scholarly journals Purification and molecular characterization of the NAD+-dependent acetaldehyde/alcohol dehydrogenase from Entamoeba histolytica

1994 ◽  
Vol 303 (3) ◽  
pp. 743-748 ◽  
Author(s):  
I Bruchhaus ◽  
E Tannich
Keyword(s):  
Alcohol Dehydrogenase ◽  
Entamoeba Histolytica ◽  
Dna Sequences ◽  
Microscopic Analysis ◽  
Kinetic Studies ◽  
Protozoan Parasite ◽  
Striking Similarity ◽  
Electron Microscopic ◽  
Catalytic Functions

A bifunctional 95 kDa polypeptide (EhADH2) harbouring acetaldehyde dehydrogenase and alcohol dehydrogenase activities was purified to homogeneity from trophozoite extracts of the protozoan parasite Entamoeba histolytica. Kinetic studies revealed that the enzyme utilizes NAD+ rather than NADP+ as cofactor. Km values for acetyl-CoA, acetaldehyde and ethanol were found to be 0.015, 0.15 and 80 mM respectively in the presence of 0.2 mM NAD+. The primary structure of EhADH2 as deduced from respective amoebic DNA sequences showed striking similarity to the trifunctional AdhE protein of Escherichia coli and the bifunctional AAD protein of Clostridium acetobutylicum. Alignment with a number of aldehyde dehydrogenases and alcohol dehydrogenases from various species suggested that the two catalytic functions of EhADH2 are located on separate parts of the molecule. By cross-linking experiments and electron-microscopic analysis, native EhADH2 was found to be organized in a homopolymeric fashion consisting of more than 20 associated promoters which form rods about 50-120 nm in length.

Electron microscopy of Achlya deoxyribonucleic acid sequence organization

1981 ◽  
Vol 1 (2) ◽  
pp. 136-143
Author(s):  
M Pellegrini ◽  
W E Timberlake ◽  
R B Goldberg
Keyword(s):  
Dna Sequences ◽  
Deoxyribonucleic Acid ◽  
Microscopic Analysis ◽  
Single Copy ◽  
Electron Microscopic ◽  
Sequence Organization ◽  
Double Stranded Dna ◽  
Copy Dna ◽  
Gene 32 ◽  
Single Copy Dna

Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single- and double-stranded DNA regions. Long (greater than 6-kilobase) DNA fragments were isolated after reannealing to two different repetitive C0t values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.

Characterization of the ryanodine receptor/channel of invertebrate muscle

1998 ◽  
Vol 274 (2) ◽  
pp. R494-R502 ◽  
Author(s):  
Kerry E. Quinn ◽  
Loriana Castellani ◽  
Karol Ondrias ◽  
Barbara E. Ehrlich
Keyword(s):  
Ryanodine Receptor ◽  
Single Channel ◽  
Microscopic Analysis ◽  
Ruthenium Red ◽  
Electron Microscopic ◽  
Release Channel ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Bell Shaped Curve

Electron-microscopic analysis was used to show that invertebrate muscle has feetlike structures on the sarcoplasmic reticulum (SR) displaying the typical four-subunit appearance of the calcium (Ca2+) release channel/ryanodine receptor (RyR) observed in vertebrate skeletal muscle (K. E. Loesser, L. Castellani, and C. Franzini-Armstrong. J. Muscle Res. Cell Motil. 13: 161–173, 1992). SR vesicles from invertebrate muscle exhibited specific ryanodine binding and single channel currents that were activated by Ca2+, caffeine, and ATP and inhibited by ruthenium red. The single channel conductance of this invertebrate RyR was lower than that of the vertebrate RyR (49 and 102 pS, respectively). Activation of lobster and scallop SR Ca2+ release channel, in response to cytoplasmic Ca2+ (1 nM–10 mM), reflected a bell-shaped curve, as is found with the mammalian RyR. In contrast to a previous report (J.-H. Seok, L. Xu, N. R. Kramarcy, R. Sealock, and G. Meissner. J. Biol. Chem. 267: 15893–15901, 1992), our results show that regulation of the invertebrate and vertebrate RyRs is quite similar and suggest remarkably similar paths in these diverse organisms.

