Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical but Functionally Different

ABSTRACT The gene encoding melamine deaminase (TriA) fromPseudomonas sp. strain NRRL B-12227 was identified, cloned into Escherichia coli, sequenced, and expressed for in vitro study of enzyme activity. Melamine deaminase displaced two of the three amino groups from melamine, producing ammeline and ammelide as sequential products. The first deamination reaction occurred more than 10 times faster than the second. Ammelide did not inhibit the first or second deamination reaction, suggesting that the lower rate of ammeline hydrolysis was due to differential substrate turnover rather than product inhibition. Remarkably, melamine deaminase is 98% identical to the enzyme atrazine chlorohydrolase (AtzA) fromPseudomonas sp. strain ADP. Each enzyme consists of 475 amino acids and differs by only 9 amino acids. AtzA was shown to exclusively catalyze dehalogenation of halo-substituted triazine ring compounds and had no activity with melamine and ammeline. Similarly, melamine deaminase had no detectable activity with the halo-triazine substrates. Melamine deaminase was active in deamination of a substrate that was structurally identical to atrazine, except for the substitution of an amino group for the chlorine atom. Moreover, melamine deaminase and AtzA are found in bacteria that grow on melamine and atrazine compounds, respectively. These data strongly suggest that the 9 amino acid differences between melamine deaminase and AtzA represent a short evolutionary pathway connecting enzymes catalyzing physiologically relevant deamination and dehalogenation reactions, respectively.

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In Vitro Study of the Effect of Osmotic Solutes on the Interactions between Cells from the Peritoneum and Peritoneal Cavity

Peritoneal Dialysis International ◽

10.1177/089686089401400210 ◽

1994 ◽

Vol 14 (2) ◽

pp. 149-154 ◽

Cited By ~ 8

Author(s):

Andrzej Breborowicz ◽

Elias Balaskas ◽

George D. Oreopoulos ◽

Leo Martis ◽

Kenneth Serkes ◽

...

Keyword(s):

Amino Acids ◽

Growth Factors ◽

Conditioned Medium ◽

In Vitro Study ◽

Mesothelial Cells ◽

Peritoneal Mesothelial Cells ◽

Inhibition Of Growth ◽

Peritoneal Leukocytes ◽

Osmotic Solutes

Objective To study how the presence of osmotic solutes in medium affects growth of the peritoneal mesothelial cells and fibroblasts and how osmotic solutes influence the production of factors regulating growth of these cells. Design The proliferation of mesothelial cells and fibroblasts was evaluated by measuring the incorporation of 3H-thymidine into the cells. Cells were exposed to osmotic solutes; the concentration of the latter in the medium was continuously lowered over the time of the experiment to simulate changes of their concentration in the dialysate. The synthesis of factors influencing the proliferation of the mesothelial cells or fibroblasts, by mesothelial cells or fibroblasts themselves, or by peritoneal leukocytes, was tested by the characteristics of the “conditioned” medium. The conditioned medium was produced by exposing standard medium to mesothelial or fibroblasts monolayer or to peritoneal leukocytes over 24 hours; following filtration it was applied to growing test cells for the study of growth factors. Results The effect of osmotic solutes on the growth of mesothelial cells is less inhibitory when their concentration is gradually lowered over the time of the study, compared to previous findings with a constant concentration. Peritoneal leukocytes produce growth factors for mesothelial cells and fibroblasts. Glucose and amino acids inhibit production of peritonealleukocyte-derived growth factors for mesothelial cells, while glycerol increases synthesis of such growth factors for fibroblasts. Mesothelial cells produce factors stimulating the proliferation of mesothelial cells and fibroblasts. In the presence of glycerol or amino acids synthesis of mesothelium derived growth factors for fibroblasts is augmented. Finally, fibroblasts produce factors that inhibit the proliferation of the mesothelial cells, and this effect is potentiated in the presence of amino acids. Conclusions Cytotoxicity of the osmotic solutes measured by the inhibition of growth of the mesothelial cells or their increased damage is significantly reduced during in vitro kinetic study when the concentration of these solutes is gradually lowered. Presence of osmotic solutes in the medium affects synthesis of growth factors derived from mesothelium, fibroblasts, or peritoneal leukocytes, which affect the proliferation of mesothelial cells or fibroblasts.

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Degradation of Amino Acids to Short-Chain Fatty Acids in Humans: An in Vitro Study

Scandinavian Journal of Gastroenterology ◽

10.3109/00365528809103964 ◽

1988 ◽

Vol 23 (2) ◽

pp. 178-182 ◽

Cited By ~ 82

Author(s):

H. S. Rasmussen ◽

K. Holtug ◽

P. B. Mortensen

Keyword(s):

Amino Acids ◽

Fatty Acids ◽

In Vitro Study ◽

Short Chain Fatty Acids ◽

Vitro Study ◽

Short Chain ◽

Chain Fatty Acids

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First identification of PODXL nonsense mutations in autosomal dominant focal segmental glomerulosclerosis

Clinical Science ◽

10.1042/cs20180676 ◽

2019 ◽

Vol 133 (1) ◽

pp. 9-21 ◽

Cited By ~ 2

Author(s):

Fu-Jun Lin ◽

Lei Yao ◽

Xue-Qing Hu ◽

Fan Bian ◽

Gang Ji ◽

...

