apollo 17 lunar module, implying that lunar module exhaust was not a primary source of amino acid precursors. Solar-wind-implanted precursors such as HCN also appear to be at most a minor contributor, given a lack of correlation between amino acid content and soil maturity (as measured by Is/FeO ratio) and the differences between the δ13C values of the amino acids and the solar wind.

Enzymatic determination of carbon-14 labeled L-alanine in biological samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serra, F.; Palou, A.; Pons, A.

A method for determination of L-alanine-specific radioactivity in biological samples is presented. This method is based on the specific enzymatic transformation of L-alanine to pyruvic acid hydrazone catalyzed by the enzyme L-alanine dehydrogenase, formation of the pyruvic acid 2,4-dinitrophenylhydrazone derivative, and quantitative trapping in Amberlite XAD-7 columns, followed by radioactivity counting of the lipophilic eluate. No interferences from other UC-labeled materials such as D-glucose, glycerol, L-lactate, L-serine, L-glutamate, L-phenylalanine, glycine, L-leucine, and L-arginine were observed. This inexpensive and high-speed method is applicable to the simultaneous determination of L-alanine-specific radioactivity for a large number of samples.

Biochemical characterization of a phosphinate inhibitor of Escherichia coli MurC.

PubMed

Marmor, S; Petersen, C P; Reck, F; Yang, W; Gao, N; Fisher, S L

2001-10-09

The bacterial UDP-N-acetylmuramyl-L-alanine ligase (MurC) from Escherichia coli, an essential, cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent ligation of L-alanine (Ala) and UDP-N-acetylmuramic acid (UNAM) to form UDP-N-acetylmuramyl-L-alanine (UNAM-Ala). The phosphinate inhibitor 1 was designed and prepared as a multisubstrate/transition state analogue. The compound exhibits mixed-type inhibition with respect to all three enzyme substrates (ATP, UNAM, Ala), suggesting that this compound forms dead-end complexes with multiple enzyme states. Results from isothermal titration calorimetry (ITC) studies supported these findings as exothermic binding was observed under conditions with free enzyme (K(d) = 1.80-2.79 microM, 95% CI), enzyme saturated with ATP (K(d) = 0.097-0.108 microM, 95% CI), and enzyme saturated with the reaction product ADP (K(d) = 0.371-0.751 microM, 95% CI). Titrations run under conditions of saturating UNAM or the product UNAM-Ala did not show heat effects consistent with competitive compound binding to the active site. The potent binding affinity observed in the presence of ATP is consistent with the inhibitor design and the proposed Ordered Ter-Ter mechanism for this enzyme; however, the additional binding pathways suggest that the inhibitor can also serve as a product analogue.

Diagnostic Dilemma for Low Viremia with Significant Fibrosis; Is HBV DNA Threshold Level a Good Indicator for Predicting Liver Damage?

PubMed

Yenilmez, Ercan; Çetinkaya, Rıza Aytaç; Tural, Ersin

2018-05-04

The most important difficulties about management of hepatitis B are still determining the liver damage and the right time to start antiviral therapy. To reveal the role of hepatitis B virus DNA threshold level for prediction of liver fibrosis and inflammation in young-aged hepatitis B e antigen negative chronic hepatitis B patients. Diagnostic accuracy study. A total of 273 hepatitis B e antigen negative young chronic hepatitis B patients with any hepatitis B virus DNA levels between 2008 and 2016, who had liver biopsy after at least 6 months follow up period, enrolled in this retrospective study. We created two groups as case and control, cases with hepatitis B virus DNA levels below 2.000 IU/mL and controls with hepatitis B virus DNA levels over 2.000 IU/mL. Having histological activity index ≥4 or/and fibrosis scores ≥2 were defined as significant histological abnormality. Then, we analyzed the relationship between these groups. We showed that significant fibrosis may occur in one third of young chronic hepatitis B patients with low viremia (30.2%, n=42/139 in cases, %55.2, n=74/134 in controls). Among the 42 cases with low viremia and significant fibrosis, 21.4% had alanine aminotransferase level between 40-59 U/L, 42.8% had alanine aminotransferase level between 60-79 U/L, and 35.7% had alanine aminotransferase level over 80 U/L. There was weak correlation between hepatitis B virus DNA threshold level and fibrosis score (p=0.000, rho=0.253). The optimum serum hepatitis B virus DNA threshold level in our study for predicting significant fibrosis was 1293 IU/mL (p=0.00, AUC: 0.657±0.034). The optimum alanine aminotransferase threshold level for predicting significant histological activity index and fibrosis was 64.5 and 59.5 U/L, respectively. The sensitivity and the specificity of 1293 vs 2000 IU/mL hepatitis B virus DNA threshold with 60 U/L alanine aminotransferase threshold level for predicting F≥2 fibrosis score were similar (sensitivity: 0.43 and 0.38, respectively; specificity: 0.76 and 0.77, respectively). Significant fibrosis may occur even in young cases with low viremia. It is not possible to define a single threshold hepatitis B virus DNA level for differentiating inactive carriers from patients with hepatitis B e antigen-negative chronic hepatitis. Diagnostic accuracy of hepatitis B virus DNA with alanine aminotransferase thresholds for the prediction of significant fibrosis is weak.

Metabolic analysis of osteoarthritis subchondral bone based on UPLC/Q-TOF-MS.

PubMed

Yang, Gang; Zhang, Hua; Chen, Tingmei; Zhu, Weiwen; Ding, Shijia; Xu, Kaiming; Xu, Zhongwei; Guo, Yanlei; Zhang, Jian

2016-06-01

Osteoarthritis (OA), one of the most widespread musculoskeletal joint diseases among the aged, is characterized by the progressive loss of articular cartilage and continuous changes in subchondral bone. The exact pathogenesis of osteoarthritis is not completely clear. In this work, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) in combination with multivariate statistical analysis was applied to analyze the metabolic profiling of subchondral bone from 42 primary osteoarthritis patients. This paper described a modified two-step method for extracting the metabolites of subchondral bone from primary osteoarthritis patients. Finally, 68 metabolites were identified to be significantly changed in the sclerotic subchondral bone compared with the non-sclerotic subchondral bone. Taurine and hypotaurine metabolism and beta-alanine metabolism were probably relevant to the sclerosis of subchondral bone. Taurine, L-carnitine, and glycerophospholipids played a vital regulation role in the pathological process of sclerotic subchondral bone. In the sclerotic process, beta-alanine and L-carnitine might be related to the increase of energy consumption. In addition, our findings suggested that the intra-cellular environment of sclerotic subchondral bone might be more acidotic and hypoxic compared with the non-sclerotic subchondral bone. In conclusion, this study provided a new insight into the pathogenesis of subchondral bone sclerosis. Our results indicated that metabolomics could serve as a promising approach for elucidating the pathogenesis of subchondral bone sclerosis in primary osteoarthritis. Graphical Abstract Metabolic analysis of osteoarthritis subchondral bone.

Enantiomeric Excesses of Acid Labile Amino Acid Precursors of the Murchison Meteorite

NASA Astrophysics Data System (ADS)

Pizzarello, Sandra

1998-10-01

Amino acids present in carbonaceous chondrite are extracted in water in part as free compounds and in approximately equal part as acid labile precursors. On the assumption that they would be free of contamination, the precursors of two Murchison amino acids that have terrestrial occurrence, alanine and glutamic acid, have been targeted for analysis of their enantiomeric ratios. Pyroglutamic acid, the precursor of glutamic acid, was found with an L-enantiomeric excess comparable to that of the free acid, while alanine's precursor, N-acetyl alanine, appears approximately racemic. Also alpha-imino propioacetic acid, a proposed end product of alanine synthesis in the meteorite, was analyzed and found racemic.

Enantiomeric Excesses of Acid Labile Amino Acid Precursors of the Murchison Meteorite

NASA Technical Reports Server (NTRS)

Pizzarello, Sandra

1998-01-01

Amino acids present in carbonaceous chondrite are extracted in water in part as free compounds and in approximately equal part as acid labile precursors. On the assumption that they would be free of contamination, the precursors of two Murchison amino acids that have terrestrial occurrence, alanine and glutamic acid, have been targeted for analysis of their enantiomeric ratios. Pyroglutamic acid, the precursor of glutamic acid, was found with an L-enantiomeric excess comparable to that of the free acid, while alanine's precursor, N-acetyl alanine, appears approximately racemic. Also alpha-imino propioacetic acid, a proposed end product of alanine synthesis in the meteorite, was analyzed and found racemic.

Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli on the L-alanine export.

PubMed

Kim, Seryoung; Ihara, Kohei; Katsube, Satoshi; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

2017-01-01

The Escherichia coli alaE gene encodes the L-alanine exporter, AlaE, that catalyzes active export of L-alanine using proton electrochemical potential. The transporter comprises only 149 amino acid residues and four predicted transmembrane domains (TMs), which contain three charged amino acid residues. The AlaE-deficient L-alanine non-metabolizing cells (ΔalaE cells) appeared hypersusceptible to L-alanyl-L-alanine showing a minimum inhibitory concentration (MIC) of 2.5 µg/ml for the dipeptide due to a toxic accumulation of L-alanine. To elucidate the mechanism by which AlaE exports L-alanine, we replaced charged amino acid residues in the TMs, glutamic acid-30 (TM-I), arginine-45 (TM-II), and aspartic acid-84 (TM-III) with their respective charge-conserved amino acid or a net neutral cysteine. The ΔalaE cells producing R45K or R45C appeared hypersusceptible to the dipeptide, indicating that arginine-45 is essential for AlaE activity. MIC of the dipeptide in the ΔalaE cells expressing E30D and E30C was 156 µg/ml and >10,000 µg/ml, respectively, thereby suggesting that a negative charge at this position is not essential. The ΔalaE cells expressing D84E or D84C showed an MIC >10,000 and 78 µg/ml, respectively, implying that a negative charge is required at this position. These results were generally consistent with that of the L-alanine accumulation experiments in intact cells. We therefore concluded that charged amino acid residues (R45 and D84) in the AlaE transmembrane domain play a pivotal role in L-alanine export. Replacement of three cysteine residues at C22, C28 (both in TM-I), and C135 (C-terminal region) with alanine showed only a marginal effect on L-alanine export.

Carbodithioic acid esters of fluoxetine, a novel class of dual-function spermicides.

PubMed

Kiran Kumar, S T V S; Kumar, Lalit; Sharma, Vishnu L; Jain, Ashish; Jain, Rajeev K; Maikhuri, Jagdamba P; Kumar, Manish; Shukla, Praveen K; Gupta, Gopal

2008-10-01

Carbodithioic acid esters of fluoxetine have been prepared by replacing the methylamino function in aminopropane chain with carbodithioic acid ester group and by adding various S-2-hydroxypropyl ester of dialkyl carbodithioic acid at 3-methylamino group. Some of these compounds showed spermicidal, antifungal and anti-Trichomonas activities. The study revealed that incorporation of carbodithioic acid residue directly into fluoxetine structure leads to compounds with better antifungal and anti-Trichomonas activities, and N-methyl-[3-phenyl-3-(4-trifluoromethyl-phenoxy)-propyl]carbodithioic acid S-(2-pyrrolidino-ethyl) ester (14) has shown better profile than both fluoxetine and nonoxynol-9. Further lead optimization may yield a potent dual-function spermicide.

l-Alanine Auxotrophy of Lactobacillus johnsonii as Demonstrated by Physiological, Genomic, and Gene Complementation Approaches

PubMed Central

van der Kaaij, Hengameh; Desiere, Frank; Mollet, Beat; Germond, Jacques-Edouard

2004-01-01

Using a chemically defined medium without l-alanine, Lactobacillus johnsonii was demonstrated to be strictly auxotrophic for that amino acid. A comparative genetic analysis showed that all known genes involved in l-alanine biosynthesis are absent from the genome of L. johnsonii. This auxotrophy was complemented by heterologous expression of the Bacillus subtilis l-alanine dehydrogenase. PMID:15006820

EPR study of gamma-irradiated N-methyl-L-alanine, DL-2-methyl glutamic acid hemihydrate and Di-leucine hydrochloride in solid state

NASA Astrophysics Data System (ADS)

Sütçü, Kerem; Osmanoğlu, Y. Emre

2017-12-01

In this study, it was aimed to investigate ɣ-irradiated powders of N-methyl-L-alanine (NMLA), DL-2-methyl glutamic acid hemihydrate (DL2MGAH), and Di-leucine hydrochloride (DLHCl) at room temperature by electron paramagnetic resonance spectroscopy. After the γ-irradiation the samples indicated the existence of the CH3ĊNHCH3COOH, HOOCCH3NH2CĊHCH2COOH·1/2H2O and (CH3)2ĊCH2CH NHCOOHCOCH (NH2HCl) CH2CH (CH3)2 radicals, respectively. The spectral parameters of the radicals were determined. The results were compared with the earlier studies and discussed accordingly.

Exchanging the minimal cell binding fragments of tetanus neurotoxin in botulinum neurotoxin A and B impacts their toxicity at the neuromuscular junction and central neurons.

PubMed

Höltje, Markus; Schulze, Sebastian; Strotmeier, Jasmin; Mahrhold, Stefan; Richter, Karin; Binz, Thomas; Bigalke, Hans; Ahnert-Hilger, Gudrun; Rummel, Andreas

2013-12-01

The modular four domain structure of clostridial neurotoxins supports the idea to reassemble individual domains from tetanus and botulinum neurotoxins to generate novel molecules with altered pharmacological properties. To treat disorders of the central nervous system drug transporter molecules based on catalytically inactive clostridial neurotoxins circumventing the passage of the blood-brain-barrier are desired. Such molecules can be produced based on the highly effective botulinum neurotoxin serotype A incorporating the retrograde axonal sorting property of tetanus neurotoxin which is supposed to be encoded within its C-terminal cell binding domain HC. The corresponding exchange of the tetanus neurotoxin HC-fragment in botulinum neurotoxin A yielded the novel hybrid molecule AATT which displayed decreased potency at the neuromuscular junction like tetanus neurotoxin but exerted equal activity in cortical neurons compared to botulinum neurotoxin A wild-type. Minimizing the tetanus neurotoxin cell binding domain to its N- or C-terminal half drastically reduced the potencies of AATA and AAAT in cortical neurons indicating that the structural motif mediating sorting of tetanus neurotoxin is predominantly encoded within the entire HC-fragment. However, the reciprocal exchange resulted in TTAA which showed a similar potency as tetanus neurotoxin at the neuromuscular junction indicating that the tetanus neurotoxin portion prevents a high potency as observed for botulinum neurotoxins. In conclusion, clostridial neurotoxin based inactivated drug transporter for targeting central neurons should contain the cell binding domain of tetanus neurotoxin to exert its tropism for the central nervous system. Copyright © 2013 Elsevier Ltd. All rights reserved.

Isolation of temperature-sensitive mutations in murC of Staphylococcus aureus.

PubMed

Ishibashi, Mihoko; Kurokawa, Kenji; Nishida, Satoshi; Ueno, Kohji; Matsuo, Miki; Sekimizu, Kazuhisa

2007-09-01

Enzymes in the bacterial peptidoglycan biosynthesis pathway are important targets for novel antibiotics. Of 750 temperature-sensitive (TS) mutants of Gram-positive Staphylococcus aureus, six were complemented by the murC gene, which encodes the UDP-N-acetylmuramic acid:l-alanine ligase. Each mutation resulted in a single amino acid substitution and, in all cases, the TS phenotype was suppressed by high osmotic stress. In mutant strains with the G222E substitution, a decrease in the viable cell number immediately after shift to the restrictive temperature was observed. These results suggest that S. aureus MurC protein is essential for cell growth. The MurC H343Y mutation is located in the putative alanine recognition pocket. Consistent with this, allele-specific suppression was observed of the H343Y mutation by multiple copies of the aapA gene, which encodes an alanine transporter. The results suggest an in vivo role for the H343 residue of S. aureus MurC protein in high-affinity binding to L-alanine.

Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues.

PubMed

Karakawa, Sachise; Shimbo, Kazutaka; Yamada, Naoyuki; Mizukoshi, Toshimi; Miyano, Hiroshi; Mita, Masashi; Lindner, Wolfgang; Hamase, Kenji

2015-11-10

A highly sensitive and selective chiral LC-MS/MS method for D-alanine, D-aspartic acid and D-serine has been developed using the precolumn derivatization reagents, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag) or p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS). The thus N-tagged enantiomers of the derivatized amino acids were nicely separated within 20min using the cinchona alkaloid-based zwittterionic ion-exchange type enantioselective column, Chiralpak ZWIX(+). The selected reaction monitoring was applied for detecting the target d-amino acids in biological matrices. By using the present chiral LC-MS/MS method, the three d-amino acids and their l-forms could be simultaneously determined in the range of 0.1-500nmol/mL. Finally, the technique was successfully applied to rat plasma and tissue samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Beta-alanine supplementation improves isometric, but not isotonic or isokinetic strength endurance in recreationally strength-trained young men.

PubMed

Bassinello, Diogo; de Salles Painelli, Vitor; Dolan, Eimear; Lixandrão, Manoel; Cajueiro, Monique; de Capitani, Mariana; Saunders, Bryan; Sale, Craig; Artioli, Guilherme G; Gualano, Bruno; Roschel, Hamilton

2018-06-15

β-Alanine (BA) supplementation may be ergogenic during high-intensity exercise, primarily due to the buffering of hydrogen cations, although the effects of beta-alanine supplementation on strength endurance are equivocal. The aim of the study was to determine the effects of 4 weeks of beta-alanine supplementation on skeletal muscle endurance using a battery of performance tests. This study employed a parallel group, repeated measures, randomised, double-blinded and placebo-controlled design. Twenty recreationally strength-trained healthy males completed tests of isotonic strength endurance (repeated bench and leg press), along with tests of isometric and isokinetic endurance conducted using an isokinetic dynamometer. Tests were performed before and after a 4 week intervention, comprising an intake of 6.4 g day -1 of BA (n = 9) or placebo (maltodextrin, n = 11). Time-to-exhaustion during the isometric endurance test improved by ~ 17% in the BA group (p < 0.01), while PL remained unchanged. No significant within-group differences (p > 0.1) were shown for any of the performance variables in the isokinetic test (peak torque, fatigue index, total work) nor for the total number of repetitions performed in the isotonic endurance tests (leg or bench press). Four weeks of BA supplementation (6.4 g day -1 ) improved isometric, but not isokinetic or isotonic endurance performance.

Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases.

PubMed

Meziane-Cherif, Djalal; Stogios, Peter J; Evdokimova, Elena; Egorova, Olga; Savchenko, Alexei; Courvalin, Patrice

2015-08-11

Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl-D-alanine target of peptidoglycan precursors with D-alanyl-D-lactate or D-alanyl-D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the L-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria. Vancomycin is one of the drugs of last resort against Gram-positive antibiotic-resistant pathogens. However, bacteria have evolved a sophisticated mechanism which remodels the drug target, the D-alanine ending precursors in cell wall synthesis, into precursors terminating with D-lactate or D-serine, to which vancomycin has less affinity. D-Ser is synthesized by VanT serine racemase, which has two unusual characteristics: (i) it is one of the few serine racemases identified in bacteria and (ii) it contains a membrane-bound domain involved in L-Ser uptake. The structure of the catalytic domain of VanTG showed high similarity to alanine racemases, and we identified three specific active site substitutions responsible for L-Ser specificity. The data provide the molecular basis for VanT evolution to a bifunctional enzyme coordinating both transport and racemization. Our findings also illustrate the evolution of the essential alanine racemase into a vancomycin resistance enzyme in response to antibiotic pressure. Copyright © 2015 Meziane-Cherif et al.

Effects of obstructive sleep apnea syndrome on serum aminotransferase levels in obese patients.

PubMed

Chin, Kazuo; Nakamura, Takaya; Takahashi, Kenichi; Sumi, Kensuke; Ogawa, Yoshihiro; Masuzaki, Hiroaki; Muro, Shigeo; Hattori, Noboru; Matsumoto, Hisako; Niimi, Akio; Chiba, Tsutomu; Nakao, Kazuwa; Mishima, Michiaki; Ohi, Motoharu; Nakamura, Takashi

2003-04-01

Obesity has been associated with obstructive sleep apnea and hepatic steatosis. We investigated the effects of obstructive sleep apnea and treatment with nasal continuous positive airway pressure (CPAP) on serum aminotransferase levels in obese patients. We studied 40 obese men with obstructive sleep apnea syndrome. None had hepatitis B antigen or C antibody, autoimmune disease, or an excessive intake of alcohol. Serum levels of aspartate aminotransferase, alanine aminotransferase, triglyceride, glucose, insulin, and leptin were determined in the afternoon and in the morning immediately after sleep, before and after nasal CPAP treatment. Aminotransferase levels were abnormal in 35% (n = 14) of patients. Before treatment, mean (+/- SD) aspartate aminotransferase levels were higher in the morning than in the previous afternoon (presleep, 34 +/- 20 IU/L; postsleep, 39 +/- 28 IU/L; P = 0.006). The overnight mean increases in aminotransferase levels were less marked after the first night of nasal CPAP treatment (aspartate aminotransferase: from 6 +/- 11 IU/L to 2 +/- 6 IU/L, P = 0.0003; alanine aminotransferase: from 5 +/- 9 IU/L to 2 +/- 6 IU/L, P = 0.006). Leptin levels (n = 23) decreased significantly after treatment (P = 0.0002), whereas insulin resistance (calculated by the homeostasis model assessment method) and triglyceride levels were unchanged. Improvements in aspartate and alanine aminotransferase levels were maintained after 1 and 6 months of nasal CPAP treatment. Nasal CPAP therapy may have beneficial effects on serum aminotransferase abnormalities in obese patients who have obstructive sleep apnea. Copyright 2003 by Excerpta Medica Inc.

Government Beta: The Value of Unscripted Testing

DTIC Science & Technology

2012-04-26

vs . Progress The Contract and Award Fee Plan Final Thoughts 1 GOVERNMENT BETA – The Value of Unscripted Testing 2...D a t a b a s e Database Layer C/C++ SQL OS Layer Presentation / Mission Layer F u n c t i o n a l D L L Application/Controller F u n c t i o n a l...Testing 20 Unscripted, not Unplanned 21 IEEE 12207 22 CUT DD MLT ELT SLT IST RTO , OUE Architecture Based DesignTM Detailed Design Code & Unit

Imparting large macroscopic changes with small changes in polypeptide composition

NASA Astrophysics Data System (ADS)

Sing, Michelle; McKinley, Gareth; Olsen, Bradley

Block copolymers composed of polypeptides provide an excellent platform for exploring the underlying physics surrounding macroscopic associative network behavior. Previous work in our group has elucidated a difference in the mechanical properties of two nearly identical elastin-like polypeptide (ELP) endblocks. In poly(ELP)s, this substitution is known to result in tighter beta turns. These beta turns exhibit slower responses to changes in temperature within the material. Under shear, the modulus for the alanine-containing ELP triblock is almost three times higher than the glycine-containing ELP. Additionally, preliminary tensile tests show higher stress and strain at break for the alanine ELP triblock. We are able to explain the reasons for this behavior using a variety of spectroscopic and analytical techniques. Small angle neutron and x-ray scattering indicate differences in ordering between the alanine and glycine containing ELP materials both in shear and in stagnant flow.

Multiagent Vaccines Vectored by Venezuelan Equine Encephalitis Virus Replicon Elicits Immune Responses to Marburg Virus and Protection Against Anthrax and Botulinum Neurotoxin in Mice

DTIC Science & Technology

2006-01-01

and protection against anthrax and botulinum neurotoxin in mice John S. Lee a,∗, Jennifer L. Groebner a, Angela G. Hadjipanayis a,1, Diane L. Negley a...botulinum. Vaccine 24:6886 - 6892 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Lee, JS Groebner , JL Hadjipanayis

Effects of the neurotoxin MPTP and pargyline protection on extracellular energy metabolites and dopamine levels in the striatum of freely moving rats.

PubMed

Bazzu, Gianfranco; Rocchitta, Gaia; Migheli, Rossana; Alvau, Maria Domenica; Zinellu, Manuel; Puggioni, Giulia; Calia, Giammario; Mercanti, Giulia; Giusti, Pietro; Desole, Maria Speranza; Serra, Pier Andrea

2013-11-13

The neurotoxin MPTP is known to induce dopamine release and depletion of ATP in the striatum of rats. Therefore, we studied the changes induced by MPTP and pargyline protection both on striatal dopamine release and on extracellular energy metabolites in freely moving rats, using dual asymmetric-flow microdialysis. A dual microdialysis probe was inserted in the right striatum of rats. MPTP (25mg/kg, 15mg/kg, 10mg/kg) was intraperitoneally administered for three consecutive days. MAO-B inhibitor pargyline (15mg/kg) was systemically administered before neurotoxin administration. The first MPTP dose induced an increase in dialysate dopamine and a decrease of DOPAC levels in striatal dialysate. After the first neurotoxin administration, increases in striatal glucose, lactate, pyruvate, lactate/pyruvate (L/P) and lactate/glucose (L/G) ratios were observed. Subsequent MPTP administrations showed a progressive reduction of dopamine, glucose and pyruvate levels with a concomitant further increase in lactate levels and L/P and L/G ratios. At day 1, pargyline pre-treatment attenuated the MPTP-induced changes in all studied analytes. Starting from day 2, pargyline prevented the depletion of dopamine, glucose and pyruvate while reduced the increase of lactate, L/P ratio and L/G ratio. These in vivo results suggest a pargyline neuroprotection role against the MPTP-induced energetic impairment consequent to mitochondrial damage. This neuroprotective effect was confirmed by TH immunostaining of the substantia nigra. © 2013 Elsevier B.V. All rights reserved.

Long residence time of ultrasound microbubbles targeted to integrin in murine tumor model.

PubMed

Jun, Hong Young; Park, Seong Hoon; Kim, Hun Soo; Yoon, Kwon-Ha

2010-01-01

The aim of this study was to evaluate the intratumoral residence time of microbubbles (MBs) targeted to alpha(v)beta(3) integrin expressed in the endothelial cells of mice during the process of tumor angiogenesis. For the preparation of MBs, decafluorobutane gas was sonically dispersed in phosphate buffer saline containing L-A-phosphatidylcholine-distearoyl, polyethylene glycol 40 stearate, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000] in a 77:15:8 molar ratio. Avidin-fluorescein isothiocyanate and biotin-cyclic arginine-glycine-aspartate-D-tyrosine-lysine (cRGD) or biotin-alanine-glycine-aspartate (AGD) conjugates were added to the reaction mixture. Adhesion testing of the targeting MBs was performed for the MS-1 cell line expressing alpha(v)beta(3) integrin in vitro. The in vivo acoustic properties of the MBs were assessed by clinical ultrasound on the HT1080 fibrosarcoma model (n = 8) for 1 hour. Cryosections were stained with hematoxylin and eosin and by immunohistochemical staining to identify expression of alpha(v)beta(3) integrin in the HT1080 tumor. The adherence of the MBs conjugated to cRGD was significantly greater than the adherence of the MBs conjugated to biotin-AGD (P < .01) for the MS-1 endothelial cell line. The acoustic enhancement on ultrasound was observed as a stable imaging window until 1 hour after injection of the MB conjugates in the mice. The MBs targeted via cRGD preferentially adhered to the vascular endothelium of the HT-1080 tumors. The findings of ultrasound imaging were correlated with immunohistochemical findings for the expression of alpha(v)beta(3) integrin on the vascular endothelium of the tumors. The prepared MBs conjugated with cRGD demonstrated a sufficient residence time to attach to the target integrin of tumor tissues. This finding suggests that the MBs are a potential molecular contrast agent that enables characterization of tumor angiogenesis and the monitoring of antitumor and antiangiogenic therapy.

Bacterial Genome Adaptation to Niches: Divergence of the Potential Virulence Genes in Three Burkholderia Species of Different Survival Strategies

DTIC Science & Technology

2005-12-01

polyketide synthase ), some TTSS genes (e.g., BMAA1617 putative hrp protein and BMAA1619 hypo- thetical protein), and cell envelope synthesis genes (e.g...License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the...burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters

Solvation effects on alanine dipeptide: A MP2/cc-pVTZ//MP2/6-31G** study of (Phi, Psi) energy maps and conformers in the gas phase, ether, and water.

PubMed

Wang, Zhi-Xiang; Duan, Yong

2004-11-15

The effects of solvation on the conformations and energies of alanine dipeptide (AD) have been studied by ab initio calculations up to MP2/cc-pVTZ//MP2/6-31G**, utilizing the polarizable continuum model (PCM) to mimic solvation effects. The energy surfaces in the gas phase, ether, and water bear similar topological features carved by the steric hindrance, but the details differ significantly due to the solvent effects. The gas-phase energy map is qualitatively consistent with the Ramachandran plot showing seven energy minima. With respect to the gas-phase map, the significant changes of the aqueous map include (1) the expanded low-energy regions, (2) the emergence of an energy barrier between C5-beta and alpha(R)-beta(2) regions, (3) a clearly pronounced alpha(R) minimum, a new beta-conformer, and the disappearance of the gas-phase global minimum, and (4) the shift of the dominant region in LEII from the gas-phase C7(ax) region to the alpha(L) region. These changes bring the map in water to be much closer to the Ramachandran plot than the gas-phase map. The solvent effects on the geometries include the elongation of the exposed N-H and C=O bonds, the shortening of the buried HN--CO peptide bonds, and the enhanced planarity of the peptide bonds. The energy surface in ether has features similar to those both in the gas phase and in water. The free energy order computed in the gas phase and in ether is in good agreement with experimental studies that concluded that C5 and C7(eq) are the dominant species in both the gas phase and nonpolar solvents. The free energy order in water is consistent with the experimental observation that the dominant C7(eq) in the nonpolar solvent was largely replaced by P(II)-like (i.e., beta) and alpha(R) in the strong polar solvents. Based on calculations on AD + 4H(2)O and other AD-water clusters, we suggest that explicit water-AD interactions may distort C5 and beta (or alpha(R) and beta) to an intermediate conformation. Our analysis also shows that the PCM calculations at the MP2/cc-pVTZ//MP2/6-31G** level give good descriptions to the bulk solvent polarization effect. The results presented in this article should be of sufficient quality to characterize the peptide bonds in the gas phase and solvents. The energy surfaces may serve as the basis for developing of strategies enabling the inclusion of solvent polarization in the force field.

Synthesis, characterization and antimicrobial activity of some novel benzimidazole derivatives

PubMed Central

Krishnanjaneyulu, Immadisetty Sri; Saravanan, Govindaraj; Vamsi, Janga; Supriya, Pamidipamula; Bhavana, Jarugula Udaya; Sunil Kumar, Mittineni Venkata

2014-01-01

A series of novel N-((1H-benzoimidazol-1-yl) methyl)-4-(1-phenyl-5-substituted-4, 5-dihydro-1-benzoimidazol-1-yl) methyl)-4-(1-phenyl-5-substituted-4, 5-dihydro-1H-pyrazol-3-yl) benzenamine were synthesized by treating various 1-(4-((1H-benzoimidazol-1-yl) methylamino) phenyl)-3-substitutedprop-2-en-1-one with phenyl hydrazine in the presence of sodium acetate through a simple ring closure reaction. The starting material, 1-(4-((1H-benzoimidazol-1-yl) methylamino) phenyl)-3-substitutedprop-2-en-1-one,-benzoimidazol-1-yl) methylamino) phenyl)-3-substitutedprop-2-en-1-one, was synthesized from o-phenylenediamine by a multistep synthesis. All the synthesized compounds were characterized by spectroscopic means and elemental analyses. The title compounds were investigated for in vitro antibacterial and antifungal properties against some human pathogenic microorganisms by employing the agar streak dilution method using Ciprofloxacin and Ketoconazole as standard drugs. All title compounds showed activity against the entire strains of microorganism. Structural activity relationship studies reveal that compounds possessing an electron-withdrawing group display better activity than the compounds containing electron-donating groups, whereas the unsubstituted derivatives display moderate activity. Based on the results obtained, N-((1H-benzoimidazol-1-yl) methyl)-4-(1-phenyl-5-(4-(trifluoromethyl) phenyl)-4,5-dihydro-1H-pyrazol-3-yl) benzenamine 5i was found to be very active compared with the rest of the compounds and standard drugs that were subjected to antimicrobial assay. PMID:24696814

Inhibition by tetanus toxin of sodium-dependent, high-affinity [3H]5-hydroxytryptamine uptake in rat synaptosomes.

PubMed

Inserte, J; Najib, A; Pelliccioni, P; Gil, C; Aguilera, J

1999-01-01

Tetanus toxin (TeTx) is a powerful clostridial neurotoxin that inhibits Ca2+-dependent neurotransmitter secretion as do the botulinum neurotoxins (BoNTs). We found that TeTx (but not BoNT/A) produced a specific time- and dose-dependent inhibition of Na+-dependent [3H]5-hydroxytryptamine (serotonin, 5-HT) uptake in rat CNS synaptosomes. This effect was found in all CNS tryptaminergic areas, being maximal in the hippocampus and occipital cortex. TeTx produced the maximum reduction in [3H]5-HT uptake after 30 min of preincubation, being significant also at lower doses (10(-12) M) or shorter incubation times (10 min). Serotonin transport inhibitors such as fenfluramine (IC50, 11.0 +/- 0.9 microM), paroxetine (IC50, 33.5 +/- 0.1 microM), and imipramine (IC50, 89.9 +/- 5.7 microM) were 3 or 4 orders of magnitude less potent than TeTx (IC50, 8.7 +/- 1.0 nM). Of the two fragments of TeTx, (the C-terminal portion of the neurotoxin heavy chain, which is responsible for the binding to the nerve tissue) was consistently more effective than the L-H(N) fragment (the light neurotoxin chain disulfide linked to the N-terminal portion of the heavy chain, which is responsible for the toxic metalloprotease action) as inhibitor of [3H]5-HT uptake in synaptosomal preparations (56 +/- 5% and 95 +/- 3% with respect to control, respectively). Antagonism of the toxin-induced [3H]5-HT uptake blockade could not be reversed by zinc chelators but did have the ability to antagonize the TeTx inhibition of basal and K+-evoked [3H]5-HT release in rat synaptosomes. The reduction in serotonin accumulation induced by TeTx could be responsible for some tetanic symptoms that have been related to the serotonergic system.

Interferon Gamma potentiates the injury caused by MPP(+) on SH-SY5Y cells, which is attenuated by the nitric oxide synthases inhibition.

PubMed

Titze-de-Almeida, Simoneide S; Lustosa, Cátia Faria; Horst, Camila Hillesheim; Bel, Elaine Del; Titze-de-Almeida, Ricardo

2014-12-01

This study examined whether the cytokine interferon (IFN) gamma plays a role in the injury of SH-SY5Y cells caused by MPP(+) (1-methyl-4-phenylpyridinium). First of all, IFN-gamma sensitized cells to the neurotoxin MPP(+), as determined by MTT (3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide) assay. MPP(+)-injured cells showed higher reactive oxygen species (ROS) levels, which was reinforced by IFN-gamma. The injury triggered a marked expression of the neuronal NOS (nNOS) enzyme. L-NAME [N(ω)-nitro-L-arginine methyl ester, a non-specific NOS inhibitor] reestablished the cell viability after IFN-gamma challenging, and recovered cells from MPP(+) injury (95.0 vs. 84.7 %; P < 0.05). Seven-NI (7-nitroindazole, a nNOS inhibitor) protected cells against the injury by MPP(+) co-administered with IFN-gamma. Both inhibitors restrained the apoptosis of SH-SY5Y cells caused by MPP(+)/IFN-gamma. Regarding oxidative stress, L-NAME and 7-NI attenuated the increase in ROS levels caused by MPP(+) (45.3 or 48.4 vs. 87.9 %, P < 0.05). Indeed, L-NAME was more effective than 7-NI for reducing oxidative stress caused by MPP(+) under IFN-gamma exposition. The nNOS gene silencing by small-interfering RNAs recovered cells challenged by IFN-gamma (24 h), or MPP(+) (8 h). In conclusion, IFN-gamma sensitizes cells to MPP(+)-induced injury, also causing an increase in ROS levels. Pretreating cells with L-NAME or 7-NI reverts both the oxidative stress and apoptosis triggered by the neurotoxin MPP(+). Taking together, our data reinforce that IFN-gamma and NOS enzymes play a role in oxidative stress and dopaminergic cell death triggered by MPP(+).

Deficiency in L-serine deaminase interferes with one-carbon metabolism and cell wall synthesis in Escherichia coli K-12.

PubMed

Zhang, Xiao; El-Hajj, Ziad W; Newman, Elaine

2010-10-01

Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes L-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S L-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of L-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C(1) units and interferes with cell wall synthesis. We suggest that at high concentrations, L-serine decreases synthesis of UDP-N-acetylmuramate-L-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high L-serine is overcome in several ways: by a large concentration of L-alanine, by overproducing MurC together with a low concentration of L-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.

Surface targeting of the dopamine transporter involves discrete epitopes in the distal C terminus but does not require canonical PDZ domain interactions.

PubMed

Bjerggaard, Christian; Fog, Jacob U; Hastrup, Hanne; Madsen, Kenneth; Loland, Claus J; Javitch, Jonathan A; Gether, Ulrik

2004-08-04

The human dopamine transporter (hDAT) contains a C-terminal type 2 PDZ (postsynaptic density 95/Discs large/zona occludens 1) domain-binding motif (LKV) known to interact with PDZ domain proteins such as PICK1 (protein interacting with C-kinase 1). As reported previously, we found that, after deletion of this motif, hDAT was retained in the endoplasmic reticulum (ER) of human embryonic kidney (HEK) 293 and Neuro2A cells, suggesting that PDZ domain interactions might be critical for hDAT targeting. Nonetheless, substitution of LKV with SLL, the type 1 PDZ-binding sequence from the beta2-adrenergic receptor, did not disrupt plasma membrane targeting. Moreover, the addition of an alanine to the hDAT C terminus (+Ala), resulting in an LKVA termination sequence, or substitution of LKV with alanines (3xAla_618-620) prevented neither plasma membrane targeting nor targeting into sprouting neurites of differentiated N2A cells. The inability of +Ala and 3xAla_618-620 to bind PDZ domains was confirmed by lack of colocalization with PICK1 in cotransfected HEK293 cells and by the inability of corresponding C-terminal fusion proteins to pull down purified PICK1. Thus, although residues in the hDAT C terminus are indispensable for proper targeting, PDZ domain interactions are not required. By progressive substitutions with beta2-adrenergic receptor sequence, and by triple-alanine substitutions in the hDAT C terminus, we examined the importance of epitopes preceding the LKV motif. Substitution of RHW(615-617) with alanines caused retention of the transporter in the ER despite preserved ability of this mutant to bind PICK1. We propose dual roles of the hDAT C terminus: a role independent of PDZ interactions for ER export and surface targeting, and a not fully clarified role involving PDZ interactions with proteins such as PICK1.

Chiral discrimination in cyclodextrin complexes of amino acid derivatives: beta-cyclodextrin/N-acetyl-L-phenylalanine and N-acetyl-D-phenylalanine complexes.

PubMed

Alexander, Jennifer M; Clark, Joanna L; Brett, Tom J; Stezowski, John J

2002-04-16

In a systematic study of molecular recognition of amino acid derivatives in solid-state beta-cyclodextrin (beta-CD) complexes, we have determined crystal structures for complexes of beta-cyclodextrin/N-acetyl-L-phenylalanine at 298 and 20 K and for N-acetyl-D-phenylalanine at 298 K. The crystal structures for the N-acetyl-L-phenylalanine complex present disordered inclusion complexes for which the distribution of guest molecules at room temperature is not resolvable; however, they can be located with considerable confidence at low temperature. In contrast, the complex with N-acetyl-D-phenylalanine is well ordered at room temperature. The latter complex presents an example of a complex in this series in which a water molecule is included deeply in the hydrophobic torus of the extended dimer host. In an effort to understand the mechanisms of molecular recognition giving rise to the dramatic differences in crystallographic order in these crystal structures, we have examined the intermolecular interactions in detail and have examined insertion of the enantiomer of the D-complex into the chiral beta-CD complex crystal lattice.

40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for the...

40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for the...

Ophiophagus hannah venom: proteome, components bound by Naja kaouthia antivenin and neutralization by N. kaouthia neurotoxin-specific human ScFv.

PubMed

Danpaiboon, Witchuda; Reamtong, Onrapak; Sookrung, Nitat; Seesuay, Watee; Sakolvaree, Yuwaporn; Thanongsaksrikul, Jeeraphong; Dong-din-on, Fonthip; Srimanote, Potjanee; Thueng-in, Kanyarat; Chaicumpa, Wanpen

2014-05-13

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

Increased levels of fucosyltransferase IX and carbohydrate Lewis(x) adhesion determinant in human NT2N neurons.

PubMed

Brito, Catarina; Escrevente, Cristina; Reis, Celso A; Lee, Virginia M-Y; Trojanowski, John Q; Costa, Júlia

2007-05-01

The expression of the fucosylated carbohydrate Lewis(x) (Le(x)) determinant (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc-R) has been found in glycoproteins, proteoglycans, and glycolipids from the nervous system. Evidence suggests its association with cell-cell recognition, neurite outgrowth, and neuronal migration during central nervous system development. In the present work, we detected increased levels of Le(x) in differentiated human NT2N neurons cultured in vitro. To identify which fucosyltransferase (FUT) synthesized the Le(x) in NT2N neurons, RT-PCR, FUT substrate specificity and Western blot analysis were carried out. Strong activity toward acceptors Galbeta4GlcNAc-O-R and Fucalpha2Galbeta4GlcNAc-O-R [R = -(CH(2))(3)NHCO(CH(2))(5)NH-biotin], together with strong FUT9 detection by Western blot and presence of transcripts showed that FUT9 was the enzyme associated with Le(x) biosynthesis in NT2N neurons. Le(x) was detected at the plasma membrane of NT2N neurons, in lysosomes marked with lysosomal-associated membrane protein 1 (LAMP-1), and it was found for the first time to colocalize with the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) that defines the TI-VAMP exocytic compartment that is involved in neurite outgrowth. Furthermore, incubation with anti-Le(x) monoclonal antibody L5 led to impaired adhesion of NT2N neurons to the surface matrix and inhibited neurite initiation. In conclusion, FUT9 and its product Le(x) are detected specifically in human NT2N neurons and our results indicate that they underlie cell differentiation, cell adhesion, and initiation of neurite outgrowth in those neurons. (c) 2007 Wiley-Liss, Inc.

[Botulism: structure and function of botulinum toxin and its clinical application].

PubMed

Oguma, Keiji; Yamamoto, Yumiko; Suzuki, Tomonori; Fatmawati, Ni Nengah Dwi; Fujita, Kumiko

2012-08-01

Clostridium botulinum produces seven immunological distinct poisonous neurotoxins, A to G, with molecular masses of approximately 150kDa. In acidic foods and culture fluid, the neurotoxins associate with non-toxic components, and form large complexes designated progenitor toxins. The progenitor toxins are found in three forms named LL, L, and M. These neurotoxins and progenitor toxins were purified, and whole nucleotide sequences of their structure genes were determined. In this manuscript, the structure and function of these toxins, and the application of these toxins to clinical usage have been described.

Buffer salt effects in off-line coupling of capillary electrophoresis and mass spectrometry.