scholarly journals An Alcohol Dehydrogenase 3 (ADH3) from Entamoeba histolytica Is Involved in the Detoxification of Toxic Aldehydes

2020 ◽  
Vol 8 (10) ◽  
pp. 1608
Author(s):  
Constantin König ◽  
Martin Meyer ◽  
Corinna Lender ◽  
Sarah Nehls ◽  
Tina Wallaschkowski ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Entamoeba Histolytica ◽  
Biochemical Characterization ◽  
Stop Codon ◽  
In Silico Analysis ◽  
Peptidase Activity ◽  
Stool Samples ◽  
Human Stool

Recently, a putative alcohol dehydrogenase 3, termed EhADH3B of the Entamoeba histolytica isolate HM-1:IMSS was identified, which is expressed at higher levels in non-pathogenic than in pathogenic amoebae and whose overexpression reduces the virulence of pathogenic amoebae. In an in silico analysis performed in this study, we assigned EhADH3B to a four-member ADH3 family, with ehadh3b present as a duplicate (ehadh3ba/ehadh3bb). In long-term laboratory cultures a mutation was identified at position 496 of ehadh3ba, which codes for a stop codon, which was not the case for amoebae isolated from human stool samples. When using transfectants that overexpress or silence ehadh3bb, we found no or little effect on growth, size, erythrophagocytosis, motility, hemolytic or cysteine peptidase activity. Biochemical characterization of the recombinant EhADH3Bb revealed that this protein forms a dimer containing Ni2+ or Zn2+ as a co-factor and that the enzyme converts acetaldehyde and formaldehyde in the presence of NADPH. A catalytic activity based on alcohols as substrates was not detected. Based on the results, we postulate that EhADH3Bb can reduce free acetaldehyde released by hydrolysis from bifunctional acetaldehyde/alcohol dehydrogenase-bound thiohemiacetal and that it is involved in detoxification of toxic aldehydes produced by the host or the gut microbiota.

scholarly journals Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the Parasite Toxoplasma gondii

2006 ◽  
Vol 17 (2) ◽  
pp. 895-906 ◽  
Author(s):  
Nivedita Sahoo ◽  
Wandy Beatty ◽  
John Heuser ◽  
David Sept ◽  
L. David Sibley
Keyword(s):  
Critical Concentration ◽  
Microscopic Analysis ◽  
Protozoan Parasite ◽  
Gliding Motility ◽  
Host Cells ◽  
Filamentous Actin ◽  
Unusual Behavior ◽  
Electron Microscopic ◽  
Unusual Form ◽  
Kinetics Of

Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans.

scholarly journals Bioinformatics structural and phylogenetic characterization of Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2)

BIOS ◽  
2019 ◽  
Vol 90 (1) ◽  
pp. 30
Author(s):  
Katie M. Lowerre ◽  
Avelina Espinosa ◽  
Guillermo Paz-y-Miño-C ◽  
Christopher Hemme
Keyword(s):  
Alcohol Dehydrogenase ◽  
Entamoeba Histolytica ◽  
Phylogenetic Characterization ◽  
Alcohol Dehydrogenase 2

scholarly journals Immunoelectron microscopic characterization of vasopressin neurons in macaques by use of formaldehyde-fixed tissues stored for several years

2021 ◽  
Author(s):  
Akito Otubo ◽  
Sho Maejima ◽  
Takumi Oti ◽  
Kaita Satoh ◽  
Yasumasa Ueda ◽  
...  
Keyword(s):  
Translational Research ◽  
Median Eminence ◽  
Glutamate Transporter ◽  
Microscopic Analysis ◽  
Nerve Endings ◽  
Size Difference ◽  
Posterior Pituitary ◽  
Electron Microscopic ◽  
Vasopressin Neurons

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed with 4% formaldehyde and stored at −25°C for several years. The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 were colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

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