In vitro metabolomic footprint of the Echinococcus multilocularis metacestode

AbstractAlveolar echinococcosis (AE) is a zoonotic disease that is deadly if left untreated. AE is caused by the larval metacestode stage of the cestode Echinococcus multilocularis. Better knowledge on the host-parasite interface could yield novel targets for improvement of the treatment against AE. We analyzed culture media incubated with in vitro grown E. multilocularis metacestodes by 1H nuclear magnetic resonance spectroscopy to identify the unknown metabolic footprint of the parasite. Moreover, we quantitatively analyzed all amino acids, acetate, glucose, lactate, and succinate in time-course experiments using liquid chromatography and enzymatic assays. The E. multilocularis metacestodes consumed glucose and, surprisingly, threonine and produced succinate, acetate, and alanine as major fermentation products. The metabolic composition of vesicle fluid (VF) from in vitro grown E. multilocularis metacestodes was different from parasite-incubated culture medium with respect to the abundance, but not the spectrum, of metabolites, and some metabolites, in particular amino acids, accumulated in the VF. Overall, this study presents the first characterization of the in vitro metabolic footprint of E. multilocularis metacestodes and VF composition, and it provides the basis for analyses of potentially targetable pathways for future drug development.

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Some biochemical effects of triamcinolone acetonide on rat liver and muscle

Biochemical Journal ◽

10.1042/bj1160349 ◽

1970 ◽

Vol 116 (3) ◽

pp. 349-355 ◽

Cited By ~ 22

Author(s):

R. F. Peters ◽

M. C. Richardson ◽

Margaret Small ◽

A. M. White

Keyword(s):

Amino Acids ◽

Triamcinolone Acetonide ◽

Time Course ◽

Muscle Protein ◽

Specific Radioactivity ◽

Synthetic Activity ◽

Tissue Homogenate ◽

Glycogen Concentration

1. The powerful anti-inflammatory glucocorticoid triamcinolone acetonide, administered to rats at 20 and 2.5mg/kg, leads to a decrease in the incorporation in vivo of [3H]uridine and [32P]orthophosphate into hind-limb skeletal muscle. 2. At the higher dose, this decrease in the rate of incorporation of precursors into RNA precedes a decrease in the incorporating ability of muscle ribosomes, which commences about 4–5h after drug administration, but is unaccompanied by any changes in the concentration of tissue ATP or free amino acids. 3. The ribosomal dysfunction extends to polyribosomes, which can only be successfully isolated from the muscle of triamcinolone-treated animals after the addition of α-amylase to the tissue homogenate to remove glycogen. 4. The specific radioactivity of muscle protein labelled in vivo with 14C-labelled amino acids does not decrease progressively after triamcinolone administration. After 2h there is an apparent stimulation of incorporation which leads to an overall discrepancy between measurements of protein-synthetic activity made in vivo and in vitro. 5. There is a significant increase in muscle-glycogen concentration between 8 and 12h after the administration of triamcinolone acetonide (20mg/kg), although a significant decrease occurs after 4h. The fall in glycogen concentration may be due to a decrease in the rate of synthesis of protein essential for glucose uptake into the tissues. 6. As judged by (a) incorporation of 14C-labelled amino acids into protein, (b) [3H]uridine and [32P]-orthophosphate incorporation into RNA, (c) the rate of induction of tryptophan pyrrolase and (d) changes in the pool sizes of taurine and tryptophan, the responses in liver followed the same time-course as those in muscle after administration of the drug.

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Effects of 2,4-D and Amino Acids on Field Bindweed in Vitro

Weed Science ◽

10.1017/s0043174500031921 ◽

1973 ◽

Vol 21 (2) ◽

pp. 135-138 ◽

Cited By ~ 5

Author(s):

R. G. Harvey ◽

T. J. Muzik

Keyword(s):