PubMed

Marák, Jozef; Stanová, Andrea

2014-05-01

In this work, the impact of buffer salts/matrix effects on the signal in direct injection MS with an electrospray interface (DI-ESI-MS) following pITP fractionation of the sample was studied. A range of buffers frequently used in CE analyses (pH 3-10) was prepared containing 10, 50, and 90% v/v of ACN, respectively. The sets of calibration solutions of cetirizine (an antihistaminic drug with an amphiprotic character) within a 0.05-2.0 mg/L concentration range were prepared in different buffers. The greatest enhancements in the MS signal (in terms of change in the slope of the calibration line) were obtained for the beta-alanine buffer (pH 3.5) in positive ionization and for the borate buffer (pH 9.2) in negative ionization, respectively. The procedure was successfully applied to the analysis of buserelin (a peptidic drug). The slope of the calibration line for solutions containing the beta-alanine buffer with 50% of ACN was 4 times higher than for water or urine, respectively. This study clearly demonstrates that the buffer salt/matrix effects in an offline combination of pITP and DI-ESI-MS can also play a positive role, as they can enhance the signal in MS. A similar influence of the above effects can also be presumed in the CE techniques combined on-line with ESI-MS.

Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity

PubMed Central

Hesse, Lars; Bostock, Julieanne; Dementin, Sebastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Chopra, Ian

2003-01-01

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):l-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:l-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (l-alanine, l-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide. PMID:14594822

Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity.

PubMed

Hesse, Lars; Bostock, Julieanne; Dementin, Sebastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Chopra, Ian

2003-11-01

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.

Evaluation of genotoxic potential of avarol, avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models.

PubMed

Kolarević, Stoimir; Milovanović, Dragana; Kračun-Kolarević, Margareta; Kostić, Jovana; Sunjog, Karolina; Martinović, Rajko; Đorđević, Jelena; Novaković, Irena; Sladić, Dušan; Vuković-Gačić, Branka

2018-01-04

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3'-methoxyavarone, 4'-(methylamino)avarone and 3'-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3'-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3'-methoxyavarone and 3'-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.

Interaction of L-alanyl-L-valine and L-valyl-L-alanine with organic vapors: thermal stability of clathrates, sorption capacity and the change in the morphology of dipeptide films.

PubMed

Ziganshin, Marat A; Gubina, Nadezhda S; Gerasimov, Alexander V; Gorbatchuk, Valery V; Ziganshina, Sufia A; Chuklanov, Anton P; Bukharaev, Anastas A

2015-08-21

The strong effect of the amino acid sequence in L-alanyl-L-valine and L-valyl-L-alanine on their sorption properties toward organic compounds and water, and the thermal stability of the inclusion compounds of these dipeptides have been found. Generally, L-valyl-L-alanine has a greater sorption capacity for the studied compounds, but the thermal stability of the L-alanyl-L-valine clathrates is higher. Unusual selectivity of L-valyl-L-alanine for vapors of few chloroalkanes was observed. The correlation between the change in the surface morphology of thin film of dipeptides and stoichiometry of their clathrates with organic compounds was found. This discovery may be used to predict the influence of vapors on the morphology of films of short-chain oligopeptides.

Determination of the anti-inflammatory and cytoprotective effects of l-glutamine and l-alanine, or dipeptide, supplementation in rats submitted to resistance exercise.

PubMed

Raizel, Raquel; Leite, Jaqueline Santos Moreira; Hypólito, Thaís Menezes; Coqueiro, Audrey Yule; Newsholme, Philip; Cruzat, Vinicius Fernandes; Tirapegui, Julio

2016-08-01

We evaluated the effects of chronic oral supplementation with l-glutamine and l-alanine in their free form or as the dipeptide l-alanyl-l-glutamine (DIP) on muscle damage, inflammation and cytoprotection, in rats submitted to progressive resistance exercise (RE). Wistar rats (n 8/group) were submitted to 8-week RE, which consisted of climbing a ladder with progressive loads. In the final 21 d before euthanasia, supplements were delivered in a 4 % solution in drinking water. Glutamine, creatine kinase (CK), lactate dehydrogenase (LDH), TNF-α, specific IL (IL-1β, IL-6 and IL-10) and monocyte chemoattractant protein-1 (MCP-1) levels were evaluated in plasma. The concentrations of glutamine, TNF-α, IL-6 and IL-10, as well as NF-κB activation, were determined in extensor digitorum longus (EDL) skeletal muscle. HSP70 level was assayed in EDL and peripheral blood mononuclear cells (PBMC). RE reduced glutamine concentration in plasma and EDL (P<0·05 v. sedentary group). However, l-glutamine supplements (l-alanine plus l-glutamine (GLN+ALA) and DIP groups) restored glutamine levels in plasma (by 40 and 58 %, respectively) and muscle (by 93 and 105 %, respectively). GLN+ALA and DIP groups also exhibited increased level of HSP70 in EDL and PBMC, consistent with the reduction of NF-κB p65 activation and cytokines in EDL. Muscle protection was also indicated by attenuation in plasma levels of CK, LDH, TNF-α and IL-1β, as well as an increase in IL-6, IL-10 and MCP-1. Our study demonstrates that chronic oral l-glutamine treatment (given with l-alanine or as dipeptide) following progressive RE induces cyprotective effects mediated by HSP70-associated responses to muscle damage and inflammation.

Role of L-alanine for redox self-sufficient amination of alcohols.

PubMed

Klatte, Stephanie; Wendisch, Volker F

2015-01-23

In white biotechnology biocatalysis represents a key technology for chemical functionalization of non-natural compounds. The plasmid-born overproduction of an alcohol dehydrogenase, an L-alanine-dependent transaminase and an alanine dehydrogenase allows for redox self-sufficient amination of alcohols in whole cell biotransformation. Here, conditions to optimize the whole cell biocatalyst presented in (Bioorg Med Chem 22:5578-5585, 2014), and the role of L-alanine for efficient amine functionalization of 1,10-decanediol to 1,10-diaminodecane were analyzed. The enzymes of the cascade for amine functionalization of alcohols were characterized in vitro to find optimal conditions for an efficient process. Transaminase from Chromobacterium violaceum, TaCv, showed three-fold higher catalytic efficiency than transaminase from Vibrio fluvialis, TaVf, and improved production at 37°C. At 42°C, TaCv was more active, which matched thermostable alcohol dehydrogenase and alanine dehydrogenase and improved the 1,10-diaminodecane production rate four-fold. To study the role of L-alanine in the whole cell biotransformation, the L-alanine concentration was varied and 1,10.diaminodecane formation tested with constant 10 mM 1,10- decanediol and 100 mM NH4Cl. Only 5.6% diamine product were observed without added L-alanine. L-alanine concentrations equimolar to that of the alcohol enabled for 94% product formation but higher L-alanine concentrations allowed for 100% product formation. L-alanine was consumed by the E. coli biocatalyst, presumably due to pyruvate catabolism since up to 16 mM acetate accumulated. Biotransformation employing E. coli strain YYC202/pTrc99a-ald-adh-ta Cv, which is unable to catabolize pyruvate, resulted in conversion with a selectivity of 42 mol-%. Biotransformation with E. coli strains only lacking pyruvate oxidase PoxB showed similar reduced amination of 1,10-decanediol indicating that oxidative decarboxylation of pyruvate to acetate by PoxB is primarily responsible for pyruvate catabolism during redox self-sufficient amination of alcohols using this whole cell biocatalyst. The replacement of the transaminase TaVf by TaCv, which showed higher activity at 42°C, in the artificial operon ald-adh-ta improved amination of alcohols in whole cell biotransformation. The addition of L-alanine, which was consumed by E. coli via pyruvate catabolism, was required for 100% product formation possibly by providing maintenance energy. Metabolic engineering revealed that pyruvate catabolism occurred primarily via oxidative decarboxylation to acetate by PoxB under the chosen biotranformation conditions.

Enhancements to PCRSM.

DTIC Science & Technology

1991-03-01

the A parameters; yhatf, to calculate the y-hat statistics; ssrf, to calculate the uncorrected SSR; sstof, to calculate the uncorrected SSTO ; matmulmm...DEGREES OF FREEDOM * int sstocdf, ssrcdf, ssecdf; float ssr, ssto , sse; /* SUM OF SQUARES * float ssrc, sstoc, ssec; float insr, insto, inse; float...Y-HAT STATSISTICS * yhatf(x,beta,stats,n,n); /* CALCULATE UNCORRECTED SSR * ssrf(beta, x, y, mn, n, ss); ssr = ss[l][l]; /* CALCULATE UNCORRECTED SSTO

Alanine infusion during hypoglycaemia partly supports cognitive performance in healthy human subjects.

PubMed

Evans, M L; Hopkins, D; Macdonald, I A; Amiel, S A

2004-05-01

To investigate the potential for the non-glucose metabolic substrate alanine to support brain function during glucose deprivation in man. Seven healthy men were studied on two occasions using a hyperinsulinaemic glucose clamp to lower arterialized plasma glucose to 2.5 mmol/l, in the presence of either 2 mmol/kg/h alanine infusion or saline, measuring counter-regulatory hormonal responses, symptoms generated and cognitive function with a mini-battery of tests sensitive to hypoglycaemia. Alanine infusion elevated plasma alanine (peak value 1481 +/- 1260 vs. 138 +/- 32 micro mol/l, P = 0.02 alanine vs. saline) and lactate (peak value 3.09 +/- 0.14 vs. 2.05 +/- 0.12 mmol/l, P = 0.02). Cognitive function assessed by the Stroop word and colour subtests deteriorated less with alanine than saline (P < 0.01 for both). Other cognitive function tests deteriorated equally and counter-regulatory hormones rose equally during hypoglycaemia in both studies (P > 0.34) except for increased glucagon with alanine (peak 260 +/- 53 vs. 91 + 8 ng/l, P = 0.03). There was no significant effect of alanine on either autonomic or neuroglycopenic symptom scores. Some, but not all, aspects of cognitive performance may be supported by an alanine infusion during hypoglycaemia. It is not clear whether alanine supports brain function directly or via increased availability of lactate. These data contribute to the growing evidence that regional metabolic differences exist in the brain's ability to use non-glucose fuels during hypoglycaemia.

Alanine increases blood pressure during hypotension

NASA Technical Reports Server (NTRS)

Conlay, L. A.; Maher, T. J.; Wurtman, R. J.

1990-01-01

The effect of L-alanine administration on blood pressure (BP) during haemorrhagic shock was investigated using anesthetized rats whose left carotid arteries were cannulated for BP measurement, blood removal, and drug administration. It was found that L-alanine, in doses of 10, 25, 50, 100, and 200 mg/kg, increased the systolic BP of hypotensive rats by 38 to 80 percent (while 100 mg/kg pyruvate increased BP by only 9.4 mmhg, not significantly different from saline). The results suggest that L-alanine might influence cardiovascular function.

The electron transport mechanism in ester and its influence on bioactivity in the anticancer drug N-(6-ferrocenyl-2-naphthoyl)-L-alanine-glycine ethyl ester(FNLAGEE)

NASA Astrophysics Data System (ADS)

Sudhi, Geethu; Rajina, S. R.; Praveen, S. G.; Xavier, T. S.; Kenny, Peter T. M.; Binoy, J.

2018-05-01

The reactivity of ester group plays key role in inducing bioactivity of many ferrocenyl biconjugated compounds. The ester reactivity can be explained, based on electron transport mechanism using vibrational spectroscopy, aided by DFT simulation. The FT IR and FT Raman spectral measurements have been carried out for N-(6-ferrocenyl-2-naphthoyl)-L-alanine-glycine ethyl ester (FNLAGEE) and the optimized geometry and vibrational spectra have been computed using DFT method, at B3LYP/LANL2DZ level of theory. The cis conformation of ester and electron transport mechanism, thus analyzed, has been correlated to the geometry and the spectral characteristics of ester. To investigate the bioactivity and binding interactions of the molecule, molecular docking simulations and UV-Vis absorption studies of FNLAGEE with BSA and DNA has been performed.

Branched-chain amino acid supplementation during bed rest: effect on recovery

NASA Technical Reports Server (NTRS)

Stein, T. P.; Donaldson, M. R.; Leskiw, M. J.; Schluter, M. D.; Baggett, D. W.; Boden, G.

2003-01-01

Bed rest is associated with a loss of protein from the weight-bearing muscle. The objectives of this study are to determine whether increasing dietary branched-chain amino acids (BCAAs) during bed rest improves the anabolic response after bed rest. The study consisted of a 1-day ambulatory period, 14 days of bed rest, and a 4-day recovery period. During bed rest, dietary intake was supplemented with either 30 mmol/day each of glycine, serine, and alanine (group 1) or with 30 mmol/day each of the three BCAAs (group 2). Whole body protein synthesis was determined with U-(15)N-labeled amino acids, muscle, and selected plasma protein synthesis with l-[(2)H(5)]phenylalanine. Total glucose production and gluconeogenesis from alanine were determined with l-[U-(13)C(3)]alanine and [6,6-(2)H(2)]glucose. During bed rest, nitrogen (N) retention was greater with BCAA feeding (56 +/- 6 vs. 26 +/- 12 mg N. kg(-1). day(-1), P < 0.05). There was no effect of BCAA supplementation on either whole body, muscle, or plasma protein synthesis or the rate of 3-MeH excretion. Muscle tissue free amino acid concentrations were increased during bed rest with BCAA (0.214 +/- 0.066 vs. 0.088 +/- 0.12 nmol/mg protein, P < 0.05). Total glucose production and gluconeogenesis from alanine were unchanged with bed rest but were significantly reduced (P < 0.05) with the BCAA group in the recovery phase. In conclusion, the improved N retention during bed rest is due, at least in part, to accretion of amino acids in the tissue free amino acid pools. The amount accreted is not enough to impact protein kinetics in the recovery phase but does improve N retention by providing additional essential amino acids in the early recovery phase.

Deficiency in l-Serine Deaminase Interferes with One-Carbon Metabolism and Cell Wall Synthesis in Escherichia coli K-12▿

PubMed Central

Zhang, Xiao; El-Hajj, Ziad W.; Newman, Elaine

2010-01-01

Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes l-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S l-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of l-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C1 units and interferes with cell wall synthesis. We suggest that at high concentrations, l-serine decreases synthesis of UDP-N-acetylmuramate-l-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high l-serine is overcome in several ways: by a large concentration of l-alanine, by overproducing MurC together with a low concentration of l-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide. PMID:20729359

Plasma beta-endorphin, beta-lipotropin and corticotropin in polycystic ovarian disease.

PubMed

Laatikainen, T; Salminen, K; Virtanen, T; Apter, D

1987-04-01

In 9 women with polycystic ovarian disease (PCOD) and in 11 control subjects at the follicular phase of the normal cycle, blood samples were collected at 15-min intervals during a 2 h period of bed rest for the assay of beta-endorphin, beta-lipotropin, corticotropin, cortisol and prolactin. During the study period, the plasma levels of these hormones decreased more significantly in the PCOD than in the control group, suggesting that the PCOD patients had a more significant stress response to the puncture of the vein than the control subjects. The second hour of the study period was considered to represent resting levels of hormones. The mean resting levels (+/- S.E.) of the hormones between the PCOD and control groups, respectively, were as follows: beta-E, 2.0 +/- 0.4 vs. 1.1 +/- 0.1 pmol/l, p less than 0.05; beta-LPH, 3.4 +/- 0.6 vs. 2.1 +/- 0.5 pmol/l, N.S.; corticotropin, 2.0 +/- 0.3 vs. 1.1 +/- 0.5 pmol/l, p less than 0.05; cortisol, 176 +/- 24 vs. 128 +/- 16, N.S.; and prolactin; 3.9 +/- 0.6 vs. 5.6 +/- 1.2 ng/ml, N.S. These results confirm the previous findings on increased circulating levels of beta-E in PCOD. A concomitant increase of the plasma level of corticotropin suggests that the basal secretion of both beta-E and corticotropin from the anterior pituitary gland is increased in women with PCOD.

Purification, crystallization and preliminary X-ray analysis of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC).

PubMed

Deva, Taru; Pryor, KellyAnn D; Leiting, Barbara; Baker, Edward N; Smith, Clyde A

2003-08-01

UDP-N-acetylmuramoyl:L-alanine ligase (MurC) is involved in the pathway leading from UDP-N-glucosamine to the UDP-N-acetylmuramoyl:pentapeptide unit, which is the building block for the peptidoglycan layer found in all bacterial cell walls. The pathways leading to the biosynthesis of the peptidoglycan layer are important targets for the development of novel antibiotics, since animal cells do not contain these pathways. MurC is the first of four similar ATP-dependent amide-bond ligases which share primary and tertiary structural similarities. The crystal structures of three of these have been determined by X-ray crystallography, giving insights into the binding of the carbohydrate substrate and the ATP. Diffraction-quality crystals of the enzyme MurC have been obtained in both native and selenomethionine forms and X-ray diffraction data have been collected at the Se edge at a synchrotron source. The crystals are orthorhombic, with unit-cell parameters a = 73.9, b = 93.6, c = 176.8 A, and diffraction has been observed to 2.6 A resolution.

MurD ligase from E. coli: Tetrahedral intermediate formation study by hybrid quantum mechanical/molecular mechanical replica path method.

PubMed

Perdih, Andrej; Hodoscek, Milan; Solmajer, Tom

2009-02-15

MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase), a three-domain bacterial protein, catalyses a highly specific incorporation of D-glutamate to the cytoplasmic intermediate UDP-N-acetyl-muramoyl-L-alanine (UMA) utilizing ATP hydrolysis to ADP and P(i). This reaction is part of a biosynthetic path yielding bacterial peptidoglycan. On the basis of structural studies of MurD complexes, a stepwise catalytic mechanism was proposed that commences with a formation of the acyl-phosphate intermediate, followed by a nucleophilic attack of D-glutamate that, through the formation of a tetrahedral reaction intermediate and subsequent phosphate dissociation, affords the final product, UDP-N-acetyl-muramoyl-L-alanine-D-glutamate (UMAG). A hybrid quantum mechanical/molecular mechanical (QM/MM) molecular modeling approach was utilized, combining the B3LYP QM level of theory with empirical force field simulations to evaluate three possible reaction pathways leading to tetrahedral intermediate formation. Geometries of the starting structures based on crystallographic experimental data and tetrahedral intermediates were carefully examined together with a role of crucial amino acids and water molecules. The replica path method was used to generate the reaction pathways between the starting structures and the corresponding tetrahedral reaction intermediates, offering direct comparisons with a sequential kinetic mechanism and the available structural data for this enzyme. The acquired knowledge represents new and valuable information to assist in the ongoing efforts leading toward novel inhibitors of MurD as potential antibacterial drugs. (c) 2008 Wiley-Liss, Inc.

Botulinum neurotoxin serotypes detected by electrochemical impedance spectroscopy.

PubMed

Savage, Alison C; Buckley, Nicholas; Halliwell, Jennifer; Gwenin, Christopher

2015-05-06

Botulinum neurotoxin is one of the deadliest biological toxins known to mankind and is able to cause the debilitating disease botulism. The rapid detection of the different serotypes of botulinum neurotoxin is essential for both diagnosis of botulism and identifying the presence of toxin in potential cases of terrorism and food contamination. The modes of action of botulinum neurotoxins are well-established in literature and differ for each serotype. The toxins are known to specifically cleave portions of the SNARE proteins SNAP-25 or VAMP; an interaction that can be monitored by electrochemical impedance spectroscopy. This study presents a SNAP-25 and a VAMP biosensors for detecting the activity of five botulinum neurotoxin serotypes (A-E) using electrochemical impedance spectroscopy. The biosensors are able to detect concentrations of toxins as low as 25 fg/mL, in a short time-frame compared with the current standard methods of detection. Both biosensors show greater specificity for their compatible serotypes compared with incompatible serotypes and denatured toxins.

Botulinum Neurotoxin Serotypes Detected by Electrochemical Impedance Spectroscopy

PubMed Central

Savage, Alison C.; Buckley, Nicholas; Halliwell, Jennifer; Gwenin, Christopher

2015-01-01

Botulinum neurotoxin is one of the deadliest biological toxins known to mankind and is able to cause the debilitating disease botulism. The rapid detection of the different serotypes of botulinum neurotoxin is essential for both diagnosis of botulism and identifying the presence of toxin in potential cases of terrorism and food contamination. The modes of action of botulinum neurotoxins are well-established in literature and differ for each serotype. The toxins are known to specifically cleave portions of the SNARE proteins SNAP-25 or VAMP; an interaction that can be monitored by electrochemical impedance spectroscopy. This study presents a SNAP-25 and a VAMP biosensors for detecting the activity of five botulinum neurotoxin serotypes (A–E) using electrochemical impedance spectroscopy. The biosensors are able to detect concentrations of toxins as low as 25 fg/mL, in a short time-frame compared with the current standard methods of detection. Both biosensors show greater specificity for their compatible serotypes compared with incompatible serotypes and denatured toxins. PMID:25954998

A novel lenticular arena to quantify locomotor competence in walking fruit flies.

PubMed

Tom Mekdara, Nalong; Goto, Joy June; Choudhury, Songita; Jerry Mekdara, Prasong; Yingst, Nicholas; Cao, Yu; Berg, Otto; Katharina Müller, Ulrike

2012-07-01

Drosophila melanogaster has become an important invertebrate model organism in biological and medical research, for mutational and genetic analysis, and in toxicological screening. Many screening assays have been developed that assess the flies' mortality, reproduction, development, morphology, or behavioral competence. In this study, we describe a new assay for locomotor competence. It comprises a circular walking arena with a lenticular floor and a flat cover (the slope of the floor increases gradually from the center to the edge of the arena) plus automated fly tracking and statistical analysis. This simple modification of a flat arena presents a graduated physical challenge, with which we can assess fine gradations of motor ability, since a fly's time average radial distance from the arena center is a direct indicator of its climbing ability. The time averaged distribution of flies as a function of slope, activity levels, and walking speed, yields a fine grained picture of locomotory ability and motivation levels. We demonstrate the strengths and weaknesses of this assay (compared with a conventional tap-down test) by observing flies treated with a neurotoxin (BMAA) that acts as a glutamate agonist. The assay proves well suited to detect dose effects and progression effects with higher statistical power than the traditional tap-down, but it has a higher detection limit, making it less sensitive to treatment effects. © 2012 WILEY PERIODICALS, INC.

Ruthenium-Nitrosyl Complexes with Glycine, l-Alanine, l-Valine, l-Proline, d-Proline, l-Serine, l-Threonine, and l-Tyrosine: Synthesis, X-ray Diffraction Structures, Spectroscopic and Electrochemical Properties, and Antiproliferative Activity

PubMed Central

2014-01-01

The reactions of [Ru(NO)Cl5]2– with glycine (Gly), l-alanine (l-Ala), l-valine (l-Val), l-proline (l-Pro), d-proline (d-Pro), l-serine (l-Ser), l-threonine (l-Thr), and l-tyrosine (l-Tyr) in n-butanol or n-propanol afforded eight new complexes (1–8) of the general formula [RuCl3(AA–H)(NO)]−, where AA = Gly, l-Ala, l-Val, l-Pro, d-Pro, l-Ser, l-Thr, and l-Tyr, respectively. The compounds were characterized by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), 1H NMR, UV–visible and ATR IR spectroscopy, cyclic voltammetry, and X-ray crystallography. X-ray crystallography studies have revealed that in all cases the same isomer type (from three theoretically possible) was isolated, namely mer(Cl),trans(NO,O)-[RuCl3(AA–H)(NO)], as was also recently reported for osmium analogues with Gly, l-Pro, and d-Pro (see Z. Anorg. Allg. Chem.2013, 639, 1590–1597). Compounds 1, 4, 5, and 8 were investigated by ESI-MS with regard to their stability in aqueous solution and reactivity toward sodium ascorbate. In addition, cell culture experiments in three human cancer cell lines, namely, A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma), were performed, and the results are discussed in conjunction with the lipophilicity of compounds. PMID:24555845

Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174.

PubMed

Arias, C A; Weisner, J; Blackburn, J M; Reynolds, P E

2000-07-01

Vancomycin resistance in Enterococcus gallinarum results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of Escherichia coli TKL-10 that requires D-Ala for growth at 42 degrees C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent. gallinarum or the alanine racemase gene (alr) of Bacillus stearothermophilus: both plasmids were able to complement E. coli TKL-10 at 42 degrees C. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 degrees C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E. coli TKL-10/pCA4(vanT): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E. coli TKL-10/pCA4(vanT) and E. coli XL-1 Blue/pCA4(vanT). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E. coli TKL-10/pJW40(alr), with a trace (1.6%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E. coli M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from E. coli was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.

Experimental and computational thermochemical study of α-alanine (DL) and β-alanine.

PubMed

da Silva, Manuel A V Ribeiro; da Silva, Maria das Dores M C Ribeiro; Santos, Ana Filipa L O M; Roux, Maria Victoria; Foces-Foces, Concepción; Notario, Rafael; Guzmán-Mejía, Ramón; Juaristi, Eusebio

2010-12-16

This paper reports an experimental and theoretical study of the gas phase standard (p° = 0.1 MPa) molar enthalpies of formation, at T = 298.15 K, of α-alanine (DL) and β-alanine. The standard (p° = 0.1 MPa) molar enthalpies of formation of crystalline α-alanine (DL) and β-alanine were calculated from the standard molar energies of combustion, in oxygen, to yield CO2(g), N2(g), and H2O(l), measured by static-bomb combustion calorimetry at T = 298.15 K. The vapor pressures of both amino acids were measured as function of temperature by the Knudsen effusion mass-loss technique. The standard molar enthalpies of sublimation at T = 298.15 K was derived from the Clausius−Clapeyron equation. The experimental values were used to calculate the standard (p° = 0.1 MPa) enthalpy of formation of α-alanine (DL) and β-alanine in the gaseous phase, Δ(f)H(m)°(g), as −426.3 ± 2.9 and −421.2 ± 1.9 kJ·mol(−1), respectively. Standard ab initio molecular orbital calculations at the G3 level were performed. Enthalpies of formation, using atomization reactions, were calculated and compared with experimental data. Detailed inspections of the molecular and electronic structures of the compounds studied were carried out.

Asymmetric synthesis of [2,3-(13)C(2),(15)N]-4-benzyloxy-5,6-diphenyl-2,3,5,6-tetrahydro-4H-oxazine-2-one via lipase TL-mediated kinetic resolution of benzoin: general procedure for the synthesis of [2,3-(13)C(2),(15)N]-L-alanine.

PubMed

Aoyagi, Y; Iijima, A; Williams, R M

2001-11-30

Lipase TL-mediated kinetic resolution of benzoin proceeded to give the corresponding optically pure (R)-benzoin (R)-1. On the other hand, (S)-benzoin O-acetate (S)-7 could be hydrolyzed without epimerization to give (S)-benzoin (S)-1 under alkaline conditions. Furthermore, both enantiomers of benzoin (1) were converted to [(15)N]-(1R,2S)- and (1S,2R)- 2-amino-1,2-diphenylethanol (3a and 3b), respectively, according to the procedure reported previously. [2,3-(13)C(2),(15)N]-(5S,6R)-4-benzyloxy-5,6-diphenyl-2,3,5,6-tetrahydro-4H-oxazine-2-one (10) was synthesized from ethyl [1,2-(13)C(2)]bromoacetate and (1R,2S)-2-amino-1,2-diphenylethanol (3b) in three steps. Finally, [2,3-(13)C(2),(15)N]-L-alanine (12) was prepared via alkylation of the lactone 10 and hydrogenation of the alkylated product 11.

Molecular Properties of neurotoxin receptors sites associated with sodium channels from mammalian brain.

PubMed

Catterall, W A; Hartshorne, R P; Beneski, D A

1982-01-01

Neurotoxins that act at specific receptor sites on voltage-sensitive sodium channels have been used as molecular probes to identify and purify protein components of sodium channels from mammalian brain. Photoreactive derivatives of scorpion toxin have been prepared and used to covalently label sodium channels in intact synaptosomes. Two polypeptides, alpha with Mr approximately 270,000 and beta with Mr approximately 38,000, are specifically labeled indicating that they are components of the scorpion toxin receptor site on the sodium channel. The sodium channel can be solubilized with retention of specific binding of [3H] saxitoxin using nonionic detergents such as Triton X-100. The solubilized saxitoxin receptor has molecular weight of 316,000 +/- 63,000 and binds 0.9 g of Triton X-100 and phospholipid per g of protein. The solubilized receptor can be purified 750-fold by ion exchange chromatography, wheat germ lectin/Sepharose chromatography and sucrose gradient sedimentation to a final specific activity of 1488 pmol/mg. Analysis of the polypeptide chain composition of the most highly purified fractions indicates that alpha and beta comprise 65% of the protein of these fractions and are only the polypeptides whose presence correlates with saxitoxin binding activity. These studies lead to a working hypothesis of sodium channel structure in which the intact channel is comprised of a complex with Mr of approximately 316,000 containing one mole of alpha (Mr approximately 270,000) and one to three moles of beta (Mr approximately 38,000).

Combination of amino acids reduces pigmentation in B16F0 melanoma cells.

PubMed

Ishikawa, Masago; Kawase, Ichiro; Ishii, Fumio

2007-04-01

Amino acids, the building blocks of proteins, play significant roles in numerous physiological events in mammals. As the effects of amino acids on melanogenesis have yet to be demonstrated, the present study was conducted to identify whether amino acids, in particular alanine, glycine, isoleucine and leucine, influence melanogenesis in B16F0 melanoma cells. Glycine and L-isoleucine, but not D-isoleucine, reduced melanogenesis in a concentration-dependent manner without any morphological changes in B16F0 melanoma cells. L-Alanine and L-leucine, but not D-alanine and D-leucine, also reduced melanogenesis without any morphological changes in B16F0 melanoma cells. However these amino acids did not show a concentration-dependency. Combination of L-alanine and the other amino acids, particularly 4 amino acids combination, had an additive effect on the inhibition of melanogenesis compared with single treatment of L-alanine. None of the amino acids affected the activity of tyrosinase, a key enzyme in melanogenesis. These results suggest that L-alanine, glycine, L-isoleucine and L-leucine, but not the D-form amino acids, have a hypopigmenting effect in B16F0 melanoma cells, and that these effects are not due to the inhibition of tyrosinase activity. Combination of these 4 amino acids had the additive effect on hypopigmentation that was as similar as that of kojic acid.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Manisha; Jamieson, Cara; Lui, Christina

β-catenin is a key mediator of Wnt signaling and its deregulated nuclear accumulation can drive cancer progression. While the central armadillo (Arm) repeats of β-catenin stimulate nuclear entry, the N- and C-terminal “tail” sequences are thought to regulate turnover and transactivation. We show here that the N- and C-tails are also potent transport sequences. The unstructured tails of β-catenin, when individually fused to a GFP-reporter, could enter and exit the nucleus rapidly in live cells. Proximity ligation assays and pull-down assays identified a weak interaction between the tail sequences and the FG-repeats of nucleoporins, consistent with a possible direct translocationmore » of β-catenin through the nuclear pore complex. Extensive alanine mutagenesis of the tail sequences revealed that nuclear translocation of β-catenin was dependent on specific uniformly distributed patches of hydrophobic residues, whereas the mutagenesis of acidic amino acids had no effect. Moreover, the mutation of hydrophobic patches within the N-tail and C-tail of full length β-catenin reduced nuclear transport rate and diminished its ability to activate transcription. We propose that the tail sequences can contribute to β-catenin transport and suggest a possible similar role for hydrophobic unstructured regions in other proteins. - Highlights: • We show that the N- and C-tails of beta-catenin possess nuclear transport activity. • Nuclear transport of the N- or C-tails requires specific hydrophobic amino acids. • Mutagenesis of the N-terminus diminished nuclear entry of full-length beta-catenin. • We propose the N-tail contributes to beta-catenin nuclear entry and transactivation.« less

[Cardioprotective effects of glutamine in patients with ischemic heart disease operated under conditions of extracorporeal blood circulation].

PubMed

Lomivorotov, V V; Efremov, S M; Shmyrev, V A; Ponomarev, D N; Sviatchenko, A V; Kniaz'kova, L G

2012-01-01

It was conducted a study of glutamine cardioptotective effects during perioperative use in patients with ischemic heart disease, operated under CB. Exclusion criteria were: left ventricular ejection fraction less than 50%, diabetes melitus, myocardial infarction less than 3 months ago, Patients of the study group (n=25) had glutamine (20% solution N(2)-L-alanine-L-glutamine ("Dipeptiven" Fresenius Kabi, Germany); 0.4 g/kg/day. Patients of control group (n=25) received placebo (0.9% NaCl solution). The main indicators were the dynamics of troponin I, as well as central hemodynamics parameters. On the 1-st day after operation the concentration of troponin I was significantly lower in the glutamine-group compared placebo-group (1.280 (0.840-2.230) 2.410 (1.060-6.600) ng/ml; p=0.035). 4 hours after CB in a glutamine-group also had significantly large indicators of cardiac index (2.58 (2.34-2.91) l/min/m2 vs 2.03 (1.76-2.32)) l/min/m2; p=0,002) and stroke index (32.8 (27.8-36.0.) ml/m2 vs 26.1 (22.6-31.8) ml/m2; p=0.023). Systemic vascular resistance index was significantly lower in glutamine-group (1942 (1828-2209) dyn x s/cm(-5)/m2 vs 2456 (2400-3265) dyn x s/cm(-5)/m2; p=0.001). Conclusion. Perioperative use of N(2)-L-alanine-L-glutamine during the first 24 hours ofperioperative period gives cardioprotective effect in patients with ischemic heart disease operated under CB.

Midbrain dopamine function in schizophrenia and depression: a post-mortem and positron emission tomographic imaging study.

PubMed

Howes, Oliver D; Williams, Matthew; Ibrahim, Kemal; Leung, Garret; Egerton, Alice; McGuire, Philip K; Turkheimer, Federico

2013-11-01

Elevated in vivo markers of presynaptic striatal dopamine activity have been a consistent finding in schizophrenia, and include a large effect size elevation in dopamine synthesis capacity. However, it is not known if the dopaminergic dysfunction is limited to the striatal terminals of dopamine neurons, or is also evident in the dopamine neuron cell bodies, which mostly originate in the substantia nigra. The aim of our studies was therefore to determine whether dopamine synthesis capacity is altered in the substantia nigra of people with schizophrenia, and how this relates to symptoms. In a post-mortem study, a semi-quantitative analysis of tyrosine hydroxylase staining was conducted in nigral dopaminergic cells from post-mortem tissue from patients with schizophrenia (n = 12), major depressive disorder (n = 13) and matched control subjects (n = 13). In an in vivo imaging study, nigral and striatal dopaminergic function was measured in patients with schizophrenia (n = 29) and matched healthy control subjects (n = 29) using (18)F-dihydroxyphenyl-L-alanine ((18)F-DOPA) positron emission tomography. In the post-mortem study we found that tyrosine hydroxylase staining was significantly increased in nigral dopaminergic neurons in schizophrenia compared with both control subjects (P < 0.001) and major depressive disorder (P < 0.001). There was no significant difference in tyrosine hydroxylase staining between control subjects and patients with major depressive disorder, indicating that the elevation in schizophrenia is not a non-specific indicator of psychiatric illness. In the in vivo imaging study we found that (18)F-dihydroxyphenyl-L-alanine uptake was elevated in both the substantia nigra and in the striatum of patients with schizophrenia (effect sizes = 0.85, P = 0.003 and 1.14, P < 0.0001, respectively) and, in the voxel-based analysis, was elevated in the right nigra (P < 0.05 corrected for family wise-error). Furthermore, nigral (18)F-dihydroxyphenyl-L-alanine uptake was positively related with the severity of symptoms (r = 0.39, P = 0.035) in patients. However, whereas nigral and striatal (18)F-dihydroxyphenyl-L-alanine uptake were positively related in control subjects (r = 0.63, P < 0.001), this was not the case in patients (r = 0.30, P = 0.11). These findings indicate that elevated dopamine synthesis capacity is seen in the nigral origin of dopamine neurons as well as their striatal terminals in schizophrenia, and is linked to symptom severity in patients.

Prolonged continuous intravenous infusion of the dipeptide L-alanine- L-glutamine significantly increases plasma glutamine and alanine without elevating brain glutamate in patients with severe traumatic brain injury

PubMed Central

2014-01-01

Introduction Low plasma glutamine levels are associated with worse clinical outcome. Intravenous glutamine infusion dose- dependently increases plasma glutamine levels, thereby correcting hypoglutaminemia. Glutamine may be transformed to glutamate which might limit its application at a higher dose in patients with severe traumatic brain injury (TBI). To date, the optimal glutamine dose required to normalize plasma glutamine levels without increasing plasma and cerebral glutamate has not yet been defined. Methods Changes in plasma and cerebral glutamine, alanine, and glutamate as well as indirect signs of metabolic impairment reflected by increased intracranial pressure (ICP), lactate, lactate-to-pyruvate ratio, electroencephalogram (EEG) activity were determined before, during, and after continuous intravenous infusion of 0.75 g L-alanine-L-glutamine which was given either for 24 hours (group 1, n = 6) or 5 days (group 2, n = 6) in addition to regular enteral nutrition. Lab values including nitrogen balance, urea and ammonia were determined daily. Results Continuous L-alanine-L-glutamine infusion significantly increased plasma and cerebral glutamine as well as alanine levels, being mostly sustained during the 5 day infusion phase (plasma glutamine: from 295 ± 62 to 500 ± 145 μmol/ l; brain glutamine: from 183 ± 188 to 549 ± 120 μmol/ l; plasma alanine: from 327 ± 91 to 622 ± 182 μmol/ l; brain alanine: from 48 ± 55 to 89 ± 129 μmol/ l; p < 0.05, ANOVA, post hoc Dunn’s test). Plasma glutamate remained unchanged and cerebral glutamate was decreased without any signs of cerebral impairment. Urea and ammonia were significantly increased within normal limits without signs of organ dysfunction (urea: from 2.7 ± 1.6 to 5.5 ± 1.5 mmol/ l; ammonia: from 12 ± 6.3 to 26 ± 8.3 μmol/ l; p < 0.05, ANOVA, post hoc Dunn’s test). Conclusions High dose L-alanine-L-glutamine infusion (0.75 g/ kg/ d up to 5 days) increased plasma and brain glutamine and alanine levels. This was not associated with elevated glutamate or signs of potential glutamate-mediated cerebral injury. The increased nitrogen load should be considered in patients with renal and hepatic dysfunction. Trial registration Clinicaltrials.gov NCT02130674. Registered 5 April 2014 PMID:24992948

Electrospray ionization tandem mass spectrometry differentiation of N-phosphoryl-[alpha]-, [beta]- and [gamma]-amino acids

NASA Astrophysics Data System (ADS)

Qiang, Liming; Cao, Shuxia; Zhao, Xiaoyang; Mao, Xiangju; Guo, Yanchun; Liao, Xincheng; Zhao, Yufen

2007-10-01

The fragmentation patterns of N-diisopropyloxyphosphoryl-l-[alpha]-Ala (DIPP-l-[alpha]-Ala), N-diisopropyloxyphosphoryl-d-[alpha]-Ala (DIPP-d-[alpha]-Ala), N-diisopropyloxyphosphoryl-[beta]-Ala (DIPP-[beta]-Ala) and N-diisopropyloxyphosphoryl-[gamma]-amino butyric acid (DIPP-[gamma]-Aba) were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). DIPP-d-[alpha]-Ala showed the same fragmentation pathways as DIPP-l-[alpha]-Ala. In the fragmentation of protonated DIPP-[beta]-Ala, the characteristic fragment ion [M + H - 2C3H6 - H2O - CH2CO]+ appeared and could be used to distinguish [beta]-Ala from l-[alpha]-Ala and d-[alpha]-Ala through tandem mass spectra, even though they possess the same molecular weight. In the fragmentation of protonated DIPP-[gamma]-Aba, the break of PN bond occurred and an interesting protonated lactam ion with five-membered ring was generated. Furthermore, in the MS3 spectrum of [M + Na - 2C3H6]+ ion of DIPP-[gamma]-Aba, a strong intensity of unique fragment ion, namely lactam-sodium adduct with five-membered ring, was observed, which could be considered as a mark for [gamma]-amino acids. The stepwise fragmentations of their [M + Na]+ ions and [M - H]- ions showed that they all underwent a PN to PO bond migration through a five-membered or six-membered or even seven-membered ring transition state, respectively, which supported the great affinity of hydroxyl for phosphoryl group.

Recombinant Expression of a Genome-encoded N-acetylmuramoyl-L-alanine Amidase that Synergistically Lyses Listeria monocytogenes Biofilms with a Protease

USDA-ARS?s Scientific Manuscript database

Listeria monocytogenes plays a significant role in human food-borne disease caused by eating food contaminated with the bacterium and although incidence is low it is a leading cause of life-threatening, bacterial food-borne disease in humans. L. monocytogenes serotypes 1/2a and 4b can form mixed-cu...

CYP3A induction and inhibition by different antiretroviral regimens reflected by changes in plasma 4beta-hydroxycholesterol levels.

PubMed

Josephson, F; Bertilsson, L; Böttiger, Y; Flamholc, L; Gisslén, M; Ormaasen, V; Sönnerborg, A; Diczfalusy, U

2008-08-01

A member of the major human cytochrome P450 superfamily of hemoproteins, CYP3A4/5, converts cholesterol into 4beta-hydroxycholesterol. We studied plasma 4beta-hydroxycholesterol levels prior to and 4 weeks after initiating antiretroviral therapy that included efavirenz, ritonavir-boosted atazanavir or ritonavir-boosted lopinavir with the aim of exploring the usefulness of plasma 4beta-hydroxycholesterol levels as an endogenous biomarker of CYP3A activity. Efavirenz is an inducer of CYP3A, whereas the ritonavir-boosted regimens are net inhibitors of CYP3A. In patients treated with efavirenz, the median plasma 4beta-hydroxycholesterol level increased by 46 ng/mL (p = 0.004; n = 11). In contrast, patients given ritonavir-boosted atazanavir showed a median decrease in plasma 4beta-hydroxycholesterol of -9.4 ng/mL (p = 0.0003; n = 22), and those given ritonavir-boosted lopinavir showed a median change from baseline of -5.8 ng/mL (p = 0.38; n = 19). There were significant between-group differences in the effects of antiretroviral treatment on plasma 4beta-hydroxycholesterol levels (p < 0.0001). Changes in plasma 4beta-hydroxycholesterol following the initiation of efavirenz- or atazanavir/ritonavir-based antiretroviral therapy reflected the respective net increase and decrease of CYP3A activity of these regimens. The plasma 4beta-hydroxycholesterol level did not indicate a net CYP3A inhibition in the lopinavir/ritonavir arm, possibly because of concomitant enzyme induction.

Use of the alr gene as a food-grade selection marker in lactic acid bacteria.

PubMed

Bron, Peter A; Benchimol, Marcos G; Lambert, Jolanda; Palumbo, Emmanuelle; Deghorain, Marie; Delcour, Jean; De Vos, Willem M; Kleerebezem, Michiel; Hols, Pascal

2002-11-01

Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.

Impact of beta-blocker treatment on the prognostic value of currently used risk predictors in congestive heart failure.

