Construction of a DNA probe to detect isoquinoline-degrading bacteria

1994 ◽  
Vol 40 (7) ◽  
pp. 561-566 ◽  
Author(s):  
N. K. Richards ◽  
H. K. Mahanty ◽  
J. Aislabie
Keyword(s):  
Degradation Pathway ◽  
Transposon Mutagenesis ◽  
Ecori Fragment ◽  
Dna Probe ◽  
Wild Type ◽  
Dna Encoding ◽  
Comamonas Acidovorans ◽  
Visual Scoring ◽  
Degrading Bacteria ◽  
Type C

To facilitate the cloning of DNA encoding isoquinoline degradation an assay was developed that allowed the rapid visual scoring of the isoquinoline degradation phenotype of single colonies. Transposon mutagenesis of one of the isolates, Comamonas acidovorans IQ3, was performed using Tn 5, and nine Isq− mutants deficient in the ability to utilise isoquinoline as the sole nitrogen source were isolated. These mutants were also incapable of utilising the first metabolite of the isoquinoline degradation pathway, 1-hydroxyisoquinoline, as the sole carbon source. For each Isq− mutant, the EcoRI fragment containing the Tn5 insertion was cloned into pBR322. Restriction and Southern analyses of the cloned DNA revealed that of the nine Isq− mutants, six contained Tn5 insertions in a common 8.9-kb EcoRI fragment derived from the wild type, C. acidovorans IQ3. The cloned DNA thought to be involved in the degradation of isoquinoline proved to be specific when used as a probe in colony hybridization to some bacteria possessing the ability to degrade isoquinoline.Key words: DNA probe, isoquinoline degradation, transposon mutagenesis, Comamonas acidovorans.

scholarly journals Effect of Yuzu (Citrus junos) Seed Limonoids and Spermine on Intestinal Microbiota and Hypothalamic Tissue in the Sandhoff Disease Mouse Model

2021 ◽  
Vol 9 (1) ◽  
pp. 17
Author(s):  
Mayumi Minamisawa ◽  
Takuma Suzumura ◽  
Sudeep Bose ◽  
Tetsuyuki Taniai ◽  
Gota Kawai ◽  
...  
Keyword(s):  
Mouse Model ◽  
Intestinal Microbiota ◽  
Neuronal Degeneration ◽  
Sandhoff Disease ◽  
Short Chain Fatty Acids ◽  
Rrna Gene ◽  
Wild Type ◽  
Degrading Bacteria ◽  
Citrus Junos ◽  
Hypothalamic Tissue

The effect of limonoids and spermine (Spm) extracted from yuzu (Citrus junos) seeds on the gut and the brain in a mouse model with Sandhoff disease (SD) was investigated. Wild-type and SD mice were fed a normal diet, or a diet supplemented with limonoid, Spm, or limonoid + Spm for 14–18 weeks, and then 16S rRNA gene amplicon sequencing with extracted DNA from their feces was executed. For SD control mice, intestinal microbiota was mostly composed of Lactobacillus and linked to dysbiosis. For SD and wild-type mice fed with limonoids + Spm or limonoids alone, intestinal microbiota was rich in mucin-degrading bacteria, including Bacteroidetes, Verrucomicrobia, and Firmicutes, and displayed a higher production of short-chain fatty acids and immunoglobulin A. Additionally, SD mice fed with limonoids + Spm or limonoids alone had less inflammation in hypothalamic tissues and displayed a greater number of neurons. Administration of limonoids and/or Spm improved the proportions of beneficial intestinal microbiota to host health and reduced neuronal degeneration in SD mice. Yuzu seed limonoids and Spermine may help to maintain the homeostasis of intestinal microbiota and hypothalamic tissue in the SD mouse model.

scholarly journals Mutant Membrane Protein of the Budding Yeast Spindle Pole Body Is Targeted to the Endoplasmic Reticulum Degradation Pathway

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 567-578 ◽  
Author(s):  
Susan McBratney ◽  
Mark Winey
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Degradation Pathway ◽  
Spindle Pole ◽  
Wild Type ◽  
Er Quality Control ◽  
Pole Body ◽  
Cytoplasmic Face ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugation

Abstract Mutation of either the yeast MPS2 or the NDC1 gene leads to identical spindle pole body (SPB) duplication defects: The newly formed SPB is improperly inserted into the nuclear envelope (NE), preventing the cell from forming a bipolar mitotic spindle. We have previously shown that both MPS2 and NDC1 encode integral membrane proteins localized at the SPB. Here we show that CUE1, previously known to have a role in coupling ubiquitin conjugation to ER degradation, is an unusual dosage suppressor of mutations in MPS2 and NDC1. Cue1p has been shown to recruit the soluble ubiquitin-conjugating enzyme, Ubc7p, to the cytoplasmic face of the ER membrane where it can ubiquitinate its substrates and target them for degradation by the proteasome. Both mps2-1 and ndc1-1 are also suppressed by disruption of UBC7 or its partner, UBC6. The Mps2-1p mutant protein level is markedly reduced compared to wild-type Mps2p, and deletion of CUE1 restores the level of Mps2-1p to nearly wild-type levels. Our data indicate that Mps2p may be targeted for degradation by the ER quality control pathway.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

Isolation and identification of 17β-estradiol degrading bacteria and its degradation pathway

2022 ◽  
Vol 423 ◽  
pp. 127185
Author(s):  
Qu Zhang ◽  
Chao Xue ◽  
Gary Owens ◽  
Zuliang Chen
Keyword(s):  
Degradation Pathway ◽  
Isolation And Identification ◽  
Degrading Bacteria ◽  
17Β Estradiol

Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis

1993 ◽  
Vol 13 (3) ◽  
pp. 1464-1470
Author(s):  
S Bagrodia ◽  
S J Taylor ◽  
D Shalloway
Keyword(s):  
Kinase Activity ◽  
Tyrosine Phosphatase ◽  
Src Kinase ◽  
Indirect Evidence ◽  
Membrane Localization ◽  
Wild Type ◽  
Src Tyrosine Kinase ◽  
Amino Terminal ◽  
Membrane Bound ◽  
Type C

The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.

scholarly journals Mapping the degradation pathway of a disease-linked aspartoacylase variant

PLoS Genetics ◽  
2021 ◽  
Vol 17 (4) ◽  
pp. e1009539
Author(s):  
Sarah K. Gersing ◽  
Yong Wang ◽  
Martin Grønbæk-Thygesen ◽  
Caroline Kampmeyer ◽  
Lene Clausen ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Proteasomal Degradation ◽  
Degradation Pathway ◽  
Wild Type ◽  
Protein Variant ◽  
Stable Conformation ◽  
Spongy Degeneration ◽  
Brain White Matter

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.

scholarly journals Φαρμακοκινητική, φαρμακοδυναμική και φαρμακογενωμική ανάλυση της μπεβασιζουμάμπης σε ασθενείς με μεταστατικό καρκίνο του παχέος εντέρου