Amino Acids ◽

Glutamic Acid ◽

Culture Media ◽

Reductase Activity ◽

Convolvulus Arvensis ◽

Field Bindweed ◽

Agar Media ◽

Differential Binding ◽

Dichlorophenoxy Acetic Acid

Two clones of field bindweed (Convolvulus arvensisL.) which differed in their susceptibility to (2,4-dichlorophenoxy)acetic acid (2,4-D) under field and greenhouse conditions also exhibited similar differences when stem cells were cultured in liquid and agar media. Amino acids added to the culture media altered the response to 2,4-D. Glutamic acid increased the tolerance of the susceptible (S) clone, but reduced the tolerance of the resistant (R) clone. Glutamine increased the susceptibility of the S clone to a much greater degree than it did the R clone. No significant differences were noted in the rates of absorption of metabolism of 2,4-D by the two clones. Glutamine increased and glutamic acid decreased 2,4-D absorption by both clones. Levels of nitrate reductase activity (NRA), soluble protein (SP), and gross RNA (GR) increased in the S tissues but decreased or remained constant in the R tissues exposed to 4.5 × 10−5M 2,4-D. Correlations between 2,4-D susceptibility and NRA demonstrated a relationship between the effects of 2,4-D and nitrogen metabolism. Differential binding of 2,4-D within the cells appears to be the most likely explanation for the differences in response to 2,4-D.

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155 QUALITY OF PORCINE EMBRYO PRODUCED IN TWO EMBRYO CULTURE MEDIA AS ASSESSED BY TOTAL CELL NUMBER AND TIME COURSE OF BLASTOCYST HATCHING

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab155 ◽

2006 ◽

Vol 18 (2) ◽

pp. 185 ◽

Cited By ~ 1

Author(s):

Y. Agca ◽

H. Men ◽

S. F. Mullen ◽

L. K. Riley ◽

R. S. Prather ◽

...

Keyword(s):

Embryo Culture ◽

Time Course ◽

Culture Media ◽

Cell Number ◽

Total Cell ◽

Total Cell Number ◽

Blastocyst Hatching ◽

Hatching Rates

The ability to produce porcine embryos of good quality will have a significant impact on a number of porcine assisted reproductive technologies, such as cloning, intracytoplasmic sperm injection, and embryo cryopreservation. However, porcine embryos resulting from current serum-free embryo culture systems differ significantly both structurally and functionally from those derived in vivo (Wang et al. 1999 Mol. Reprod. Dev. 53, 99-107). In this experiment, the quality of porcine embryos produced by North Carolina State University (NCSU)-23 medium (Petters and Wells 1993 J. Reprod. Fertil. Suppl. 1993, 48, 61-73) and porcine zygote medium (PZM)-1 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) were compared by assessing the total cell number and the time course of in vitro blastocyst hatching. Porcine embryos were produced by in vitro maturation and fertilization using serum-free systems. After fertilization, presumptive zygotes were randomly allocated to either PZM-1 or NCSU-23 for subsequent development. On Day 4 of culture, the embryo culture media were supplemented with 10% fetal bovine serum (FBS). Day 6 blastocysts from each group were counted and the blastocysts were subsequently fixed in 4% formalin for counting the total cell number. The cell number in each embryo was determined by counting the nuclei after staining with bisbenzimide (Hoechst 33342). To assess the hatching ability of blastocysts, Day 6 blastocysts were cultured until Day 9 and hatched blastocysts were counted daily. Day 6 blastocyst rates (ratio of blastocysts to oocytes) and total cell number count were replicated three times. The time course of blastocyst hatching experiment was repeated four times. The data were analyzed using a chi-square test, Fisher's exact test, or Student's t-test. The blastocyst rate from culture in PZM-3 was 19.4 � 0.96% (mean � SEM), which was similar to that (16.7 � 3.2%) resulting from culture in NCSU-23 (P > 0.05). However, the total cell number in Day 6 blastocysts cultured in PZM-3 was significantly higher than for blastocysts cultured in NCSU-23 (57 � 3.1 vs. 46 � 1.7; P < 0.01). The total hatching rates (ratio of hatched blastocysts to total blastocysts) by Day 9 were similar between the two culture systems (50.1 � 9.1% vs. 50.7 � 4.1%; P > 0.05). However, on Day 6, 2.1% of blastocysts from PZM-3 culture hatched whereas no blastocysts from NCSU-23 culture hatched. The cumulative hatching rates from PZM-3 culture on Day 7 were significantly higher than those from NCSU-23 culture (15.1 � 3.8% vs. 2.6 � 1.1%; P < 0.01). In conclusion, these data suggest that blastocysts produced in PZM-3 medium have better quality than blastocysts produced in the NCSU-23 culture system as assessed by the total cell number and the time course of blastocyst hatching. This project was supported by a grant from the National Institutes of Health (U42 RR 018877).