PubMed

Zugck, Christian; Haunstetter, Armin; Krüger, Carsten; Kell, Robert; Schellberg, Dieter; Kübler, Wolfgang; Haass, Markus

2002-05-15

This prospective study tested the impact of beta-blocker treatment on currently used risk predictors in congestive heart failure (CHF). Given the survival benefit obtained by beta-blockade, risk stratification by factors established in the "pre-beta-blocker era" may be questioned. The study included 408 patients who had CHF with left ventricular ejection fraction (LVEF) <45%, all treated with an angiotensin-converting enzyme inhibitor or angiotensin type 1 receptor antagonist, who were classified into those receiving a beta-blocker (n = 165) and those who were not (n = 243). In all patients, LVEF, peak oxygen consumption (peakVO(2)), plasma norepinephrine (NE) and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were determined. Although the New York Heart Association functional class (2.2 +/- 0.7 vs. 2.3 +/- 0.7), peakVO(2) (14.4 +/- 5.2 ml/min per kg vs. 14.4 +/- 5.5 ml/min per kg) and NT-proBNP (337 +/- 360 pmol/l vs. 434 +/- 538 pmol/l) were similar in the groups with and without beta-blocker treatment, the group with beta-blocker treatment had a lower heart rate (68 +/- 30 beats/min vs. 76 +/- 30 beats/min), lower NE (1.7 +/- 1.2 nmol/l vs. 2.5 +/- 2.2 nmol/l) and higher LVEF (24 +/- 10% vs. 21 +/- 9%; all p < 0.05). Within one year, 34% of patients without beta-blocker treatment, but only 16% of those with beta-blocker treatment (p < 0.001), reached the combined end point, defined as hospital admission due to worsening CHF and/or cardiac death. A beneficial effect of beta-blocker treatment was most obvious in the advanced stages of CHF, because the end-point rates were markedly lower (all p < 0.05) in the group with beta-blocker treatment versus the group without it, as characterized by peakVO(2) <10 ml/min per kg (26% vs. 64%), LVEF < or = 20% (25% vs. 45%), NE >2.24 nmol/l (18% vs. 40%) and NT-proBNP >364 pmol/l (27% vs. 45%), although patients with beta-blocker treatment received only 37 +/- 21% of the maximal recommended beta-blocker dosages. The prognostic value of variables used for risk stratification of patients with CHF is markedly influenced by beta-blocker treatment. Therefore, in the beta-blocker era, a re-evaluation of the selection criteria for heart transplantation is warranted.

Conformational study of 13C-enriched fibroin in the solid state, using the cross polarization nuclear magnetic resonance method.

PubMed

Fujiwara, T; Kobayashi, Y; Kyogoku, Y; Kataoka, K

1986-01-05

Silk fibroin with the alanyl carboxyl carbon enriched with 13C was obtained by giving a diet containing 13C-enriched alanine to the larvae of Bombyx mori and Antheraea pernyi at the fifth instar. Sericin-free fibroin fibers were prepared from cocoons, and gut was made from the liquid silk in the gland. The final 13C content was about 13%. Cross polarization/magic angle sample spinning spectra at 25 MHz and 75 MHz were measured for each sample at different orientations. Spectra were simulated using the principal values and orientations of the shielding tensor in the alanine crystal. The results indicate that the beta-structure of the fibroin may be a little more flattened than the typical pleated sheet beta-structure.

Underexpression of the Na+-dependent neutral amino acid transporter ASCT2 in the spontaneously hypertensive rat kidney.

PubMed

Pinho, Maria João; Pinto, Vanda; Serrão, Maria Paula; Jose, Pedro A; Soares-da-Silva, Patrício

2007-07-01

This study examined the inward transport of l-[(14)C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. l-[(14)C]alanine uptake was highly dependent on extracellular Na(+). Replacement of NaCl by LiCl or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na(+)-dependent l-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Na(+)-dependent l-alanine transport under V(max) conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar K(m) values. Differences in magnitude of Na(+)-dependent l-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na(+)-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells.

l-glutamine and l-alanine supplementation increase glutamine-glutathione axis and muscle HSP-27 in rats trained using a progressive high-intensity resistance exercise.

PubMed

Leite, Jaqueline Santos Moreira; Raizel, Raquel; Hypólito, Thaís Menezes; Rosa, Thiago Dos Santos; Cruzat, Vinicius Fernandes; Tirapegui, Julio

2016-08-01

In this study we investigated the chronic effects of oral l-glutamine and l-alanine supplementation, either in their free or dipeptide form, on glutamine-glutathione (GLN-GSH) axis and cytoprotection mediated by HSP-27 in rats submitted to resistance exercise (RE). Forty Wistar rats were distributed into 5 groups: sedentary; trained (CTRL); and trained supplemented with l-alanyl-l-glutamine, l-glutamine and l-alanine in their free form (GLN+ALA), or free l-alanine (ALA). All trained animals were submitted to a 6-week ladder-climbing protocol. Supplementations were offered in a 4% drinking water solution for 21 days prior to euthanasia. Plasma glutamine, creatine kinase (CK), myoglobin (MYO), and erythrocyte concentration of reduced GSH and glutathione disulfide (GSSG) were measured. In tibialis anterior skeletal muscle, GLN-GSH axis, thiobarbituric acid reactive substances (TBARS), and the expression of heat shock factor 1 (HSF-1), 27-kDa heat shock protein (HSP-27), and glutamine synthetase were determined. In CRTL animals, high-intensity RE reduced muscle glutamine levels and increased GSSG/GSH rate and TBARS, as well as augmented plasma CK and MYO levels. Conversely, l-glutamine-supplemented animals showed an increase in plasma and muscle levels of glutamine, with a reduction in GSSG/GSH rate, TBARS, and CK. Free l-alanine administration increased plasma glutamine concentration and lowered muscle TBARS. HSF-1 and HSP-27 were high in all supplemented groups when compared with CTRL (p < 0.05). The results presented herein demonstrate that l-glutamine supplemented with l-alanine, in both a free or dipeptide form, improve the GLN-GSH axis and promote cytoprotective effects in rats submitted to high-intensity RE training.

Food toxins, ampa receptors, and motor neuron diseases.

PubMed

Spencer, P S

1999-08-01

Environmental chemicals involved in the etiology of human neurodegenerative disorders are challenging to identify. Described here is research designed to determine the etiology and molecular pathogenesis of nerve cell degeneration in two little known corticomotoneuronal diseases with established environmental triggers. Both conditions are toxic-nutritional disorders dominated by persistent spastic weakness of the legs and degeneration of corresponding corticospinal pathways. Lathyrism, a disease caused by dietary dependence on grass pea (Lathyrus sativus), is mediated by a stereospecific plant amino acid (beta-N-oxalylamino-L-alanine) that serves as a potent agonist at the (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) subclass of neuronal glutamate receptors. A neurologically similar disorder, konzo ("tied legs"), is found among protein-poor African communities that rely for food on cyanogen-containing cassava roots. Thiocyanate, the principal metabolite of cyanide, is an attractive etiologic candidate for konzo because it selectively promotes the action of glutamate at AMPA receptors. Studies are urgently needed to assess the health effects of cassava and other cyanogenic plants, components of which are widely used as food.

Sequence-specific unusual (1-->2)-type helical turns in alpha/beta-hybrid peptides.

PubMed

Prabhakaran, Panchami; Kale, Sangram S; Puranik, Vedavati G; Rajamohanan, P R; Chetina, Olga; Howard, Judith A K; Hofmann, Hans-Jörg; Sanjayan, Gangadhar J

2008-12-31

This article describes novel conformationally ordered alpha/beta-hybrid peptides consisting of repeating l-proline-anthranilic acid building blocks. These oligomers adopt a compact, right-handed helical architecture determined by the intrinsic conformational preferences of the individual amino acid residues. The striking feature of these oligomers is their ability to display an unusual periodic pseudo beta-turn network of nine-membered hydrogen-bonded rings formed in the forward direction of the sequence by 1-->2 amino acid interactions both in solid-state and in solution. Conformational investigations of several of these oligomers by single-crystal X-ray diffraction, solution-state NMR, and ab initio MO theory suggest that the characteristic steric and dihedral angle restraints exerted by proline are essential for stabilizing the unusual pseudo beta-turn network found in these oligomers. Replacing proline by the conformationally flexible analogue alanine (Ala) or by the conformationally more constrained alpha-amino isobutyric acid (Aib) had an adverse effect on the stabilization of this structural architecture. These findings increase the potential to design novel secondary structure elements profiting from the steric and dihedral angle constraints of the amino acid constituents and help to augment the conformational space available for synthetic oligomer design with diverse backbone structures.

Time-resolved FT EPR and optical spectroscopy study on photooxidation of aliphatic alpha-amino acids in aqueous solutions; electron transfer from amino vs carboxylate functional group.

PubMed

Tarabek, Peter; Bonifacić, Marija; Beckert, Dieter

2006-06-08

Using time-resolved Fourier transform electron paramagnetic resonance, FT EPR, and optical spectroscopy, the photooxidation of glycine, alpha-alanine, alpha-aminoisobutyric acid, and model compounds beta-alanine, methylamine and sodium acetate, by excited triplets of anthraquinone-2,6-disulfonate dianion was studied in aqueous solutions in the pH range 5-13. Anthraquinone radical trianions showing strong emissive spin-polarization (CIDEP) were formed, indicating fast electron transfer from the quenchers to the spin-polarized quinone triplet as the primary reaction. None of the primary radicals formed upon one-electron oxidation of quenchers could be detected at the nanosecond time scale of FT EPR measurements because of their very fast transformation into secondary products. The latter were identified to be decarboxylated alpha-aminoalkyl radicals for alpha-amino acids anions and zwitterions, beta-aminoalkyl radicals for beta-alanine zwitterions, and methyl radicals for acetate anions; corresponding aminyl radicals were the first EPR detectable products from beta-alanine anions and methylamine. Thus, anthraquinone-2,6-disulfonate triplet can take an electron from both NH(2)- and -CO(2)(-) functional groups forming aminium ((+*)NH(2)-) and acyloxyl (-CO(2)(*)) radicals, respectively. Aminium radicals derived from beta-alanine anions and CH(3)-NH(2) stabilize by deprotonation into aminyl radicals, whereas these derived from alpha-amino acids anions are known to suffer ultrafast decarboxylation (tau approximately 10 ps). Analysis of the polarization patterns revealed that decarboxylation from acyloxyl radicals are considerably slower (ns < tau < 0.1 micros). Therefore, in the case of alpha-amino acids, the isoelectronic structures NH(2)-CR(2)-CO(2)(*) and (+*)NH(2)-CR(2)-CO(2)(-) probably do not constitute resonance mesomeric forms of one and the same species and the decarboxylation of aminium radicals is not preceded by the intramolecular carboxylate to amino group electron transfer. Absolute triplet quenching rate constants at zero ionic strength were in the range of 2 x 10(8) to 2 x 10(9) M(-1) s(-1) for R-NH(2) and 2 x 10(7) to 10(8) M(-1) s(-1) for R-CO(2)(-) type of electron donors, reflecting in principle their standard reduction potentials. The strengths of acids: (+)NH(3)-(*)CH(2), (+)NH(3)-(*)C(CH(3))H, and (+)NH(3)-(*)C(CH(3))(2), pK(a) <4, >6, and >7, respectively, were found to be remarkably strongly dependent on alpha-C substitution. The conjugate bases of these alpha-aminoalkyl radicals reduce anthraquinone-2,6-disulfonate dianion ground state with k(sec) = 3 x 10(9) M(-1) s(-1).

AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar

2012-08-17

Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protectsmore » against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.« less

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-beta, and H2O2.

PubMed

Lehmann, Petra; Rank, Petra; Hallfeldt, Klaus L J; Krebs, Bjarne; Gärtner, Roland

2006-08-01

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), as well as H2O2 and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-beta (5 ng/mL), or H2O2 (100 muM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. Asignificant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 microM), thyroidstimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 microM iodine, EGF (5 ng/mL) and TGF-beta (5 ng/mL), or H2O2 (100 microM) (p < 0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H2O2-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4 +/- 0.2 to 2.25 +/- 0.4 mU/microg DNA with 10 nMselenite and 2.6 + 0.4 mU/microg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-beta, EGF, iodide, and even H2O2.

Extraterrestrial amino acids in Orgueil and Ivuna: Tracing the parent body of CI type carbonaceous chondrites

PubMed Central

Ehrenfreund, Pascale; Glavin, Daniel P.; Botta, Oliver; Cooper, George; Bada, Jeffrey L.

2001-01-01

Amino acid analyses using HPLC of pristine interior pieces of the CI carbonaceous chondrites Orgueil and Ivuna have found that β-alanine, glycine, and γ-amino-n-butyric acid (ABA) are the most abundant amino acids in these two meteorites, with concentrations ranging from ≈600 to 2,000 parts per billion (ppb). Other α-amino acids such as alanine, α-ABA, α-aminoisobutyric acid (AIB), and isovaline are present only in trace amounts (<200 ppb). Carbon isotopic measurements of β-alanine and glycine and the presence of racemic (D/L ≈ 1) alanine and β-ABA in Orgueil suggest that these amino acids are extraterrestrial in origin. In comparison to the CM carbonaceous chondrites Murchison and Murray, the amino acid composition of the CIs is strikingly distinct, suggesting that these meteorites came from a different type of parent body, possibly an extinct comet, than did the CM carbonaceous chondrites. PMID:11226205

Peptide fragments of a beta-defensin derivative with potent bactericidal activity.

PubMed

Reynolds, Natalie L; De Cecco, Martin; Taylor, Karen; Stanton, Chloe; Kilanowski, Fiona; Kalapothakis, Jason; Seo, Emily; Uhrin, Dusan; Campopiano, Dominic; Govan, John; Macmillan, Derek; Barran, Perdita; Dorin, Julia R

2010-05-01

Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this beta-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide.

CRYSTAL STRUCTURE OF CLOSTRIDIUM BOTULINUM NEUROTOXIN SEROTYPE B.

DOE Office of Scientific and Technical Information (OSTI.GOV)

SWAMINATHAN,S.; ESWARAMOORTHY,S.

2001-11-19

The toxigenic strains of Clostridium botulinum produce seven serologically distinct types of neurotoxins labeled A - G (EC 3.4.24.69), while Clostridium tetani produces tetanus neurotoxin (EC 3.4.24.68). Botulinum and tetanus neurotoxins (BoNTs and TeNT) are produced as single inactive chains of molecular mass of approximately 150 kDa. Most of these neurotoxins are released after being cleaved into two chains, a heavy chain (HI) of 100 kDa and a light chain (L) of 50 kDa held together by an interchain disulfide bond, by tissue proteinases. BoNT/E is released as a single chain but cleaved by host proteinases [1]. Clostvidium botulinum neurotoxinsmore » are extremely poisonous proteins with their LD{sub 50} for humans in the range of 0.1 - 1 ng kg{sup -1} [2]. Botulinum neurotoxins are responsible for neuroparalytic syndromes of botulism characterized by serious neurological disorders and flaccid paralysis. BoNTs block the release of acetylcholine at the neuromuscular junction causing flaccid paralysis while TeNT blocks the release of neurotransmitters like glycine and {gamma}-aminobutyric acid (GABA) in the inhibitory interneurons of the spinal cord resulting in spastic paralysis. In spite of different clinical symptoms, their aetiological agents intoxicate neuronal cells in the same way and these toxins have similar structural organization [3].« less

Special feature of kinetics of ZcE isomerization of β-N-methylaminovinyl trifluoromethyl ketone in Ar matrix exposed to UV radiation and spontaneous E⇌Z isomerization of α-methyl-β-N-methylaminovinyl trifluoromethyl ketone.

PubMed

Vdovenko, Sergey I; Gerus, Igor I; Pagacz-Kostrzewa, Magdalena; Wierzejewska, Maria; Zhuk, Yuri I; Kukhar, Valery P

2018-06-15

Although it is well known that reactivity of α,β-unsaturated enaminoketones is closely associated with spatial and electronic structure but until now little attention was devoted to quantitative investigation of interconversion of different stereoisomeric forms of enaminoketones. In present work we studied peculiarities of kinetics of Z⇌E isomerization of enaminoketone 4-(N-methylamino)-1,1,1-trifluorobut-3-en-2-one F 3 C-COCHCHNH(CH 3 ) (1) in Ar-matrix exposed to UV-radiation (λ=340nm) with IR Fourier and 2D correlation spectroscopy and we found that Z-s-Z-s-trans isomer transforms primarily into two E-isomers, E-s-E-s-trans and E-s-Z-s-trans which further turn into the E-s-E-s-cis and E-s-Z-s-cis conformers all interconversion rate constants being comparable in magnitude. Along with this process long-term exposure to the UV-radiation results in proton transfer from nitrogen of methylamino group to carbonyl oxygen with simultaneous isomerization of 'cyclic' iminoenol form into 'linear'one. In solution of enaminoketone 4-(N-methylamino)-1,1,1-trifluoro-3-methylbut-3-en-2-one F 3 C-CO-C(CH 3 )CH-NH(CH 3 ) (2) we observed reversed process, namely, spontaneous interconversion of the E-s-E-s-trans and E-s-Z-s-trans conformers into the Z-s-Z-trans isomer. It was found that rate constants of the dimeric forms of the E-s-E-s-trans and E-s-Z-s-trans conformers are higher than those of the monomers and are independent on total enaminoketone concentration. Addition of highly polar HMPA promotes proton transfer from nitrogen to oxygen in the Z-s-Z-s-trans isomer of 2 with subsequent isomerization into the linear imino-enol product but the rate constant of this transformation is ten-fold smaller than that for 1 in the Ar matrix exposed to UV radiation. Copyright © 2018 Elsevier B.V. All rights reserved.

Taurine transport into fetal cord blood cells: inhibition by cyclosporine A.

PubMed

Speake, Paul F; Zipitis, Christos S; Houston, Angela; D'Souza, Stephen

2004-10-01

Pregnant women undergoing long-term organ transplant treatment have an increased incidence of delivering infants with intrauterine growth restriction (IUGR). Cyclosporine A is used as an immunosuppressant in such women and indirect evidence suggests that IUGR might result from an effect of cyclosporine A on amino acid transport by the placenta. In this study we tested the hypothesis that the transport of an essential amino acid, taurine, by fetal tissue other than the placenta is modulated by cyclosporine A. Cord blood cells (CBCs) were used to test this hypothesis as an easily obtainable fetal tissue. Transport of taurine into CBCs was measured using standard tracer flux assays. Uptake of [(3)H] taurine by CBCs was linear over 15 minutes (76.2 +/- 16.6 fmol/10(6) cells/min, mean +/- SEM, n = 6) and inhibitable by 10 mM beta-alanine, a substrate of the system-beta taurine transport protein (6.7 +/- 1.0 fmol/10(6) cells/min, n = 6, P <.05, paired Student t test). Pre-incubation with cyclosporine A (5 microM) inhibited [(3)H] taurine uptake by 29.3%-5.3% (n = 8, P <.05, paired Student t test). These data show that amino acid transport via system-beta can be measured in CBCs and may be a useful model for amino acid transport studies in fetal cells. We also show that system-beta was inhibited by the immunosuppressant, cyclosporine A. This suggests that the increased incidence of IUGR reported in mothers treated with cyclosporine A may be due partially to effects on taurine uptake into fetal cells outside the placenta.

omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines.

PubMed

Yun, Hyungdon; Lim, Seongyop; Cho, Byung-Kwan; Kim, Byung-Gee

2004-04-01

Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed omega-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic beta-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for L-beta-amino-n-butyric acid (L-beta-ABA). The enzyme converts various beta-amino acids and amines to the corresponding beta-keto acids and ketones by using pyruvate as an amine acceptor. The apparent K(m) and V(max) for L-beta-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-beta-ABA, the apparent K(m) and V(max) for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM D,L-beta-ABA, producing optically pure D-beta-ABA (99% enantiomeric excess) with 53% conversion.

30 CFR 57.5006 - Air Quality-Surface Only [Reserved

Code of Federal Regulations, 2010 CFR

2010-07-01

... acceptable to the Secretary. (a) Carbon tetrachloride, (b) Phenol, (c) 4-Nitrobiphenyl, (d) Alpha..., (h) Bis (chloromethyl) ether, (i) Beta-napthylamine, (j) Benzidine, (k) 4-Aminodiphenyl, (l) Ethyleneimine, (m) Beta-propiolactone, (n) 2-Acetylaminofluorene, (o) 4-Dimethylaminobenzene, and (p) N...

30 CFR 57.5006 - Air Quality-Surface Only [Reserved

Code of Federal Regulations, 2011 CFR

2011-07-01

... acceptable to the Secretary. (a) Carbon tetrachloride, (b) Phenol, (c) 4-Nitrobiphenyl, (d) Alpha..., (h) Bis (chloromethyl) ether, (i) Beta-napthylamine, (j) Benzidine, (k) 4-Aminodiphenyl, (l) Ethyleneimine, (m) Beta-propiolactone, (n) 2-Acetylaminofluorene, (o) 4-Dimethylaminobenzene, and (p) N...

Direct interaction of Ski with either Smad3 or Smad4 is necessary and sufficient for Ski-mediated repression of transforming growth factor-beta signaling.

PubMed

Ueki, Nobuhide; Hayman, Michael J

2003-08-29

The oncoprotein Ski represses transforming growth factor (TGF)-beta signaling in an N-CoR-independent manner. However, the molecular mechanism(s) underlying this event has not been elucidated. Here, we identify an additional domain in Ski that mediates interaction with Smad3 which is important for this repression. This domain is distinct from the previously reported N-terminal Smad3 binding domain in Ski. Individual alanine substitution of several residues in the domain significantly affected Ski-Smad3 interaction. Furthermore, combined mutations within this domain, together with those in the previously identified Smad3 binding domain, can completely abolish the interaction of Ski with Smad3, while mutation in each domain alone retained partial interaction. By introducing those mutations that abolish direct interaction with Smad3 or Smad4 individually, or in combination, we show that interaction of Ski with either Smad3 or Smad4 is sufficient for Ski-mediated repression of TGF-beta signaling. Furthermore our results clearly demonstrate that Ski does not disrupt Smad3-Smad4 heteromer formation, and recruitment of Ski to the Smad3/4 complex through binding to either Smad3 or Smad4 is both necessary and sufficient for repression.

Comprehensive analysis of pyrimidine metabolism in 450 children with unspecific neurological symptoms using high-pressure liquid chromatography-electrospray ionization tandem mass spectrometry.

PubMed

Schmidt, C; Hofmann, U; Kohlmüller, D; Mürdter, T; Zanger, U M; Schwab, M; Hoffmann, G F

2005-01-01

To evaluate the significance of inborn metabolic disorders of the pyrimidine degradation pathway, 450 children with unspecific neurological symptoms were comprehensively studied; 200 healthy children were recruited as controls. Uracil and thymine as well as their degradation products in urine were determined with an improved method based on reversed-phase HPLC coupled with electrospray ionization tandem mass spectrometry and detection by multiple-reaction monitoring using stable-isotope-labelled reference compounds as internal standards. From the results of the control group we established age-related reference ranges of all pyrimidine degradation products. In the patient group, two children with dihydropyrimidine dehydrogenase (DPYD) deficiency were identified; one of these was homozygous for the exon 14-skipping mutation of the DPYD gene. In addition, two patients with high uracil, dihydrouracil and beta-ureidopropionate were found to have ornithine transcarbamylase deficiency. In the urine of 9 patients, beta-alanine was markedly elevated owing to treatment with vigabatrin, an irreversible inhibitor of GABA transaminase, which interferes with beta-alanine breakdown. Four patients had exclusively high levels of beta-aminoisobutyrate (beta-AIB) due to a low activity of the D-beta-AIB-pyruvate aminotransferase, probably without clinical significance. In conclusion, quantitative investigation of pyrimidine metabolites in children with unexplained neurological symptoms, particularly epileptic seizures with or without psychomotor retardation, can be recommended as a helpful tool for diagnosis in clinical practice. Sensitive methods and age-related reference ranges enable the detection of partial enzyme deficiencies.

Atomic Layer Deposition of L-Alanine Polypeptide

DOE PAGES

Fu, Yaqin; Li, Binsong; Jiang, Ying-Bing; ...

2014-10-30

L-Alanine polypeptide thin films were synthesized via atomic layer deposition (ALD). Rather, instead of using an amino acid monomer as the precursor, an L-alanine amino acid derivatized with a protecting group was used to prevent self-polymerization, increase the vapor pressure, and allow linear cycle-by-cycle growth emblematic of ALD. Moreover, the successful deposition of a conformal polypeptide film has been confirmed by FTIR, TEM, and Mass Spectrometry, and the ALD process has been extended to polyvaline.

LIQUID CHROMATOGRAPHIC DETERMINATION OF 5-(METHYLAMINO)-2-PHENYL-4-[3-(TRIFLUROMETHYL) PHENYL]-3-(2H)-FURANONE IN SOIL

EPA Science Inventory

A rapid, sensitive method is described for the determination of 5-(methylamino)-2-phenyl-4-[3-(trifluromethyl)phenyl]-3-(2H)-furanone RE-40885) concentrations in three soil types. he method consists of extraction of soil samples with methanol, filtration, liquid chromatographic s...

Several novel N-donor tridentate ligands formed in chemical studies of new fac-Re(CO)3 complexes relevant to fac-99mTc(CO)3 radiopharmaceuticals: attack of a terminal amine on coordinated acetonitrile.

PubMed

Perera, Theshini; Marzilli, Patricia A; Fronczek, Frank R; Marzilli, Luigi G

2010-03-01

To evaluate syntheses of fac-[Re(CO)(3)L](+) complexes in organic solvents, we treated fac-[Re(CO)(3)(CH(3)CN)(3)]PF(6)/BF(4) in acetonitrile with triamine ligands (L). When L had two primary or two tertiary terminal amine groups, the expected fac-[Re(CO)(3)L](+) complexes formed. In contrast, N,N-dimethyldiethylenetriamine (N,N-Me(2)dien) formed an unusual compound, fac-[Re(CO)(3)(DAE)]BF(4) {DAE = (Z)-N'-(2-(2-(dimethylamino)ethylamino)ethyl)acetimidamide = (Me(2)NCH(2)CH(2))NH(CH(2)CH(2)N=C(NH(2))Me)}. DAE is formed by addition of acetonitrile to the N,N-Me(2)dien terminal primary amine, converting this sp(3) nitrogen to an sp(2) nitrogen with a double bond to the original acetonitrile sp carbon. The three Ns bound to Re derive from N,N-Me(2)dien. The pathway to fac-[Re(CO)(3)(DAE)]BF(4) is suggested by a second unusual compound, fac-[Re(CO)(3)(MAE)]PF(6) {MAE = N-methyl-N-(2-(methyl-(2-(methylamino)ethyl)amino)ethyl)acetimidamide = MeN(H)-CH(2)CH(2)-N(Me)-CH(2)CH(2)-N(Me)-C(Me)=NH}, isolated after treating fac-[Re(CO)(3)(CH(3)CN)(3)]PF(6) with N,N',N''-trimethyldiethylenetriamine (N,N',N''-Me(3)dien). MAE chelates via a terminal and a central sp(3) N from N,N',N''-Me(3)dien and via one sp(2) NH in a C(Me)=NH group. This group is derived from acetonitrile by addition of the other N,N',N''-Me(3)dien terminal amine to the nitrile carbon. This addition creates an endocyclic NMe group within a seven-membered chelate ring. The structure and other properties of fac-[Re(CO)(3)(MAE)]PF(6) allow us to propose a reaction scheme for the formation of the unprecedented DAE ligand. The new compounds advance our understanding of the spectral and structural properties of Re analogues of (99m)Tc radiopharmaceuticals.

Chemical characterization of the oligosaccharides in Bryde's whale (Balaenoptera edeni) and Sei whale (Balaenoptera borealis lesson) milk.

PubMed

Urashima, Tadasu; Kobayashi, Mami; Asakuma, Sadaki; Uemura, Yusuke; Arai, Ikichi; Fukuda, Kenji; Saito, Tadao; Mogoe, Toshihiro; Ishikawa, Hajime; Fukui, Yutaka

2007-02-01

Samples of milk from a Bryde's whale and a Sei whale contained 2.7 g/100 mL and 1.7 g/100 mL of hexose, respectively. Both contained lactose as the dominant saccharide along with small amounts of Neu5Ac(alpha2-3)Gal(beta1-4)Glc (3'-N-acetylneuraminyllactose), Neu5Ac(alpha2-6)Gal(beta1-4)Glc (6'-N-acetylneuraminyllactose) and Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LST c). The dominance of lactose in the carbohydrate of these milks is similar to that of Minke whale milk and bottlenose dolphin colostrum, but the oligosaccharide patterns are different from those of these two species, illustrating the heterogeneity of milk oligosaccharides among the Cetacea.

Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (l-Ala-P)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Au, Kinfai; Ren, Jingshan; Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN

2008-05-01

Structures of BA0252, an alanine racemase from B. anthracis, in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (l-Ala-P) and determined by X-ray crystallography to resolutions of 2.1 and 1.47 Å, respectively, are described. Bacillus anthracis, the causative agent of anthrax, has been targeted by the Oxford Protein Production Facility to validate high-throughput protocols within the Structural Proteomics in Europe project. As part of this work, the structures of an alanine racemase (BA0252) in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (l-Ala-P) have determined by X-ray crystallo@@graphy to resolutions of 2.1 and 1.47 Å, respectively. Difficulties inmore » crystallizing this protein were overcome by the use of reductive methylation. Alanine racemase has attracted much interest as a possible target for anti-anthrax drugs: not only is d-alanine a vital component of the bacterial cell wall, but recent studies also indicate that alanine racemase, which is accessible in the exosporium, plays a key role in inhibition of germination in B. anthracis. These structures confirm the binding mode of l-Ala-P but suggest an unexpected mechanism of inhibition of alanine racemase by this compound and could provide a basis for the design of improved alanine racemase inhibitors with potential as anti-anthrax therapies.« less

Bank security dye packs: synthesis, isolation, and characterization of chlorinated products of bleached 1-(methylamino)anthraquinone.

PubMed

Egan, James M; Rickenbach, Michael; Mooney, Kim E; Palenik, Chris S; Golombeck, Rebecca; Mueller, Karl T

2006-11-01

Banknote evidence is often submitted after a suspect has attempted to disguise or remove red dye stain that has been released because of an anti-theft device that activates after banknotes have been unlawfully removed from bank premises. Three chlorinated compounds have been synthesized as forensic chemical standards to indicate bank security dye bleaching as a suspect's intentional method for masking a robbery involving dye pack release on banknotes. A novel, facile synthetic method to provide three chlorinated derivatives of 1-(methylamino)anthraquinone (MAAQ) is presented. The synthetic route involved Ultra Clorox bleach as the chlorine source, iron chloride as the catalyst, and MAAQ as the starting material and resulted in a three-component product mixture. Two mono-chlorinated isomers (2-chloro-1-(methylamino)anthraquinone and 4-chloro-1-(methylamino)anthraquinone) and one di-chlorinated compound (2,4-dichloro-1-(methylamino)anthraquinone) of the MAAQ parent molecule were detected by gas chromatography mass spectrometry (GC-MS), and subsequently isolated by liquid chromatography (LC) with postcolumn fraction collection. Although GC-MS is sensitive enough to detect all of the chlorinated products, it is not definitive enough to identify the structural isomers. Liquid-state nuclear magnetic resonance (NMR) spectroscopy was utilized to elucidate structurally the ortho- and para-mono-chlorinated isomers once enough material was properly isolated. A reaction mechanism involving iron is proposed to explain the presence of chlorinated MAAQ species on stolen banknotes after attempted bleaching.

Atomistic Model for the Polyamide Formation from β-Lactam Catalyzed by Candida Antarctica Lipase B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baum, Iris; Elsasser, Brigitta M.; Schwab, Leendert

2011-04-01

Candida antarctica lipase B (CALB) is an established biocatalyst for a variety of transesterification, amidation, and polymerization reactions. In contrast to polyesters, polyamides are not yet generally accessible via enzymatic polymerization. In this regard, an enzyme-catalyzed ring-opening polymerization of {beta}-lactam (2-azetidinone) using CALB is the first example of an enzymatic polyamide formation yielding unbranched poly({beta}-alanine), nylon 3. The performance of this polymerization, however, is poor, considering the maximum chain length of 18 monomer units with an average length of 8, and the molecular basis of the reaction so far is not understood. We have employed molecular modeling techniques using dockingmore » tools, molecular dynamics, and QM/MM procedures to gain insight into the mechanistic details of the various reaction steps involved. As a result, we propose a catalytic cycle for the oligomerization of {beta}-lactam that rationalizes the activation of the monomer, the chain elongation by additional {beta}-lactam molecules, and the termination of the polymer chain. In addition, the processes leading to a premature chain termination are studied. Particularly, the QM/MM calculation enables an atomistic description of all eight steps involved in the catalytic cycle, which features an in situ-generated {beta}-alanine as the elongating monomer and which is compatible with the experimental findings.« less

30 CFR 56.5006 - Restricted use of chemicals.

Code of Federal Regulations, 2011 CFR

2011-07-01

... recognized agency acceptable to the Secretary. (a) Carbon tetrachloride. (b) Phenol, (c) 4-Nitrobiphenyl, (d... Dichlorobenzidine, (h) Bis (chloromethyl) ether, (i) Beta-napthylamine, (j) Benzidine, (k) 4-Aminodiphenyl, (l) Ethyleneimine, (m) Beta-propiolactone, (n) 2-Acetylaminofluorene, (o) 4-Dimethylaminobenzene, and (p) N...

30 CFR 56.5006 - Restricted use of chemicals.

Code of Federal Regulations, 2010 CFR

2010-07-01

... recognized agency acceptable to the Secretary. (a) Carbon tetrachloride. (b) Phenol, (c) 4-Nitrobiphenyl, (d... Dichlorobenzidine, (h) Bis (chloromethyl) ether, (i) Beta-napthylamine, (j) Benzidine, (k) 4-Aminodiphenyl, (l) Ethyleneimine, (m) Beta-propiolactone, (n) 2-Acetylaminofluorene, (o) 4-Dimethylaminobenzene, and (p) N...

Ultra-Rapid Crystallization of L-alanine Using Monomode Microwaves, Indium Tin Oxide and Metal-Assisted and Microwave-Accelerated Evaporative Crystallization.

PubMed

Lansiquot, Carisse; Boone-Kukoyi, Zainab; Shortt, Raquel; Thompson, Nishone; Ajifa, Hillary; Kioko, Bridgit; Constance, Edward Ned; Clement, Travis; Ozturk, Birol; Aslan, Kadir

2017-01-01

The use of indium tin oxide (ITO) and focused monomode microwave heating for the ultra-rapid crystallization of L-alanine (a model amino acid) is reported. Commercially available ITO dots (< 5 mm) attached to blank poly(methyl)methacrylate (PMMA, 5 cm in diameter with 21-well silicon isolators: referred to as the iCrystal plates) were found to withstand prolonged microwave heating during crystallization experiments. Crystallization of L-alanine was performed at room temperature (a control experiment), with the use of two microwave sources: a 2.45 GHz conventional microwave (900 W, power level 1, a control experiment) and 8 GHz (20 W) solid state, monomode microwave source with an applicator tip that focuses the microwave field to a 5-mm cavity. Initial appearance of L-alanine crystals and on iCrystal plates with ITO dots took 47 ± 2.9 min, 12 ± 7.6 min and 1.5 ± 0.5 min at room temperature, using a conventional microwave and focused monomode microwave heating, respectively. Complete evaporation of the solvent using the focused microwaves was achieved in 3.2 ± 0.5 min, which is ~52-fold and ~172-fold faster than that observed at room temperature and using conventional microwave heating, respectively. The size and number of L-alanine crystals was dependent on the type of the 21-well iCrystal plates and the microwave heating method: 33 crystals of 585 ± 137 μm in size at room temperature > 37 crystals of 542 ± 100 μm in size with conventional microwave heating > 331 crystals of 311 ± 190 μm in size with focused monomode microwave. FTIR, optical microscopy and powder X-ray diffraction analysis showed that the chemical composition and crystallinity of the L-alanine crystals did not change when exposed to microwave heating and ITO surfaces. In addition, theoretical simulations for the binding of L-alanine molecules to ITO and other metals showed the predicted nature of hydrogen bonds formed between L-alanine and these surfaces.

Ultra-Rapid Crystallization of L-alanine Using Monomode Microwaves, Indium Tin Oxide and Metal-Assisted and Microwave-Accelerated Evaporative Crystallization

PubMed Central

Lansiquot, Carisse; Boone-Kukoyi, Zainab; Shortt, Raquel; Thompson, Nishone; Ajifa, Hillary; Kioko, Bridgit; Constance, Edward Ned; Clement, Travis; Ozturk, Birol; Aslan, Kadir

2018-01-01

The use of indium tin oxide (ITO) and focused monomode microwave heating for the ultra-rapid crystallization of L-alanine (a model amino acid) is reported. Commercially available ITO dots (< 5 mm) attached to blank poly(methyl)methacrylate (PMMA, 5 cm in diameter with 21-well silicon isolators: referred to as the iCrystal plates) were found to withstand prolonged microwave heating during crystallization experiments. Crystallization of L-alanine was performed at room temperature (a control experiment), with the use of two microwave sources: a 2.45 GHz conventional microwave (900 W, power level 1, a control experiment) and 8 GHz (20 W) solid state, monomode microwave source with an applicator tip that focuses the microwave field to a 5-mm cavity. Initial appearance of L-alanine crystals and on iCrystal plates with ITO dots took 47 ± 2.9 min, 12 ± 7.6 min and 1.5 ± 0.5 min at room temperature, using a conventional microwave and focused monomode microwave heating, respectively. Complete evaporation of the solvent using the focused microwaves was achieved in 3.2 ± 0.5 min, which is ~52-fold and ~172-fold faster than that observed at room temperature and using conventional microwave heating, respectively. The size and number of L-alanine crystals was dependent on the type of the 21-well iCrystal plates and the microwave heating method: 33 crystals of 585 ± 137 μm in size at room temperature > 37 crystals of 542 ± 100 μm in size with conventional microwave heating > 331 crystals of 311 ± 190 μm in size with focused monomode microwave. FTIR, optical microscopy and powder X-ray diffraction analysis showed that the chemical composition and crystallinity of the L-alanine crystals did not change when exposed to microwave heating and ITO surfaces. In addition, theoretical simulations for the binding of L-alanine molecules to ITO and other metals showed the predicted nature of hydrogen bonds formed between L-alanine and these surfaces. PMID:29657884

Changes in the free amino acid contents of honeys during storage at ambient temperature.

PubMed

Iglesias, M Teresa; Martín-Alvarez, Pedro J; Polo, M Carmen; de Lorenzo, Cristina; Gonzalez, Montserrat; Pueyo, Encarnación

2006-11-29

This study was carried out to establish the changes in the free amino acid contents of floral honeys, honeydew honeys, and blend honeys during storage at room temperature and to test the capacity of the amino acids to distinguish the origin of the honeys after storage. For this purpose, 54 artisanal honeys (39 floral, 5 honeydew, and 10 blend) were studied. Samples were taken from recently collected honeys and at 3, 6, 9, 12, 16, 20, and 24 months after harvesting. The contents of most of the free amino acids were found to decrease with storage time, with the greatest reduction observed in the first 9 months. The contents of the amino acids aspartic acid, beta-alanine, and proline increased in the first few months after storage, reaching maximum values at 6 months, suggesting the possible existence of enzymatic activities. The application of stepwise discriminant analysis to the free amino acid content data demonstrated that the contents of the amino acids valine, beta-alanine, gamma-aminobutyric acid, serine, isoleucine, alpha-alanine, ornithine, and glutamine correctly assigned 87% of honeys to their group of origin: floral, honeydew, or blend.

Influence of nitrogen sources on growth and fermentation performance of different wine yeast species during alcoholic fermentation.

PubMed

Kemsawasd, Varongsiri; Viana, Tiago; Ardö, Ylva; Arneborg, Nils

2015-12-01

In this study, the influence of twenty different single (i.e. 19 amino acids and ammonium sulphate) and two multiple nitrogen sources (N-sources) on growth and fermentation (i.e. glucose consumption and ethanol production) performance of Saccharomyces cerevisiae and of four wine-related non-Saccharomyces yeast species (Lachancea thermotolerans, Metschnikowia pulcherrima, Hanseniaspora uvarum and Torulaspora delbrueckii) was investigated during alcoholic fermentation. Briefly, the N-sources with beneficial effects on all performance parameters (or for the majority of them) for each yeast species were alanine, arginine, asparagine, aspartic acid, glutamine, isoleucine, ammonium sulphate, serine, valine and mixtures of 19 amino acids and of 19 amino acids plus ammonium sulphate (for S. cerevisiae), serine (for L. thermotolerans), alanine (for H. uvarum), alanine and asparagine (for M. pulcherrima), arginine, asparagine, glutamine, isoleucine and mixture of 19 amino acids (for T. delbrueckii). Furthermore, our results showed a clear positive effect of complex mixtures of N-sources on S. cerevisiae and on T. delbrueckii (although to a lesser extent) as to all performance parameters studied, whereas for L. thermotolerans, H. uvarum and M. pulcherrima, single amino acids affected growth and fermentation performance to the same extent as the mixtures. Moreover, we found groups of N-sources with similar effects on the growth and/or fermentation performance of two or more yeast species. Finally, the influences of N-sources observed for T. delbrueckii and H. uvarum resembled those of S. cerevisiae the most and the least, respectively. Overall, this work contributes to an improved understanding of how different N-sources affect growth, glucose consumption and ethanol production of wine-related yeast species under oxygen-limited conditions, which, in turn, may be used to, e.g. optimize growth and fermentation performance of the given yeast upon N-source supplementation during wine fermentations.

The effects of lower than conventional doses of oral nadolol on relative beta 1/beta 2-adrenoceptor blockade.

PubMed

Wheeldon, N M; McDevitt, D G; Lipworth, B J

1994-08-01

1. The aim of the present study was to evaluate the relative beta 1/beta 2 antagonist selectivity of the beta-adrenoceptor blocker nadolol, in lower than conventional clinical doses. 2. Eight normal volunteers received single oral doses of either placebo (PL), nadolol 5 mg (N5), 20 mg (N20) or 80 mg (N80) in a single-blind, randomised crossover design. beta 1-adrenoceptor antagonism was assessed by attenuation of exercise tachycardia, and beta 2-adrenoceptor blockade by effects on salbutamol-induced chronotropic, hypokalaemic and finger tremor responses. The relative percentage attenuation of beta 2 and beta 1-mediated responses was calculated and expressed as beta 2:beta 1 selectivity ratios. 3. Nadolol produced dose-related reductions in exercise tachycardia in keeping with increasing beta 1-adrenoceptor blockade; mean % reduction (95% CI) compared with placebo: N5 10.7 (6.6 to 14.8), N20 21.4 (17.3 to 25.4), N80 38.9 (34.8 to 42.9). However, even the lowest dose of nadolol (5 mg) produced almost complete blunting of beta 2-mediated effects and significantly increase exercise hyperkalaemia; peak exercise hyperkalaemia (mmol l-1) (means and 95% CI): PL 4.88 (4.68 to 5.07), N5 5.36 (5.17 to 5.55), N20 5.48 (5.28 to 5.67), N80 5.42 (5.22 to 5.61). beta 2:beta 1 selectivity ratios significantly increased as the dose of nadolol was reduced. 4. These data suggest that whereas in the clinical dose range nadolol behaves as a non-selective beta-adrenoceptor antagonist, as the dose is reduced this drug demonstrates an increasing degree of selectivity for the beta 2-adrenoceptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection with Recombinant Clostridium botulinum C1 and D Binding Domain Subunit (Hc) Vaccines Against C and D Neurotoxins

DTIC Science & Technology

2007-03-16

A p o p f b a © K 1 c s r h a d t f f r i 0 d Vaccine 25 (2007) 4273–4282 Protection with recombinant Clostridium botulinum C1 and D binding domain...reserved. r-bindin s r t r w i t l s eywords: Botulinum neurotoxin subtypes; Recombinant vaccine; Recepto . Introduction Botulism is the collective term...amedd.army.mil (L.A. Smith). e c t B i T c s 264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. oi:10.1016/j.vaccine

Low levels of serum serotonin and amino acids identified in migraine patients.