2019 ◽  
Author(s):  
Απόστολος Παπαχρήστος
Keyword(s):  
Wild Type ◽  
Type C

Το όφελος στη θεραπεία διαφόρων όγκων από τη μπεβασιζουμάμπη έχει διαπιστωθεί από διάφορες μελέτες. Ωστόσο, τα κλινικά αποτελέσματα εμφανίζουν μεταβλητότητα, με ορισμένους ασθενείς να ανταποκρίνονται αξιοσημείωτα καλά, ενώ κάποιοι άλλοι όχι. Οι φαρμακοκινητικές παράμετροι συγκαταλέγονται μεταξύ των σημαντικότερων παραμέτρων, οι οποίες επηρεάζουν τη δράση του φαρμάκου και την κλινική ανταπόκριση.Για την αξιολόγηση της επίδρασης διαφόρων φυσιολογικών, φαρμακοκινητικών, φαρμακοδυναμικών και γενετικών παραγόντων στα κλινικά αποτελέσματα που παρατηρούνται σε ασθενείς που λαμβάνουν πρώτης γραμμής θεραπεία με μπεβασιζουμάμπη σε συνδυασμό με χημειοθεραπεία για μεταστατικό καρκίνο του παχέος εντέρου και την προβλεπτική τους ικανότητα διεξήχθη μια προοπτική, μη παρεμβατική μελέτη παρατήρησης. Η μελέτη έγινε σε 46 ασθενείς με επιβεβαιωμένο καρκίνο παχέος εντέρου σταδίου 4 που έλαβαν πρώτης γραμμής θεραπεία με μπεβασιζουμάμπη και χημειοθεραπεία. Η διαδικασία ένταξης των ασθενών στη μελέτη διήρκησε ένα έτος. Ο χρόνος παρακολούθησης των ασθενών μετά την ένταξή τους στη μελέτη ήταν πέντε έτη. Έγινε αξιολόγηση της ανταπόκρισης στη θεραπεία, του διαστήματος ελεύθερου νόσου και της συνολικής επιβίωσης και προσδιορίστηκε η συγκέντρωση της ολικής μπεβασιζουμάμπης και του ελεύθερου VEFG165 σε ορό αίματος. Έγινε επίσης ανίχνευση των μονονουκλεοτιδικών πολυμορφισμών για τα γονίδια VEG-A και ICAM-1 σε ολικό αίμα και για τα γονίδια KRAS, NRAS και BRAF σε δείγμα καρκινικού ιστού. Η ανταπόκριση στη θεραπεία δεν επηρεάστηκε από οποιοδήποτε πολυμορφισμό στα γονίδια VEGF-A, ICAM-1, KRAS και NRAS. Αντίθετα οι μεταλλάξεις στο BRAF συνδέονταν στατιστικά σημαντικά (p=0.026) με μη ανταπόκριση στη θεραπεία. Ασθενείς ομόζυγοι για το VEGF-A rs699947 A/A παρουσίαζαν στατιστικά σημαντικά παρατεταμένο PFS και OS σε σχέση με τους ασθενείς που παρουσίαζαν την wild-type C/C μορφή του γονιδίου. Όσον αφορά το γονίδιο ICAM-1 οι ασθενείς που έφεραν το G/A αλληλόμορφο του rs1799969 παρουσίασαν στατιστικά σημαντικά μεγαλύτερη OS σε σχέση με τους φορείς του G/G. Αναφορικά με το γονίδιο BRAF βρέθηκε ότι οι ασθενείς που είχαν όγκους με τον wild-type τύπο του γονιδίου είχαν στατιστικά σημαντικά παρατεταμένη επιβίωση σε σχέση με τους ασθενείς που είχαν μεταλλαγμένο BRAF. Παρατηρήθηκε ισχυρά αρνητική συσχέτιση μεταξύ των επιπέδων ολικής μπεβασιζουμάμπης με τα επίπεδα του μοριακού της στόχου VEGF165. Ο παράγοντας που επηρέαζε κυρίως το μοντέλο πρόσδεσης ήταν το αλληλόμορφο του VEGF-Α rs699947 A/A (p=0.002). Τέλος, παρατηρήθηκε ότι όσο υψηλότερες ήταν ο συγκεντρώσεις της μπεβασιζουμάμπης τόσο αυξανόταν η OS (p=0.0003).Εάν αυτά τα ευρήματα επιβεβαιωθούν με περαιτέρω μελέτες, αυτοί οι βιοδείκτες θα μπορούσαν να ενσωματωθούν και να χρησιμοποιηθούν για την επιλογή ασθενών με mCRC, οι οποίοι θα μπορούσαν να επωφεληθούν από τις θεραπείες με μπεβασιζουμάμπη.

scholarly journals Degradation of Anthracene by Mycobacterium sp. Strain LB501T Proceeds via a Novel Pathway, through o-Phthalic Acid

2003 ◽  
Vol 69 (1) ◽  
pp. 186-190 ◽  
Author(s):  
René van Herwijnen ◽  
Dirk Springael ◽  
Pieter Slot ◽  
Harrie A. J. Govers ◽  
John R. Parsons
Keyword(s):  
Energy Source ◽  
Type Strain ◽  
Phthalic Acid ◽  
Protocatechuic Acid ◽  
Wild Type Strain ◽  
Degradation Pathway ◽  
Wild Type ◽  
Catabolic Pathway ◽  
Naphthalene Degradation

ABSTRACT Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.