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60 IMPROVEMENT IN EFFICIENCY AND QUALITY OF CLONED AND PARTHENOGENETIC PORCINE EMBRYOS BY AMINO ACIDS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab60 ◽

2007 ◽

Vol 19 (1) ◽

pp. 147

Author(s):

H. T. Lee ◽

J. M. Jang ◽

S. H. Lee ◽

M. K. Gupta

Keyword(s):

Amino Acids ◽

Culture Media ◽

Cell Number ◽

Control Group ◽

In Vitro Production ◽

Cleavage Rate ◽

Fetal Fibroblasts ◽

Blastocyst Rate

In vitro production of cloned porcine embryos by somatic cell nuclear transfer (SCNT) has become routine in several laboratories but the efficiency and quality of the resultant blastocysts remains sub-optimal. Cloned porcine blastocysts show low cell number, high fragmentation rate, and apoptosis which results in lower pregnancy rates upon embryo transfer. Earlier we reported that supplementation of culture media with amino acids benefit pre-implantation embryo development of in vivo- as well as in vitro-fertilized porcine embryos (Koo et al. 1997 Theriogenology 48, 791–802). This study evaluated how exogenous amino acids could affect pre-implantation development and quality of cloned or parthenogenetic porcine embryos. The effects of commercially available amino acids, referred to as Eagle's non-essential amino acids (NEAA), added or not added (control) to NCSU23 medium containing fatty acid-free BSA were studied. Oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro and parthenogenetically activated (PA) or nuclear-transferred with fetal fibroblasts (SCNT), as described earlier (Uhm et al. 2000 Mol. Reprod. Dev. 57, 331–337). At 168 h post-activation, blastocysts were harvested for assessment of embryo quality by TUNEL labeling, Hoechst 33342 staining, and gene expression analysis. Results showed that, in the PA group, the cleavage rate was not affected by the supplementation of NEAA. However, the blastocyst rate was significantly improved when NEAA was present in the medium compared to that of the control group (38.9 ± 0.3 vs. 27.5 ± 0.3&percnt;, respectively) throughout the culture period. The supplementation during the pre-compaction period alone gave better results than during the post-compaction period alone (59.5 ± 0.9 vs. 33.4 ± 0.3&percnt;, respectively). In the SCNT group, however, both cleavage (73.6 ± 0.2 vs. 64.2 ± 0.4&percnt;) and blastocyst rate (18.7 ± 0.2 vs. 13.8 ± 0.3&percnt;) were improved by NEAA supplementation. Furthermore, these blastocysts had higher hatching ability (30.0 ± 1.8 vs. 14.6 ± 4.9&percnt;) than those of control group (P < 0.05). Supplementation of NEAA also increased the mean nuclei number of PA-derived (76.1 ± 4.9 vs. 66.5 ± 3.3) as well as SCNT-derived (43.1 ± 2.6 vs. 31.8 ± 1.9) blastocysts and reduced the time during which blastocysts formed. TUNEL assay revealed that incidence of nuclear fragmentation and apotosis was reduced by NEAA. Real-time qRT-PCR for Bax and Bcl-XL transcripts revealed that the relative abundance of Bax was reduced while that of Bcl-XL was increased. These effects were more pronounced when NEAA was present during the pre-compaction period alone. Thus, our data suggest that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion and positively modulating the total cell number and apoptosis. These data may have implications for understanding the nutritional needs of cloned porcine embryos produced in vitro and for optimizing the composition of culture media to support their development. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

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Localization of amino acid neurotransmitters following in vitro ischemia and anoxia in the rat retina

Visual Neuroscience ◽

10.1017/s0952523801183082 ◽

2001 ◽

Vol 18 (3) ◽

pp. 413-427 ◽

Cited By ~ 29

Author(s):