PubMed

Ren, Caixia; Liu, Jia; Zhou, Juntuo; Liang, Hui; Wang, Yayun; Sun, Yinping; Ma, Bin; Yin, Yuxin

2018-02-05

Migraine is a highly disabling primary headache associated with a high socioeconomic burden and a generally high prevalence. The clinical management of migraine remains a challenge. This study was undertaken to identify potential serum biomarkers of migraine. Using Liquid Chromatography coupled to Mass Spectrometry (LC-MS), the metabolomic profile of migraine was compared with healthy individuals. Principal component analysis (PCA) and Orthogonal partial least squares-discriminant analysis (orthoPLS-DA) showed the metabolomic profile of migraine is distinguishable from controls. Volcano plot analysis identified 10 serum metabolites significantly decreased during migraine. One of these was serotonin, and the other 9 were amino acids. Pathway analysis and enrichment analysis showed tryptophan metabolism (serotonin metabolism), arginine and proline metabolism, and aminoacyl-tRNA biosynthesis are the three most prominently altered pathways in migraine. ROC curve analysis indicated Glycyl-l-proline, N-Methyl-dl-Alanine and l-Methionine are potential sensitive and specific biomarkers for migraine. Our results show Glycyl-l-proline, N-Methyl-dl-Alanine and l-Methionine may be as specific or more specific for migraine than serotonin which is the traditional biomarker of migraine. We propose that therapeutic manipulation of these metabolites or metabolic pathways may be helpful in the prevention and treatment of migraine. Copyright © 2018 Elsevier Inc. All rights reserved.

Crosslinking of aromatic polyamides via pendant propargyl groups

NASA Technical Reports Server (NTRS)

St.clair, A. K.; St.clair, T. L.; Barrick, J. D.; Campbell, F. J.

1980-01-01

Methods for crosslinking N-methyl substituted aromatic polyamides were investigated in an effort to improve the applicability of these polymers as matrix resins for Kavlar trademark fiber composites. High molecular weight polymers were prepared from isophthaloyl dichloride and 4,4'- bis(methylamino)diphenylmethane with varying proportions of the N,N'bispropargyl diamine incorporated as a crosslinking agent. The propargylcontaining diamines were crosslinked thermally and characterized by infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analysis. Attempts were also made to crosslink polyamide films by exposure to ultraviolet light, electron beam, and gamma radiation.

Mechanism of substrate recognition by the novel Botulinum Neurotoxin subtype F5.

PubMed

Guo, Jiubiao; Chan, Edward Wai Chi; Chen, Sheng

2016-01-22

Botulinum Neurotoxins (BoNTs) are the causative agents of botulism, which act by potently inhibiting the neurotransmitter release in motor neurons. Seven serotypes of BoNTs designated as BoNT/A-G have been identified. Recently, two novel types of Botulinum neurotoxins, which cleave a novel scissile bond, L(54)-E(55), of VAMP-2 have been reported including BoNT/F subtype F5 and serotype H. However, little has been known on how these BoNTs recognize their substrates. The present study addressed for the first time the unique substrate recognition mechanism of LC/F5. Our data indicated that the optimal peptide required for efficient LC/F5 substrate cleavage is VAMP-2 (20-65). Interestingly, the overall mode of substrate recognition adopted by LC/F5 was similar to LC/F1, except that its recognition sites were shifted one helix toward the N-terminus of VAMP-2 when compared to that of LC/F1. The composition of LC/F5 pockets were found to have changed accordingly to facilitate specific recognition of these new sites of VAMP-2, including the P2', P1', P2, P3, B3, B2 and B1 sites. The study provides direct evidence of the evolutionary adaption of BoNT to recognize its substrate which is useful for effective antitoxin and inhibitor development.

Five months of fasting in king penguin chicks: body mass loss and fuel metabolism.

PubMed

Cherel, Y; Le Maho, Y

1985-10-01

When king penguin chicks are 3-4 mo old, they enter a season of interrupted growth due to long periods of fasting, because they are irregularly fed in winter. Nine captive chicks [mean initial body mass (m) = 12.5 kg] had fasted an average of 5 mo at the end of the experiment; m was then 4.0 kg, a 68% decrease. They probably could have fasted longer, since chicks of parents delayed in the return to the colony die from starvation at an m of 3.0 kg. The long fast could be divided into three periods based on the changes in rate of decrease of m. The remarkable resistance of king penguin chicks to starvation may be partly explained by their ability to maintain protein sparing for as much as 4 mo, the duration of period II; plasma concentrations of uric acid, urea, and alanine were then minimum, 0.1, 0.4, and 0.4 mmol X l-1 respectively. Particular changes during this period, i.e., progressive increase of beta-hydroxybutyrate and decrease of glucose concentrations, might contribute to the efficiency of protein sparing. Period III was marked by a rise in protein utilization, plasma concentrations of uric acid, urea, and alanine increasing to 0.7, 1.5, and 0.8 mmol X l-1, respectively.

Interleukin-1beta-induced extracellular matrix degradation and glycosaminoglycan release is inhibited by curcumin in an explant model of cartilage inflammation.

PubMed

Clutterbuck, Abigail L; Mobasheri, Ali; Shakibaei, Mehdi; Allaway, David; Harris, Pat

2009-08-01

Osteoarthritis (OA) is a degenerative and inflammatory disease of synovial joints that is characterized by the loss of articular cartilage, for which there is increasing interest in natural remedies. Curcumin (diferuloylmethane) is the main polyphenol in the spice turmeric, derived from rhizomes of the plant Curcuma longa. Curcumin has potent chemopreventive properties and has been shown to inhibit nuclear factor kappaB-mediated inflammatory signaling in many cell types, including chondrocytes. In this study, normal articular cartilage was harvested from metacarpophalangeal and metatarsophalangeal joints of eight horses, euthanized for reasons other than research purposes, to establish an explant model mimicking the inflammatory events that occur in OA. Initially, cartilage explants (N= 8) were stimulated with increasing concentrations of the proinflammatory cytokine IL-1beta to select effective doses for inducing cartilage degeneration in the explant model. Separate cartilage explants were then cotreated with IL-1beta at either 10 ng/mL (n= 3) or 25 ng/mL (n= 3) and curcumin (0.1 micromol/L, 0.5 micromol/L, 1 micromol/L, 10 micromol/L, and 100 micromol/L). After 5 days, the percentage of glycosaminoglycan (GAG) release from the explants was assessed using a dimethylmethylene blue colorimetric assay. Curcumin (100 micromol/L) significantly reduced IL-1beta-stimulated GAG release in the explants by an average of 20% at 10 ng/mL and 27% at 25 ng/mL back to unstimulated control levels (P < 0.001). Our results suggest that this explant model effectively simulates the proinflammatory cytokine-mediated release of articular cartilage components seen in OA. Furthermore, the evidence suggests that the inflammatory cartilage explant model is useful for studying the effects of curcumin on inflammatory pathways and gene expression in IL-1beta-stimulated chondrocytes.

Photochemical redox reactions of copper(II)-alanine complexes in aqueous solutions.

PubMed

Lin, Chen-Jui; Hsu, Chao-Sheng; Wang, Po-Yen; Lin, Yi-Liang; Lo, Yu-Shiu; Wu, Chien-Hou

2014-05-19

The photochemical redox reactions of Cu(II)/alanine complexes have been studied in deaerated solutions over an extensive range of pH, Cu(II) concentration, and alanine concentration. Under irradiation, the ligand-to-metal charge transfer results in the reduction of Cu(II) to Cu(I) and the concomitant oxidation of alanine, which produces ammonia and acetaldehyde. Molar absorptivities and quantum yields of photoproducts for Cu(II)/alanine complexes at 313 nm are characterized mainly with the equilibrium Cu(II) speciation where the presence of simultaneously existing Cu(II) species is taken into account. By applying regression analysis, individual Cu(I) quantum yields are determined to be 0.094 ± 0.014 for the 1:1 complex (CuL) and 0.064 ± 0.012 for the 1:2 complex (CuL2). Individual quantum yields of ammonia are 0.055 ± 0.007 for CuL and 0.036 ± 0.005 for CuL2. Individual quantum yields of acetaldehyde are 0.030 ± 0.007 for CuL and 0.024 ± 0.007 for CuL2. CuL always has larger quantum yields than CuL2, which can be attributed to the Cu(II) stabilizing effect of the second ligand. For both CuL and CuL2, the individual quantum yields of Cu(I), ammonia, and acetaldehyde are in the ratio of 1.8:1:0.7. A reaction mechanism for the formation of the observed photoproducts is proposed.

Neurotoxins from Clostridium botulinum (serotype A) isolated from the soil of Mendoza (Argentina) differ from the A-Hall archetype and from that causing infant botulism.

PubMed

Caballero, P; Troncoso, M; Patterson, S I; López Gómez, C; Fernandez, R; Sosa, M A

2016-10-01

The type A of neurotoxin produced by Clostridium botulinum is the prevalent serotype in strains of Mendoza. The soil is the main reservoir for C.botulinum and is possibly one of the infection sources in infant botulism. In this study, we characterized and compared autochthonous C. botulinum strains and their neurotoxins. Bacterial samples were obtained from the soil and from fecal samples collected from children with infant botulism. We first observed differences in the appearance of the colonies between strains from each source and with the A Hall control strain. In addition, purified neurotoxins of both strains were found to be enriched in a band of 300 kDa, whereas the A-Hall strain was mainly made up of a band of ∼600 kDa. This finding is in line with the lack of hemagglutinating activity of the neurotoxins under study. Moreover, the proteolytic activity of C. botulinum neurotoxins was evaluated against SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins from rat brain. It was observed that both, SNAP 25 (synaptosomal-associated protein 25) and VAMP 2 (vesicle-associated membrane protein) were cleaved by the neurotoxins isolated from the soil strains, whereas the neurotoxins from infant botulism strains only induced a partial cleavage of VAMP 2. On the other hand, the neurotoxin from the A-Hall strain was able to cleave both proteins, though at a lesser extent. Our data indicate that the C.botulinum strain isolated from the soil, and its BoNT, exhibit different properties compared to the strain obtained from infant botulism patients, and from the A-Hall archetype. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phytochemical investigation of the seeds of Althea officinalis L.

PubMed

Rani, Sunita; Khan, Suroor A; Ali, M

2010-09-01

Phytochemical investigation of the seeds of Althea officinalis L. (Malvaceae) led to the isolation of three new phytoconstituents, identified as n-hexacos-2-enyl-1,5-olide (altheahexacosanyl lactone), 2beta-hydroxycalamene (altheacalamene) and 5,6-dihydroxycoumarin-5-dodecanoate-6beta-D-glucopyranoside (altheacoumarin glucoside), along with the known phytoconstituents lauric acid, beta-sitosterol and lanosterol. The structures of these compounds were established on the basis of spectral analysis and chemical reactions.

Bioactive metabolites from the fungus Nectria galligena, the main apple canker agent in Chile.

PubMed

Gutiérrez, Margarita; Theoduloz, Cristina; Rodríguez, Jaime; Lolas, Mauricio; Schmeda-Hirschmann, Guillermo

2005-10-05

The phytopathogenic fungus Nectria galligena Bres. is the most common canker disease agent of hardwood trees. The terpenoids colletochlorin B, colletorin B, ilicicolin C, E, and F, as well as the phytotoxin alpha,beta-dehydrocurvularin have been isolated from liquid cultures of N. galligena obtained from the xylem of infected apple trees in central Chile. Ilicicolin C and F and alpha,beta-dehydrocurvularin were active against Pseudomonas syringae with IC50 values of 28.5, 28.5, and 14.2 microg/mL, respectively, in the same range as streptomycin and penicillin G (11 and 15 microg/mL, respectively). All of the compounds showed moderate inhibitory activity toward the enzymes acetylcholinesterase (AChE) and beta-glucuronidase. The most active enzyme inhibitors were colletochlorin B and ilicicolin C and E, with IC50 values of 30-36 microg/mL in the AChE assay and 32-43 microg/mL in the beta-glucuronidase test. All of the chlorinated compounds showed some toxicity toward human lung fibroblasts, with IC50 values in the range of 64-120 microg/mL. alpha,beta-Dehydrocurvularin proved to be the most toxic compound, showing IC50 values less than 12 microg/mL. The effect of the isolated compounds on seed germination and radicle and epicotyl growth was assessed in lettuce and millet seeds. At 100 and 200 microg/disk, alpha,beta-dehydrocurvularin significantly reduced radicle length and epicotyl growth in Lactuca sativa. This is the first report on the occurrence of colletochlorin B, colletorin B, ilicicolin C, E, and F, as well as alpha,beta-dehydrocurvularin associated to N. galligena.

Dietary choline and folate relationships with serum hepatic inflammatory injury markers in Taiwanese adults.

PubMed

Cheng, Chin-Pao; Chen, Chien-Hung; Kuo, Chang-Sheng; Kuo, Hsing-Tao; Huang, Kuang-Ta; Shen, Yu-Li; Chang, Chin-Hao; Huang, Rwei Fen S

The relationships of dietary choline and folate intake with hepatic function have yet to be established in the Taiwanese population. We investigated the associations of choline and folate intake with hepatic inflammatory injury in Taiwanese adults. Blood samples and data on dietary choline components and folate intake from 548 Taiwanese adults without pathological liver disease were collected. Dietary intake was derived using a semiquantitative food-frequency questionnaire. Serum liver injury markers of alanine transaminase, aspartate transaminase, and hepatitis viral infection were measured. Elevated serum hepatic injury markers (>40 U/L) were associated with low folate and free choline intake (p<0.05). Folate intake was the most significant dietary determinant of serum aspartate transaminase concentration (beta=-0.05, p=0.04), followed by free choline intake (beta=-0.249, p=0.055). Folate intake exceeding the median level (268 μg/d) was correlated with a reduced rate of hepatitis viral infection (p=0.032) and with normalized serum aspartate transaminase (odds ratio [OR]=0.998, 95% confidence interval [CI]=0.996-1, p=0.042) and alanine transaminase (OR=0.998, 95% CI=0.007-1, p=0.019). Total choline intake exceeding the median level (233 mg/d) was associated with normalized serum aspartate transaminase (OR=0.518, 95% CI=0.360-0.745, p=0.018). The newly established relationships of dietary intake of total choline and folate with normalized hepatic inflammatory markers can guide the development of dietary choline and folate intake recommendations for Taiwanese adults.

Relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-beta1 levels.

PubMed

Hueso, Miguel; Navarro, Estanis; Moreso, Francesc; Beltrán-Sastre, Violeta; Ventura, Francesc; Grinyó, Josep M; Serón, Daniel

2006-05-27

Transforming growth factor (TGF)-beta(1) is increased in allograft rejection and its production is associated with single nucleotide polymorphisms (SNPs). The contribution of SNPs at codons 10 and 25 of the TGF-beta(1) gene to renal allograft damage was assessed in 6-month protocol biopsies and their association with TGF-beta(1) production. TGF-beta(1) genotypes were evaluated by polymerase chain reaction (PCR)/restriction fragment length polymorphism. Intragraft TGF-beta(1) messenger RNA (mRNA) was measured by real-time PCR and TGF-beta(1) plasma levels were assessed by enzyme-linked immunosorbent assay. Eighty consecutive patients were included. Allele T at codon 10 (risk ratio, 6.7; P = 0.02) and an episode of acute rejection before protocol biopsy (risk ratio, 6.2; P = 0.01) were independent predictors of subclinical rejection (SCR). TGF-beta(1) plasma levels, but not those of TGF-beta(1) mRNA, were increased in patients with SCR (2.59 ng/mL +/- 0.91 [n = 22] vs. 2.05 ng/mL +/- 0.76 [n = 43]; P = 0.01). There was no association between allele T and TGF-beta(1) plasma or intragraft levels. Allele T at codon 10 of the TGF-beta(1) gene is associated with a higher incidence of SCR.

Nanoelectrospray with ion-trap mass spectrometry for the determination of beta-casomorphins in derived milk products.

PubMed

Juan-García, Ana; Font, Guillermina; Juan, Cristina; Picó, Yolanda

2009-11-15

Beta-casomorphins (b-CMs) are bioactive peptides derived from casein with opioid agonist effects similar to morphine. The use of electrospray (ESI) with quadrupole ion-trap mass spectrometry (QIT-MS) for these compounds in two matrices, cheese and milk, was examined. It was compared to a liquid chromatography (LC) coupled to mass spectrometry (LC-MS), and a "soft" ionisation technique, NanoMate, with selected ion monitoring (SIM), which are unreliable for the determination of trace casomorphins in derived milk products. b-CM mass fragmentation pathways were done for the four most common b-CMs: beta-casomorphin (1-5) bovine (b-CM-5), beta-casomorphin (1-7) bovine (b-CM-7), [D-Ala2, D-Pro4,Tyr5]-beta-casomorphin (1-5) amide (b-CM-10) and beta-casomorphin (1-5) amide [D-Ala2,Hyp4,Tyr5] (b-CM-11). The major product ions obtained in QIT-MS were used to construct fragmentation pathways for b-CMs. The different collision energies using automated nanoelectrospray ion source NanoMate and conventional LC in QIT-MS were studied. Calibration data for b-CMs, using spiked milk or cheese samples (10 g or 10 mL), were: NanoMate/MS (25-1000 microg/L), r(2)=0.998; NanoMate/MS(2) (5-1000 microg/L), r(2)=0.9992; NanoMate/MS(3) (2.5-1000 microg/L), r(2)=0.9998. Reproducibility data (% RSD, N=5) for NanoMate/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L.

Genetic Mapping of a Mutant Defective in d, l-Alanine Racemase in Bacillus subtilis 168

PubMed Central

Dul, Michael J.; Young, Frank E.

1973-01-01

Genetic analysis of a d-alanine requiring mutant (dal) of Bacillus subtilis reveals that the gene that codes for d,l-alanine racemase is linked to purB. The order of genes in this region of the chromosome is purB, pig, tsi, dal. Thus there are at least two clusters of genes that regulate cell wall biosynthesis in B. subtilis. PMID:4199510

On the role of endogenous neurotoxins and neuroprotection in Parkinson's disease.

PubMed

Segura-Aguilar, Juan

2017-06-01

For 50 years ago was introduced L-3,4-dihydroxyphenylalanine (L-dopa) in Parkinson's disease treatment and during this significant advances has been done but what trigger the degeneration of the nigrostriatal system remain unknown. There is a general agreement in the scientific community that mitochondrial dysfunction, protein degradation dysfunction, alpha-synuclein aggregation to neurotoxic oligomers, neuroinflammation, oxidative and endoplasmic reticulum stress are involved in the loss of dopaminergic neurons containing neuromelanin in Parkinson's disease. The question is what triggers these mechanisms. The age of normal onset in idiopathic Parkinson's disease suggests that environmental factors such as metals, pollutants or genetic mutations cannot be involved because these factors are related to early onset of Parkinsonism. Therefore, we have to search for endogenous neurotoxins and neuroprotection in order to understand what trigger the loss of dopaminergic neurons. One important feature of Parkinson's disease is the rate of the degenerative process before the motor symptoms are evident and during the disease progression. The extremely slow rate of Parkinson's disease suggests that the neurotoxins and the neuroprotection have to be related to dopamine metabolism. Possible candidates for endogenous neurotoxins are alpha-synuclein neurotoxic oligomers, 4-dihydroxyphenylacetaldehyde and ortho-quinones formed during dopamine oxidation to neuromelanin. Vesicular monoamine transporter-2, DT-diaphorase and glutathione transferase M2-2 seems to be the most important neuroprotective mechanism to prevent neurotoxic mechanism during dopamine oxidation.

New estradiol-linked nitrosoureas: can the pharmacokinetic properties help to explain the pharmacodynamic activities?

PubMed

Betsch, B; Berger, M R; Spiegelhalder, B; Eisenbrand, G; Schmähl, D

1989-01-01

The pharmacokinetics of 1-(2-chloroethyl)-1-nitrosocarbamoyl-L-alanine-estradiol-17-ester (CNC-alanine-estradiol-17-ester) a new estradiol-linked anticancer drug and the unlinked DNA-crosslinking agent 1-(2-chloroethyl)-1-nitrosocarbamoyl-L-alanine (CNC-alanine) have been studied in methylnitrosourea-induced female Sprague-Dawley rats after equimolar intravenous and oral administration. In comparison with the unlinked single agent, the CNC-alanine-estradiol-17-ester showed a 3-fold longer halflife in plasma and a three times larger volume of distribution. The distribution after intravenous administration was nearly three times faster. The absorption after peroral administration was likewise two times faster. The bioavailability of the estradiol-linked drug was determined to be 52%. After application of CNC-alanine-estradiol-17-ester the cytostatic metabolite CNC-alanine was found, indicating the cleavage of the ester bond. CNC-alanine generated from CNC-alanine-estradiol-17-ester showed a 50% longer halflife than when applied directly. The results indicate that linking 2-chloroethyl-nitrosoureas to estradiol can result in new anticancer agents with modified properties in comparison to the unlinked single agent. The higher antineoplastic activity of the hormone-linked drug can mainly be attributed to differences in the pharmacokinetic behaviour.

Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors.

PubMed

Farroni, Jeffrey S; McCool, Brian A

2004-08-09

Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The beta-amino acid taurine possessed 30-50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for beta-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology.

Isolation and structure determination of malevamide E, a dolastatin 14 analogue, from the marine cyanobacterium Symploca laete-viridis.

PubMed

Adams, Beatrice; Pörzgen, Peter; Pittman, Emily; Yoshida, Wesley Y; Westenburg, Hans E; Horgen, F David

2008-05-01

A new depsipeptide, malevamide E (1), was isolated from field-collected colonies of the filamentous cyanobacterium Symploca laete-viridis. The gross structure of 1 was determined by spectroscopic analyses, including one- and two-dimensional NMR and accurately measured MS/MS. Chiral HPLC analyses of an acid hydrolysate of 1 allowed the stereochemical assignments of its amino acid residues, which include N-methyl-L-alanine, alpha-N,gamma-N-dimethyl-L-asparagine, N-methyl-L-phenylalanine, L-proline, D-valine, and N-methyl-L-valine. LC-MS/MS analysis of S. laete-viridis fractions established the co-occurrence of malevamide E (1) and its homologue dolastatin 14 (2), which was previously reported in low yield from the sea hare Dolabella auricularia. Malevamide E (1) demonstrated a dose-dependent (2-45 microM) inhibition of store-operated Ca(2+) entry in thapsigargin-treated human embryonic kidney (HEK) cells, indicating an inhibitory effect on Ca(2+) release-activated Ca(2+) (CRAC) channels.

Non-enzymic beta-decarboxylation of aspartic acid.

NASA Technical Reports Server (NTRS)

Doctor, V. M.; Oro, J.

1972-01-01

Study of the mechanism of nonenzymic beta-decarboxylation of aspartic acid in the presence of metal ions and pyridoxal. The results suggest that aspartic acid is first converted to oxalacetic acid by transamination with pyridoxal which in turn is converted to pyridoxamine. This is followed by decarboxylation of oxalacetic acid to form pyruvic acid which transaminates with pyridoxamine to form alanine. The possible significance of these results to prebiotic molecular evolution is briefly discussed.

Molecular immune recognition of botulinum neurotoxin B. The light chain regions that bind human blocking antibodies from toxin-treated cervical dystonia patients. Antigenic structure of the entire BoNT/B molecule.

PubMed

Atassi, M Zouhair; Jankovic, Joseph; Steward, Lance E; Aoki, K Roger; Dolimbek, Behzod Z

2012-01-01

We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs. Copyright © 2011. Published by Elsevier GmbH.

Characterization of Clostridium botulinum Type B Neurotoxin Associated with Infant Botulism in Japan

PubMed Central

Kozaki, Shunji; Kamata, Yoichi; Nishiki, Tei-ichi; Kakinuma, Hiroaki; Maruyama, Hiromi; Takahashi, Hiroaki; Karasawa, Tadahiro; Yamakawa, Kiyotaka; Nakamura, Shinichi

1998-01-01

The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that 125I-labeled 111/NT bound to a single binding site with a dissociation constant (Kd) of 2.5 nM; the value was rather lower than that (0.42 nM) of 125I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b, 125I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the Kd values accorded with those of the low- and high-affinity binding sites in synaptosomes. However, 125I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity. PMID:9746583

Inhibitors of the bacterial cell wall biosynthesis enzyme MurC.

PubMed

Reck, F; Marmor, S; Fisher, S; Wuonola, M A

2001-06-04

A series of phosphinate transition-state analogues of the L-alanine adding enzyme (MurC) of bacterial peptidoglycan biosynthesis was prepared and tested as inhibitors of the Escherichia coli enzyme. Compound 4 was identified as a potent inhibitor of MurC from Escherichia coli with an IC(50) of 49nM.

Reactive carriers of immobilized compounds.

PubMed

Coupek, J; Labský, J; Kálal, J; Turková, J; Valentová, O

1977-04-12

Sphericanl macroporous reactive carriers capable of forming covalent bonds with amino acids and proteins were prepared by the suspension copolymerization of 2-hydroxyethyl methacrylate, ethylene dimethacrylate and p-nitrophenyl esters of methacrylic acid and methacryloyl derivatives of glycine, beta-alanine and epsilon-aminocaproic acid. The effect of the spacer length, pH and the type of the buffer used, concentration of reactive groups in the copolymer, concentration of the ligand and the participation of the hydrolytic and aminolytic reaction of p-nitrophenyl functional groups in the attachment of glycine, D,L-phenylalanine and serumalbumin was studied. Macroporous copolymers containing reactive functional groups can be used as active enzyme carriers, if their activity is not blocked by the presence of p-nitrophenol split off in the attachment reaction.

HPLC purification and re-evaluation of chemical identity of two circular bacteriocins, gassericin A and reutericin 6.

PubMed

Arakawa, K; Kawai, Y; Ito, Y; Nakamura, K; Chujo, T; Nishimura, J; Kitazawa, H; Saito, T

2010-04-01

The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6. Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse-phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2-propanol. Mass analysis, N-terminal sequencing and bacteriocin assay of the HPLC-purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H](+) and both had the same specific activity. D/L-amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)-1-(9-fluorenyl)ethyl chloroformate and O-phthalaldehyde/N-acetyl-L-cystein revealed neither gassericin A nor reutericin 6 contained D-alanine residues contrary to our previous results. Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no D-alanine residues. The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.

Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131.

PubMed

Kobayashi, Jyumpei; Yukimoto, Jotaro; Shimizu, Yasuhiro; Ohmori, Taketo; Suzuki, Hirokazu; Doi, Katsumi; Ohshima, Toshihisa

2015-01-01

Many strains of lactic acid bacteria produce high concentrations of d-amino acids. Among them, Lactobacillus salivarius UCC 118 produces d-alanine at a relative concentration much greater than 50 % of the total d, l-alanine (100d/d, l-alanine). We characterized the L. salivarius alanine racemase (ALR) likely responsible for this d-alanine production and found that the enzyme was activated by carboxylates, which is an unique characteristic among ALRs. In addition, alignment of the amino acid sequences of several ALRs revealed that A131 of L. salivarius ALR is likely involved in the activation. To confirm that finding, an L. salivarius ALR variant with an A131K (ALR(A131K)) substitution was prepared, and its properties were compared with those of ALR. The activity of ALR(A131K) was about three times greater than that of ALR. In addition, whereas L. salivarius ALR was strongly activated by low concentrations (e.g., 1 mM) of short chain carboxylates, and was inhibited at higher concentrations (e.g., 10 mM), ALR(A131K) was clearly inhibited at all carboxylate concentrations tested (1-40 mM). Acetate also increased the stability of ALR such that maximum activity was observed at 35 °C and pH 8.0 without acetate, but at 50 °C in the presence of 1 mM acetate. On the other hand, maximum ALR(A131K) activity was observed at 45 °C and around pH 9.0 with or without acetate. It thus appears that A131 mediates the activation and stabilization of L. salivarius ALR by short chain carboxylates.

Leap-dynamics: efficient sampling of conformational space of proteins and peptides in solution.

PubMed

Kleinjung, J; Bayley, P; Fraternali, F

2000-03-31

A molecular simulation scheme, called Leap-dynamics, that provides efficient sampling of protein conformational space in solution is presented. The scheme is a combined approach using a fast sampling method, imposing conformational 'leaps' to force the system over energy barriers, and molecular dynamics (MD) for refinement. The presence of solvent is approximated by a potential of mean force depending on the solvent accessible surface area. The method has been successfully applied to N-acetyl-L-alanine-N-methylamide (alanine dipeptide), sampling experimentally observed conformations inaccessible to MD alone under the chosen conditions. The method predicts correctly the increased partial flexibility of the mutant Y35G compared to native bovine pancreatic trypsin inhibitor. In particular, the improvement over MD consists of the detection of conformational flexibility that corresponds closely to slow motions identified by nuclear magnetic resonance techniques.

21 CFR 172.540 - DL-Alanine.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Agents and Related Substances § 172.540 DL-Alanine. DL-Alanine (a racemic mixture of D- and L-alanine; CAS Reg. No. 302-72-7) may be safely used as a flavor enhancer for sweeteners in pickling mixtures at a level not to exceed 1 percent of the pickling spice that is added to the pickling brine. [56 FR...

Inhibition of GSK-3beta ameliorates hepatic ischemia-reperfusion injury through GSK-3beta/beta-catenin signaling pathway in mice.

PubMed

Xia, Yong-Xiang; Lu, Ling; Wu, Zheng-Shan; Pu, Li-Yong; Sun, Bei-Cheng; Wang, Xue-Hao

2012-06-01

Glycogen synthase kinase (GSK)-3beta/beta-catenin signaling regulates ischemia-reperfusion (I/R)-induced apoptosis and proliferation, and inhibition of GSK-3beta has beneficial effects on I/R injury in the heart and the central nervous system. However, the role of this signaling in hepatic I/R injury remains unclear. The present study aimed to investigate the effects and mechanism of GSK-3beta/beta-catenin signaling in hepatic I/R injury. Male C57BL/6 mice (weighing 22-25 g) were pretreated with either SB216763, an inhibitor of GSK-3beta, or vehicle. These mice were subjected to partial hepatic I/R. Blood was collected for test of alanine aminotransferase (ALT), and liver specimen for assays of phosphorylation at the Ser9 residue of GSK-3beta, GSK-3beta activity, axin 2 and the anti-apoptotic factors Bcl-2 and survivin, as well as the proliferative factors cyclin D1 and proliferating cell nuclear antigen, and apoptotic index (TUNEL). Real-time PCR, Western blotting and immunohistochemical staining were used. SB216763 increased phospho-GSK-3beta levels and suppressed GSK-3beta activity (1880+/-229 vs 3280+/-272 cpm, P<0.01). ALT peaked at 6 hours after reperfusion. Compared with control, SB216763 decreased ALT after 6 hours of reperfusion (4451+/-424 vs 7868+/-845 IU/L, P<0.01), and alleviated hepatocyte necrosis and vacuolization. GSK-3beta inhibition led to the accumulation of beta-catenin in the cytosol (0.40+/-0.05 vs 1.31+/-0.11, P<0.05) and nucleus (0.62+/-0.14 vs 1.73+/-0.12, P<0.05), beta-catenin further upregulated the expression of axin 2. Upregulation of GSK-3beta/beta-catenin signaling increased Bcl-2, survivin and cyclin D1. Serological and histological analyses showed that SB216763 alleviated hepatic I/R-induced injury by reducing apoptosis (1.4+/-0.2% vs 3.6+/-0.4%, P<0.05) and enhanced liver proliferation (56+/-8% vs 19+/-4%, P<0.05). Inhibition of GSK-3beta ameliorates hepatic I/R injury through the GSK-3beta/beta-catenin signaling pathway.

Amino Acid Chemistry as a Link Between Small Solar System Bodies and Carbonaceous Chondrites

NASA Technical Reports Server (NTRS)

Glavin, Daniel P.; Ehrenfreund, Pascale; Botta, Oliver; Cooper, George; Bada, Jeffrey L.

2000-01-01

Establishing chemical links between meteorites and small solar system bodies, such as comets and asteroids, provides a tool for investigating the processes that occurred during the formation of the solar system. Carbonaceous meteorites are of particular interest, since they may have seeded the early Earth with a variety of prebiotic organic compounds including amino acids, purines and pyrimidines, which are thought to be necessary for the origin of life. Here we report the results of high-performance liquid chromatography (HPLC) based amino acid analyses of the acid-hydrolyzed hot water extracts from pristine interior pieces of the CI carbonaceous chondrites Orgueil and Ivuna and the CM meteorites Murchison and Murray. We found that the CI meteorites Orgueil and Ivuna contained high abundances of beta-alanine and glycine, while only traces of other amino acids like alanine, alpha-amino-n-butryic acid (ABA) and alpha-aminoisobutyric acid (AIB) were detected in these meteorites. Carbon isotopic measurements of beta-alanine and glycine in Orgueil by gas chromatography combustion-isotope ratio mass spectrometry clearly indicate an extraterrestrial origin of these amino acids. The amino acid composition of Orgueil and Ivuna was strikingly different from the CM chondrites Murchison and Murray. The most notable difference was the high relative abundance of B-alanine in Orgueil and Ivuna compared to Murchison and Murray. Furthermore, AIB, which is one of the most abundant amino acids found in Murchison and Murray, was present in only trace amounts in Orgueil and Ivuna. Our amino acid data strongly suggest that the CI meteorites Orgueil and Ivuna came from a different type of parent body than the CM meteorites Murchison and Murray, possibly from an extinct comet. It is generally thought that carbonaceous meteorites are fragments of larger asteroidal bodies delivered via near Earth objects (NEO). Orbital and dynamic studies suggest that both fragments of main belt asteroids and comets replenish the NEO population, therefore extinct comets may contribute up to half of all NEO's. A comparison of an amino acid analysis of a returned NEO sample to CI and CM carbonaceous chondrites would help establish a link between small solar system bodies and meteorites. Based on our amino acid measurements of CI and CM chondrites, amino acid chemistry can be included as an additional set of criteria to constrain the nature of meteorite parent bodies.

Glucose is necessary to maintain neurotransmitter homeostasis during synaptic activity in cultured glutamatergic neurons.

PubMed

Bak, Lasse K; Schousboe, Arne; Sonnewald, Ursula; Waagepetersen, Helle S

2006-10-01

Glucose is the primary energy substrate for the adult mammalian brain. However, lactate produced within the brain might be able to serve this purpose in neurons. In the present study, the relative significance of glucose and lactate as substrates to maintain neurotransmitter homeostasis was investigated. Cultured cerebellar (primarily glutamatergic) neurons were superfused in medium containing [U-13C]glucose (2.5 mmol/L) and lactate (1 or 5 mmol/L) or glucose (2.5 mmol/L) and [U-13C]lactate (1 mmol/L), and exposed to pulses of N-methyl-D-aspartate (300 micromol/L), leading to synaptic activity including vesicular release. The incorporation of 13C label into intracellular lactate, alanine, succinate, glutamate, and aspartate was determined by mass spectrometry. The metabolism of [U-13C]lactate under non-depolarizing conditions was high compared with that of [U-13C]glucose; however, it decreased significantly during induced depolarization. In contrast, at both concentrations of extracellular lactate, the metabolism of [U-13C]glucose was increased during neuronal depolarization. The role of glucose and lactate as energy substrates during vesicular release as well as transporter-mediated influx and efflux of glutamate was examined using preloaded D-[3H]aspartate as a glutamate tracer and DL-threo-beta-benzyloxyaspartate to inhibit glutamate transporters. The results suggest that glucose is essential to prevent depolarization-induced reversal of the transporter (efflux), whereas vesicular release was unaffected by the choice of substrate. In conclusion, the present study shows that glucose is a necessary substrate to maintain neurotransmitter homeostasis during synaptic activity and that synaptic activity does not induce an upregulation of lactate metabolism in glutamatergic neurons.

Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lentz, T.L.

1991-11-12

Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of {sup 125}I-{alpha}-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b and the structurally similarmore » segment of CVS rabies virus glycoprotein. These affinities were comparable to those of d-tubocurarine and suberyldicholine. These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.« less

Special feature of kinetics of ZcE isomerization of β-N-methylaminovinyl trifluoromethyl ketone in Ar matrix exposed to UV radiation and spontaneous E ⇌ Z isomerization of α-methyl-β-N-methylaminovinyl trifluoromethyl ketone

NASA Astrophysics Data System (ADS)

Vdovenko, Sergey I.; Gerus, Igor I.; Pagacz-Kostrzewa, Magdalena; Wierzejewska, Maria; Zhuk, Yuri I.; Kukhar, Valery P.

2018-06-01

Although it is well known that reactivity of α,β-unsaturated enaminoketones is closely associated with spatial and electronic structure but until now little attention was devoted to quantitative investigation of interconversion of different stereoisomeric forms of enaminoketones. In present work we studied peculiarities of kinetics of Z ⇌ E isomerization of enaminoketone 4-(N-methylamino)-1,1,1-trifluorobut-3-en-2-one F3C-COsbnd CHdbnd CHsbnd NH(CH3) (1) in Ar-matrix exposed to UV-radiation (λ = 340 nm) with IR Fourier and 2D correlation spectroscopy and we found that Z-s-Z-s-trans isomer transforms primarily into two E-isomers, E-s-E-s-trans and E-s-Z-s-trans which further turn into the E-s-E-s-cis and E-s-Z-s-cis conformers all interconversion rate constants being comparable in magnitude. Along with this process long-term exposure to the UV-radiation results in proton transfer from nitrogen of methylamino group to carbonyl oxygen with simultaneous isomerization of 'cyclic' iminoenol form into 'linear'one. In solution of enaminoketone 4-(N-methylamino)-1,1,1-trifluoro-3-methylbut-3-en-2-one F3C-CO-C(CH3)dbnd CH-NH(CH3) (2) we observed reversed process, namely, spontaneous interconversion of the E-s-E-s-trans and E-s-Z-s-trans conformers into the Z-s-Z-trans isomer. It was found that rate constants of the dimeric forms of the E-s-E-s-trans and E-s-Z-s-trans conformers are higher than those of the monomers and are independent on total enaminoketone concentration. Addition of highly polar HMPA promotes proton transfer from nitrogen to oxygen in the Z-s-Z-s-trans isomer of 2 with subsequent isomerization into the linear imino-enol product but the rate constant of this transformation is ten-fold smaller than that for 1 in the Ar matrix exposed to UV radiation. Special feature of kinetics of Z ⇌ E isomerization of β-N-methylaminovinyl trifluoromethyl ketone in Ar matrix exposed to UV radiation and spontaneous E ⇌ Z isomerization of α-Methyl-, β-N-methylaminovinyl trifluoromethyl ketone. Sergey I. Vdovenko, Igor I. Gerus, Magdalena Pagacz-Kostrzewa, Maria Wierzejewska, Yuri I. Zhuk and Valery P. Kukhar.

The Effects of Alkali and Temperature on the Hydrolysis Rate of N-methylpyrrolidone

NASA Astrophysics Data System (ADS)

Ou, Yu Jing; Wang, Xiao Mei; Lei Li, Chun; Zhu, Ya Long; Li, Xiao Long

2017-12-01

By studying the hydrolysis of N-methylpyrrolidone, it was found that the effects of NaOH concentration and temperature on N-methylpyrrolidone's hydrolysis were remarkable. Fourier transform infrared (FTIR) and Gel Permeation Chromatography (GPC) detected that the mainly hydrolyzate was 4-(methylamino)butyric acid, and the hydrolyzate can generate polymers, which of molecular weight increases with temperature rising. The results of Gas Chromatography (GC) and moisture meter test showed that adding alkaline and raising temperature can aggravate hydrolysis of NMP. This study provide theoretical basis for recycling solvent (NMP) in the production of polyphenylene sulfide (PPS).

Evaluation of mysids and sea urchins exposed to saxitoxins.

PubMed

Bernardi Bif, Mariana; Yunes, João Sarkis; Resgalla, Charrid

2013-11-01

Saxitoxins are neurotoxins produced by dinoflagellates and cyanobacteria that form toxic blooms in waters. The impact of saxitoxins to the most vulnerable taxa and environment are not well understood. The experimental model was based on the use of toxic cell extracts containing saxitoxins. This extract was utilized for acute and chronic tests with Mysidopsis juniae. Chronic tests were also done with Lytechinus variegatus and Arbacia lixula larvae. Acute test with mysids had a LC₅₀=2.34 μg/L. The chronic test with sea urchins showed morphological abnormalities resulting in malformation of larval appendices at low concentrations of the toxin (EC₅₀=2.96 μg/L for L. variegatus and 2.06 μg/L for A. lixula). Although saxitoxins are considered neurotoxins, both species of sea urchins showed symptoms not related to nerve cells. A. lixula was more sensitive than L. variegatus, proving that its sensitivity should be taken in consideration to be another option to toxicological tests. Copyright © 2013. Published by Elsevier B.V.

Sensory-guided decomposition of roasted cocoa nibs (Theobroma cacao) and structure determination of taste-active polyphenols.

PubMed

Stark, Timo; Bareuther, Sabine; Hofmann, Thomas

2005-06-29

Sequential application of solvent extraction, gel permeation chromatography, and RP-HPLC in combination with taste dilution analyses, followed by LC-MS and 1D/2D-NMR experiments and thiolytic degradation, revealed that, besides theobromine and caffeine, the flavan-3-ols epicatechin, catechin, procyanidin B-2, procyanidin B-5, procyanidin C-1, [epicatechin-(4beta-->8)](3)-epicatechin, and [epicatechin-(4beta-->8)](4)-epicatechin were among the key compounds contributing to the bitter taste as well as the astringent mouthfeel imparted upon consumption of roasted cocoa. In addition, a series of quercetin, naringenin, luteolin, and apigenin glycopyranosides as well as a family of not previously identified amino acid amides, namely, (+)-N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine, (+)-N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-aspartic acid, and (+)-N-(E)-cinnamoyl-L-aspartic acid, have been identified as key astringent compounds of roasted cocoa. Furthermore, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-3-hydroxy-l-tyrosine (clovamide), (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-tyrosine (deoxyclovamide), and (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tyrosine, reported previously as antioxidants, have been found as contributors of cocoa's astringent taste. By means of the half-tongue test, the taste thresholds of flavan-3-ols and glycosides have been determined.