Purification and physical properties of nematode mini-titins and their relation to twitchin

1991 ◽  
Vol 98 (4) ◽  
pp. 491-496
Author(s):  
R. Nave ◽  
D. Furst ◽  
U. Vinkemeier ◽  
K. Weber
Keyword(s):  
Caenorhabditis Elegans ◽  
Immunofluorescence Microscopy ◽  
Apparent Molecular Weight ◽  
The Body ◽  
Wild Type ◽  
Normal Muscle ◽  
Insect Muscle ◽  
C Elegans ◽  
Type C ◽  
Beta Structure

We have isolated mini-titin from the nematodes Ascaris lumbricoides and Caenorhabditis elegans under native conditions using a modification in the procedure to prepare this protein from insect muscle. The proteins have an apparent molecular weight of 600,000 and appear in oriented specimens as flexible thin rods with a length around 240–250 nm. The circular dichroism spectrum of the Ascaris protein is dominated by beta-structure. The proteins react with antibodies to insect mini-titin and also with antibodies raised against peptides contained in the sequence predicted for twitchin, the product of the Caenorhabditis elegans unc-22 gene. Antibodies to insect mini-titin decorate the body musculature as well as the pharynx of wild-type C. elegans in immunofluorescence microscopy. In the twitchin mutant E66 only the pharynx is decorated. We conclude that the mini-titins of invertebrate muscles defined earlier by ultrastructural criteria are very likely to be twitchins, i.e. molecules necessary for normal muscle contraction. We discuss the molecular properties of the proteins in the light of the sequence established for twitchin.

scholarly journals Steroid Degradation in Comamonas testosteroni TA441: Identification of the Entire β-Oxidation Cycle of the Cleaved B Ring

2019 ◽  
Vol 85 (20) ◽  
Author(s):  
Masae Horinouchi ◽  
Hiroyuki Koshino ◽  
Michal Malon ◽  
Hiroshi Hirota ◽  
Toshiaki Hayashi
Keyword(s):  
Gene Clusters ◽  
Degradation Process ◽  
Degradation Pathway ◽  
Ring Cleavage ◽  
Comamonas Testosteroni ◽  
Content Type ◽  
Coa Transferase ◽  
Degrading Bacteria ◽  
Globally Distributed

ABSTRACT Comamonas testosteroni TA441 degrades steroids via aromatization of the A ring, followed by degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, mainly by β-oxidation. In this study, we revealed that 7β,9α-dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-coenzyme A (CoA) ester is dehydrogenated by (3S)-3-hydroxylacyl CoA-dehydrogenase, encoded by scdE (ORF27), and then the resultant 9α-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is converted by 3-ketoacyl-CoA transferase, encoded by scdF (ORF23). With these results, the whole cycle of β-oxidation on the side chain at C-8 of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid is clarified; 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is dehydrogenated at C-6 by ScdC1C2, followed by hydration by ScdD. 7β,9α-Dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-CoA ester then is dehydrogenated by ScdE to be converted to 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid-CoA ester and acetyl-CoA by ScdF. ScdF is an ortholog of FadA6 in Mycobacterium tuberculosis H37Rv, which was reported as a 3-ketoacyl-CoA transferase involved in C ring cleavage. We also obtained results suggesting that ScdF is also involved in C ring cleavage, but further investigation is required for confirmation. ORF25 and ORF26, located between scdF and scdE, encode enzymes belonging to the amidase superfamily. Disrupting either ORF25 or ORF26 did not affect steroid degradation. Among the bacteria having gene clusters similar to those of tesB to tesR, some have both ORF25- and ORF26-like proteins or only an ORF26-like protein, but others do not have either ORF25- or ORF26-like proteins. ORF25 and ORF26 are not crucial for steroid degradation, yet they might provide clues to elucidate the evolution of bacterial steroid degradation clusters. IMPORTANCE Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in relation to human health is attracting attention. The overall aerobic degradation of the four basic steroidal rings has been proposed; however, there is still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in Comamonas testosteroni TA441 as a model of steroid-degrading bacteria. C. testosteroni is one of the most studied representative steroid-degrading bacteria and is suitable for exploring the degradation pathway, because the involvement of degradation-related genes can be determined by gene disruption. Here, we elucidated the entire β-oxidation cycle of the cleaved B ring. This cycle is essential for the following C and D ring cleavage.

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