GENEVIEVE A. NAPPER ◽

MICHAEL J. PIANTA ◽

MICHAEL KALLONIATIS

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cellular Localization ◽

Time Course ◽

Enzymatic Reactions ◽

Glutamate Metabolism ◽

Accumulation Time ◽

Glucose Levels ◽

Acid Loading

Glutamate and γ-aminobutyric acid (GABA) are two of the dominant neurotransmitters in the retina and brain. The production/degradation of glutamate and GABA involves an intricate interrelationship between neurons and glia, as well as aerobic and anaerobic metabolic pathways. The aim of this work was to develop an in vitro model of retinal ischemia/anoxia and determine the changes in cellular localization of glutamate and GABA and the time course for such changes. After anoxic/ischemic insult, glutamate and GABA rapidly accumulate within glia with GABA showing a quicker time course and larger magnitude change. The accumulation time constant for both glutamate and GABA under anoxic conditions was dependent upon glucose concentration: high glucose levels resulted in delayed glial amino acid loading. The differences in time constants between GABA and glutamate glial loading most likely reflect the multitude of glutamate degradation pathways compared to the single aerobically dependent GABA pathway. Oxygen availability and reduced glucose (hypoglycemia) lead to an almost immediate increase (within 1 min) of glutamate and GABA labelling within glia. In addition, altered labelling patterns were found under anoxic/ischemic conditions for amino acids involved in glutamate transamination reactions: aspartate, leucine, alanine, and ornithine. These changes are consistent with alterations of equilibria of enzymatic reactions involved in glutamate metabolism, and thus support a role for all four amino acids in glutamate metabolism within a variety of retinal neurons.

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Review: Role of tubal environment in preimplantation embryogenesis: application to co-culture assays

Zygote ◽

10.1017/s0967199410000092 ◽

2010 ◽

Vol 19 (1) ◽

pp. 47-54 ◽

Cited By ~ 11

Author(s):

Pierre Guérin ◽

Yves Ménézo

Keyword(s):

Amino Acids ◽

Embryo Culture ◽

Metabolic Flux ◽

Cultured Cells ◽

Culture Media ◽

Culture Conditions ◽

Antioxidant Compounds ◽

Beneficial Effects ◽

Stage Embryo

SummaryThe culture of early preimplantation stage embryo is still delicate and the metabolic pathways of embryos are not completely understood. Embryo needs are evolutionary during the preimplantation development, consequently it is difficult to meet embryo needs in vitro. Culture conditions have to respect several physical and chemical equilibria: such as redox potential, pH, osmotic pressure, metabolic flux of energetic compounds, endogenous pools of amino acids and transcripts, etc. Embryo culture media are generally supplemented with amino acids, glucose, other energetic metabolites and antioxidant compounds, vitamin, and growth factors etc. Furthermore autocrine and paracrine regulation of embryo development probably exist. In fact embryo culture conditions have to be as non-toxic as possible. Various types of co-culture systems have been devised to overcome these problems. Complex interrelations exist between embryos and co-cultured cells. The beneficial effects of co-cultured cells may be due to continuous modifications of the culture medium, i.e. the elimination of toxic compounds and/or the supply of embryotrophic factors.

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FURTHER STUDIES ON THE PHYSIOLOGY OF CLADOSPORIUM FULVUM CKE. AND RESISTANCE IN TOMATO

Canadian Journal of Botany ◽

10.1139/b64-132 ◽

1964 ◽

Vol 42 (10) ◽

pp. 1365-1386 ◽

Cited By ~ 11

Author(s):

Ruth L. Lowther

Keyword(s):

Culture Media ◽

Carbon Sources ◽

Amino Nitrogen ◽

Cladosporium Fulvum ◽

Host Parasite Interactions ◽

Race 1 ◽

Species Characteristics ◽

Pathogenic Races ◽

Host Parasite

Some host–parasite interactions of the C. fulvum – tomato leaf complex have been correlated with degrees of utilization in vitro by races of the pathogen of metabolites which have been shown to occur in different amounts in some tomato hosts which react differentially to them. Similarities and differences in the behavior of three pathogenic races were noted when the plants were grown in a number of amino nitrogen and sugar carbon sources. Similarities are interpreted as representing species characteristics; on the other hand, differences, including colony colors in glutamine and γ-aminobutyric acid, appear to be racial characteristics. Further evidence suggesting that certain host metabolites modify or condition the pathogenic expression was obtained from studies of the effect on pathogenicity of culture media differing in amino nitrogen content. From a comparison of the metabolites of host–pathogen complexes of differing reaction types, some insight was gained into probable nutritional requirements for a race 1 susceptible response. Conversely, where these differed in other reaction-type complexes, clues were obtained regarding possible reasons for resistance to this race.