On the output factor measurements of the CyberKnife iris collimator small fields: Experimental determination of the k{sub Q{sub c{sub l{sub i{sub n,Q{sub m{sub s{sub r}{sup f{sub c}{sub l}{sub i}{sub n},f{sub m}{sub s}{sub r}}}}}}}}} correction factors for microchamber and diode detectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pantelis, E.; Moutsatsos, A.; Zourari, K.

Purpose: To measure the output factors (OFs) of the small fields formed by the variable aperture collimator system (iris) of a CyberKnife (CK) robotic radiosurgery system, and determine the k{sub Q{sub c{sub l{sub i{sub n,Q{sub m{sub s{sub r}{sup f{sub c}{sub l}{sub i}{sub n},f{sub m}{sub s}{sub r}}}}}}}}} correction factors for a microchamber and four diode detectors. Methods: OF measurements were performed using a PTW PinPoint 31014 microchamber, four diode detectors (PTW-60017, -60012, -60008, and the SunNuclear EDGE detector), TLD-100 microcubes, alanine dosimeters, EBT films, and polymer gels for the 5 mm, 7.5 mm, 10 mm, 12.5 mm, and 15 mm irismore » collimators at 650 mm, 800 mm, and 1000 mm source to detector distance (SDD). The alanine OF measurements were corrected for volume averaging effects using the 3D dose distributions registered in polymer gel dosimeters. k{sub Q{sub c{sub l{sub i{sub n,Q{sub m{sub s{sub r}{sup f{sub c}{sub l}{sub i}{sub n},f{sub m}{sub s}{sub r}}}}}}}}} correction factors for the PinPoint microchamber and the diode dosimeters were calculated through comparison against corresponding polymer gel, EBT, alanine, and TLD results. Results: Experimental OF results are presented for the array of dosimetric systems used. The PinPoint microchamber was found to underestimate small field OFs, and a k{sub Q{sub c{sub l{sub i{sub n,Q{sub m{sub s{sub r}{sup f{sub c}{sub l}{sub i}{sub n},f{sub m}{sub s}{sub r}}}}}}}}} correction factor ranging from 1.127 {+-} 0.022 (for the 5 mm iris collimator) to 1.004 {+-} 0.010 (for the 15 mm iris collimator) was determined at the reference SDD of 800 mm. The PinPoint k{sub Q{sub c{sub l{sub i{sub n,Q{sub m{sub s{sub r}{sup f{sub c}{sub l}{sub i}{sub n},f{sub m}{sub s}{sub r}}}}}}}}} correction factor was also found to increase with decreasing SDD; k{sub Q{sub c{sub l{sub i{sub n,Q{sub m{sub s{sub r}{sup f{sub c}{sub l}{sub i}{sub n},f{sub m}{sub s}{sub r}}}}}}}}} values equal to 1.220 {+-} 0.028 and 1.077 {+-} 0.016 were obtained for the 5 mm iris collimator at 650 mm and 1000 mm SDD, respectively. On the contrary, diode detectors were found to overestimate small field OFs and a correction factor equal to 0.973 {+-} 0.006, 0.954 {+-} 0.006, 0.937 {+-} 0.007, and 0.964 {+-} 0.006 was measured for the PTW-60017, -60012, -60008 and the EDGE diode detectors, respectively, for the 5 mm iris collimator at 800 mm SDD. The corresponding correction factors for the 15 mm iris collimator were found equal to 0.997 {+-} 0.010, 0.994 {+-} 0.009, 0.988 {+-} 0.010, and 0.986 {+-} 0.010, respectively. No correlation of the diode k{sub Q{sub c{sub l{sub i{sub n,Q{sub m{sub s{sub r}{sup f{sub c}{sub l}{sub i}{sub n},f{sub m}{sub s}{sub r}}}}}}}}} correction factors with SDD was observed. Conclusions: This work demonstrates an experimental procedure for the determination of the k{sub Q{sub c{sub l{sub i{sub n,Q{sub m{sub s{sub r}{sup f{sub c}{sub l}{sub i}{sub n},f{sub m}{sub s}{sub r}}}}}}}}} correction factors required to obtain small field OF results of increased accuracy.« less

An elevated serum beta-2-microglobulin level is an adverse prognostic factor for overall survival in patients with early-stage Hodgkin disease.

PubMed

Chronowski, Gregory M; Wilder, Richard B; Tucker, Susan L; Ha, Chul S; Sarris, Andreas H; Hagemeister, Fredrick B; Barista, Ibrahim; Hess, Mark A; Cabanillas, Fernando; Cox, James D

2002-12-15

The relative importance of prognostic factors in patients with early-stage Hodgkin disease remains controversial. The purpose of this study was to evaluate prognostic factors among patients who received chemotherapy before radiotherapy. From 1987 to 1995, 217 consecutive patients ranging in age from 16 to 88 years (median, 28 years) with Ann Arbor Stage I (n = 55) or II (n = 162) Hodgkin disease underwent chemotherapy before radiotherapy at a single center. Most were treated on prospective studies. Patients received a median of three cycles of induction chemotherapy. Mitoxantrone, vincristine, vinblastine, and prednisone (NOVP), doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD), mechlorethamine, vincristine, procarbazine, and prednisone (MOPP), cyclophosphamide, vinblastine, procarbazine, prednisone, doxorubicin, bleomycin, dacarbazine, and CCNU (CVPP/ABDIC), or other chemotherapeutic regimens were given to 160, 18, 15, 10, and 14 patients, respectively. The median radiotherapy dose was 40 Gy. Serum beta-2-microglobulin (beta-2M) levels ranged from 1.0 to 4.1 mg/L (median, 1.7 mg/L; upper limit of normal, 2.0 mg/L). We studied univariate and multivariate associations between survival and the following clinical features: serum beta-2M level above 1.25 times the upper limit of normal (n = 12), male gender (n = 113), hypoalbuminemia (n = 11), and bulky mediastinal disease (n = 94). Follow-up of surviving patients ranged from 0.9 to 13.4 years (median, 6.6 years) and 92% were observed for 3.0 or more years. Nineteen patients have died. Only elevation of the serum beta-2M level was an independent adverse prognostic factor for overall survival (P = 0.0009). The prognostic significance of a simple, widely available, and inexpensive blood test, beta-2M, has not been studied routinely in patients with Hodgkin disease and should be tested prospectively in large, cooperative group trials. Copyright 2002 American Cancer Society.DOI 10.1002/cncr.10998

β-alanine Supplementation Fails to Increase Peak Aerobic Power or Ventilatory Threshold in Aerobically Trained Males.

PubMed

Greer, Beau Kjerulf; Katalinas, Matthew E; Shaholli, Danielle M; Gallo, Paul M

2016-01-01

The purpose of the present study was to determine the effect of 30 days of β-alanine supplementation on peak aerobic power and ventilatory threshold (VT) in aerobically fit males. Fourteen males (28.8 ± 9.8 yrs) were assigned to either a β-alanine (SUPP) or placebo (PLAC) group; groups were matched for VT as it was the primary outcome measure. β-alanine supplementation consisted of 3 g/day for 7 days, and 6 g/day for the remaining 23 days. Before and after the supplementation period, subjects performed a continuous, graded cycle ergometry test to determine VO2 peak and VT. Metabolic data were analyzed using a 2 × 2 ANOVA with repeated measures. Thirty days of β-alanine supplementation (SUPP) did not increase VO2 peak (4.05 ± 0.6 vs. 4.14 ± 0.6 L/min) as compared to the placebo (PLAC) group (3.88 ± 0.2 vs. 3.97 ± 0.2 L/min) (p > .05). VT did not significantly improve in either the SUPP (3.21 ± 0.5 vs. 3.33 ± 0.5 L/min) or PLAC (3.19 ± 0.1 vs. 3.20 ± 0.1 L/min) group (p > .05). In conclusion, 30 days of β-alanine supplementation had no effect on VO2 peak or VT in aerobically trained athletes.

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

PubMed Central

Heyne, R I; de Vrij, W; Crielaard, W; Konings, W N

1991-01-01

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism. PMID:1670936

Tarantula huwentoxin-IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the domain ii voltage sensor in the closed configuration.

PubMed

Xiao, Yucheng; Bingham, Jon-Paul; Zhu, Weiguo; Moczydlowski, Edward; Liang, Songping; Cummins, Theodore R

2008-10-03

Peptide toxins with high affinity, divergent pharmacological functions, and isoform-specific selectivity are powerful tools for investigating the structure-function relationships of voltage-gated sodium channels (VGSCs). Although a number of interesting inhibitors have been reported from tarantula venoms, little is known about the mechanism for their interaction with VGSCs. We show that huwentoxin-IV (HWTX-IV), a 35-residue peptide from tarantula Ornithoctonus huwena venom, preferentially inhibits neuronal VGSC subtypes rNav1.2, rNav1.3, and hNav1.7 compared with muscle subtypes rNav1.4 and hNav1.5. Of the five VGSCs examined, hNav1.7 was most sensitive to HWTX-IV (IC(50) approximately 26 nM). Following application of 1 microm HWTX-IV, hNav1.7 currents could only be elicited with extreme depolarizations (>+100 mV). Recovery of hNav1.7 channels from HWTX-IV inhibition could be induced by extreme depolarizations or moderate depolarizations lasting several minutes. Site-directed mutagenesis analysis indicated that the toxin docked at neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II. Mutations E818Q and D816N in hNav1.7 decreased toxin affinity for hNav1.7 by approximately 300-fold, whereas the reverse mutations in rNav1.4 (N655D/Q657E) and the corresponding mutations in hNav1.5 (R812D/S814E) greatly increased the sensitivity of the muscle VGSCs to HWTX-IV. Our data identify a novel mechanism for sodium channel inhibition by tarantula toxins involving binding to neurotoxin receptor site 4. In contrast to scorpion beta-toxins that trap the IIS4 voltage sensor in an outward configuration, we propose that HWTX-IV traps the voltage sensor of domain II in the inward, closed configuration.

Evidence for paralytic shellfish poisons in the freshwater cyanobacterium Lyngbya wollei (Farlow ex Gomont) comb. nov.

PubMed

Carmichael, W W; Evans, W R; Yin, Q Q; Bell, P; Moczydlowski, E

1997-08-01

Lyngbya wollei (Farlow ex Gomont) comb. nov., a perennial mat-forming filamentous cyanobacterium prevalent in lakes and reservoirs of the southeastern United States, was found to produce a potent, acutely lethal neurotoxin when tested in the mouse bioassay. Signs of poisoning were similar to those of paralytic shellfish poisoning. As part of the Tennessee Valley Authority master plan for Guntersville Reservoir, the mat-forming filamentous cyanobacterium L. wollei, a species that had recently invaded from other areas of the southern United States, was studied to determine if it could produce any of the known cyanotoxins. Of the 91 field samples collected at 10 locations at Guntersville Reservoir, Ala., on the Tennessee River, over a 3-year period, 72.5% were toxic. The minimum 100% lethal doses of the toxic samples ranged from 150 to 1,500 mg kg of lyophilized L. wollei cells-1, with the majority of samples being toxic at 500 mg kg-1. Samples bioassayed for paralytic shellfish toxins by the Association of Official Analytical Chemists method exhibited saxitoxin equivalents ranging from 0 to 58 micrograms g (dry weight)-1. Characteristics of the neurotoxic compound(s), such as the lack of adsorption by C18 solid-phase extraction columns, the short retention times on C18 high-performance liquid chromatography (HPLC) columns, the interaction of the neurotoxins with saxiphilin (a soluble saxitoxin-binding protein), and external blockage of voltage-sensitive sodium channels, led to our discovery that this neurotoxin(s) is related to the saxitoxins, the compounds responsible for paralytic shellfish poisonings. The major saxitoxin compounds thus far identified by comparison of HPLC fluorescence retention times are decarbamoyl gonyautoxins 2 and 3. There was no evidence of paralytic shellfish poison C toxins being produced by L. wollei. Fifty field samples were placed in unialgal culture and grown under defined culture conditions. Toxicity and signs of poisoning for these laboratory-grown strains of L. wollei were similar to those of the field collection samples.

[Establishing biological reference intervals of alanine transaminase for clinical laboratory stored database].

PubMed

Guo, Wei; Song, Binbin; Shen, Junfei; Wu, Jiong; Zhang, Chunyan; Wang, Beili; Pan, Baishen

2015-08-25

To establish an indirect reference interval based on the test results of alanine aminotransferase stored in a laboratory information system. All alanine aminotransferase results were included for outpatients and physical examinations that were stored in the laboratory information system of Zhongshan Hospital during 2014. The original data were transformed using a Box-Cox transformation to obtain an approximate normal distribution. Outliers were identified and omitted using the Chauvenet and Tukey methods. The indirect reference intervals were obtained by simultaneously applying nonparametric and Hoffmann methods. The reference change value was selected to determine the statistical significance of the observed differences between the calculated and published reference intervals. The indirect reference intervals for alanine aminotransferase of all groups were 12 to 41 U/L (male, outpatient), 12 to 48 U/L (male, physical examination), 9 to 32 U/L (female, outpatient), and 8 to 35 U/L (female, physical examination), respectively. The absolute differences when compared with the direct results were all smaller than the reference change value of alanine aminotransferase. The Box-Cox transformation combined with the Hoffmann and Tukey methods is a simple and reliable technique that should be promoted and used by clinical laboratories.

Difference in the structures of alanine tri- and tetra-peptides with antiparallel β-sheet assessed by X-ray diffraction, solid-state NMR and chemical shift calculations by GIPAW.

PubMed

Asakura, Tetsuo; Yazawa, Koji; Horiguchi, Kumiko; Suzuki, Furitsu; Nishiyama, Yusuke; Nishimura, Katsuyuki; Kaji, Hironori

2014-01-01

Alanine oligomers provide a key structure for silk fibers from spider and wild silkworms.We report on structural analysis of L-alanyl-L-alanyl-L-alanyl-L-alanine (Ala)4 with anti-parallel (AP) β-structures using X-ray and solid-state NMR. All of the Ala residues in the (Ala)4 are in equivalent positions, whereas for alanine trimer (Ala)3 there are two alternative locations in a unit cell as reported previously (Fawcett and Camerman, Acta Cryst., 1975, 31, 658-665). (Ala)4 with AP β-structure is more stable than AP-(Ala)3 due to formation of the stronger hydrogen bonds. The intermolecular structure of (Ala)4 is also different from polyalanine fiber structure, indicating that the interchain arrangement of AP β-structure changes with increasing alanine sequencelength. Furthermore the precise (1)H positions, which are usually inaccesible by X-ray diffraction method, are determined by high resolution (1)H solid state NMR combined with the chemical shift calculations by the gauge-including projector augmented wave method. Copyright © 2013 Wiley Periodicals, Inc.

Effects of Running Wheel Activity and Dietary HMB and β-alanine Co-Supplementation on Muscle Quality in Aged Male Rats.

PubMed

Russ, D W; Acksel, C; McCorkle, K W; Edens, N K; Garvey, S M

2017-01-01

Loss of skeletal muscle function is linked to increased risk for loss of health and independence in older adults. Dietary interventions that can enhance aging muscle function, alone or in combination with exercise, may offer an effective way to reduce these risks. The goal of this study was to evaluate the muscular effects of beta-hydroxy-beta-methylbutyrate (HMB) and beta-alanine (β-Ala) co-supplementation in aged Sprague-Dawley rats with voluntary access to running wheels (RW). Aged (20 months) rats were housed with ad libitum access to RW while on a purified diet for 4 weeks, then balanced for RW activity and assigned to either a control or an experimental diet (control + HMB and β-Ala) for the next 4 weeks (n = 10/group). At the end of the study, we assessed muscle size, in situ force and fatigability in the medial gastrocnemius muscles, as well as an array of protein markers related to various age- and activity-responsive signaling pathways. Dietary HMB+β-Ala did not improve muscle force or fatigue resistance, but a trend for increased muscle cross-sectional area (CSA) was observed (P = 0.077). As a result, rats on the experimental diet exhibited reduced muscle quality (force/CSA; P = 0.032). Dietary HMB+β-Ala reduced both the abundance of PGC1-α (P = 0.050) and the ratio of the lipidated to non-lipidated forms of microtubule-associated protein 1 light chain 3 beta (P = 0.004), markers of mitochondrial biogenesis and autophagy, respectively. Some alterations in myostatin signaling also occurred in the dietary HMB+β-Ala group. There was an unexpected difference (P = 0.046) in RW activity, which increased throughout the study in the animals on the control diet, but not in animals on the experimental diet. These data suggest that the short-term addition of dietary HMB+β-Ala to modest physical activity provided little enhancement of muscle function in this model of uncomplicated aging.

Glycosidases in Brachionus plicatilis (Rotifera).

PubMed

Kühle, K; Kleinow, W

1990-01-01

1. Tests for glycosidases were performed in homogenates of Brachionus plicatilis. 2. Hydrolytic activity was detected with the following substrates: (a) with synthetic substrates (NP = 4-nitrophenyl): NP-alpha- and NP-beta-D-glucopyranoside, NP-alpha- and NP-beta-D-galactopyranoside, NP-N-acetyl-beta-D-glucosaminide, NP-N-acetyl-beta-D-galactosaminide, NP-alpha- and NP-beta-D-mannopyranoside and NP-alpha-L-fucopyranoside; (b) with disaccharides: sucrose, maltose, trehalose, isomaltose, cellobiose, gentiobiose and lactose; (c) with polysaccharides: laminarine, carboxymethyl-cellulose, avicel, Micrococcus luteus (for lysozyme) and 4-nitrophenyl-alpha-D-maltoheptaoside (for amylase). 3. The pH dependence of the glycosidase activities was determined. 4. The distribution of enzyme activities within fractions from the homogenate was studied in order to localize them within the cell. 5. Proteins from Brachionus homogenate were separated by SDS-gel electrophoresis and the positions of the following glycosidase activities were detected by assays performed on the gels (estimated molecular weights in parentheses): alpha-glucosidase (250,000); beta-glucosidase (200,000); beta-galactosidase (70,000); N-acetyl-beta-glucosaminidase (60,000).

Elevated CSF-lactate is a reliable marker of mitochondrial disorders in children even after brief seizures.

PubMed

Magner, Martin; Szentiványi, Karol; Svandová, Ivana; Ješina, Pavel; Tesařová, Markéta; Honzík, Tomáš; Zeman, Jiří

2011-03-01

Increased lactate is an important biochemical marker in diagnosis of children with suspicion of mitochondrial disorders. A diagnostic dilemma may originate if analyses are performed after seizures, when the increased lactate levels may be considered to result from the seizures. To address this problem, we ascertained the diagnostic value of lactate and alanine in blood (B) and cerebrospinal fluid (CSF) in children with mitochondrial disorders (n = 24), epilepsy (n = 32), psychomotor retardation (n = 23), meningitis (n = 12) and meningism (n = 16). Lactate concentration was measured using a spectrophotometric method. Amino acids in serum and CSF were analyzed by ion exchange chromatography with ninhydrin detection. Average blood and CSF-lactate levels were significantly higher in children with mitochondrial disorders (3.87 ± 0.48 and 4.43 ± 0.55 mmol/l) and meningitis (2.77 ± 0.45 and 8.58 ± 1.08 mmol/l) than in children with epilepsy (1.72 ± 0.13 and 1.62 ± 0.04 mmol/l), psychomotor retardation (1.79 ± 1.40 and 1.68 ± 0.06 mmol/l) or meningism (1.70 ± 0.13 and 1.64 ± 0.07 mmol/l). Blood and CSF-alanine levels were also higher in children with mitochondrial disorders (558 ± 44 and 51 ± 8 μmol/l) than in children with epilepsy (327 ± 23 and 27 ± 3 μmol/l) or psychomotor retardation (323 ± 27 and 26 ± 3 μmol/l). The CSF-lactate levels of children with epilepsy were similar whether the samples were obtained 3 ± 0.6 h after an attack of brief seizures or from children without history of recent seizures. Elevated cerebrospinal fluid lactate level is a reliable marker pointing to mitochondrial origin of disease, even in children who have recently suffered short-lasting seizures. Some children with mitochondrial disorders manifest only mild or intermittent elevation of lactate levels. Copyright © 2010 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

ALK and TGF-Beta Resistance in Breast Cancer

DTIC Science & Technology

2017-10-01

and H.F. Lodish, Role of transforming growth factor beta in human disease. N Engl J Med, 2000. 342(18): p. 1350-8. 3. Massague, J., S.W. Blain, and... Transforming growth factor-beta signaling in normal and malignant hematopoiesis. Leukemia, 2003. 17(9): p. 1731-7. 5. Lehman, H.L., et al., Modeling and

PROGRESS ON THE STUDY OF BETA TREATMENT OF URANIUM, DECEMBER 1, 1960-MARCH 30, 1961

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russell, R.B.

The variables affecting the beta treatment of uranium are described. Results are included on the effects of time at beta temperature, the influence of air delay before quenching, and the effects of 800 l C annealing. A description of x-ray automation equipment is presented. (N.W.R.)

Evaluation of Lateral-Flow Clostridium botulinum Neurotoxin Detection Kits for Food Analysis

DTIC Science & Technology

2005-02-08

toxic proteins. Annu. Rev. Microbiol. 53:551–575. 19. Ketema, F., C. Zeh, D. C. Edelman, R. Saville, and N. T. Constantine. 2001. Assessment of the...H. H., and F. Sun. 2001. Assessing cyanogen content in cassava -based food using the enzyme-dipstick method. Food Chem. Toxicol. 39:649–653. VOL. 71, 2005 RAPID DETECTION OF C. BOTULINUM NEUROTOXIN 3941

Extreme Sensitivity of Botulinum Neurotoxin Domains Toward Mild Agitation

DTIC Science & Technology

2009-09-01

Department Molecular Biology, Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick...hydroxyethylpiperazine-N0-2-ethanesulfo- cular dichroism. AL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 9, SEPTapplications. Botulinum neurotoxins (BoNTs) are...Dr. S. Ashraf Ahmed (Telephone: 301- 619-6299; Fax: 301-619-2348; E-mail: syed.ahmed@amedd.army.mil) Journal of Pharmaceutical Sciences, Vol. 98

Component effect of stem cell-loaded thermosensitive polypeptide hydrogels on cartilage repair.

PubMed

Liu, He; Cheng, Yilong; Chen, Jinjin; Chang, Fei; Wang, Jincheng; Ding, Jianxun; Chen, Xuesi

2018-06-01

Biophysical properties of the desired biomimetic scaffolds, such as porosity and elasticity, have been proven associated with the efficacy of cartilage regeneration. In this work, the copolymers of poly(l-alanine)-block-poly(ethylene glycol)-block-poly(l-alanine) (PA-PEG-PA) and poly(l-alanine-co-l-phenylalanine)-block-poly(ethylene glycol)-block-poly(l-alanine-co-l-phenylalanine) (PAF-PEG-PAF) with different ratios of alanine to phenylalanine were synthesized. The introduction of a hydrophobic amino acid, i.e., phenylalanine, into polyalanine-based thermosensitive hydrogel led to the enhanced gelation behaviors and upregulated mechanical properties. Moreover, the increase of phenylalanine content resulted in the enlarged pore size and enhanced mechanical strength of PAF-PEG-PAF thermogel, followed by the regeneration of hyaline-like cartilage with reduced fibrous tissue formation in vivo. The findings indicated the great potential of thermosensitive polypeptide hydrogels in cartilage tissue engineering. Articular cartilage defect has limited self-repair ability due to the lack of blood supply and innervation, which may lead to knee osteoarthritis afterwards. Injectable hydrogels are demonstrated possessing outstanding properties as biomimetic scaffolds in cartilage tissue engineering, while the effect of biophysical properties on the efficacy of cartilage regeneration has not been revealed. Herein, the poly(ethylene glycol)-polypeptide triblock copolymers with different ratios of alanine to phenylalanine were synthesized. The sol-to-gel transition temperature and the critical gelation concentration decreased as the increased amount of phenylalanine unit, resulting in the enlarged pore size and enhanced mechanical strength. These features lead to better regeneration of hyaline-like cartilage with reduced fibrous tissue formation, indicating great potential of thermosensitive polypeptide hydrogels for efficient cartilage repair. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Functional Identification and Structure Determination of Two Novel Prolidases from cog1228 in the Amidohydrolase Superfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, Dao Feng; Patskovsky, Yury; Xu, Chengfu

2010-12-07

Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed, and purified to homogeneity. The two proteins, Sgx9260c (gi|44242006) and Sgx9260b (gi|44479596), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of L-amino acids. Sgx9260c catalyzes the hydrolysis of Gly-L-Pro, L-Ala-L-Pro, and N-acyl derivatives of L-Pro. The best substrate identified to date is N-acetyl-L-Pro with a value of k{sub cat}/K{sub m} of 3 x 10{sup 5} M{sup -1} s{sup -1}. Sgx9260b catalyzes the hydrolysismore » of L-hydrophobic L-Pro dipeptides and N-acyl derivatives of L-Pro. The best substrate identified to date is N-propionyl-L-Pro with a value of k{sub cat}/K{sub m} of 1 x 10{sup 5} M{sup -1} s{sup -1}. Three-dimensional structures of both proteins were determined by X-ray diffraction methods (PDB codes 3MKV and 3FEQ). These proteins fold as distorted ({beta}/{alpha})8-barrels with two divalent cations in the active site. The structure of Sgx9260c was also determined as a complex with the N-methylphosphonate derivative of L-Pro (PDB code 3N2C). In this structure the phosphonate moiety bridges the binuclear metal center, and one oxygen atom interacts with His-140. The {alpha}-carboxylate of the inhibitor interacts with Tyr-231. The proline side chain occupies a small substrate binding cavity formed by residues contributed from the loop that follows {beta}-strand 7 within the ({beta}/{alpha})8-barrel. A total of 38 other proteins from cog1228 are predicted to have the same substrate profile based on conservation of the substrate binding residues. The structure of an evolutionarily related protein, Cc2672 from Caulobacter crecentus, was determined as a complex with the N-methylphosphonate derivative of L-arginine (PDB code 3MTW).« less

Structures of Adnectin/Protein Complexes Reveal an Expanded Binding Footprint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramamurthy, Vidhyashankar; Krystek, Jr., Stanley R.; Bush, Alexander

2014-10-02

Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin ({sup 10}Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three {sup 10}Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibodymore » combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the {beta} strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these {beta} strand interactions, indicating that these nonloop residues can expand the available binding footprint.« less

Effect of metformin therapy on circulating amino acids in a randomized trial: the CAMERA study.

PubMed

Preiss, D; Rankin, N; Welsh, P; Holman, R R; Kangas, A J; Soininen, P; Würtz, P; Ala-Korpela, M; Sattar, N

2016-11-01

To investigate whether metformin therapy alters circulating aromatic and branched-chain amino acid concentrations, increased levels amino acid concentrations, increased levels of which have been found to predict Type 2 diabetes. In the Carotid Atherosclerosis: Metformin for Insulin Resistance (CAMERA) study (NCT00723307), 173 individuals without Type 2 diabetes, but with coronary disease, were randomized to metformin (n=86) or placebo (n=87) for 18 months. Plasma samples, taken every 6 months, were analysed using quantitative nuclear magnetic resonance spectroscopy. Ten metabolites consisting of eight amino acids [three branched-chain (isoleucine, leucine, valine), three aromatic (tyrosine, phenylalanine, histidine) and two other amino acids (alanine, glutamine)], lactate and pyruvate were quantified and analysed using repeated-measures models. On-treatment analyses were conducted to investigate whether amino acid changes were dependent on changes in weight, fat mass or insulin resistance estimated using homeostasis model assessment (HOMA-IR). Tyrosine decreased [-6.1 μmol/l (95% CI -8.5, -3.7); P<0.0001], while alanine [42 umol/l (95% CI 25, 59); P<0.0001] increased in the metformin-treated group compared with the placebo-treated group. Decreases in phenylalanine [-2.0 μmol/l (95% CI -3.6, -0.3); P=0.018] and increases in histidine [2.3 μmol/l (95% CI 0.1, 4.6); P=0.045] were also observed in the metformin group, although these changes were less statistically robust. Changes in these four amino acids were not accounted for by changes in weight, fat mass or HOMA-IR values. Levels of branched-chain amino acids, glutamine, pyruvate and lactate were not altered by metformin therapy. Metformin therapy results in a sustained and specific pattern of changes in aromatic amino acid and alanine concentrations. These changes are independent of any effects on weight and insulin sensitivity. Any causal link to metformin's unexplained cardiometabolic benefit requires further study. © 2016 Diabetes UK.

Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris.

PubMed

Driessen, A J; Hellingwerf, K J; Konings, W N

1987-09-15

The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1.

Overexpression of SnoN/SkiL, amplified at the 3q26.2 locus, in ovarian cancers: A role in ovarian pathogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nanjundan, Meera; Cheng, Kwai Wa; Zhang, Fan

2008-07-18

High-resolution array comparative genomic hybridization of 235 serous epithelial ovarian cancers demonstrated a regional increase at 3q26.2 encompassing SnoN/SkiL, a coregulator of SMAD/TGF{beta} signaling. SnoN RNA transcripts were elevated in {approx}80% of advanced stage serous epithelial ovarian cancers. In both immortalized normal (TIOSE) and ovarian carcinoma cell lines (OVCA), SnoN RNA levels were increased by TGF{beta} stimulation and altered by LY294002 and JNK II inhibitor treatment suggesting that the PI3K and JNK signaling pathways may regulate TGF{beta}-induced increases in SnoN RNA. In TIOSE, SnoN protein levels were reduced 15min post TGF{beta}-stimulation, likely by proteosome-mediated degradation. In contrast, in OVCA, SnoNmore » levels were elevated 3h post-stimulation potentially as a result of inhibition of the proteosome. To elucidate the role of SnoN in ovarian tumorigenesis, we explored the effects of both increasing and decreasing SnoN levels. In both TIOSE and OVCA, SnoN siRNA decreased cell growth between 20 and 50% concurrent with increased p21 levels. In TIOSE, transient expression of SnoN repressed TGF{beta} induction of PAI-1 promoters with little effect on the p21 promoter or resultant cell growth. In contrast to the effects of transient expression, stable expression of SnoN in TIOSE led to growth arrest through induction of senescence. Collectively, these results implicate SnoN levels in multiple roles during ovarian carcinogenesis: promoting cellular proliferation in ovarian cancer cells and as a positive mediator of cell cycle arrest and senescence in non-transformed ovarian epithelial cells.« less

Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl-L-alanine amidase as a potential antimicrobial to control the bacterium

USDA-ARS?s Scientific Manuscript database

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to iden...

Effect of chronic hypo and hypervitaminosis C on the brush border enzymes and the intestinal uptake of glucose and alanine.

PubMed

Mahmood, A; Chauhan, V P; Lyall, V; Sarkar, A K

1979-08-15

Brush border sucrase and alkaline phosphatase activities are considerably enhanced in the intestine of ascorbic acid deficient guinea-pigs. Similar increase in the uptake of D-glucose and L-alanine also occurs in chronic vitamin C deficiency. However the permeability of D-glucose and L-alanine in the intestine of animals fed with large doses of vitamin C is severely depressed, with a reduction in the levels of sucrase and alkaline phosphatase activities.

Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

PubMed

Fujimi, T J; Nakajyo, T; Nishimura, E; Ogura, E; Tsuchiya, T; Tamiya, T

2003-08-14

The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

An Ultrasensitive Gold Nanoparticle-based Lateral Flow Test for the Detection of Active Botulinum Neurotoxin Type A

NASA Astrophysics Data System (ADS)

Liu, Jing; Gao, Shan; Kang, Lin; Ji, Bin; Xin, Wenwen; Kang, Jingjing; Li, Ping; Gao, Jie; Wang, Hanbin; Wang, Jinglin; Yang, Hao

2017-03-01

Botulism is a severe and potentially lethal paralytic disease caused by several botulinum neurotoxin-producing Clostridia spp. In China, the majority of the cases caused by botulism were from less-developed rural areas. Here, we designed specific substrate peptides and reconfigured gold nanoparticle-based lateral flow test strip (LFTS) to develop an endopeptidase-based lateral flow assay for the diagnosis of botulism. We performed this lateral flow assay on botulinum neurotoxin-spiked human serum samples. The as-prepared LFTS had excellent performance in the detection of botulinum neurotoxin using only 1 μL of simulated serum, and its sensitivity and specificity were comparable to that of mouse lethality assay. Moreover, the assay takes only half a day and does not require highly trained laboratory staff, specialized facility, or equipment. Finally, our LFTS can be potentially extended to other serotypes of BoNTs by designing specific substrate peptides against the different types of BoNTs. Overall, we demonstrate a strategy by which LFTS and endopeptidase activity assays can be integrated to achieve facile and economic diagnosis of botulism in resource-limited settings.

Extraterrestrial Amino Acids in the Almahata Sitta Meteorite

NASA Technical Reports Server (NTRS)

Glavin, Daniel P.; Aubrey, Andrew D.; Callahan, Michael P.; Dworkin, Jason P.; Elsila, Jamie E.; Parker, Eric T.; Bada, Jeffrey L.

2009-01-01

Amino acid analysis of a meteorite fragment of asteroid 2008 TC(sub 3) called Almahata Sitta was carried out using reverse-phase high-perfo rmance liquid chromatography coupled with UV fluorescence detection a nd time-of-flight mass spectrometry (HPLC-FD/ToF-MS) as part of a sam ple analysis consortium. HPLC analyses of hot-water extracts from the meteorite revealed a complex distribution of two- to six-carbon aliph atic amino acids and one- to three carbon amines with abundances rang ing from 0.5 to 149 parts-per-billion (ppb). The enantiomeric ratios of the amino acids alanine, Beta-amino-n-butyric acid (Beta-ABA), 2-amino-2- methylbutanoic acid (isovaline), and 2-aminopentanoic acid (no rvaline) in the meteorite were racemic (D/L approximately 1), indicat ing that these amino acids are indigenous to the meteorite and not te rrestrial contaminants. Several other non-protein amino acids were also identified in the meteorite above background levels including alpha -aminoisobutyric acid (alpha-AIB), 4-amino-2- methybutanoic acid, 4-a mino-3-methylbutanoic acid, and 3-, 4-, and 5-aminopentanoic acid. Th e total abundances of isovaline and AlB in Almahata Sitta are approximately 1000 times lower than the abundances of these amino acids found in the CM carbonaceous meteorite Murchison. The extremely love abund ances and unusual distribution of five carbon amino acids in Almahata Sitta compared to Cl, CM, and CR carbonaceous meteorites and may be due to extensive thermal alteration of amino acids on the parent aster oid by partial melting during formation or impact shock heating.

Alpha-helical hydrophobic polypeptides form proton-selective channels in lipid bilayers

NASA Technical Reports Server (NTRS)

Oliver, A. E.; Deamer, D. W.

1994-01-01

Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two populations of conductive events, one in the 0.1-0.5 pA range, and one in the 1.0-5.0 pA range, whereas nearly all events caused by poly-L-alanine were in the 0.1-0.5 pA range at an applied voltage of +60 mV. The channel-like activity appeared to switch between conductive and nonconductive states, with most open-times in the range of 50-200 ms. We conclude that hydrophobic polyamino acids produce proton-conducting defects in lipid bilayers that may be used to model functional proton channels in biological membranes.

[Chiral separation of five beta-blockers using di-n-hexyl L-tartrate-boric acid complex as mobile phase additive by reversed-phase liquid chromatography].

PubMed

Yang, Juan; Wang, Lijuan; Guo, Qiaoling; Yang, Gengliang

2012-03-01

A reversed-phase high performance liquid chromatographic (HPLC) method using the di-n-hexyl L-tartrate-boric acid complex as a chiral mobile phase additive was developed for the enantioseparation of five beta-blockers including propranolol, esmolol, metoprolol, bisoprolol and sotalol. In order to obtain a better enantioseparation, the influences of concentrations of di-n-butyl L-tartrate and boric acid, the type, concentration and pH of the buffer, methanol content as well as the molecular structure of analytes were extensively investigated. The separation of the analytes was performed on a Venusil MP-C18 column (250 mm x 4.6 mm, 5 microm). The mobile phase was 15 mmol/L ammonium acetate-methanol containing 60 mmol/L boric acid, 70 mmol/L di-n-hexyl L-tartrate (pH 6.00). The volume ratios of 15 mmol/L ammonium acetate to methanol were 20: 80 for propranolol, esmolol, metoprolol, bisoprolol and 30: 70 for sotalol. The flow rate was 0.5 mL/min and the detection wavelength was set at 214 nm. Under the optimized conditions, baseline enantioseparation was obtained separately for the five pairs of analytes.

Interferon-alpha receptor 1 mRNA expression in peripheral blood mononuclear cells is associated with response to interferon-alpha therapy of patients with chronic hepatitis C.

PubMed

Massirer, K B; Hirata, M H; Silva, A E B; Ferraz, M L G; Nguyen, N Y; Hirata, R D C

2004-05-01

Interferon (IFN)-alpha receptor mRNA expression in liver of patients with chronic hepatitis C has been shown to be a response to IFN-alpha therapy. The objective of the present study was to determine whether the expression of mRNA for subunit 1 of the IFN-alpha receptor (IFNAR1) in peripheral blood mononuclear cells (PBMC) is associated with the response to IFN-alpha in patients with chronic hepatitis C. Thirty patients with positive anti-HCV and HCV-RNA, and abnormal levels of alanine aminotransferase in serum were selected and treated with IFN-alpha 2b for one year. Those with HBV or HIV infection, or using alcohol were not included. Thirteen discontinued the treatment and were not evaluated. The IFN-alpha response was monitored on the basis of alanine aminotransferase level and positivity for HCV-RNA in serum. IFNAR1-mRNA expression in PBMC was measured by reverse transcription-polymerase chain reaction before and during the first three months of therapy. The results are reported as IFNAR1-mRNA/beta-actin-mRNA ratio (mean +/- SD). Before treatment, responder patients had significantly higher IFNAR1-mRNA expression in PBMC (0.67 +/- 0.15; N = 5; P < 0.05) compared to non-responders (0.35 +/- 0.17; N = 12) and controls (0.30 +/- 0.16; N = 9). Moreover, IFNAR1-mRNA levels were significantly reduced after 3 months of treatment in responders, whereas there were no differences in IFNAR1 expression in non-responders during IFN-alpha therapy. Basal IFNAR1-mRNA expression was not correlated with the serum level of alanine and aspartate aminotransferases or the presence of cirrhosis. The present results suggest that IFNAR1-mRNA expression in PBMC is associated with IFN-alpha response to hepatitis C and may be useful for monitoring therapy in patients with chronic hepatitis C.

Atherosclerosis and Liver Function Tests in Coronary Angiography Patients.

PubMed

Doganer, Y C; Rohrer, J E; Aydogan, U; Agerter, D C; Cayci, T; Barcin, C

2015-09-01

Elevated aminotransferase levels indicating liver function, even in the normal range, have attracted great concern as potential novel markers of cardiovascular risk assessment. We hypothesized the possibility that liver function test variations in the normal range might be meaningfully associated to coronary artery disease (CAD). Eighty-eight patients were randomly selected from those who underwent coronary angiography from June 2010 to June 2011 after applying to the outpatient cardiology clinic in Gulhane Military Medical Academy. According to the results of angiographies, patients were classified into three groups as normal, non-critical (< 50% involvement in coronaries), and critical (≥ 50% involvement in coronaries). In addition to angiographic intervention, measurements of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations, albumin and the other serum parameters were performed in all patients. The patient groups of CAD were balanced (28 critical cases, 30 non-critical cases and 30 normal cases). Mean age was 51.93 ± 9.3 (range 32-65) years and 19.3 per cent (n = 17) were females. Multiple linear regression analysis of all three liver function tests explained a significant portion of the variance, but adjusted r-squares were small (AST = 0.174, ALT = 0.242, albumin = 0.124). Albumin was significantly higher for patients with critical CAD than for patients with no CAD (beta = 3.205, p = 0.002). Non-critical CAD was not significantly different from no CAD for any of the dependent variables. Mean AST was significantly higher for patients taking aspirin (beta = 0.218, p = 0.049), as was mean ALT (beta = 0.264, p = 0.015). Alanine aminotransferase and AST may not be associated with angiographically determined coronary atherosclerosis. Albumin may be more sensitive to demonstrate the burden of atherosclerosis. These results indicate that the association between the liver function tests and coronary atherosclerosis may be more complex than generally appreciated.

Evaluation of drug-induced tissue injury by measuring alanine aminotransferase (ALT) activity in silkworm hemolymph

PubMed Central

2012-01-01

Background Our previous studies suggest silkworms can be used as model animals instead of mammals in pharmacologic studies to develop novel therapeutic medicines. We examined the usefulness of the silkworm larvae Bombyx mori as an animal model for evaluating tissue injury induced by various cytotoxic drugs. Drugs that induce hepatotoxic effects in mammals were injected into the silkworm hemocoel, and alanine aminotransferase (ALT) activity was measured in the hemolymph 1 day later. Results Injection of CCl4 into the hemocoel led to an increase in ALT activity. The increase in ALT activity was attenuated by pretreatment with N-acetyl-L-cysteine. Injection of benzoic acid derivatives, ferric sulfate, sodium valproate, tetracycline, amiodarone hydrochloride, methyldopa, ketoconazole, pemoline (Betanamin), N-nitroso-fenfluramine, and D-galactosamine also increased ALT activity. Conclusions These findings indicate that silkworms are useful for evaluating the effects of chemicals that induce tissue injury in mammals. PMID:23137391

Membrane transport of amino acid enantiomers in protoscoleces of Echinococcus granulosus (Cestoda).

PubMed

Allen, J T; Arme, C

1991-02-01

Protoscoleces of Echinococcus granulosus absorb both L- and D-alanine. Concentration ratios exceed 1 with values for D-alanine exceeding those for the L-isomer, suggesting that both are absorbed by active mechanisms. Uptake of both isomers involves both diffusion and carrier-mediated components. Values for the diffusion component (Kd) for L- and D-alanine were 0.21 and 0.38 nmol mg-1 protein/1.5 min mM-1 respectively, and values for Kt, the transport constants, 0.17 mM and 0.21 mM respectively. Uptake of both isomers was inhibited competitively by a number of other amino acids.

Biochemical characterization of an inhibitor of Escherichia coli UDP-N-acetylmuramyl-l-alanine ligase.

PubMed

Ehmann, David E; Demeritt, Julie E; Hull, Kenneth G; Fisher, Stewart L

2004-05-06

UDP-N-acetylmuramyl-l-alanine ligase (MurC) is an essential bacterial enzyme involved in peptidoglycan biosynthesis and a target for the discovery of novel antibacterial agents. As a result of a high-throughput screen (HTS) against a chemical library for inhibitors of MurC, a series of benzofuran acyl-sulfonamides was identified as potential leads. One of these compounds, Compound A, inhibited Escherichia coli MurC with an IC(50) of 2.3 microM. Compound A exhibited time-dependent, partially reversible inhibition of E. coli MurC. Kinetic studies revealed a mode of inhibition consistent with the compound acting competitively with the MurC substrates ATP and UDP-N-acetyl-muramic acid (UNAM) with a K(i) of 4.5 microM against ATP and 6.3 microM against UNAM. Fluorescence binding experiments yielded a K(d) of 3.1 microM for the compound binding to MurC. Compound A also exhibited high-affinity binding to bovine serum albumin (BSA) as evidenced by a severe reduction in MurC inhibition upon addition of BSA. This finding is consistent with the high lipophilicity of the compound. Advancement of this compound series for further drug development will require reduction of albumin binding.

R76 in transmembrane domain 3 of the aspartate:alanine transporter AspT is involved in substrate transport.

PubMed

Suzuki, Satomi; Nanatani, Kei; Abe, Keietsu

2016-01-01

The L-aspartate:L-alanine antiporter of Tetragenococcus halophilus (AspT) possesses an arginine residue (R76) within the GxxxG motif in the central part of transmembrane domain 3 (TM3)-a residue that has been estimated to transport function. In this study, we carried out amino acid substitutions of R76 and used proteoliposome reconstitution for analyzing the transport function of each substitution. Both l-aspartate and l-alanine transport assays showed that R76K has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than the AspT-WT. These results suggest that R76 is involved in AspT substrate transport.

A novel antagonist, phenylbenzene omega-phosphono-alpha-amino acid, for strychnine-sensitive glycine receptors in the rat spinal cord.

PubMed Central

Saitoh, T; Ishida, M; Maruyama, M; Shinozaki, H

1994-01-01

1. 3-[2'-Phosphonomethyl[1,1'-biphenyl]-3-yl]alanine (PMBA) is a novel glycine antagonist at strychnine-sensitive receptors. The chemical structure of PMBA, possessing both a glycine moiety and a phosphono group, is quite different from that of strychnine. 2. In the spinal motoneurone of newborn rats, glycine (100 microM-1 mM) induced depolarizing responses in a concentration-dependent manner. PMBA effectively inhibited depolarizing responses to glycine and other agonists, such as taurine and beta-alanine. The dose-response curves for glycine were shifted to the right in an almost parallel manner (pA2 value: 5.30 +/- 0.23, n = 5) by PMBA which was about 60 times less potent than strychnine (pA2 value: 7.08 +/- 0.21, n = 5) as a glycine antagonist. 3. PMBA (1-100 microM) did not interact with modulatory glycine sites on N-methyl-D-aspartate (NMDA) receptors, which suggests a high selectivity of PMBA for strychnine-sensitive glycine receptors. At considerably high concentrations (0.1 mM-1 mM), PMBA depressed responses to GABA (pA2 value: 3.57 +/- 0.24, n = 3). 4. PMBA inhibited the binding of [3H]-strychnine to synaptosomes from adult rat spinal cords; the IC50 values of PMBA, glycine and strychnine were 8 +/- 2, 9 +/- 3 and 0.08 +/- 0.04 microM, respectively (n = 5) for [3H]-strychnine (4.8 nM). 5. PMBA is a central excitant drug with relatively high potency and selectivity and should be useful as a pharmacological probe for analysing the mechanisms underlying physiological functions of glycine receptors. PMID:7812607

Beta-oxidation as channeled reaction linked to citric acid cycle: evidence from measurements of mitochondrial pyruvate oxidation during fatty acid degradation.

PubMed

Förster, M E; Staib, W

1992-07-01

1. The kinetics of mitochondrial mammalian pyruvate dehydrogenase multienzyme complex (PDHC) is studied by the formation of CO2 using tracer amounts of [1-14C]pyruvate. It is found that the Hill plot results in a (pseudo-)cooperativity with a transition of n-1----3 at a pyruvate concentration about Ks. 2. Addition of L-carnitine, octanoate, palmitoyl-CoA or palmitate + L-carnitine + fatty acid-binding protein results in a Hill coefficient of n = 2 following the kinetics of pyruvate oxidation. 3. Addition of fatty acid-binding protein to an assay system oxidizing palmitate in presence of L-carnitine alters the pattern of the kinetics in the Hill plot so that an apparently lower level of L-carnitine is necessary for the reaction course of beta-degradation. 4. It is concluded that beta-degradation is a coordinated, multienzyme-complex based mechanism tightly linked to citric acid cycle and it is proposed that L-carnitine is actively involved into the reaction and not only functioning as carrier-molecule for transmembrane transport.

Glycogen synthase kinase-3beta (GSK-3beta) inhibitors AR-A014418 and B6B3O prevent human immunodeficiency virus-mediated neurotoxicity in primary human neurons.

PubMed

Nguyen, Timothy B; Lucero, Ginger R; Chana, Gursharan; Hult, Britta J; Tatro, Erick T; Masliah, Eliezer; Grant, Igor; Achim, Cristian L; Everall, Ian P

2009-09-01

Glycogen synthase kinase-3beta (GSK3beta) role in human immunodeficiency virus(HIV)-associated neurodegeneration has been evidenced by previous investigations. In this study, we investigated the specificity of two GSK3beta-specific inhibitors, AR-A014418 (A) and B6B30 (B) to prevent direct neurotoxicity in primary human neurons exposed to HIV (BaL). Neurons were exposed to HIV (500 pg/ml) for 12-h and 6-day periods in the presence and absence of A (1 microM, 100 nM, 10 nM) and B (50 nM, 5 nM, 500 pM) to investigate acute and ongoing mechanisms of HIV neurotoxicity. Using an lactate dehydrogenase (LDH) assay to assess cytotoxicity, we observed a significant neurotoxic effect of HIV from control values (P < .01) that was not restored via coexposures of all concentrations of A and B. Additionally, no change in LDH levels were observed after 6 days. However, activity of the acute proapoptotic markers caspases 3 and 7 using a luminescence assay were measured and found to be increased by exposure to HIV (BaL) compared to controls (P = .022). This effect was ameliorated via coexposure to all concentrations of A and 50 nM B after 12 h (P < .01) and to all concentrations of A and B after 6 days (P < .01). Overall, the results from this study provide further evidence for the ability of GSK3beta inhibition to be neuroprotective against HIV-associated neurotoxicity by reducing HIV associated procaspase induction. These data support a role for GSK3beta as a potential therapeutic target and may have important clinical implications for treatment of HIV-associated neurocognitive disorder.

Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by site-directed mutagenesis in the UDP-MurNAc:L-alanine ligase from Escherichia coli.

PubMed

Bouhss, A; Mengin-Lecreulx, D; Blanot, D; van Heijenoort, J; Parquet, C

1997-09-30

The comparison of the amino acid sequences of 20 cytoplasmic peptidoglycan synthetases (MurC, MurD, MurE, MurF, and Mpl) from various bacterial organisms has allowed us to detect common invariants: seven amino acids and the ATP-binding consensus sequence GXXGKT/S all at the same position in the alignment. The Mur synthetases thus appeared as a well-defined class of closely functionally related proteins. The conservation of a constant backbone length between certain invariants suggested common structural motifs. Among the other enzymes catalyzing a peptide bond formation driven by ATP hydrolysis to ADP and Pi, only folylpoly-gamma-l-glutamate synthetases presented the same common conserved amino acid residues, except for the most N-terminal invariant D50. Site-directed mutageneses were carried out to replace the K130, E174, H199, N293, N296, R327, and D351 residues by alanine in the MurC protein from Escherichia coli taken as model. For this purpose, plasmid pAM1005 was used as template, MurC being highly overproduced in this genetic setting. Analysis of the Vmax values of the mutated proteins suggested that residues K130, E174, and D351 are essential for the catalytic process whereas residues H199, N293, N296, and R327 were not. Mutations K130A, H199A, N293A, N296A, and R327A led to important variations of the Km values for one or more substrates, thereby indicating that these residues are involved in the structure of the active site and suggesting that the binding order of the substrates could be ATP, UDP-MurNAc, and alanine. The various mutated murC plasmids were tested for their effects on the growth, cell morphology, and peptidoglycan cell content of a murC thermosensitive strain at 42 degrees C. The observed effects (complementation, altered morphology, and reduced peptidoglycan content) paralleled more or less the decreased values of the MurC activity of each mutant.

Complete structural characterization of the lipid A fraction of a clinical strain of B. cepacia genomovar I lipopolysaccharide.

PubMed

Silipo, Alba; Molinaro, Antonio; Cescutti, Paola; Bedini, Emiliano; Rizzo, Roberto; Parrilli, Michelangelo; Lanzetta, Rosa

2005-05-01

Burkholderia cepacia, a Gram-negative bacterium ubiquitous in the environment, is a plant pathogen causing soft rot of onions. This microorganism has recently emerged as a life-threatening multiresistant pathogen in cystic fibrosis patients. An important virulence factor of B. cepacia is the lipopolysaccharide (LPS) fraction. Clinical isolates and environmental strains possess LPS of high inflammatory nature, which induces a high level production of cytokines. For the first time, the complete structure of the lipid A components isolated from the lipopolysaccharide fraction of a clinical strain of B. cepacia is described. The structural studies carried out by selective chemical degradations, MS, and NMR spectroscopy revealed multiple species differing in the acylation and in the phosphorylation patterns. The highest mass species was identified as a penta-acylated tetrasaccharide backbone containing two phosphoryl-arabinosamine residues in addition to the archetypal glucosamine disaccharide [Arap4N-l-beta-1-P-4-beta-D-GlcpN-(1-6)-alpha-D-GlcpN-1-P-1-beta-L-Arap4N]. Lipid A fatty acids substitution was also deduced, with two 3-hydroxytetradecanoic acids 14:0 (3-OH) in ester linkage, and two 3-hydroxyhexadecanoic acids 16:0 (3-OH) in amide linkage, one of which was substituted by a secondary 14:0 residue at its C-3. Other lipid A species present in the mixture and exhibiting lower molecular weight lacked one or both beta-L-Arap4N residues.

Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria.

PubMed

Martino, P Di; Fursy, R; Bret, L; Sundararaju, B; Phillips, R S

2003-07-01

We demonstrated previously that genetic inactivation of tryptophanase is responsible for a dramatic decrease in biofilm formation in the laboratory strain Escherichia coli S17-1. In the present study, we tested whether the biochemical inhibition of tryptophanase, with the competitive inhibitor oxindolyl-L-alanine, could affect polystyrene colonization by E. coli and other indole-producing bacteria. Oxindolyl-L-alanine inhibits, in a dose-dependent manner, indole production and biofilm formation by strain S17-1 grown in Luria-Bertani (LB) medium. Supplementation with indole at physiologically relevant concentrations restores biofilm formation by strain S17-1 in the presence of oxindolyl-L-alanine and by mutant strain E. coli 3714 (S17-1 tnaA::Tn5) in LB medium. Oxindolyl-L-alanine also inhibits the adherence of S17-1 cells to polystyrene for a 3-h incubation time, but mutant strain 3714 cells are unaffected. At 0.5 mg/mL, oxindolyl-L-alanine exhibits inhibitory activity against biofilm formation in LB medium and in synthetic urine for several clinical isolates of E. coli, Klebsiella oxytoca, Citrobacter koseri, Providencia stuartii, and Morganella morganii but has no affect on indole-negative Klebsiella pneumoniae strains. In conclusion, these data suggest that indole, produced by the action of tryptophanase, is involved in polystyrene colonization by several indole-producing bacterial species. Indole may act as a signalling molecule to regulate the expression of adhesion and biofilm-promoting factors.

Elucidating the pH-Dependent Structural Transition of T7 Bacteriophage Endolysin.

PubMed

Sharma, Meenakshi; Kumar, Dinesh; Poluri, Krishna Mohan

2016-08-23

Bacteriophages are the most abundant and diverse biological entities on earth. Bacteriophage endolysins are unique peptidoglycan hydrolases and have huge potential as effective enzybiotics in various infectious models. T7 bacteriophage endolysin (T7L), also known as N-acetylmuramoyl-l-alanine amidase or T7 lysozyme, is a 17 kDa protein that lyses a range of Gram-negative bacteria by hydrolyzing the amide bond between N-acetylmuramoyl residues and the l-alanine of the peptidoglycan layer. Although the activity profiles of several of the T7 family members have been known for many years, the molecular basis for their pH-dependent differential activity is not clear. In this study, we explored the pH-induced structural, stability, and activity characteristics of T7L by applying a variety of biophysical techniques and protein nuclear magnetic resonance (NMR) spectroscopy. Our studies established a reversible structural transition of T7L below pH 6 and the formation of a partially denatured conformation at pH 3. This low-pH conformation is thermally stable and exposed its hydrophobic pockets. Further, NMR relaxation measurements and structural analysis unraveled that T7L is highly dynamic in its native state and a network of His residues are responsible for the observed pH-dependent conformational dynamics and transitions. As bacteriophage chimeric and engineered endolysins are being developed as novel therapeutics against multiple drug resistance pathogens, we believe that our results are of great help in designing these entities as broadband antimicrobial and/or antibacterial agents.

Detection of buphedrone in biological and non-biological material--two case reports.

PubMed

Zuba, Dariusz; Adamowicz, Piotr; Byrska, Bogumiła

2013-04-10

Buphedrone (2-(methylamino)-1-phenylbutan-1-one, α-methylamino-butyrophenone, MABP) is a positional isomer of mephedrone. In Poland, it was marketed in the second half of 2010 after the banning of mephedrone. Buphedrone is a stimulant that is snorted, smoked or taken orally. This substance was identified in 15 products seized by law enforcement after August 2010 and analysed in the Institute of Forensic Research (IFR). Buphedrone was the sole psychoactive substance in only 5 products. It was mixed mainly with 4-MEC and MDPV. This paper presents two cases in which both biological and non-biological materials were delivered to the IFR for toxicological analysis. In the first case, a passenger car crashed into a truck. The car driver suffered severe injuries resulting in his death. During external inspection of the deceased the police discovered several packages containing a white powder. In the second case, a man was arrested for possession of illicit drugs. Analysis of powders was carried out using gas chromatography-mass spectrometry (GC-MS) and high-pressure liquid chromatography with diode array detection (HPLC-DAD). The purity of buphedrone found in powder samples was in the range of 58-68%. Analyses of blood were carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Buphedrone was found in the blood of the deceased at a concentration of 127 ng/mL and of the drug user/seller at 3 ng/mL. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The catalytic effect of L- and D-histidine on alanine and lysine peptide formation.

PubMed

Fitz, Daniel; Jakschitz, Thomas; Rode, Bernd M

2008-12-01

A starting phase of chemical evolution on our ancient Earth around 4 billion years ago was the formation of amino acids and their combination to peptides and proteins. The salt-induced peptide formation (SIPF) reaction has been shown to be appropriate for this condensation reaction under moderate and plausible primitive Earth conditions, forming short peptides from amino acids in aqueous solution containing sodium chloride and Cu(II) ions. In this paper we report results about the formation of dialanine and dilysine from their monomers in this reaction. The catalytic influence of l- and d-histidine dramatically increases dialanine yields when starting from lower alanine concentrations, but also dilysine formation is markedly boosted by these catalysts. Attention is paid to measurable preferences for one enantiomeric form of alanine and lysine in the SIPF reaction. Alanine, especially, shows stereospecific behaviour, mostly in favour of the l-form.

Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors

PubMed Central

Farroni, Jeffrey S; McCool, Brian A

2004-01-01

Background Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. Results While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for β-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Conclusions Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology. PMID:15301692

Year-long changes in protein metabolism in elderly men and women supplemented with a nutrition cocktail of beta-hydroxy-beta-methylbutyrate (HMB), L-arginine, and L-lysine.

PubMed

Baier, Shawn; Johannsen, Darcy; Abumrad, Naji; Rathmacher, John A; Nissen, Steven; Flakoll, Paul

2009-01-01

A major contributing factor to the loss of mobility in elderly people is the gradual and continuous loss of lean body mass. To determine whether supplementation of an amino acid cocktail daily for 1 year could improve the age-associated changes in protein turnover and lean body mass in elderly people. Elderly (76+/-1.6 years) women (n=39) and men (n=38) were recruited for a double-blinded controlled study. Study participants were randomly assigned to either an isonitrogenous control-supplement (n=37) or a treatment-supplement (HMB/Arg/Lys) consisting of beta-hydroxy-beta-methylbutyrate, L-arginine, and L-lysine (n=40) for the 1-year study. Lean tissue mass was measured using both bioelectrical-impedance analysis (BIA) and dual energy x-ray absorptiometry (DXA). Rates of whole-body protein turnover were estimated using primed/intermittent oral doses of 15N-glycine. In subjects taking the HMB/Arg/Lys supplement, lean tissue increased over the year of study while in the control group, lean tissue did not change. Compared with control, HMB/Arg/Lys increased body cell mass (BIA) by 1.6% (P=.002) and lean mass (DXA) by 1.2% (P=.05). The rates of protein turnover were significantly increased 8% and 12% in the HMB/Arg/Lys-supplemented group while rates of protein turnover decreased 11% and 9% in the control-supplemented subjects (P<.01), at 3 and 12 months, respectively. Consumption of a simple amino acid-related cocktail increased protein turnover and lean tissue in elderly individuals in a year-long study.

[Synthesis and biological activity of 2,3-secotriterpene acid mono- and diamides].

PubMed

Tolmacheva, I A; Igosheva, E V; Vikharev, Iu B; Grishko, V V; Savinova, O V; Boreko, E I; Eremin, V F

2013-01-01

Four types of amide (C3; C28; C3-C28) conjugates based on 2,3-seco-18alphaH-oleanane and 2,3-secolupane mono- and dicarboxylic acids were synthesized. The range of diamide derivatives was supplemented with C3-C3' and C28-C28' dicondensed amides with two A-secotriterpene backbones educed by reacting monocarboxylic A-secoacids with biogenic amino acid lysine. Compounds with inhibitory action against herpes virus reproduction (EC50 8.7 and 4.1 McM) were found among the synthesized mono- and diamide derivatives containing an ethyl-beta-alaninate fragment. It has been ascertained that diamide with ethyl-beta-alaninate fragment combines anti-herpes virus properties and anti-HIV activity (EC50 5.1 McM). For active compounds, the maximum non-toxic concentration (MNTC)/EC50 ratios ranges from 9.7 to 40.8. The synthesized amide conjugates do not exhibit any marked cytotoxic effects against human tumor cell lines rabdomiosarcoma RD TE32, A549 lung carcinoma and melanoma MS.

Physiological and chemical analysis of neurotransmitter candidates at a fast excitatory synapse in the jellyfish Cyanea capillata (Cnidaria, Scyphozoa).

PubMed

Anderson, Peter A V; Trapido-Rosenthal, H G

2009-12-01

Motor nerve net (MNN) neurons in the jellyfish Cyanea capillata communicate with one another by way of fast, bidirectional excitatory chemical synapses. As is the case with almost all identified chemical synapses in cnidarians, the identity of the neurotransmitter at these synapses is unclear. MNN neurons are large enough for stable intracellular recordings. This, together with the fact that they can be exposed, providing unlimited access to them and to their synapses, prompted a study of the action of a variety of neurotransmitter candidates, including those typically associated with fast synapses in higher animals. Only the amino acids taurine and beta-alanine produced physiological responses consistent with those of the normal EPSP in these cells. Moreover, chemical analysis revealed that both taurine and beta-alanine are present in the neurons and released by depolarization. These various findings strongly suggest that either or both of these amino acids, or a closely related compound is the neurotransmitter at the fast chemical synapses between MNN neurons.

Solute kinetics with short-daily home hemodialysis using slow dialysate flow rate.

PubMed

Kohn, Orly F; Coe, Fredric L; Ing, Todd S

2010-01-01

"NxStage System One()" is increasingly used for daily home hemodialysis. The ultrapure dialysate volumes are typically between 15 L and 30 L per dialysis, substantially smaller than the volumes used in conventional dialysis. In this study, the impact of the use of low dialysate volumes on the removal rates of solutes of different molecular weights and volumes of distribution was evaluated. Serum measurements before and after dialysis and total dialysate collection were performed over 30 times in 5 functionally anephric patients undergoing short-daily home hemodialysis (6 d/wk) over the course of 8 to 16 months. Measured solutes included beta(2) microglobulin (beta(2)M), phosphorus, urea nitrogen, and potassium. The average spent dialysate volume (dialysate plus ultrafiltrate) was 25.4+/-4.7 L and the dialysis duration was 175+/-15 min. beta(2) microglobulin clearance of the polyethersulfone dialyzer averaged 53+/-14 mL/min. Total beta(2)M recovered in the dialysate was 106+/-42 mg per treatment (n=38). Predialysis serum beta(2)M levels remained stable over the observation period. Phosphorus removal averaged 694+/-343 mg per treatment with a mean predialysis serum phosphorus of 5.2+/-1.8 mg/dL (n=34). Standard Kt/V averaged 2.5+/-0.3 per week and correlated with the dialysate-based weekly Kt/V. Weekly beta(2)M, phosphorus, and urea nitrogen removal in patients dialyzing 6 d/wk with these relatively low dialysate volumes compared favorably with values published for thrice weekly conventional and with short-daily hemodialysis performed with machines using much higher dialysate flow rates. Results of the present study were achieved, however, with an average of 17.5 hours of dialysis per week.

Ultraviolet radiation induces stress in etiolated Landoltia punctata, as evidenced by the presence of alanine, a universal stress signal: a ¹⁵N NMR study.

PubMed

Monselise, E B-I; Levkovitz, A; Kost, D

2015-01-01

Analysis with (15) N NMR revealed that alanine, a universal cellular stress signal, accumulates in etiolated duckweed plants exposed to 15-min pulsed UV light, but not in the absence of UV irradiation. The addition of 10 mm vitamin C, a radical scavenger, reduced alanine levels to zero, indicating the involvement of free radicals. Free D-alanine was detected in (15) N NMR analysis of the chiral amino acid content, using D-tartaric acid as solvent. The accumulation of D-alanine under stress conditions presents a new perspective on the biochemical processes taking place in prokaryote and eukaryote cells. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Computer modeling of the neurotoxin binding site of acetylcholine receptor spanning residues 185 through 196

NASA Technical Reports Server (NTRS)

Garduno-Juarez, R.; Shibata, M.; Zielinski, T. J.; Rein, R.

1987-01-01

A model of the complex between the acetylcholine receptor and the snake neurotoxin, cobratoxin, was built by molecular model building and energy optimization techniques. The experimentally identified functionally important residues of cobratoxin and the dodecapeptide corresponding to the residues 185-196 of acetylcholine receptor alpha subunit were used to build the model. Both cis and trans conformers of cyclic L-cystine portion of the dodecapeptide were examined. Binding residues independently identified on cobratoxin are shown to interact with the dodecapeptide AChR model.

Growth and toxin production by Clostridium botulinum in steamed rice aseptically packed under modified atmosphere.

PubMed

Kasai, Yoshiaki; Kimura, Bon; Kawasaki, Susumu; Fukaya, Tetsuya; Sakuma, Kinya; Fujii, Tateo

2005-05-01

Sales and consumption of ready-to-eat aseptic steamed rice products have increased manyfold in Japan over the past 10 years. To determine the safety of steamed rice (water content 60%, pH 6.5) aseptically packaged under modified atmosphere, challenge studies were performed using a mixture of Clostridium botulinum proteolytic strains (five strains of type A and five strains of type B). Atmospheric conditions of 0 and 15% oxygen (with 5% CO2 and 5% N2 as the balance) were used. No neurotoxins were detected, and organoleptically acceptable conditions persisted for 24 weeks at 15% oxygen conditions. However, botulinum neurotoxin was found in one of three samples at 12 weeks and in one of two samples at 24 weeks at 0% oxygen and 30 degrees C. When samples were inoculated with C. botulinum with amylase (0% oxygen), neurotoxin and sample spoilage was detected after only 1 week of storage. Challenge studies using proteolytic strains of C. botulinum mixed with Bacillus subtilis (amylase formers) also were performed with atmosphere conditions of oxygen at 0, 5, 10, and 15% (with 5% CO2 and 5% N2 as the balance). Under 10 and 15% oxygen conditions, neurotoxin was not detected after 1 week of storage, but sample spoilage was detected after the same period. Under 0% oxygen conditions, neurotoxin was detected at 1 week, but the sample remained organoleptically acceptable even after 2 weeks of storage. Both neurotoxin and sample spoilage were detected at 1 week of storage under 5% oxygen conditions. Based on these results, cocontamination of amylase-producing Bacillus with C. botulinum would increase the risk of foodborne botulism when aseptic rice samples are packed under low-oxygen conditions (<5%). Therefore, to ensure the safety of these products, packing under atmospheric containing more than 10% oxygen is recommended.

Low breast milk TGF-beta2 is induced by Lactobacillus reuteri supplementation and associates with reduced risk of sensitization during infancy.

PubMed

Böttcher, Malin Fagerås; Abrahamsson, Thomas Robert; Fredriksson, Mats; Jakobsson, Ted; Björkstén, Bengt

2008-09-01

The immunological composition of breast milk differs between mothers. The reasons for these differences and the consequences for the breast-fed infants are poorly understood. The aim of this study was to evaluate the effect of probiotic Lactobacillus reuteri supplementation on the immunological composition of breast milk in relation to sensitization and eczema in the babies. Total IgA, secretory IgA (SIgA), TGF-beta1, TGF-beta2, IL-10, TNF, soluble CD14 (sCD14), and Na/K ratios were analyzed in colostrum and mature milk obtained from women treated with L. reuteri (n = 54) or placebo (n = 55) from gestational week 36 until delivery. Bacteriological analyses of L. reuteri were performed in faecal samples of the mothers. The infants were followed prospectively for 2 yr regarding development of eczema and sensitization as defined by a positive skin prick test and/or circulating allergen-specific IgE antibodies at 6, 12, and 24 months of age. Supplementation of L. reuteri during pregnancy was associated with low levels of TGF-beta2 and slightly increased levels of IL-10 in colostrum. For TGF-beta2, this association was most pronounced in mothers with detectable L. reuteri in faeces. Infants receiving breast milk with low levels of TGF-beta2 were less likely to become sensitized during their first 2 yr of life. A similar trend was observed for development of IgE-associated eczema. The levels of total IgA, SIgA, TGF-beta1, TNF, sCD14, and Na/K ratios in breast milk were not affected by the intake of L. reuteri. None of these parameters correlated with sensitization or development of eczema in the infant, except for high Na/K ratios that associated with increased risk of sensitization. Supplementation with L. reuteri during late pregnancy reduces breast milk levels of TGF-beta2, and low levels of this cytokine are associated with less sensitization and possibly less IgE-associated eczema in breast-fed infants.

Low alanine aminotransferase levels and higher number of cardiovascular events in people with Type 2 diabetes: analysis of the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study.

PubMed

Williams, K H; Sullivan, D R; Veillard, A S; O'Brien, R; George, J; Jenkins, A J; Young, S; Ehnholm, C; Duffield, A; Twigg, S M; Keech, A C

2016-03-01

To determine whether alanine aminotransferase or gamma-glutamyltransferase levels, as markers of liver health and non-alcoholic fatty liver disease, might predict cardiovascular events in people with Type 2 diabetes. Data from the Fenofibrate Intervention and Event Lowering in Diabetes study were analysed to examine the relationship between liver enzymes and incident cardiovascular events (non-fatal myocardial infarction, stroke, coronary and other cardiovascular death, coronary or carotid revascularization) over 5 years. Alanine aminotransferase measure had a linear inverse relationship with the first cardiovascular event occurring in participants during the study period. After adjustment, for every 1 sd higher baseline alanine aminotransferase measure (13.2 U/l), the risk of a cardiovascular event was 7% lower (95% CI 4-13; P = 0.02). Participants with alanine aminotransferase levels below and above the reference range 8-41 U/l for women and 9-59 U/l for men, had hazard ratios for a cardiovascular event of 1.86 (95% CI 1.12-3.09) and 0.65 (95% CI 0.49-0.87), respectively (P = 0.001). No relationship was found for gamma-glutamyltransferase. The data may indicate that in people with Type 2 diabetes, which is associated with higher alanine aminotransferase levels because of prevalent non-alcoholic fatty liver disease, a low alanine aminotransferase level is a marker of hepatic or systemic frailty rather than health. © 2015 The Authors. Diabetic Medicine © 2015 Diabetes UK.

Transport of extraterrestrial biomolecules to the Earth: problem of thermal stability.

PubMed

Basiuk, V A; Douda, J; Navarro-Gonzalez, R

1999-01-01

The idea of extraterrestrial delivery of organic matter to the early Earth is especially attractive at present and is strongly supported by the detection of a large variety of organic compounds, including amino acids and nucleobases, in carbonaceous chondrites. Whether these compounds can be delivered by other space bodies is unclear and depends primarily on capability of the biomolecules to survive high temperatures during atmospheric deceleration and impacts to the terrestrial surface. In the present study we estimated survivability of simple amino acids (alpha-aminoisobutyric acid, L-alanine, L-valine and L-leucine), purines (adenine and guanine) and pyrimidines (uracil and cytosine) under rapid heating to temperatures of 400 to 1000 degrees C under N2 or CO2 atmosphere. We have found that most of the compounds studied cannot survive the temperatures substantially higher than 700 degrees C; however at 500-600 degrees C, the recovery can be at a per cent level (or even 10%-level for adenine, uracil, alanine, and valine). Implications of the data for extraterrestrial delivery of the biomolecules are discussed.

[Effects of ß-alanine supplementation on wingate tests in university female footballers].

PubMed

Rodríguez Rodríguez, Fernando; Delgado Ormeño, Alex; Rivera Lobos, Patricio; Tapia Aranda, Víctor; Cristi-Montero, Carlos

2014-11-01

Football is a sport that develops actions intermittent high-intensity exercise using the anaerobic pathway, for that reason, the muscle fatigue would produce primarily by increasing acidosis. Carnosine, which is formed from L-histidine, ß-alanine, has proven to produce an effect "buffer" of acidosis. To determine the effect of ß-alanine supplementation, on three successive Wingate tests and compare the average power, maximum power and lactate blood in selected female college soccer. We evaluated 10 football players who were three Wingate, 5 min rest between each sprint, determining the average power, maximum and lactate at the end of each test, then consumed 2,4 gr/day of ß-alanine for 30 days and repeated the tests. The control group (n=8) performed the same tests, but without consuming the supplement. Monark cycle ergometer was used (Ergomedic 874E) and to measure lactate the Lactate Pro 2. The group with supplementation significantly improved mean power difference from the control group. The maximum power improved only in the first sprint unlike the control group and Lactate did not differ. The evidence shows that the ß-alanine improves performance on tests of more than 30 second long, but in our study improves average power and peak power even when performing consecutive sprint, being able to emulate the reality of the football game. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain

PubMed Central

Wang, Jiafu; Meng, Jianghui; Nugent, Marc; Tang, Minhong; Dolly, J. Oliver

2017-01-01

Botulinum neurotoxins (BoNTs) are the most toxic proteins known, due to inhibiting the neuronal release of acetylcholine and causing flaccid paralysis. Most BoNT serotypes target neurons by binding to synaptic vesicle proteins and gangliosides via a C-terminal binding sub-domain (HCC). However, the role of their conserved N-terminal sub-domain (HCN) has not been established. Herein, we created a mutant form of recombinant BoNT/A lacking HCN (rAΔHCN) and showed that the lethality of this mutant is reduced 3.3 × 104-fold compared to wild-type BoNT/A. Accordingly, low concentrations of rAΔHCN failed to bind either synaptic vesicle protein 2C or neurons, unlike the high-affinity neuronal binding obtained with 125I-BoNT/A (Kd = 0.46 nM). At a higher concentration, rAΔHCN did bind to cultured sensory neurons and cluster on the surface, even after 24 h exposure. In contrast, BoNT/A became internalised and its light chain appeared associated with the plasmalemma, and partially co-localised with vesicle-associated membrane protein 2 in some vesicular compartments. We further found that a point mutation (W985L) within HCN reduced the toxicity over 10-fold, while this mutant maintained the same level of binding to neurons as wild type BoNT/A, suggesting that HCN makes additional contributions to productive internalization/translocation steps beyond binding to neurons. PMID:28295026

Initial transport validation studies using NSTX-U L-mode plasmas

NASA Astrophysics Data System (ADS)

Guttenfelder, Walter; Battaglia, D.; Bell, R. E.; Boyer, M. D.; Crocker, N.; Diallo, A.; Ferraro, N.; Gerhardt, S. P.; Kaye, S. M.; Leblanc, B. P.; Liu, D.; Menard, J. E.; Mueller, D.; Myer, C.; Podesta, M.; Raman, R.; Ren, Y.; Sabbagh, S.; Smith, D.

2016-10-01

A variety of stationary L-mode plasmas have been successfully developed in NSTX-U for physics validation studies. The plasmas span a range of density (1-4 ×1019 m-3) , plasma current (0.65-1.0 MA), and neutral beam heating power (<=4 MW), taking advantage of new, more tangential neutral beam sources to vary rotation profiles. Transport analysis (TRANSP) and turbulence measurements (BES, reflectometry) of these plasmas will be illustrated and compared with initial microstability and transport predictions. In particular, the normalized beta of these L-modes range between βN = 1-2, providing a valuable bridge in parameter space between (i) H-modes at comparable beta in conventional tokamaks (R/a 3, βN 2), where transport models have been largely developed and tested, and (ii) low-aspect-ratio H-modes at higher beta (R/a 1.5-1.7, βN 5), where transport models are less tested and challenged by stronger electromagnetic and equilibrium effects. This work is supported by US DOE contract DE-AC02-09CH11466.

Deconfinement in Yang-Mills Theory through Toroidal Compactification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simic, Dusan; Unsal, Mithat; /Stanford U., Phys. Dept. /SLAC

2011-08-12

We introduce field theory techniques through which the deconfinement transition of four-dimensional Yang-Mills theory can be moved to a semi-classical domain where it becomes calculable using two-dimensional field theory. We achieve this through a double-trace deformation of toroidally compactified Yang-Mills theory on R{sup 2} x S{sub L}{sup 1} x S{sub {beta}}{sup 1}. At large N, fixed-L, and arbitrary {beta}, the thermodynamics of the deformed theory is equivalent to that of ordinary Yang-Mills theory at leading order in the large N expansion. At fixed-N, small L and a range of {beta}, the deformed theory maps to a two-dimensional theory with electricmore » and magnetic (order and disorder) perturbations, analogs of which appear in planar spin-systems and statistical physics. We show that in this regime the deconfinement transition is driven by the competition between electric and magnetic perturbations in this two-dimensional theory. This appears to support the scenario proposed by Liao and Shuryak regarding the magnetic component of the quark-gluon plasma at RHIC.« less

Automated in-tube solid-phase microextraction coupled with liquid chromatography/electrospray ionization mass spectrometry for the determination of beta-blockers and metabolites in urine and serum samples.

PubMed

Kataoka, H; Narimatsu, S; Lord, H L; Pawliszyn, J

1999-10-01

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was evaluated for the determination of beta-blockers in urine and serum samples. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. LC/MS analyses of beta-blockers were initially performed by liquid injection onto a LC column. Nine beta-blockers tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M + H]+ were observed for all beta-blockers. The beta-blockers were separated with a Hypersil BDS C18 column using acetonitrile/methanol/water/acetic acid (15:15:70:1) as a mobile phase. To optimize the extraction of beta-blockers, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 30 microL of sample in 100 mM Tris-HCl (pH 8.5) at a flow rate of 100 microL/min using an Omegawax 250 capillary (Supelco, Bellefonte, PA). The beta-blockers extracted by the capillary were easily desorbed by mobile-phase flow, and carryover of beta-blockers was not observed. Using in-tube SPME/LC/ESI-MS with selected ion monitoring, the calibration curves of beta-blockers were linear in the range from 2 to 100 ng/mL with correlation coefficients above 0.9982 (n = 18) and detection limits (S/N = 3) of 0.1-1.2 ng/mL. This method was successfully applied to the analysis of biological samples without interference peaks. The recoveries of beta-blockers spiked into human urine and serum samples were above 84 and 71%, respectively. A serum sample from a patient administrated propranolol was analyzed using this method and both propranolol and its metabolites were detected.

Water-Solubilized, Cap-Stabilized, Helical Polyalanines: Calibration Standards for NMR and CD Analyses

PubMed Central

Heitmann, Björn; Job, Gabriel E.; Kennedy, Robert J.; Walker, Sharon M.; Kemp, Daniel S.

2006-01-01

NMR and CD studies are reported for two length series of solubilized, spaced, highly helical polyalanines that are N-capped by the optimal helix stabilizer βAsp-Hel and C-capped by β-aminoalanine beta and that are studied in water at 2 °C, pH 1–8. NMR analysis yields a structural characterization of the peptide AcβAspHelAla8betaNH2 and selected members of one βAspHelAlanbeta series. At pH > 4.5 the βAspHel cap provides a preorganized triad of carboxylate anion and two amide residues that is complementary to the helical polyalanine N-terminus. The C-terminal β-aminoalanine assumes a helix-stabilizing conformation consistent with literature precedents. H(N)CO NMR experiments applied to capped, uniformly 13C- and 15N-labeled Ala8 and Ala12 peptides define Alan hydrogen bonding signatures as α-helical without detectable 310 character. Relative NH→ND exchange rates yield site protection factors PFi that define uniquely high fractional helicities FH for the peptide Alan regions. These Alan calibration series, studied in water and lacking helix-stabilizing tertiary structure, yield the first 13C NMR chemical shifts, 3JHNHα coupling constants, and CD ellipticities [θMolar]λ,n characteristic of a fully helical alanine within an Alan context. CD data are used to assign parameters X and [θ]λ,∞, required for rigorous calculation of FH values from CD ellipticities. PMID:15701003

40 CFR 180.505 - Emamectin; tolerances for residues.

Code of Federal Regulations, 2011 CFR

2011-07-01

... residues of emamectin (a mixture of a minimum of 90% 4′-epi-methylamino-4′-deoxyavermectin B1a and maximum of 10% 4′-epi-methylamino-4′-deoxyavermectin B1b) and its metabolites 8,9-isomer of the B1a and B1b component of the parent (8,9-ZMA), or 4′-deoxy-4′-epi-amino-avermectin B1a and 4′-deoxy-4′-epi-amino...

Involvement of Smad3 phosphoisoform-mediated signaling in the development of colonic cancer in IL-10-deficient mice.

PubMed

Hachimine, Daisaku; Uchida, Kazushige; Asada, Masanori; Nishio, Akiyoshi; Kawamata, Seiji; Sekimoto, Go; Murata, Miki; Yamagata, Hideo; Yoshida, Katsunori; Mori, Shigeo; Tahashi, Yoshiya; Matsuzaki, Koichi; Okazaki, Kazuichi

2008-06-01

Chronic inflammation predisposes to cancer. Transforming growth factor (TGF)-beta, a multifunctional protein, suppresses the growth of normal colonic epithelial cells, whereas it stimulates the proliferation of cancer cells. Interleukin (IL)-10-deficient mice, which develop colitis and colorectal cancer, show an increased level of plasma TGF-beta. Although TGF-beta may be a key molecule in the development of colon cancer arising from chronic colitis in IL-10-deficient mice, the role of TGF-beta still remains unclear. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), which converts the mediator Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). We studied C57BL/6-IL-10-deficient mice (n=18) at 4 to 32 weeks of age. We investigated histology, and pSmad2/3L, pSmad2/3C, and p53 by immunohistochemistry. pSmad3L staining was detected in the cancer cells in all 10 mice with colonic cancer and in the epithelial cells in 7 of 12 mice with colonic dysplasia, but not in the normal or colitic mice. pSmad3c was detected without any significant difference between stages. p53 was weakly stained in a few cancer cells in 5 out of 10 mice. Smad3L signaling plays an important role in the carcinogenesis of chronic colitis in IL-10-deficient mice.

The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton.

PubMed

Hohaus, Annette; Person, Veronika; Behlke, Joachim; Schaper, Jutta; Morano, Ingo; Haase, Hannelore

2002-08-01

Ahnak is a ubiquitously expressed giant protein of 5643 amino acids implicated in cell differentiation and signal transduction. In a recent study, we demonstrated the association of ahnak with the regulatory beta2 subunit of the cardiac L-type Ca2+ channel. Here we identify the most carboxyl-terminal ahnak region (aa 5262-5643) to interact with recombinant beta2a as well as with beta2 and beta1a isoforms of native muscle Ca2+ channels using a panel of GST fusion proteins. Equilibrium sedimentation analysis revealed Kd values of 55 +/- 11 nM and 328 +/- 24 nM for carboxyl-terminal (aa 195-606) and amino-terminal (aa 1-200) truncates of the beta2a subunit, respectively. The same carboxyl-terminal ahnak region (aa 5262-5643) bound to G-actin and cosedimented with F-actin. Confocal microscopy of human left ventricular tissue localized the carboxyl-terminal ahnak portion to the sarcolemma including the T-tubular system and the intercalated disks of cardiomyocytes. These results suggest that ahnak provides a structural basis for the subsarcolemmal cytoarchitecture and confers the regulatory role of the actin-based cytoskeleton to the L-type Ca2+ channel.

Nitric oxide synthase modulates CFA-induced thermal hyperalgesia through cytokine regulation in mice.

PubMed

Chen, Yong; Boettger, Michael K; Reif, Andreas; Schmitt, Angelika; Uçeyler, Nurcan; Sommer, Claudia

2010-03-02

Although it has been largely demonstrated that nitric oxide synthase (NOS), a key enzyme for nitric oxide (NO) production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process. Intraperitoneal (i.p.) pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor), aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor), L-N(G)-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor), but not L-N(5)-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor), significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1beta), and interleukin-10 (IL-10) gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1beta. The increase of the anti-inflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO) mice had lower gene expression of TNF, IL-1beta, and IL-10 following CFA, overall corroborating the inhibitor data. These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: III. Alanine Is the Product of Anaerobic Ammonium Assimilation.

PubMed

Vanlerberghe, G C; Joy, K W; Turpin, D H

1991-02-01

We have determined the flow of (15)N into free amino acids of the N-limited green alga Selenastrum minutum (Naeg.) Collins after addition of (15)NH(4) (+) to aerobic or anaerobic cells. Under aerobic conditions, only a small proportion of the N assimilated was retained in the free amino acid pool. However, under anaerobic conditions almost all assimilated NH(4) (+) accumulates in alanine. This is a unique feature of anaerobic NH(4) (+) assimilation. The pathway of carbon flow to alanine results in the production of ATP and reductant which matches exactly the requirements of NH(4) (+) assimilation. Alanine synthesis is therefore an excellent strategy to maintain energy and redox balance during anaerobic NH(4) (+) assimilation.

Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae.

PubMed

Yano, Akira; Kikuchi, Sayaka; Nakagawa, Yuko; Sakamoto, Yuichi; Sato, Toshitsugu

2009-01-01

The shiitake mushroom, Lentinula edodes, has an extracelluar secretory-type laccase, Lcc1, and a fruiting-body-accumulation-type laccase, Lcc4. We previously reported the production of Lcc1 by plant cells, but had difficulty producing Lcc4. Here, we report the production of Lcc1 and Lcc4 by Aspergillus oryzae and the extracellular secretory production of Lcc4 using a modified secretion signal peptide (SP) from Lcc1. Sp-Lcc4 produced by A. oryzae had biochemical activities similar to Lcc4 produced by L. edodes. Lcc1 did not react with beta-(3,4-dihydroxyphenol) alanine (DOPA), but Lcc4 from L. edodes and A. oryzae could oxidize DOPA. K(M) values for the substrates 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate), 2,6-dimethoxyphenol, guaiacol, pyrogallol, and catechol were similar for Lcc4 and Sp-Lcc4. In conclusion, a non-secretory-type fungal laccase is secreted into the culture media with its original enzymatic properties by exploiting modified secretory signal peptide. 2008 Elsevier GmbH.