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In vitro culture of Echinococcus multilocularis and Echinococcus vogeli metacestodes: studies on the host–parasite interface

Acta Tropica ◽

10.1016/s0001-706x(02)00220-6 ◽

2003 ◽

Vol 85 (2) ◽

pp. 145-155 ◽

Cited By ~ 18

Author(s):

Andrew Hemphill ◽

Marianne Stettler ◽

Mirjam Walker ◽

Mar Siles-Lucas ◽

Renate Fink ◽

...

Keyword(s):

In Vitro Culture ◽

Echinococcus Multilocularis ◽

Host Parasite ◽

Echinococcus Vogeli

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Human and mouse embryonic development, metabolism and gene expression are altered by an ammonium gradient in vitro

Reproduction ◽

10.1530/rep-12-0348 ◽

2013 ◽

Vol 146 (1) ◽

pp. 49-61 ◽

Cited By ~ 30

Author(s):

D K Gardner ◽

R Hamilton ◽

B McCallie ◽

W B Schoolcraft ◽

M G Katz-Jaffe

Keyword(s):

Gene Expression ◽

Amino Acids ◽

Transcriptome Analysis ◽

Human Embryo ◽

Culture Media ◽

Expression Profiles ◽

Human Embryos ◽

Embryo Metabolism ◽

Human And Mouse

Ammonium is generated in culture media by the spontaneous deamination of amino acids at 37 °C and through the metabolism of amino acids by human embryos. The appearance of ammonium is a time-dependent phenomenon and can compromise embryo physiology, development and viability. In this study, the effects of a gradient of ammonium on the development, metabolism and transcriptome of human and mouse embryos were investigated. Pronucleate oocytes were cultured in the presence of an ammonium gradient that mimicked the spontaneous deamination of Eagle's amino acids together with 1 mM glutamine. All embryos were cultured in sequential media G1/G2 at 5% O2, 6% CO2 and 89% N2. Human embryo metabolism was assessed through a non-invasive fluorometric analysis of pyruvate consumption. Transcriptome analysis was performed on the resultant blastocysts from both species using a microarray technology. Embryo development prior to compaction was negatively affected by the presence of low levels of ammonium in both species. Human embryo metabolism was significantly inhibited after just 24 and 48 h of culture. Transcriptome analysis of blastocysts from both species revealed significantly altered gene expression profiles, both decreased and increased. Functional annotation of the altered genes revealed the following over represented biological processes: metabolism, cell growth and/or maintenance, transcription, cell communication, transport, development and transcription regulation. These data emphasize the enhanced sensitivity of the cleavage-stage embryo to its environment and highlight the requirement to renew culture media at frequent intervals in order to alleviate the in vitro induced effects of ammonium build-up in the environment surrounding the embryo.

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Analysis of the roles of cysteine proteinases of Leishmania mexicana in the host–parasite interaction

Parasitology ◽

10.1017/s0031182099006435 ◽

2000 ◽

Vol 121 (4) ◽

pp. 367-377 ◽

Cited By ~ 41

Author(s):

M. J. FRAME ◽

J. C. MOTTRAM ◽

G. H. COOMBS

Keyword(s):

Time Course ◽

Cysteine Proteinase ◽

Mammalian Host ◽

Leishmania Mexicana ◽

Wild Type ◽

Large Numbers ◽

Host Parasite

Promastigotes of Leishmania mexicana mutants lacking the multicopy CPB cysteine proteinase genes (ΔCPB) are markedly less able than wild-type parasites to infect macrophages in vitro. ΔCPB promastigotes invade macrophages in large numbers but are unable to survive in the majority of the cells. In contrast, ΔCPB amastigotes invade and survive within macrophages in vitro. This extreme in vitro stage-specific difference was not mimicked in vivo; both promastigotes and amastigotes of ΔCPB produced lesions in BALB/c mice, but in each case the lesions grew considerably more slowly than those caused by wild-type parasites and only small lesions resulted. Inhibition of CPB in situ using cell-permeant peptidyldiazomethylketones had no measurable effect on parasite growth or differentiation axenically in vitro. In contrast, N-benzoyloxycarbonyl-phe-ala-diazomethylketone reduced the infectivity of wild-type parasites to macrophages by 80%. Time-course experiments demonstrated that application of the inhibitor caused effects not seen with ΔCPB, suggesting that CPB may not be the prime target of this inhibitor. The data show that the CPB genes of L. mexicana encode enzymes that have important roles in intracellular survival of the parasite and more generally in its interaction with its mammalian host.

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