Mechanical stability analysis of the protein L immunoglobulin-binding domain by full alanine screening using molecular dynamics simulations.

PubMed

Glyakina, Anna V; Likhachev, Ilya V; Balabaev, Nikolay K; Galzitskaya, Oxana V

2015-03-01

This article is the first to study the mechanical properties of the immunoglobulin-binding domain of protein L (referred to as protein L) and its mutants at the atomic level. In the structure of protein L, each amino acid residue (except for alanines and glycines) was replaced sequentially by alanine. Thus, 49 mutants of protein L were obtained. The proteins were stretched at their termini at constant velocity using molecular dynamics simulations in water, i.e. by forced unfolding. 19 out of 49 mutations resulted in a large decrease of mechanical protein stability. These amino acids were affecting either the secondary structure (11 mutations) or loop structures (8 mutations) of protein L. Analysis of mechanical unfolding of the generated protein that has the same topology as protein L but consists of only alanines and glycines allows us to suggest that the mechanical stability of proteins, and specifically protein L, is determined by interactions between certain amino acid residues, although the unfolding pathway depends on the protein topology. This insight can now be used to modulate the mechanical properties of proteins and their unfolding pathways in the desired direction for using them in various biochips, biosensors and biomaterials for medicine, industry, and household purposes. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of 13CO2 breath tests for the detection of fructose malabsorption.

PubMed

Hoekstra, J H; van den Aker, J H; Kneepkens, C M; Stellaard, F; Geypens, B; Ghoos, Y F

1996-03-01

Breath hydrogen (H2) studies have made clear that small intestinal absorption of fructose is limited, especially in toddlers. Malabsorption of fructose may be a cause of recurrent abdominal pain and chronic nonspecific diarrhea (toddler's diarrhea). Fructose absorption is facilitated by equimolar doses of glucose and, as we have found, amino acids (especially L-alanine); the mechanism underlying this effect remains unclear. To study fructose absorption in a more direct way, we combined breath H2 studies with breath 13CO2 studies. Gastric emptying was studied by using L-glycine-1-13C in 4 children from 12.1 to 16.0 years of age. After 25 gm of fructose and 27.5 gm of glucose, when given together, gastric emptying was significantly (p<0.05) slower than with either sugar alone. In a second series of experiments, 5 children from 12.0 to 15.9 years of age were tested with 25 gm of fructose, alone and with equimolar doses of glucose and L-alanine, and 4 younger children from 3.1 to 6.1 years of age were tested with 2 gm/kg (max 37.5 gm) fructose, alone or with an equimolar dose of L-alanine. All fructose solutions were enriched with 15 mg of D-fructose-13C-6. In all 9 children, fructose was malabsorbed as judged by breath H2 increases > or = 20 ppm, and the addition of glucose or L-alanine resulted in significantly lower breath H2 increases (p < or = 0.005 for glucose, p < or = 0.001 for alanine). In contrast, the addition of alanine or glucose did not change the pattern of breath 13CO2 excretion in the 5 older children, whereas in the 4 younger children (with relatively higher doses), L-alanine addition resulted in significantly lower increases in breath 13CO2. In the latter group, for each time point, breath H2 and 13CO2 concentrations after fructose were compared with those after fructose plus L-alanine; in 20 out of 24 points, both H2 and 13CO2 were higher after fructose. These results suggest that 13CO2 not only originated from the oxidation of absorbed substrate but also, at least in part, from colonic bacterial metabolism. For the detection of [correction of or] fructose malabsorption--as opposed to, for instance, lactose--the 13CO2 breath test seems to be of limited value.

Synthesis, characterization and monoamine transporter activity of the new psychoactive substance mexedrone and its N-methoxy positional isomer, N-methoxymephedrone

PubMed Central

McLaughlin, Gavin; Morris, Noreen; Kavanagh, Pierce V.; Power, John D.; Dowling, Geraldine; Twamley, Brendan; O'Brien, John; Talbot, Brian; Walther, Donna; Partilla, John S.; Baumann, Michael H.; Brandt, Simon D.

2017-01-01

3-Methoxy-2-(methylamino)-1-(4-methylphenyl)propan-1-one (mexedrone) appeared in 2015 and was advertised by UK Internet retailers as a non-controlled mephedrone derivative (2-(methylamino)-1-(4-methylphenyl)propan-1-one), which was of particular interest to countries who operate generic drugs legislation. This study describes the synthesis and analytical characterization of mexedrone and the differentiation from its isomer, N-methoxymephedrone, which was predicted to be a suitable candidate before the identity of mexedrone was revealed. A full analytical characterization is described using various chromatographic, spectroscopic and mass spectrometric platforms and X-ray crystal structure analysis. The analytical data obtained for a vendor sample were consistent with the synthesized mexedrone reference standard and analytical differentiation between the mexedrone and N-methoxymephedrone positional isomers was achieved. Furthermore, α-chloromethylmephedrone was identified as a by-product during mexedrone synthesis. All three substances were also studied for their uptake and releasing properties at dopamine transporters (DAT), norepinephrine transporters (NET) and serotonin transporters (SERT) using in vitro monoamine transporter assays in rat brain synaptosomes and compared to mephedrone. Mexedrone was a weak non-selective uptake blocker with IC50 values in the low μM range. It was also devoid of releasing activity at DAT and NET but displayed weak releasing activity at SERT (EC50= 2.5 μM). The isomer N-methoxymephedrone was found to be a weak uptake blocker at DAT, NET and SERT, as well as a fully efficacious substrate-type releasing agent across all three transporters with EC50 values in the low micromolar range. The synthesis by-product α-chloromethylmephedrone was inactive in all assays. PMID:27524685

Synthesis, characterization and monoamine transporter activity of the new psychoactive substance mexedrone and its N-methoxy positional isomer, N-methoxymephedrone.

PubMed

McLaughlin, Gavin; Morris, Noreen; Kavanagh, Pierce V; Power, John D; Dowling, Geraldine; Twamley, Brendan; O'Brien, John; Talbot, Brian; Walther, Donna; Partilla, John S; Baumann, Michael H; Brandt, Simon D

2017-03-01

3-Methoxy-2-(methylamino)-1-(4-methylphenyl)propan-1-one (mexedrone) appeared in 2015 and was advertised by UK Internet retailers as a non-controlled mephedrone derivative (2-(methylamino)-1-(4-methylphenyl)propan-1-one), which was of particular interest to countries who operate generic drugs legislation. This study describes the synthesis and analytical characterization of mexedrone and the differentiation from its isomer, N-methoxymephedrone, which was predicted to be a suitable candidate before the identity of mexedrone was revealed. A full analytical characterization is described using various chromatographic, spectroscopic and mass spectrometric platforms and X-ray crystal structure analysis. The analytical data obtained for a vendor sample were consistent with the synthesized mexedrone reference standard and analytical differentiation between the mexedrone and N-methoxymephedrone positional isomers was achieved. Furthermore, α-chloromethylmephedrone was identified as a by-product during mexedrone synthesis. All three substances were also studied for their uptake and releasing properties at dopamine transporters (DAT), norepinephrine transporters (NET) and serotonin transporters (SERT) using in vitro monoamine transporter assays in rat brain synaptosomes and compared to mephedrone. Mexedrone was a weak non-selective uptake blocker with IC 50 values in the low μM range. It was also devoid of releasing activity at DAT and NET but displayed weak releasing activity at SERT (EC 50  = 2.5 μM). The isomer N-methoxymephedrone was found to be a weak uptake blocker at DAT, NET and SERT, as well as a fully efficacious substrate-type releasing agent across all three transporters with EC 50 values in the low micromolar range. The synthesis by-product α-chloromethylmephedrone was inactive in all assays. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Molecular organization of amyloid protofilament-like assembly of betabellin 15D: helical array of beta-sandwiches.

PubMed Central

Inouye, Hideyo; Bond, Jeremy E; Deverin, Sean P; Lim, Amareth; Costello, Catherine E; Kirschner, Daniel A

2002-01-01

Betabellin is a 32-residue peptide engineered to fold into a four-stranded antiparallel beta-sheet protein. Upon air oxidation, the betabellin peptides can fold and assemble into a disulfide-bridged homodimer, or beta-sandwich, of 64 residues. Recent biophysical and ultrastructural studies indicate that betabellin 15D (B15D) (a homodimer of HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p = DPro, k = DLys, and h = DHis) forms unbranched, 35-A wide assemblies that resemble the protofilaments of amyloid fibers. In the present study, we have analyzed in detail the X-ray diffraction patterns of B15D prepared from acetonitrile. The fiber diffraction analysis indicated that the B15D fibril was composed of a double helix defined by the selection rule l = n + 7m (where l is even, and n and m are any integers), and having a 199-A period and pitch, 28-A rise per unit, and 10-A radius. This helical model is equivalent to a reverse-handed, single helix with half the period and defined by the selection rule l = -3n + 7m (where l is any integer). The asymmetric unit is the single B15D beta-sandwich molecule. These results suggest that the betabellin assembly that models the protofilaments of amyloid fibers is made up of discrete subunits on a helical array. Multiple intersheet hydrogen bonds in the axial direction and intersandwich polar interactions in the lateral direction stabilize the array. PMID:12202394

Perturbative momentum transport in MAST L-mode plasmas

DOE PAGES

Guttenfelder, W.; Field, A. R.; Lupelli, I.; ...

2017-03-28

Non-axisymmetric magnetic fields are used to perturbatively probe momentum transport physics in MAST L-mode plasmas. The low beta L-mode target was chosen to complement previous experiments conducted in high beta NSTX H-mode plasmas (beta N = 3.5-4.6) where an inward momentum pinch was measured. In those cases quasi-linear gyrokinetic simulations of unstable ballooning micro-instabilities predict weak or outward momentum convection, in contrast to the measurements. The weak pinch was predicted to be due to both electromagnetic effects at high beta and low aspect ratio minimizing the symmetry-breaking of the instabilities responsible for momentum transport. In an attempt to lessen thesemore » electromagnetic effects at low aspect ratio, perturbative experiments were run in MAST L-mode discharges at lower beta (beta N = 2). The perturbative transport analysis used the time-dependent response following the termination of applied 3D fields that briefly brake the plasma rotation ( similar to the NSTX H-mode experiments). Assuming time-invariant diffusive (chi(phi))and convective (V-phi) transport coefficients, an inward pinch is inferred with magnitudes, (RV phi/chi(phi)) = (-1)-(-9), similar to those found in NSTX H-modes and in conventional tokamaks. However, if experimental uncertainties due to non-stationary conditions during and after the applied 3D field are considered, a weak pinch or even outward convection is inferred, ( RV phi/chi(phi)) = (-1)-(+5). Linear gyrokinetic simulations indicate that for these lower beta L-modes, the predicted momentum pinch is predicted to be relatively small, ( RV phi/chi(phi))(sim) approximate to -1. While this falls within the experimentally inferred range, the uncertainties are practically too large to quantitatively validate the predictions. Challenges and implications for this particular experimental technique are discussed, as well as additional possible physical mechanisms that may be important in understanding momentum transport in these low aspect ratio plasmas.« less

Perturbative momentum transport in MAST L-mode plasmas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guttenfelder, W.; Field, A. R.; Lupelli, I.

Non-axisymmetric magnetic fields are used to perturbatively probe momentum transport physics in MAST L-mode plasmas. The low beta L-mode target was chosen to complement previous experiments conducted in high beta NSTX H-mode plasmas (beta N = 3.5-4.6) where an inward momentum pinch was measured. In those cases quasi-linear gyrokinetic simulations of unstable ballooning micro-instabilities predict weak or outward momentum convection, in contrast to the measurements. The weak pinch was predicted to be due to both electromagnetic effects at high beta and low aspect ratio minimizing the symmetry-breaking of the instabilities responsible for momentum transport. In an attempt to lessen thesemore » electromagnetic effects at low aspect ratio, perturbative experiments were run in MAST L-mode discharges at lower beta (beta N = 2). The perturbative transport analysis used the time-dependent response following the termination of applied 3D fields that briefly brake the plasma rotation ( similar to the NSTX H-mode experiments). Assuming time-invariant diffusive (chi(phi))and convective (V-phi) transport coefficients, an inward pinch is inferred with magnitudes, (RV phi/chi(phi)) = (-1)-(-9), similar to those found in NSTX H-modes and in conventional tokamaks. However, if experimental uncertainties due to non-stationary conditions during and after the applied 3D field are considered, a weak pinch or even outward convection is inferred, ( RV phi/chi(phi)) = (-1)-(+5). Linear gyrokinetic simulations indicate that for these lower beta L-modes, the predicted momentum pinch is predicted to be relatively small, ( RV phi/chi(phi))(sim) approximate to -1. While this falls within the experimentally inferred range, the uncertainties are practically too large to quantitatively validate the predictions. Challenges and implications for this particular experimental technique are discussed, as well as additional possible physical mechanisms that may be important in understanding momentum transport in these low aspect ratio plasmas.« less

Smad3 phosphoisoform-mediated signaling during sporadic human colorectal carcinogenesis.

PubMed

Matsuzaki, K

2006-06-01

Transforming growth factor-beta (TGF-beta) signaling occurring during human colorectal carcinogenesis involves a shift in TGF-beta function, reducing the cytokine's antiproliferative effect, while increasing actions that promote invasion and metastasis. TGF-beta signaling involves phosphorylation of Smad3 at serine residues 208 and 213 in the linker region and serine residues 423 and 425 in the C-terminal region. Exogenous TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). Either pSmad3C or pSmad3L oligomerizes with Smad4, and translocates into nuclei. While the TbetaRI/pSmad3C pathway inhibits growth of normal epithelial cells in vivo, JNK/pSmad3L-mediated signaling promotes tumor cell invasion and extracellular matrix synthesis by activated mesenchymal cells. Furthermore, hepatocyte growth factor signaling interacts with TGF-beta to activate the JNK/pSmad3L pathway, accelerating nuclear transport of cytoplasmic pSmad3L. This reduces accessibility of unphosphorylated Smad3 to membrane-anchored TbetaRI, preventing Smad3C phosphorylation, pSmad3C-mediated transcription, and antiproliferative effects of TGF-beta on epithelial cells. As neoplasia progresses from normal colorectal epithelium through adenoma to invasive adenocarcinoma with distant metastasis, nuclear pSmad3L gradually increases while pSmad3C decreases. The shift from TbetaRI/pSmad3C-mediated to JNK/pSmad3L-mediated signaling is a major mechanism orchestrating a complex transition of TGF-beta signaling during sporadic human colorectal carcinogenesis. This review summarizes the recent understanding of Smad3 phosphoisoform-mediated signaling, particularly 'cross-talk' between Smad3 and JNK pathways that cooperatively promote oncogenic activities. Understanding of these actions should help to develop more effective therapy against human colorectal cancer, involving inhibition of JNK/pSmad3L pathway.

Prebiotic Peptide (Amide) Bond Synthesis Accelerated by Glycerol and Bicarbonate Under Neutral to Alkaline Dry-Down Conditions

NASA Astrophysics Data System (ADS)

Forsythe, J. G.; Weber, A. L.

2017-07-01

We report a new process for robust peptide bond synthesis in the pH 6–10 range that involves dry-down heating of amino acids in the presence of glycerol and bicarbonate (substrates: L-alanine, L-2-aminobutyric acid, β-alanine, isoserine).

Synthesis and characterization of novel chiral ionic liquids and investigation of their enantiomeric recognition properties.

PubMed

Bwambok, David K; Marwani, Hadi M; Fernand, Vivian E; Fakayode, Sayo O; Lowry, Mark; Negulescu, Ioan; Strongin, Robert M; Warner, Isiah M

2008-02-01

We report the synthesis and characterization of amino acid ester based chiral ionic liquids, derived from L- and D-alanine tert butyl ester chloride. The synthesis was accomplished via an anion metathesis reaction between commercially available L- and D-alanine tert butyl ester chloride using a variety of counterions such as lithium bis (trifluoromethane) sulfonimide, silver nitrate, silver lactate, and silver tetrafluoroborate. Both enantiomeric forms were obtained as confirmed by bands of opposite sign in the circular dichroism spectra. The L- and D-alanine tert butyl ester bis (trifluoromethane) sulfonimide were obtained as liquids at room temperature and intriguingly exhibited the highest thermal stability (up to 263 degrees C). In addition, the ionic liquids demonstrated enantiomeric recognition ability as evidenced by splitting of racemic Mosher's sodium salt signal using a liquid state (19)F nuclear magnetic resonance (NMR) and fluorescence spectroscopy. The L- and D-alanine tert butyl ester chloride resulted in solid salts with nitrate, lactate, and tetrafluoroborate anions. This illustrates the previously observed tunability of ionic liquid synthesis, resulting in ionic liquids of varying properties as a function of varying the anion.

Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faraci, W.S.; Walsh, C.T.

1988-05-03

Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L ..-->.. D and D..-->.. L directions for all three enzymes to assess the degree to which abstraction of the ..cap alpha..-proton or protonation of substratemore » PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of ..cap alpha..-/sup 3/H from substrate to product and solvent exchange/substrate conversion experiments in /sup 3/H/sub 2/O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.« less

Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide.

PubMed

von Ossowski, Ingemar; Oksanen, Esko; von Ossowski, Lotta; Cai, Chunlin; Sundberg, Maria; Goldman, Adrian; Keinänen, Kari

2006-11-01

Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser/Thr-x-phi-COOH where phi is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97(PDZ2)). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97(PDZ2) variant in complex with an 18-mer C-terminal peptide (residues 890-907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8-2.44 A resolutions. SAP97(PDZ2) folds to a compact globular domain comprising six beta-strands and two alpha-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR-A are visible, and align as an antiparallel beta-strand in the binding groove of SAP97(PDZ2). The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The betaAlpha-betaBeta loop moves away from the C-terminal end of alphaB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in betaAlpha-betaBeta loop with Asp396 or Thr394 in betaBeta. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a 'classical' type I PDZ interaction between SAP97 and GluR-A C-terminus.

Computational studies on non-succinimide-mediated stereoinversion mechanism of aspartic acid residues assisted by phosphate

NASA Astrophysics Data System (ADS)

Nakayoshi, Tomoki; Fukuyoshi, Shuichi; Takahashi, Ohgi; Oda, Akifumi

2018-03-01

Although nearly all of the amino acids that constitute proteins are l-amino acids, d-amino acid residues in human proteins have been recently reported. d-amino acid residues cause a change in the three-dimensional structure of proteins, and d-aspartic acid (Asp) residues are considered to be one of the causes of age-related diseases. The stereoinversion of Asp residues in peptides and proteins is thought to proceed via a succinimide intermediate; however, it has been reported that stereoinversion can occur even under conditions where a succinimide intermediate cannot be formed. In order to elucidate the non-succinimide-mediated stereoinversion pathway, we investigated the stereoinversion of l-Asp to d-Asp catalysed by phosphate and estimated the activation barrier using B3LYP/6-31+G(d,p) density functional theory (DFT) calculations. For the DFT calculations, a model compound in which the Asp residue is capped with acetyl and methyl-amino groups on the N- and C-termini, respectively, was used. The calculated activation barrier was not excessively high for the stereoinversion to occur in vivo. Therefore, this stereoinversion mechanism may compete with the succinimide-mediated mechanism.

Effect of Nitrogen on Cellular Production and Release of the Neurotoxin Anatoxin-A in a Nitrogen-Fixing Cyanobacterium

PubMed Central

Gagnon, Alexis; Pick, Frances R.

2012-01-01

Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and implicated in lethal poisonings of domesticated animals and wildlife. The factors leading to its production in nature and in culture are not well understood. Resource availability may influence its cellular production as suggested by the carbon-nutrient hypothesis, which links the amount of secondary metabolites produced by plants or microbes to the relative abundance of nutrients. We tested the effects of nitrogen supply (as 1, 5, and 100% N of standard cyanobacterial medium corresponding to 15, 75, and 1500 mg L−1 of NaNO3 respectively) on ANTX production and release in a toxic strain of the planktonic cyanobacterium Aphanizomenon issatschenkoi (Nostocales). We hypothesized that nitrogen deficiency might constrain the production of ANTX. However, the total concentration and more significantly the cellular content of anatoxin-a peaked (max. 146 μg/L and 1683 μg g−1 dry weight) at intermediate levels of nitrogen supply when N-deficiency was evident based on phycocyanin to chlorophyll a and carbon to nitrogen ratios. The results suggest that the cellular production of anatoxin-a may be stimulated by moderate nitrogen stress. Maximal cellular contents of other cyanotoxins have recently been reported under severe stress conditions in another Nostocales species. PMID:22701451

Utility of the FIB-4 Index for hepatocarcinogenesis in hepatitis C virus carriers with normal alanine aminotransferase levels.

PubMed

Ito, T; Kumada, T; Toyoda, H; Tada, T; Kiriyama, S; Tanikawa, M; Hisanaga, Y; Kanamori, A; Kitabatake, S

2015-10-01

The FIB-4 index is a simple formula using age, aspartate aminotransferase, alanine aminotransferase (ALT) and platelet count to evaluate liver fibrosis. We investigated the ability of the FIB-4 index for hepatocarcinogenesis in hepatitis C virus (HCV) carriers with normal ALT levels. A total of 516 patients with ALT levels persistently at or below 40 IU/L during an observation period of over 3 years were included. Factors associated with the development of HCC were determined. Hepatocellular carcinoma (HCC) developed in 60 of 516 patients (11.6%). The incidence rate of HCC at 5 and 10 years was 2.6% and 17.6%, respectively. When patients were categorized according to the FIB-4 index as ≤ 2.0 (n = 226), >2.0 and ≤ 4.0 (n = 169), and > 4.0 (n = 121), the cumulative incidence of HCC at 5 years was 0.5%, 1.3% and 8.0%, respectively, and 2.8%, 25.6% and 37.1% at 10 years, respectively. Patients with FIB-4 index >4.0 were at the highest risk (P < 0.001). Factors that were significantly associated with HCC in the multivariate analysis were FIB-4 index >2.0 (hazard ratio (HR), 7.690), FIB-4 index >4.0 (HR, 8.991), α-fetoprotein (AFP) >5 ng/mL (HR, 2.742), AFP >10 ng/mL (HR, 4.915) and total bilirubin >1.2 mg/dL (HR, 2.142). A scoring system for hepatocarcinogenesis that combines the FIB-4 index and AFP predicted patient outcomes with excellent discriminative ability. The FIB-4 index is strongly associated with the risk of HCC in HCV carriers with normal ALT levels. © 2015 John Wiley & Sons Ltd.

Association of biochemical markers with the severity of pre-eclampsia.

PubMed

Maged, Ahmed M; Aid, Gamal; Bassiouny, Nehal; Eldin, Doaa S; Dahab, Sherif; Ghamry, Nevein K

2017-02-01

To assess the association between pre-eclampsia severity and biochemical and ultrasonography markers. A retrospective study was undertaken of women with severe pre-eclampsia (group 1, n=90), mild pre-eclampsia (group 2, n=90), or a normal pregnancy (group 3, n=90) who attended a hospital in Egypt in October 2013-April 2015. Associations between pre-eclampsia and biochemical, cardiotocography, and ultrasonography markers were investigated. There were significant differences between the groups in C-reactive protein (331.44±112.38, 251.43±59.05, and 23.81±16.19 nmol/L; P≤0.05 for all), platelet count (113.40±36.72, 172.93±57.60, and 212.68±70.00×10 9 /L; P≤0.05 for group 1 comparisons), alanine transaminase (52.24±14.83, 38.34±13.12, and 23.11±6.92 U/L; P≤0.05 for group 1 comparisons), and serum uric acid (600.80±117.19, 481.83±118.97, and 243.89±53.54 μmol/L; P=0.050 for group 3 comparisons). Cardiotocography score was worse among women with severe pre-eclampsia than among those in the other two groups (P=0.039 for both comparisons). Biophysical profile score and umbilical artery resistance index differed by group (P≤0.05 for all). Middle cerebral artery resistance index was lower among women with severe pre-eclampsia (P≤0.05). The levels of C-reactive protein, blood urea nitrogen, serum uric acid, and alanine transaminase, and the platelet count were linked with the presence and severity of pre-eclampsia. © 2016 International Federation of Gynecology and Obstetrics.

Evidence for paralytic shellfish poisons in the freshwater cyanobacterium Lyngbya wollei (Farlow ex Gomont) comb. nov.

PubMed Central

Carmichael, W W; Evans, W R; Yin, Q Q; Bell, P; Moczydlowski, E

1997-01-01

Lyngbya wollei (Farlow ex Gomont) comb. nov., a perennial mat-forming filamentous cyanobacterium prevalent in lakes and reservoirs of the southeastern United States, was found to produce a potent, acutely lethal neurotoxin when tested in the mouse bioassay. Signs of poisoning were similar to those of paralytic shellfish poisoning. As part of the Tennessee Valley Authority master plan for Guntersville Reservoir, the mat-forming filamentous cyanobacterium L. wollei, a species that had recently invaded from other areas of the southern United States, was studied to determine if it could produce any of the known cyanotoxins. Of the 91 field samples collected at 10 locations at Guntersville Reservoir, Ala., on the Tennessee River, over a 3-year period, 72.5% were toxic. The minimum 100% lethal doses of the toxic samples ranged from 150 to 1,500 mg kg of lyophilized L. wollei cells-1, with the majority of samples being toxic at 500 mg kg-1. Samples bioassayed for paralytic shellfish toxins by the Association of Official Analytical Chemists method exhibited saxitoxin equivalents ranging from 0 to 58 micrograms g (dry weight)-1. Characteristics of the neurotoxic compound(s), such as the lack of adsorption by C18 solid-phase extraction columns, the short retention times on C18 high-performance liquid chromatography (HPLC) columns, the interaction of the neurotoxins with saxiphilin (a soluble saxitoxin-binding protein), and external blockage of voltage-sensitive sodium channels, led to our discovery that this neurotoxin(s) is related to the saxitoxins, the compounds responsible for paralytic shellfish poisonings. The major saxitoxin compounds thus far identified by comparison of HPLC fluorescence retention times are decarbamoyl gonyautoxins 2 and 3. There was no evidence of paralytic shellfish poison C toxins being produced by L. wollei. Fifty field samples were placed in unialgal culture and grown under defined culture conditions. Toxicity and signs of poisoning for these laboratory-grown strains of L. wollei were similar to those of the field collection samples. PMID:9251196

Asymmetric adsorption by quartz - A model for the prebiotic origin of optical activity

NASA Technical Reports Server (NTRS)

Bonner, W. M.; Kavasmaneck, P. R.; Martin, F. S.; Flores, J. J.

1975-01-01

One mechanism previously proposed for the abiotic accumulation of molecules of one chirality in nature is asymmetric adsorption on the chiral surfaces of optically active quartz crystals. Earlier literature in this field is reviewed, with the conclusion that previous investigations of this phenomenon, using optical rotation criteria, have afforded ambiguous results. We now have studied the adsorption of radioactive D- and L-alanine on powdered d- and l-quartz, using change in radioactivity level as a criterion for both gross and differential adsorption, d-Quartz preferentially adsorbed D-alanine from anhydrous dimethyl-formamide solution, and l-quartz L-alanine. The differential adsorption varied between 1.0 and 1.8%. The implications of these observations are discussed from the viewpoint of early chemical evolution and the origin of optically active organic compounds in nature.

A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica.

PubMed

Hilbert, F; García-del Portillo, F; Groisman, E A

1999-04-01

The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.

A Novel Surface Plasmon Resonance Biosensor for the Rapid Detection of Botulinum Neurotoxins

PubMed Central

Patel, Kruti; Halevi, Shmuel; Melman, Paul; Schwartz, John; Cai, Shuowei; Singh, Bal Ram

2017-01-01

Botulinum neurotoxins (BoNTs) are Category A agents on the NIAID (National Institute of Allergy and Infectious Diseases) priority pathogen list owing to their extreme toxicity and the relative ease of production. These deadly toxins, in minute quantities (estimated human i.v. lethal dose LD50 of 1–2 ng/kg body weight), cause fatal flaccid paralysis by blocking neurotransmitter release. The current gold standard detection method, the mouse-bioassay, often takes days to confirm botulism. Furthermore, there are no effective antidotes known to reverse the symptoms of botulism, and as a result, patients with severe botulism often require meticulous care during the prolonged paralytic illness. To combat potential bio-terrorism incidents of botulinum neurotoxins, their rapid detection is paramount. Surface plasmon resonance (SPR) is a very sensitive technique to examine bio-molecular interactions. The label-free, real-time analysis, with high sensitivity and low sample consumption makes this technology particularly suitable for detection of the toxin. In this study, we demonstrated the feasibility in an assay with a newly designed SPR instrument for the rapid detection of botulinum neurotoxins. The LOD (limit of detection) of the Newton Photonics (NP) SPR based assay is 6.76 pg/mL for Botulinum Neurotoxin type A Light Chain (BoNT/A LC). We established that the detection sensitivity of the system is comparable to the traditional mouse LD50 bioassay in BoNT/A using this SPR technology. PMID:28783115

Topology of AspT, the Aspartate:Alanine Antiporter of Tetragenococcus halophilus, Determined by Site-Directed Fluorescence Labeling▿ †

PubMed Central

Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C.; Abe, Keietsu

2007-01-01

The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of l-aspartate (Asp) with release of l-alanine (Ala) and CO2. The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an l-aspartate-β-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity. PMID:17660287

SU-E-T-608: Perturbation Corrections for Alanine Dosimeters in Different Phantom Materials in High-Energy Photon Beams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voigts-Rhetz, P von; Czarnecki, D; Anton, M

Purpose: Alanine dosimeters are often used for in-vivo dosimetry purposes in radiation therapy. In a Monte Carlo study the influence of 20 different surrounding/phantom materials for alanine dosimeters was investigated. The investigations were performed in high-energy photon beams, covering the whole range from {sup 60}Co up to 25 MV-X. The aim of the study is the introduction of a perturbation correction k{sub env} for alanine dosimeters accounting for the environmental material. Methods: The influence of different surrounding materials on the response of alanine dosimeters was investigated with Monte Carlo simulations using the EGSnrc code. The photon source was adapted withmore » BEAMnrc to a {sup 60}Co unit and an Elekta (E{sub nom}=6, 10, 25 MV-X) linear accelerator. Different tissue-equivalent materials ranging from cortical bone to lung were investigated. In addition to available phantom materials, some material compositions were taken and scaled to different electron densities. The depth of the alanine detectors within the different phantom materials corresponds to 5 cm depth in water, i.e. the depth is scaled according to the electron density (n{sub e}/n{sub e,w}) of the corresponding phantom material. The dose was scored within the detector volume once for an alanine/paraffin mixture and once for a liquid water voxel. The relative response, the ratio of the absorbed dose to alanine to the absorbed dose to water, was calculated and compared to the corresponding ratio under reference conditions. Results: For each beam quality the relative response r and the correction factor for the environment kenv was calculated. k{sub env}=0.9991+0.0049 *((n{sub e}/n{sub e,w})−0.7659){sup 3} Conclusion: A perturbation correction factor k{sub env} accounting for the phantom environment has been introduced. The response of the alanine dosimeter can be considered independent of the surrounding material for relative electron densities (n{sub e}/n{sub e,w}) between 1 and 1.4. For denser materials such as bone or much less dense surroundings such as lung, a small correction would be appropriate.« less

Purification and characterization of l,(l/d)-aminopeptidase from Guinea pig serum.

PubMed

Krstanović, Marina; Brgles, Marija; Halassy, Beata; Frkanec, Ruza; Vrdoljak, Anto; Branović, Karmen; Tomasić, Jelka; Benedetti, Fabio

2006-01-01

Mammalian sera contain enzymes that catalyze the hydrolytic degradation of peptidoglycans and molecules of related structure and are relevant for the metabolism of peptidoglycans. We now report on a novel L,(L/D)-aminopeptidase found in human and mammalian sera. The enzyme hydrolyses the pentapeptide L-Ala-D-iso-Gln-meso-DAP(omegaNH(2))-D-Ala-D-Ala yielding the free L-alanine and the respective tetrapeptide (K(M) 18 mM). L,(L/D)-aminopeptidase from guinea pig serum was highly purified in four chromatographic steps, up to 700-fold. Molecular weight of the enzyme was estimated by HPLC to be approximately 175,000. The configuration of alanine obtained by hydrolysis of the pentapeptide was determined by oxidation with L-amino acid oxidase. The amino acids sequence in the respective tetrapeptide was deduced from the results of mass spectrometry. The novel L,(L/D)-aminopeptidase also hydrolyzed alanine-4-nitroanilide (K(M)=0.6 mM) and several peptides comprising L-amino acids. Peptides containing D-amino acid at the amino end and L-Asp-L-Asp were not the substrates for this enzyme. The purified enzyme also exhibited enkephalin degrading activity, hydrolyzing enkephalins comprising L,L- and L,D-peptide bonds. The enzyme was inhibited strongly by metal chelating agents, bestatin and amastatin.

Genetics Home Reference: beta-propeller protein-associated neurodegeneration

MedlinePlus

... SE, Amemiya A, Bean LJH, Bird TD, Ledbetter N, Mefford HC, Smith RJH, Stephens K, editors. GeneReviews® [ ... TB, Kurian MA, Houlden HH, Anderson J, Boddaert N, Sanford L, Harik SI, Dandu VH, Nardocci N, ...

Preparation of polydimethylsiloxane/beta-cyclodextrin/divinylbenzene coated "dumbbell-shaped" stir bar and its application to the analysis of polycyclic aromatic hydrocarbons and polycyclic aromatic sulfur heterocycles compounds in lake water and soil by high performance liquid chromatography.

PubMed

Yu, Chunhe; Yao, Zhimin; Hu, Bin

2009-05-08

A "dumbbell-shaped" stir bar was proposed to prevent the friction loss of coating during the stirring process, and thus prolonged the lifetime of stir bars. The effects of the coating components, including polydimethylsiloxane (PDMS), beta-cyclodextrin (beta-CD) and divinylbenzene (DVB) were investigated according to an orthogonal experimental design, using three polycyclic aromatic hydrocarbons (PAHs) and four polycyclic aromatic sulfur heterocycles (PASHs) as model analytes. Four kinds of stir bars coated with PDMS, PDMS/beta-CD, PDMS/DVB and PDMS/beta-CD/DVB were prepared and their extraction efficiencies for the target compounds were compared. It was demonstrated that PDMS/beta-CD/DVB-coated stir bar showed the best affinity to the studied compounds. The preparation reproducibility of PDMS/beta-CD/DVB-coated stir bar ranged from 3.2% to 15.2% (n = 6) in one batch, and 5.2% to 13.4% (n = 6) among batches. The "dumbbell-shaped" stir bar could be used for about 40 times, which were 10 extractions more than a normal stir bar. The prepared PDMS/beta-CD/DVB-coated "dumbbell-shaped" stir bar was used for stir bar sorptive extraction (SBSE) of PAHs and PASHs and the desorbed solution was introduced into HPLC-UV for subsequent analysis. The limits of detection of the proposed method for seven target analytes ranged from 0.007 to 0.103 microg L(-1), the relative standard deviations were in the range of 6.3-12.9% (n = 6, c = 40 microg L(-1)), and the enrichment factors were 19-86. The proposed method was successfully applied to the analysis of seven target analytes in lake water and soil samples.

Self-Assembly, Supramolecular Organization, and Phase Behavior of L-Alanine Alkyl Esters (n = 9-18) and Characterization of Equimolar L-Alanine Lauryl Ester/Lauryl Sulfate Catanionic Complex.

PubMed

Sivaramakrishna, D; Swamy, Musti J

2015-09-08

A homologous series of l-alanine alkyl ester hydrochlorides (AEs) bearing 9-18 C atoms in the alkyl chain have been synthesized and characterized with respect to self-assembly, supramolecular structure, and phase transitions. The CMCs of AEs bearing 11-18 C atoms were found to range between 0.1 and 10 mM. Differential scanning calorimetric (DSC) studies showed that the transition temperatures (Tt), enthalpies (ΔHt) and entropies (ΔSt) of AEs in the dry state exhibit odd-even alternation, with the odd-chain-length compounds having higher Tt values, but the even-chain-length homologues showing higher values of ΔHt and ΔSt. In DSC measurements on hydrated samples, carried out at pH 5.0 and pH 10.0 (where they exist in cationic and neutral forms, respectively), compounds with 13-18 C atoms in the alkyl chain showed sharp gel-to-liquid crystalline phase transitions, and odd-even alternation was not seen in the thermodynamic parameters. The molecular structure, packing properties, and intermolecular interactions of AEs with 9 and 10 C atoms in the alkyl chain were determined by single crystal X-ray diffraction, which showed that the alkyl chains are packed in a tilted interdigitated bilayer format. d-Spacings obtained from powder X-ray diffraction studies exhibited a linear dependence on the alkyl chain length, suggesting that the other AEs also adopt an interdigitated bilayer structure. Turbidimetric, fluorescence spectroscopic, and isothermal titration calorimetric (ITC) studies established that in aqueous dispersions l-alanine lauryl ester hydrochloride (ALE·HCl) and sodium dodecyl sulfate (SDS) form an equimolar complex. Transmission electron microscopic and DSC studies indicate that the complex exists as unilamellar liposomes, which exhibit a sharp phase transition at ∼39 °C. The aggregates were disrupted at high pH, suggesting that the catanionic complex would be useful to develop a base-labile drug delivery system. ITC studies indicated that ALE·HCl forms a strong complex with DNA, suggesting that the AEs may find use in DNA therapeutics as well.

The effect of chaya (Cnidoscolus aconitifolius) leaf meal and of exogenous enzymes on amino acid digestibility in broilers.

PubMed

Sarmiento-Franco, L; McNab, J M; Pearson, A; Belmar-Casso, R

2003-07-01

1. The apparent ileal nitrogen (N) and amino acid digestibilities in chaya leaf meal (CLM) (Cnidoscolus aconitifolius) with added enzymes, and the same variables in diets containing different amounts of CLM were studied in chickens. 2. In the first experiment pectinase, beta-glucanase, and pectinase + beta-glucanase were added to CLM. In the second experiment, there were three diets based on maize and soybean: 0, 150 and 250 g/kg CLM. 3. Pectinase significantly increased both lysine and overall amino acid digestibilities in CLM. 4. In experiment 2, the amino acid digestibility in birds fed on CLM250 was lower than that from birds fed on either control or CLM150. Only the digestibilities of alanine, arginine and proline were lower in birds fed on CLM150 than in those fed on the control diet. Nitrogen digestibility was lower in birds fed on the CLM250 diet than on either control or CLM150 diets. These findings were attributed to the increasing concentration of fibre with increasing dietary CLM.

Endoplasmic Reticulum Stress as a Mediator of Neurotoxin-Induced Dopamine Neuron Death

DTIC Science & Technology

2007-07-01

the pathogenesis and treatment of disease. Science 267, 1456−1462. Tournier, C., Dong, C., Turner, T. K., Jones, S . N., Flavell, R. A., & Davis, R. J...are those of the author( s ) and should not be construed as an official Department of the Army position, policy or decision unless so designated by...Stress as a Mediator of Neurotoxin-Induced Dopamine Neuron Death 5b. GRANT NUMBER DAMD17-03-1-0492 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR( S ) 5d

Experimental determination of the carboxylate oxygen electric-field-gradient and chemical shielding tensors in L-alanine and L-phenylalanine

NASA Astrophysics Data System (ADS)

Yamada, Kazuhiko; Asanuma, Miwako; Honda, Hisashi; Nemoto, Takahiro; Yamazaki, Toshio; Hirota, Hiroshi

2007-10-01

We report a solid-state 17O NMR study of the 17O electric-field-gradient (EFG) and chemical shielding (CS) tensors for each carboxylate group in polycrystalline L-alanine and L-phenylalanine. The magic angle spinning (MAS) and stationary 17O NMR spectra of these compounds were obtained at 9.4, 14.1, and 16.4 T. Analyzes of these 17O NMR spectra yielded reliable experimental NMR parameters including 17O CS tensor components, 17O quadrupole coupling parameters, and the relative orientations between the 17O CS and EFG tensors. The extensive quantum chemical calculations at both the restricted Hartree-Fock and density-functional theories were carried out with various basis sets to evaluate the quality of quantum chemical calculations for the 17O NMR tensors in L-alanine. For 17O CS tensors, the calculations at the B3LYP/D95 ∗∗ level could reasonably reproduce 17O CS tensors, but they still showed some discrepancies in the δ11 components by approximately 36 ppm. For 17O EFG calculations, it was advantageous to use calibrated Q value to give acceptable CQ values. The calculated results also demonstrated that not only complete intermolecular hydrogen-bonding networks to target oxygen in L-alanine, but also intermolecular interactions around the NH3+ group were significant to reproduce the 17O NMR tensors.

Redox self-sufficient whole cell biotransformation for amination of alcohols.

PubMed

Klatte, Stephanie; Wendisch, Volker F

2014-10-15

Whole cell biotransformation is an upcoming tool to replace common chemical routes for functionalization and modification of desired molecules. In the approach presented here the production of various non-natural (di)amines was realized using the designed whole cell biocatalyst Escherichia coli W3110/pTrc99A-ald-adh-ta with plasmid-borne overexpression of genes for an l-alanine dehydrogenase, an alcohol dehydrogenase and a transaminase. Cascading alcohol oxidation with l-alanine dependent transamination and l-alanine dehydrogenase allowed for redox self-sufficient conversion of alcohols to the corresponding amines. The supplementation of the corresponding (di)alcohol precursors as well as amino group donor l-alanine and ammonium chloride were sufficient for amination and redox cofactor recycling in a resting buffer system. The addition of the transaminase cofactor pyridoxal-phosphate and the alcohol dehydrogenase cofactor NAD(+) was not necessary to obtain complete conversion. Secondary and cyclic alcohols, for example, 2-hexanol and cyclohexanol were not aminated. However, efficient redox self-sufficient amination of aliphatic and aromatic (di)alcohols in vivo was achieved with 1-hexanol, 1,10-decanediol and benzylalcohol being aminated best. Copyright © 2014 Elsevier Ltd. All rights reserved.

Precision and sensitivity of the measurement of 15N enrichment in D-alanine from bacterial cell walls using positive/negative ion mass spectrometry

NASA Technical Reports Server (NTRS)

Tunlid, A.; Odham, G.; Findlay, R. H.; White, D. C.

1985-01-01

Sensitive detection of cellular components from specific groups of microbes can be utilized as 'signatures' in the examination of microbial consortia from soils, sediments or biofilms. Utilizing capillary gas chromatography/mass spectrometry and stereospecific derivatizing agents, D-alanine, a component localized in the prokaryotic (bacterial) cell wall, can be detected reproducibly. Enrichments of D-[15N]alanine determined in E. coli grown with [15N]ammonia can be determined with precision at 1.0 atom%. Chemical ionization with methane gas and the detection of negative ions (M - HF)- and (M - F or M + H - HF)- formed from the heptafluorobutyryl D-2 butanol ester of D-alanine allowed as little as 8 pg (90 fmol) to be detected reproducibly. This method can be utilized to define the metabolic activity in terms of 15N incorporation at the level of 10(3)-10(4) cells, as a function of the 15N-14N ratio.

Development and validation of a rapid, selective, and sensitive LC-MS/MS method for simultaneous determination of D- and L-amino acids in human serum: application to the study of hepatocellular carcinoma.

PubMed

Han, Minlu; Xie, Mengyu; Han, Jun; Yuan, Daoyi; Yang, Tian; Xie, Ying

2018-04-01

A validated liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of D- and L-amino acids in human serum. Under the optimum conditions, except for DL-proline, L-glutamine, and D-lysine, the enantioseparation of the other 19 enantiomeric pairs of proteinogenic amino acids and nonchiral glycine was achieved with a CROWNPAK CR-I(+) chiral column within 13 min. The lower limits of quantitation for L-amino acids (including glycine) and D-amino acids were 5-56.25 μM and 0.625-500 nM, respectively, in human serum. The intraday precision and interday precision for all the analytes were less than 15%, and the accuracy ranged from -12.84% to 12.37% at three quality control levels. The proposed method, exhibiting high rapidity, enantioresolution, and sensitivity, was successfully applied to the quantification of D- and L-amino acid levels in serum from hepatocellular carcinoma patients and healthy individuals. The serum concentrations of L-arginine, L-isoleucine, L-aspartate, L-tryptophan, L-alanine, L-methionine, L-serine, glycine, L-valine, L-leucine, L-phenylalanine, L-threonine, D-isoleucine, D-alanine, D-glutamate, D-glutamine, D-methionine, and D-threonine were significantly reduced in the hepatocellular carcinoma patients compared with the healthy individuals (P < 0.01). D-Glutamate and D-glutamine were identified as the most downregulated serum markers (fold change greater than 1.5), which deserves further attention in hepatocellular carcinoma research. Graphical abstract Simultaneous determination of D- and L-amino acids in human serum from hepatocellular carcinoma patients and healthy individuals. AA amino acid, HCC hepatocellular carcinoma, LC liquid chromatography, MS/MS tandem mass spectrometry, NC normal control, TIC total ion chromatogram.

Fabrication of a Novel Highly Sensitive and Selective Immunosensor for Botulinum Neurotoxin Serotype A Based on an Effective Platform of Electrosynthesized Gold Nanodendrites/Chitosan Nanoparticles.

PubMed

Sorouri, Rahim; Bagheri, Hasan; Afkhami, Abbas; Salimian, Jafar

2017-05-09

In this work, a novel nanocomposite consisting of electrosynthesized gold nanodendrites and chitosan nanoparticles (AuNDs/CSNPs) has been prepared to fabricate an impedimetric immunosensor based on a screen printed carbon electrode (SPCE) for the rapid and sensitive immunoassay of botulinum neurotoxin A (BoNT/A). BoNT/A polyclonal antibody was immobilized on the nanocomposite-modified SPCE for the signal amplification. The structure of the prepared nanocomposite was investigated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The charge transfer resistance (R CT ) changes were used to detect BoNT/A as the specific immuno-interactions at the immunosensor surface that efficiently limited the electron transfer of Fe(CN)₆ 3-/4- as a redox probe at pH = 7.4. A linear relationship was observed between the %∆ R CT and the concentration logarithm of BoNT/A within the range of 0.2 to 230 pg·mL -1 with a detection limit (S/N = 3) of 0.15 pg·mL -1 . The practical applicability of the proposed sensor was examined by evaluating the detection of BoNT/A in milk and serum samples with satisfactory recoveries. Therefore, the prepared immunosensor holds great promise for the fast, simple and sensitive detection of BoNT/A in various real samples.

Cyclic mu-opioid receptor ligands containing multiple N-methylated amino acid residues.

PubMed

Adamska-Bartłomiejczyk, Anna; Janecka, Anna; Szabó, Márton Richárd; Cerlesi, Maria Camilla; Calo, Girolamo; Kluczyk, Alicja; Tömböly, Csaba; Borics, Attila

2017-04-15

In this study we report the in vitro activities of four cyclic opioid peptides with various sequence length/macrocycle size and N-methylamino acid residue content. N-Methylated amino acids were incorporated and cyclization was employed to enhance conformational rigidity to various extent. The effect of such modifications on ligand structure and binding properties were studied. The pentapeptide containing one endocyclic and one exocyclic N-methylated amino acid displayed the highest affinity to the mu-opioid receptor. This peptide was also shown to be a full agonist, while the other analogs failed to activate the mu opioid receptor. Results of molecular docking studies provided rationale for the explanation of binding properties on a structural basis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Peptidase activity in various species of dairy thermophilic lactobacilli.

PubMed

Gatti, M; Fornasari, M E; Lazzi, C; Mucchetti, G; Neviani, E

2004-01-01

The aim of the present work was to evaluate the enzymatic potential manifested by aminopeptidase activity of different thermophilic Lactobacillus biotypes and to measure the influence of cell growth phase on enzyme expression. The activities were evaluated by the hydrolysis of beta-naphthylamide substrates for both whole and mechanically disrupted cells of L. helveticus, L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis strains, collected from both the exponential and the stationary growth phase. In general, activities were higher for cells in the exponential rather than in the stationary phase and the disrupted cells showed higher activities than the whole cells. The highest activity expressed by all strains corresponded to X-prolyl-dipeptidyl aminopeptidase while a moderate activity was observed towards Arg-betaNa, Lys-betaNa and Leu-betaNa. The lowest activity was observed for Pro-betaNa. It may be inferred that the cell structure and the cell physiology are crucial to define the level of efficiency of expression for aminopeptidase activity. The two species may be characterized by a different enzymatic system that hydrolyses N-terminal leucine. The differences of peptidase activities in L. helveticus and L. delbrueckii species acquires an importance to comprehend their role in the biochemical events occurring in cheese ripening.

Metabolic engineering of Corynebacterium glutamicum ATCC13869 for L-valine production.

PubMed

Chen, Cheng; Li, Yanyan; Hu, Jinyu; Dong, Xunyan; Wang, Xiaoyuan

2015-05-01

In this study, an L-valine-producing strain was developed from Corynebacterium glutamicum ATCC13869 through deletion of the three genes aceE, alaT and ilvA combined with the overexpression of six genes ilvB, ilvN, ilvC, lrp1, brnF and brnE. Overexpression of lrp1 alone increased L-valine production by 16-fold. Deletion of the aceE, alaT and ilvA increased L-valine production by 44-fold. Overexpression of the six genes ilvB, ilvN, ilvC, lrp1, brnE and brnF in the triple deletion mutant WCC003 further increased L-valine production. The strain WCC003/pJYW-4-ilvBNC1-lrp1-brnFE produced 243mM L-valine in flask cultivation and 437mM (51g/L) L-valine in fed-batch fermentation and lacked detectable amino-acid byproduct such as l-alanine and l-isoleucine that are usually found in the fermentation of L-valine-producing C. glutamicum. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Mutual orientation of three magnetic tensors in a polycrystalline dipeptide by dipole-modulated 15N chemical shift spectroscopy

NASA Astrophysics Data System (ADS)

Hartzell, C. J.; Pratum, T. K.; Drobny, G.

1987-10-01

This study demonstrates the mutual orientation of three tensor interactions in a single NMR experiment. The orientation of the 15N chemical shift tensor relative to the molecular frame has thus been determined in polycrystalline L-[1-13C] alanyl-L-[15N] alanine. The 13C-15N and 15N-1H dipole interactions are determined using the 1H dipole-modulated, 13C dipole-coupled 15N spectrum obtained as a transform of the data in t2. From simulations of the experimental spectra, two sets of polar angles have been determined relating the 13C-15N and 15N-1H dipoles to the 15N chemical shift tensor. The values determined are βCN =106°, αCN =5° and βNH =-19°, αNH =12°. The experiment verifies, without reference to single crystal data, that σ33 lies in the peptide plane and σ22 is nearly perpendicular to the plane.

Amyloid-beta peptide oligomers disrupt axonal transport through an NMDA receptor-dependent mechanism that is mediated by glycogen synthase kinase 3beta in primary cultured hippocampal neurons.

PubMed

Decker, Helena; Lo, Karen Y; Unger, Sandra M; Ferreira, Sergio T; Silverman, Michael A

2010-07-07

Disruption of axonal transport is a hallmark of several neurodegenerative diseases, including Alzheimer's disease (AD). Even though defective transport is considered an early pathologic event, the mechanisms by which neurodegenerative insults impact transport are poorly understood. We show that soluble oligomers of the amyloid-beta peptide (AbetaOs), increasingly recognized as the proximal neurotoxins in AD pathology, induce disruption of organelle transport in primary hippocampal neurons in culture. Live imaging of fluorescent protein-tagged organelles revealed a marked decrease in axonal trafficking of dense-core vesicles and mitochondria in the presence of 0.5 microm AbetaOs. NMDA receptor (NMDAR) antagonists, including d-AP5, MK-801, and memantine, prevented the disruption of trafficking, thereby identifying signals for AbetaO action at the cell membrane. Significantly, both pharmacological inhibition of glycogen synthase kinase-3beta (GSK-3beta) and transfection of neurons with a kinase-dead form of GSK-3beta prevented the transport defect. Finally, we demonstrate by biochemical and immunocytochemical means that AbetaOs do not affect microtubule stability, indicating that disruption of transport involves a more subtle mechanism than microtubule destabilization, likely the dysregulation of intracellular signaling cascades. Results demonstrate that AbetaOs negatively impact axonal transport by a mechanism that is initiated by NMDARs and mediated by GSK-3beta and establish a new connection between toxic Abeta oligomers and AD pathology.

Influence of the composition of aqueous dimethylsulfoxide solvent on thermodynamics of complexing between 18-crown-6-ether and D,L-alanine

NASA Astrophysics Data System (ADS)

Usacheva, T. R.; Kuzmina, I. A.; Sharnin, V. A.; Chernov, I. V.; Matteoli, E.

2012-07-01

Standard thermodynamic parameters (log K o, Δr H o, TΔr S o) of complexing 18-crown-6 ether (18C6) with D,L-alanine (Ala) in mixed water-dimethysulfoxide (H2O-DMSO) solvents are calculated on the basis of calorimetric titration results. A rise in the DMSO concentration in mixed solvent is found to increase stability and increase the exothermicity of the formation of [Ala-18C6] molecular complex. Changes in the reaction energetic are shown to be determined by changes in the solvation state of 18C6 that is the characteristic of the reactions of molecular complex formation between 18C6 and D,L-alanine or glycine in water-organic solvents.

Uncovering the proteome response of murine neuroblastoma cells against low-dose exposure to saxitoxin.

PubMed

Chen, Xiao; Sun, Ye; Huang, Haiyan; Liu, Wei; Hu, Panpan; Huang, Xinfeng; Zou, Fei; Liu, Jianjun

2018-06-01

The potent neurotoxin saxitoxin produced by both marine and freshwater phytoplankton causes paralytic shellfish poisoning syndrome. The toxicity and mode of action of the acute exposure of high-dose saxitoxin have been intensively studied for decades; however, the potential risk of exposure of low-dose saxitoxin remained to be uncovered. Here we present a proteomics study of murine neuroblastoma N2A cell with low-dose saxitoxin exposure (1 nM and 10 nM, 24-h intoxication). Differential proteins were profiled by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). A total of 9 proteins, including 14-3-3 beta (1433B), alpha enolase (ENO1) and cofilin 2 (CFL2), were altered by the low-dose saxitoxin exposure. We further validated the expressions of 1433B, ENO1 and CFL2 by Western blot analysis and the enzyme-linked immunosorbent assay. These 9 proteins involve cell apoptotic pathways, cell skeleton maintenance, membrane potentials and mitochondrial functions. Modulation of these 9 proteins by low-dose saxitoxin exposure could correlate to the reports on genotoxicity and neurotoxicity induced by saxitoxin. This study also suggested other potential risks of saxitoxin.

Triterpene glycosides from the tubers of Anemone coronaria.

PubMed

Mimaki, Yoshihiro; Watanabe, Kazuki; Matsuo, Yukiko; Sakagami, Hiroshi

2009-07-01

Six new triterpene glycosides (1-6), together with 11 known ones (7-17), have been isolated from a glycoside-enriched fraction prepared from the tubers of Anemone coronaria L. (Ranunculaceae). On the basis of extensive spectroscopic analysis, including 2D NMR data, and the results of hydrolytic cleavage, the structures of 1-6 were determined to be 3beta-[(O-beta-D-glucopyranosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-2beta,23-dihydroxyolean-12-en-28-oic acid (1), 3beta-[(O-beta-D-glucopyranosyl-(1-->3)-O-alpha-L-rhamnopyranosyl-(1-->2)-O-[beta-D-glucopyranosyl-(1-->4)]-alpha-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid (2), 3beta-[(O-beta-D-glucopyranosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (3), 3beta-[(O-beta-D-glucopyranosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-2beta,23-dihydroxyolean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (4), 3beta-[(O-beta-D-glucopyranosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-2beta-hydroxyolean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (5), and 3beta-[(O-beta-D-glucopyranosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-23-hydroxyolean-18-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (6), respectively. Furthermore, the isolated compounds were evaluated for their cytotoxic activity against HSC-2 cells.

Advanced drug delivery of N-acetylcarnosine (N-acetyl-beta-alanyl-L-histidine), carcinine (beta-alanylhistamine) and L-carnosine (beta-alanyl-L-histidine) in targeting peptide compounds as pharmacological chaperones for use in tissue engineering, human disease management and therapy: from in vitro to the clinic.

PubMed

Babizhayev, Mark A; Yegorov, Yegor E

2010-11-01

A pharmacological chaperone is a relatively new concept in the treatment of certain chronic disabling diseases. Cells maintain a complete set of functionally competent proteins normally and in the face of injury or environmental stress with the use of various mechanisms, including systems of proteins called molecular chaperones. Proteins that are denatured by any form of proteotoxic stress are cooperatively recognized by heat shock proteins (HSP) and directed for refolding or degradation. Under non-denaturing conditions HSP have important functions in cell physiology such as in transmembrane protein transport and in enabling assembly and folding of newly synthesized polypeptides. Besides cellular molecular chaperones, which are stress-induced proteins, there have been recently reported chemical, or so-called pharmacological chaperones with demonstrated ability to be effective in preventing misfolding of different disease causing proteins, specifically in the therapeutic management of sight-threatening eye diseases, essentially reducing the severity of several neurodegenerative disorders (such as age-related macular degeneration), cataract and many other protein-misfolding diseases. This work reviews the biological and therapeutic activities protected with the patents of the family of imidazole-containing peptidomimetics Carcinine (β-alanylhistamine), N-acetylcarnosine (N-acetyl-β-alanylhistidine) and Carnosine (β-alanyl-L-histidine) which are essential constituents possessing diverse biological and pharmacological chaperone properties in human tissues.

Identification of novel psychoactive substances 25B-NBOMe and 4-CMC in biological material using HPLC-Q-TOF-MS and their quantification in blood using UPLC-MS/MS in case of severe intoxications.

PubMed

Wiergowski, Marek; Aszyk, Justyna; Kaliszan, Michał; Wilczewska, Kamila; Anand, Jacek Sein; Kot-Wasik, Agata; Jankowski, Zbigniew

2017-01-15

This paper describes cases of poisoning caused by new psychoactive substances such as: 25B-NBOMe (2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine) and 4-CMC (1-(4-chlorophenyl)-2-(methylamino)-1-propanone). The analytical procedure includes rapid and selective method for the extraction and determination of 4-CMC and 25B-NBOMe in blood samples using UPLC-MS/MS technique. To the best of our knowledge, this is the first report, that involves a fully validated method for quantification of new-designer drug - 4-CMC in postmortem blood samples. The biological material was also analyzed with the use of routine analytical methods: immunochemical techniques, gas chromatography with flame ionization detection and gas chromatography with electron impact mass spectrometry. The results of real samples analyses correspond to possible toxicological effects: death resulting from 25B-NBOMe - mediated hallucinations (661ng/mL of 25B-NBOMe and 0.887ng/mL of 4-CMC), fatal overdose of 25B-NBOMe and 4-CMC (66.5ng/mL of 25B-NBOMe and 2.14ng/mL of 4-CMC) and non-fatal intoxication of these drugs (38.4ng/mL of 25B NBOMe and 0.181ng/mL of 4-CMC). Additionally, O-demethylathed O, O-bis-demethylathed and glucuronidated metabolites of 25B-NBOMe in biological specimens were detected. Copyright © 2016 Elsevier B.V. All rights reserved.

Neurotoxin and Epitope Structural Studies

DTIC Science & Technology

1991-01-25

N., Oguma, K., Yokosawa , N., Kimura, K., and Tsuzuki, K., 1988, AppI. Environ. Microbiol., 54, 69-73. 15. Gimenez, J., Foley, J., and DasGupta, B.R...Wernars, K., and Niemann H., 1990, J. Biol. Chem., 265, 9153-9158. 18. Kimura, K., Fujii, N., Tsuzuki, K., Murakami, T., Indoh, T., Yokosawa , N

Anaerobic metabolism in the N-limited green alga Selenastrum minutum. 3. Alanine is the product of anaerobic ammonium assimilation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vanlerberghe, G.C.; Turpin, D.H.; Joy, K.W.

The authors have determined the flow of {sup 15}N into free amino acids of the N-limited green alga Selenastrum minutum (Naeg.) Collins after addition of {sup 15}NH{sub 4}{sup +} to aerobic or anaerobic cells. Under aerobic conditions, only a small proportion of the N assimilated was retained in the free amino acid pool. However, under anaerobic conditions almost all assimilated NH{sub 4}{sup +} accumulates in alanine. This is a unique feature of anaerobic NH{sub 4}{sup +} assimilation. The pathway of carbon flow to alanine results in the production of ATP and reductant which matches exactly the requirements of NH{sub 4}{supmore » +} assimilation. Alanine synthesis is therefore an excellent strategy to maintain energy and redox balance during anaerobic NH{sub 4}{sup +} assimilation.« less

Transpeptidation reactions of a specific substrate catalyzed by the streptomyces R61 DD-peptidase: characterization of a chromogenic substrate and acyl acceptor design.

PubMed

Kumar, Ish; Pratt, R F

2005-08-02

The Streptomyces R61 dd-peptidase, a functional model for penicillin-binding proteins, catalyzes the hydrolysis and aminolysis of d-alanyl-d-alanine-terminating peptides by specific amines. In vivo, this reaction completes bacterial cell wall biosynthesis. For in vitro studies of this enzyme to date, various nonspecific acyl-donor substrates have been employed. Recently, however, a peptidoglycan-mimetic peptide substrate, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, has been described that is much more specific for this enzyme. In this paper, we describe the synthesis and kinetic characterization of an analogous thiolester substrate, 3-(N-glycyl-l-cysteinyl)-propanoyl-d-alanyl-d-thiolactate, that the enzyme hydrolyzes and aminolyzes very efficiently (k(cat)/K(m) = 1.0 x 10(7) s(-)(1) M(-)(1)). Direct or indirect, by means of a thiol trap, spectrophotometric monitoring of the reactions of this substrate is readily achieved. Deacylation of the enzyme is rate-determining under substrate saturation conditions, and therefore the aminolysis reaction can be directly studied. The results show that d-amino acids and certain Gly-l-Xaa dipeptides and tripeptides may act as acyl acceptors at the active site of the enzyme. d-Phenylalanine and Gly-l-Phe were the most effective d-amino acid and dipeptide acceptors, respectively. On the basis of the dual specificity of the active site for acceptors (d-amino acids and Gly-l-Xaa peptides), "dual function" acceptors were designed and synthesized. Two of these, aminomalon-(N-ethyl)amide and aminomalon-(N-phenethyl)amide, were particularly effective. It did seem, however, that the observed rates of reaction of these very effective acceptors may be limited by some common, possibly physical, step. More extended, peptidoglycan-like, acceptors were found to be essentially unreactive. The reasons for this counterintuitive behavior are discussed.

Gamow-Teller strength observed in the 48Ti(n, p) 48Sc reaction: Implications for the double beta decay of 48Ca

NASA Astrophysics Data System (ADS)

Alford, W. P.; Helmer, R. L.; Abegg, R.; Celler, A.; Frekers, D.; Green, P.; Häusser, O.; Henderson, R.; Hicks, K.; Jackson, K. P.; Jeppesen, R.; Miller, C. A.; Trudel, A.; Vetterli, M.; Yen, S.; Pourang, R.; Watson, J.; Brown, B. A.; Engel, J.

1990-07-01

Cross sections for the 48Ti(n, p) reaction have been measured at angles of 0°, 6°, and 12° at an energy of 200 MeV. The measurements are compared with results of DWIA calculations to obtain estimates of transition strengths for L = 0, 1, and ⩾2 up to an excitation energy of 25 MeV. Gamow-Teller strength ( L = 0) is peaked between 3 and 4 MeV excitation energy, with a significant distribution extending to about 12 MeV. The L = 1 strength is found mainly between 6 and 20 MeV while the cross section for transitions with L ⩾2 increases from 10 MeV to the upper limit of the measurements. The distribution of Gamow-Teller strength is in poor agreement with theoretical distributions used to calculate the lifetime for double beta decay of 48Ca.

Modulation of TGF-beta signaling during progression of chronic liver diseases.

PubMed

Matsuzaki, Koichi

2009-01-01

A large body of work has established roles for epithelial cells as important mediators of progressive fibrosis and carcinogenesis. Transforming growth factor-beta (TGF-beta) and pro-inflammatory cytokines are important inducers of fibro-carcinogenesis. TGF-beta signaling involves phosphorylation of Smad3 at middle linker and/or C-terminal regions. Reversible shifting of Smad3-dependent signaling between tumor-suppression and oncogenesis in hyperactive Ras-expressing epithelial cells indicates that Smad3 phosphorylated at the C-terminal region (pSmad3C) transmits a tumor-suppressive TGF-beta signal, while oncogenic activities such as cell proliferation and invasion are promoted by Smad3 phosphorylated at the linker region (pSmad3L). Notably, pSmad3L-mediated signaling promotes extracellular matrix deposition by activated mesenchymal cells. During progression of chronic liver diseases, hepatic epithelial hepatocytes undergo transition from the tumor-suppressive pSmad3C pathway to the fibrogenic/oncogenic pSmad3L pathway, accelerating liver fibrosis and increasing risk of hepatocellular carcinoma. c-Jun N-terminal kinase activated by pro-inflammatory cytokines is mediating this perturbed hepatocytic TGF-beta signaling. Thus, TGF-beta signaling of hepatocytes affected by chronic inflammation offers a general framework for understanding the molecular mechanisms of human fibro-carcinogenesis during progression of chronic liver diseases.

Silver-nanoparticle-coated biliary stent inhibits bacterial adhesion in bacterial cholangitis in swine.

PubMed

Wen, Wei; Ma, Li-Mei; He, Wei; Tang, Xiao-Wei; Zhang, Yin; Wang, Xiang; Liu, Li; Fan, Zhi-Ning

2016-02-01

One of the major limitations of biliary stents is the stent occlusion, which is closely related to the over-growth of bacteria. This study aimed to evaluate the feasibility of a novel silver-nanoparticle-coated polyurethane (Ag/PU) stent in bacterial cholangitis model in swine. Ag/PU was designed by coating silver nanoparticles on polyurethane (PU) stent. Twenty-four healthy pigs with bacterial cholangitis using Ag/PU and PU stents were randomly divided into an Ag/PU stent group (n=12) and a PU stent group (n=12), respectively. The stents were inserted by standard endoscopic retrograde cholangiopancreatography. Laboratory assay was performed for white blood cell (WBC) count, alanine aminotransferase (ALT), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) at baseline time, 8 hours, 1, 2, 3, and 7 days after stent placements. The segment of bile duct containing the stent was examined histologically ex vivo. Implanted biliary stents were examined by a scan electron microscope. The amount of silver release was also measured in vitro. The number of inflammatory cells and level of ALT, IL-1beta and TNF-alpha were significantly lower in the Ag/PU stent group than in the PU stent group. Hyperplasia of the mucosa was more severe in the PU stent group than in the Ag/PU stent group. In contrast to the biofilm of bacteria on the PU stent, fewer bacteria adhered to the Ag/PU stent. PU biliary stents modified with silver nanoparticles are able to alleviate the inflammation of pigs with bacterial cholangitis. Silver-nanoparticle-coated stents are resistant to bacterial adhesion.

Structural and catalytic roles of amino acid residues located at substrate-binding pocket in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase.

PubMed

Chen, Je-Hsin; Tsai, Li-Chu; Huang, Hsiao-Chuan; Shyur, Lie-Fen

2010-10-01

We created 12 mutant enzymes (E11L, F40I, Y42L, N44L, N44Q, E47I, L62G, K64A, K64M, R137M, R137Q, and N139A) from the truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (TF-glucanase). The enzymes were used to investigate the structural and catalytic roles of specific amino acid residues located at the catalytic pocket and having direct interactions with glucose subsites of the product beta-1,3-1,4-cellotriose (CLTR). Fluorescence spectrometry showed no discernible changes in secondary structures among purified TF-glucanase and the mutants. Kinetic analyses showed E11L, F40I, Y42L, R137M, and R137Q with a >10-fold decrease of specific activity (11.2- to 67.4-fold), and E11L, N44Q, E47I, K64M, R137M, R137Q, and N139A with a 2.17- to 4.3-fold increase of K(m) value when compared with TF-glucanase. Notably, E11L, R137Q, R137M, F40I, and N139A showed the most significant decrease in catalytic efficiency relative to TF-glucanase, by 2155-, 84.9-, 48.5-, 41.1-, and 19.1-fold, respectively; the five mutants showed the greatest changes in comparative energy DeltaDeltaG(b), with values of 1.94 to 4.92 kcal/mol. Combined with results from kinetic and structure modeling analyses of all mutant enzymes and X-ray crystallography of F40I, we elucidate that Glu11, Phe40, Arg137, and Asn139 play a crucial role in the catalysis of TF-glucanase owing to their local and direct interaction through hydrogen bonds or van der Waals stacking interaction by aromatic rings onto the glucose subsites -3, -2, and -1 of CLTR/substrate. The overall globular structures in the wild-type and mutant F40I enzymes do not differ. 2010 Wiley-Liss, Inc.

Synthesis and studies of polypeptide materials: Enantioselective polymerization of gamma-benzyl glutamate-N-carboxyanhydride and synthesis of optically active poly(beta-peptides)

NASA Astrophysics Data System (ADS)

Cheng, Jianjun

A class of zero-valent transition metal complexes have been developed by Deming et al for the controlled polymerization of alpha-aminoacid-N-carboxyanhydrides (alpha-NCAs). This discovery provided a superior starting point for the development of enantioselective polymerizations of racemic alpha-NCAs. Bidentate chiral ligands were synthesized and tested for their abilities to induce enantioselective polymerization of gamma-benzyl-glutamate NCA (Glu NCA) when they were coordinated to zero-valent nickel complexes. When optically active 2-pyridinyl oxazoline ligands were mixed with bis(1,5-cyclooctadiene)nickel in THF, chiral nickel complexes were formed that selectively polymerized one enantiomer of Glu NCA over the other. The highest selectivity was observed with the nickel complex of (S)-4-tert-butyl-2-pyridinyl oxazoline, which gave a ratio of enantiomeric polymerization rate constants (kD/kL) of 5.2. It was found that subtle modification of this ligand by incorporation of additional substituents had a substantial impact on initiator enantioselectivities. In separate efforts, methodology was developed for the general synthesis of optically active beta-aminoacid-N-carboxyanhydrides (beta-NCAs) via cyclization of Nbeta-Boc- or Nbeta-Cbz-beta-amino acids using phosphorus tribromide. The beta-NCA molecules could be polymerized in good yields using strong bases or transition metal complexes to give optically active poly(beta-peptides) bearing proteinogenic side chains. The resulting poly(beta-peptides), which have moderate molecular weights, adopt stable helical conformations in solution. Poly(beta-homoglutamate and poly(beta-homolysine), the side-chain deprotected polymers, were found to display pH dependent helix-coil conformation transitions in aqueous solution, similar to their alpha-analogs. A novel method for poly(beta-aspartate) synthesis was developed via the polymerization of L-aspartate alkyl ester beta lactams using metal-amido complexes. Poly(beta-aspartates) bearing short ethylene glycol side chains were obtained with controlled molecular weights and narrow molecular weight distributions when Sc(N(TMS)2)3 was used as initiator for the beta-lactam polymerizations. Polymer chain lengths could be controlled by both stoichiometry and monomer conversion, characteristic of a living polymerization system. Di- and tri-block copoly(beta-peptides) with desired chain lengths were also synthesized using this method. It was found that these techniques were generally applicable for the synthesis of poly(beta-peptides), bearing other proteinogetic side chains. Synthesis and studies of polypeptide materials were extended to unexplored areas by incorporation of both alpha- and beta-amino acid residues into single polymer chains. Two sequence specific polypeptides bearing alternating beta-alpha, or beta-alpha-alpha amino acid residues were synthesized. Both polymers were found to adopt unprecedented stable conformations in solution.

Factors associated with elevated serum alanine aminotransferase in patients with type 1 diabetes mellitus.

PubMed

Hatanaka, S A; Silva, N O; Colombo, B S; Correa, C G; Alcaire, B P; Coral, M H; Schiavon, L L; Narciso-Schiavon, J L

2015-09-01

Metabolic syndrome and type 2 diabetes are associated with insulin resistance and hepatic steatosis, which are common causes of alanine aminotransferase (ALT) elevation. This study aims to identify variables associated with altered ALT in type 1 diabetic (DM1) subjects. A cross-sectional study conducted in the outpatient endocrinology clinic of a university hospital. Patients with DM1 were seen between December 2012 and September 2013; clinical variables were collected from medical records. Fifty-six patients were included aged 27 ± 10.1 years; 60.7% were men. The study subjects exhibited an average ALT of 36.7 ± 10.3 U/L (median = 35 U/L) and their average Body Mass Index (BMI) was 23.8 ± 3.8 kg/m2. When comparing individuals with elevated ALT > 35 U/L (N. = 27) with those ALT ≤ 35 U/L (N. = 29), we found that individuals with ALT values > 35 U/L showed a higher proportion of men (77.8% vs. 44.8%, P = 0.012) and a higher mean age (30.2 ± 12.3 vs. 24.6 ± 6.9 years, P = 0.046). When new ALT reference values were applied (19 U/L for women and 30 U/L for men), five individuals had normal ALT values. Individuals with elevated ALT had higher BMI (24.3 vs. 20.9; P = 0.036), fasting glucose (194.8 ± 101.2 vs. 123.6 ± 42.0 mg/dL; P = 0.013) and higher HbA1c (9.9 ± 2.8 vs. 7.8 ± 0.7%; P < 0.001) levels. In Pearson correlation analysis, ALT values ​correlated with HbA1c (r = 0.285; P = 0.033). In patients with DM1, elevated ALT values ​​are associated with BMI, fasting glucose and HbA1c.

Dose-effect relationship between drinking water fluoride levels and damage to liver and kidney functions in children.

PubMed

Xiong, Xianzhi; Liu, Junling; He, Weihong; Xia, Tao; He, Ping; Chen, Xuemin; Yang, Kedi; Wang, Aiguo

2007-01-01

Although a dose-effect relationship between water fluoride levels and damage to liver and kidney functions in animals has been reported, it was not demonstrated in humans. To evaluate the effects of drinking water fluoride levels on the liver and kidney functions in children with and without dental fluorosis, we identified 210 children who were divided into seven groups with 30 each based on different drinking water fluoride levels in the same residential area. We found that the fluoride levels in serum and urine of these children increased as the levels of drinking water fluoride increased. There were no significant differences in the levels of total protein (TP), albumin (ALB), aspartate transamine (AST), and alanine transamine (ALT) in serum among these groups. However, the activities of serum lactic dehydrogenase (LDH), urine N-acetyl-beta-glucosaminidase (NAG), and urine gamma-glutamyl transpeptidase (gamma-GT) in children with dental fluorosis and having water fluoride of 2.15-2.96 mg/L and in children having water fluoride of 3.15-5.69 mg/L regardless of dental fluorosis were significantly higher than children exposed to water fluoride of 0.61-0.87 mg/L in a dose-response manner. In contrast to children with dental fluorosis and having water fluoride of 2.15-2.96 and 3.10-5.69 mg/L, serum LDH activity of children without dental fluorosis but exposed to the same levels of water fluoride as those with dental fluorosis were also markedly lower, but the activities of NAG and gamma-GT in their urine were not. Therefore, our results suggest that drinking water fluoride levels over 2.0mg/L can cause damage to liver and kidney functions in children and that the dental fluorosis was independent of damage to the liver but not the kidney. Further studies on the mechanisms and significance underlying damage to the liver without dental fluorosis in the exposed children are warranted.

Calorimetric and spectroscopic studies of the thermotropic phase behavior of lipid bilayer model membranes composed of a homologous series of linear saturated phosphatidylserines.

PubMed Central

Lewis, R N; McElhaney, R N

2000-01-01

The thermotropic phase behavior of lipid bilayer model membranes composed of the even-numbered, N-saturated 1,2-diacyl phosphatidylserines was studied by differential scanning calorimetry and by Fourier-transform infrared and (31)P-nuclear magnetic resonance spectroscopy. At pH 7.0, 0.1 M NaCl and in the absence of divalent cations, aqueous dispersions of these lipids, which have not been incubated at low temperature, exhibit a single calorimetrically detectable phase transition that is fully reversible, highly cooperative, and relatively energetic, and the transition temperatures and enthalpies increase progressively with increases in hydrocarbon chain length. Our spectroscopic observations confirm that this thermal event is a lamellar gel (L(beta))-to-lamellar liquid crystalline (L(alpha)) phase transition. However, after low temperature incubation, the L(beta)/L(alpha) phase transition of dilauroyl phosphatidylserine is replaced by a higher temperature, more enthalpic, and less cooperative phase transition, and an additional lower temperature, less enthalpic, and less cooperative phase transition appears in the longer chain phosphatidylserines. Our spectroscopic results indicate that this change in thermotropic phase behavior when incubated at low temperatures results from the conversion of the L(beta) phase to a highly ordered lamellar crystalline (L(c)) phase. Upon heating, the L(c) phase of dilauroyl phosphatidylserine converts directly to the L(alpha) phase at a temperature slightly higher than that of its original L(beta)/L(alpha) phase transition. Calorimetrically, this process is manifested by a less cooperative but considerably more energetic, higher-temperature phase transition, which replaces the weaker L(beta)/L(alpha) phase transition alluded to above. However, with the longer chain compounds, the L(c) phase first converts to the L(beta) phase at temperatures some 10-25 degrees C below that at which the L(beta) phase converts to the L(alpha) phase. Our results also suggest that shorter chain homologues form L(c) phases that are structurally related to, but more ordered than, those formed by the longer chain homologues, but that these L(c) phases are less ordered than those formed by other phospholipids. These studies also suggest that polar/apolar interfaces of the phosphatidylserine bilayers are more hydrated than those of other glycerolipid bilayers, possibly because of interactions between the polar headgroup and carbonyl groups of the fatty acyl chains. PMID:11023908

Molecular composition and extinction coefficient of native botulinum neurotoxin complex produced by Clostridium botulinum hall A strain.

PubMed

Bryant, Anne-Marie; Davis, Jenny; Cai, Shuowei; Singh, Bal Ram

2013-02-01

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)(-1)cm(-1). These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.

Cell Communication during Aggregation and Development of the Cellular Slime Mould Distyostelium discoideum.

DTIC Science & Technology

1985-01-01

of actin protein xg relative centrifugal force glorin N-propionyl- Y -L-glutawyl-L-ornithine- S- lactam ethyl ester [3 H]FA [7,9,3’,5 ’-3H]folic acid...solubilize the pellet and radioactivity was measured on a LKB Rack Beta scintillation counter. cAMP Binding to Whole Cells. This assay followed the well...inserts, pre-filled with 4ml of Unisolve I scintillant, and radioactivity measured on a LKB Rack Beta scintillation counter. Controls included: a) no

The effect of additives on red cell 2,3 diphosphoglycerate levels in CPDA preservatives.

PubMed

Vora, S; West, C; Beutler, E

1989-01-01

Forty-two chemical substances, chosen because they might influence red cell metabolism, were screened for effect on red cell adenosine triphosphate and 2,3 diphosphoglycerate (2,3 DPG) levels during storage in CPD or CPDA-1 at 4 degrees C. Of these substances, six appeared on initial screening to increase 2,3 DPG levels during storage; on repeated examination, four compounds, i.e., oxalate, glyoxalate, ethyl oxaloacetate, and L-phenylalanyl-L-alanine, consistently increased 2,3 DPG levels during storage. It was shown that glyoxalate was converted rapidly to oxalate in blood, presumably through the lactate dehydrogenase reaction. Ethyl oxaloacetate is known to hydrolyze, giving rise to oxalate. Thus, the effect of both glyoxalate and ethyl oxaloacetate can be explained by the formation of oxalate, a compound already known to increase 2,3 DPG levels. The effect of L-phenylalanyl-L-alanine remains to be explained, but it may be hydrolyzed to L-alanine and L-phenylalanine, both of which are thought to have the capacity to increase red cell 2,3 DPG levels by inhibiting pyruvate kinase activity.

Effects of cafestol and kahweol from coffee grounds on serum lipids and serum liver enzymes in humans.

PubMed

Urgert, R; Schulz, A G; Katan, M B

1995-01-01

The diterpenes cafestol and kahweol are present in unfiltered coffee in oil droplets and floating fines. They elevate serum cholesterol and alanine aminotransferase (ALT). We measured fines in coffee brews, and examined diterpene availability from spent grounds in healthy volunteers. Turkish or Scandinavian boiled coffee contained 2-5 g fines/L and French press coffee contained 1.5 g fines/L. An intake of 8 g fine grounds/d for 3 wk increased cholesterol by 0.65 mmol/L (95% CI 0.41-0.89 mmol/L) and ALT by 18 U/L (95% CI 4-32 U/L) relative to control subjects (n = 7/group). In a crossover study (n = 15), mean serum cholesterol was 4.9 mmol/L after consumption of both fine and coarse grounds for 10 d (P = 0.43). Serum ALT activities were 29 U/L on fine and 21 U/L on coarse grounds (P = 0.02). Floating fines could contribute substantially to the hyperlipidemic and ALT-elevating effect of unfiltered coffee. Diterpene measurements in coffee brews should include the contribution of fines.

Evaluation of Lubrication Systems for Isothermal Forging of Alpha-Beta and Beta Titanium Alloys.

DTIC Science & Technology

1977-11-01

in u i u , ’ F~~~U BEVCSR E CC’W11F.TJNG PORNI ~\\ ~ - I~JI~*lNUllS1IIl a, AccI$$IOM N ICIPIENT - ~~~~~~~~~ I _ _ _( 4 T ITL E (~~~ AOlIU ~J

Convergent synthesis of a trisaccharide as its 2-(trimethylsilyl)ethyl glycoside related to the flavonoid triglycoside from Gymnema sylvestre.

PubMed

Mukhopadhyay, Balaram; Field, Robert A

2006-07-24

The glycone part of the flavonoid triglycoside, kaempferol 3-O-beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-galactopyranoside, has been synthesized in good yield and stereoselectivity using N-iodosuccinimide and HClO4-silica promoted glycosylations of thioglycoside donors.

The influence of feeding and fasting on plasma metabolites in the dogfish shark (Squalus acanthias).

PubMed

Wood, Chris M; Walsh, Patrick J; Kajimura, Makiko; McClelland, Grant B; Chew, Shit F

2010-04-01

Dogfish sharks are opportunistic predators, eating large meals at irregular intervals. Here we present a synthesis of data from several previous studies on responses in plasma metabolites after natural feeding and during prolonged fasting (up to 56days), together with new data on changes in plasma concentrations of amino acids and non-esterified fatty acids. Post-prandial and long-term fasting responses were compared to control sharks fasted for 7days, a typical inter-meal interval. A feeding frenzy was created in which dogfish were allowed to feed naturally on dead teleosts at two consumed ration levels, 2.6% and 5.5% of body weight. Most responses were more pronounced at the higher ration level. These included increases in urea and TMAO concentrations at 20h, followed by stability through to 56days of fasting. Ammonia levels were low and exhibited little short-term response to feeding, but declined to very low values during the extended fast. Glucose and beta-hydroxybutyrate both fell after feeding, the latter to a greater and more prolonged extent (up to 60h), whereas acetoacetate did not change. During prolonged fasting, glucose concentrations were well regulated, but beta-hydroxybutyrate increased to 2-3-fold control levels. Total plasma amino acid concentrations increased in a biphasic fashion, with peaks at 6-20h, and 48-60h after the meal, followed by homeostasis during the extended fast. Essential and non-essential amino acids generally followed this same pattern, though some exhibited different trends after feeding: taurine, beta-alanine, and glycine (decreases or stability), alanine and glutamine (modest prolonged increases), and threonine, serine, asparagine, and valine (much larger short-term increases). Plasma non-esterified fatty acid concentrations declined markedly through 48h after the 2.6% meal. These data are interpreted in light of companion studies showing elevations in aerobic metabolic rate, urea production, rectal gland function, metabolic base excretion, and activation of ornithine-urea cycle and aerobic enzymes after the meal, and muscle N-depletion but maintenance of osmolality and urea production during long-term fasting.

The effects of purine compounds on the isolated aorta of the frog Rana temporaria.

PubMed Central

Knight, G. E.; Burnstock, G.

1996-01-01

1. In the isolated aorta of the frog, Rana temporaria, adenosine concentration-dependently, endothelium-independently relaxed adrenaline pre-constricted vessels. None of the adenosine analogues including D-5'-(N-ethylcarboxamide) adenosine (NECA), R- and S-N6-(2-phenylisopropyl) adenosine (R-and S-PIA) and 2-chloroadenosine (2-CA), or the more selective A1, A2 and A3 agonists cyclopentyladenosine (CPA), CGS 21680 and N6-(3-iodobenzyl) adenosine-5'-N-methylcarboxamide (IB-MECA) respectively, had any effect. 2. The non-selective adenosine antagonist, 8-p-sulphophenyl-theophylline (8-pSPT; 30 microM) failed to inhibit adenosine relaxations, as did NG-nitro-L-arginine methyl ester (L-NAME; 0.1 mM) and indomethacin (30 microM). 3. Adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP (alpha, beta-MeATP), beta, gamma-methylene ATP (beta, gamma-MeATP), 2-methylthio ATP (2-MeSATP) and uridine 5'-triphosphate (UTP) all concentration-dependently contracted the frog aorta. ATP and alpha, beta-MeATP were equipotent and more potent than UTP and beta, gamma-MeATP; 2-MeSATP had little activity. 4. The P2-purinoceptor antagonist, suramin (0.1 mM) inhibited contractions to alpha, beta-MeATP but not to ATP. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 30 microM) also inhibited contractions to alpha, beta-MeATP but not to ATP. Contractions to ATP were, however, inhibited by indomethacin (30 microM). 5. In conclusion, in the frog aorta there appears to be a novel subclass of P1-purinoceptor mediating vasodilatation, although like the A3 subclass it is not blocked by methylxanthines; a P2-purinoceptor mediates vasconstriction which resembles a P2x subtype, based on the agonist potency of alpha, beta-MeATP being more potent than 2-MeSATP (UTP has moderate activity) and PPADS is an effective antagonist. There is no evidence for the presence of a P2y-purinoceptor, mediating vasodilatation, in this preparation. PMID:8851504