Keyword(s):

Focal Segmental Glomerulosclerosis ◽

Autosomal Dominant ◽

In Vitro Study ◽

Peripheral Blood Cell ◽

Fiber Formation ◽

Causative Role ◽

Gene Encoding ◽

Nonsense Mutations ◽

Segmental Glomerulosclerosis

Abstract Recently, a novel heterozygous missense mutation c.T1421G (p. L474R) in the PODXL gene encoding podocalyxin was identified in an autosomal dominant focal segmental glomerulosclerosis (AD-FSGS) pedigree. However, this PODXL mutation appeared not to impair podocalyxin function, and it is necessary to identify new PODXL mutations and determine their causative role for FSGS. In the present study, we report the identification of a heterozygous nonsense PODXL mutation (c.C976T; p. Arg326X) in a Chinese pedigree featured by proteinuria and renal insufficiency with AD inheritance by whole exome sequencing (WES). Total mRNA and PODXL protein abundance were decreased in available peripheral blood cell samples of two affected patients undergoing hemodialysis, compared with those in healthy controls and hemodialysis controls without PODXL mutation. We identified another novel PODXL heterozygous nonsense mutation (c.C1133G; p.Ser378X) in a British–Indian pedigree of AD-FSGS by WES. In vitro study showed that, human embryonic kidney 293T cells transfected with the pEGFP-PODXL-Arg326X or pEGFP-PODXL-Ser378X plasmid expressed significantly lower mRNA and PODXL protein compared with cells transfected with the wild-type plasmid. Blocking nonsense-mediated mRNA decay (NMD) significantly restored the amount of mutant mRNA and PODXL proteins, which indicated that the pathogenic effect of PODXL nonsense mutations is likely due to NMD, resulting in podocalyxin deficiency. Functional consequences caused by the PODXL nonsense mutations were inferred by siRNA knockdown in cultured podocytes and podocalyxin down-regulation by siRNA resulted in decreased RhoA and ezrin activities, cell migration and stress fiber formation. Our results provided new data implicating heterozygous PODXL nonsense mutations in the development of FSGS.

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The tra Region of the Conjugative Plasmid pIP501 Is Organized in an Operon with the First Gene Encoding the Relaxase

Journal of Bacteriology ◽

10.1128/jb.184.6.1801-1805.2002 ◽

2002 ◽

Vol 184 (6) ◽

pp. 1801-1805 ◽

Cited By ~ 26

Author(s):

Brigitta Kurenbach ◽

Dagmar Grothe ◽

María Eugenia Farías ◽

Ulrich Szewzyk ◽

Elisabeth Grohmann

Keyword(s):

Amino Acids ◽

Transcriptional Start Site ◽

Conjugative Plasmid ◽

Start Codon ◽

Start Site ◽

Gene Encoding ◽

Cleavage Activity ◽

Reverse Transcription Pcr ◽

Single Operon

ABSTRACT The tra genes orf1 to orf11 of pIP501 were shown to be transcribed as a single operon of 11.3 kb in Enterococcus faecalis by reverse transcription-PCR. The transcriptional start site of the tra mRNA was mapped at 110 bp upstream from the predicted TTG start codon of the first gene of the operon, the traA relaxase. The TraA protein (660 amino acids) and a C-terminally truncated version of the TraA protein (293 amino acids) were purified as fusions with glutathione S-transferase. oriT cleavage activity of both TraA proteins was demonstrated in vitro on supercoiled plasmid pVA2241 DNA containing oriTpIP501 . The activity of the DNA relaxase TraA is strictly dependent on the presence of Mg2+ or Mn2+ and is highest at temperatures of between 42 and 45°C.

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Physicochemical characterization of carbamylated human serum albumin: an in vitro study

RSC Advances ◽

10.1039/c9ra05875c ◽

2019 ◽

Vol 9 (63) ◽

pp. 36508-36516

Author(s):

Asim Badar ◽

Zarina Arif ◽

Shireen Naaz Islam ◽

Khursheed Alam

Keyword(s):

Amino Acids ◽

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Physicochemical Characterization ◽

In Vitro Study ◽

Terminal Amino ◽

Carbamyl Amino Acids

Carbamylation is an ubiquitous process in which cyanate (OCN−) reacts with the N-terminal amino or ε-amino moiety and generates α-carbamyl amino acids and ε-carbamyl-lysine (homocitrulline).

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Postnatal development of the chemosensitivity of rat cerebellar purkinje cells to excitatory amino acids. An in vitro study

Developmental Brain Research ◽

10.1016/0165-3806(87)90195-7 ◽

1987 ◽

Vol 34 (1) ◽

pp. 59-68 ◽

Cited By ~ 99

Author(s):

Jean-Luc Dupont ◽

Robert Gardette ◽

Francis Crepel

Keyword(s):

Amino Acids ◽

Postnatal Development ◽

Purkinje Cells ◽

Excitatory Amino Acids ◽

In Vitro Study ◽

Vitro Study ◽

Cerebellar Purkinje Cells

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Positive Selection of Mutations Leading to Loss or Reduction of Transcriptional Activity of PrfA, the Central Regulator ofListeria monocytogenes Virulence

Journal of Bacteriology ◽

10.1128/jb.183.19.5562-5570.2001 ◽

2001 ◽

Vol 183 (19) ◽

pp. 5562-5570 ◽

Cited By ~ 23

Author(s):

M. Herler ◽

A. Bubert ◽

M. Goetz ◽

Y. Vega ◽

J. A. Vazquez-Boland ◽

...

Keyword(s):

Amino Acids ◽

Positive Selection ◽

Transcriptional Activity ◽

Leucine Zipper ◽

Stable Complex ◽

Gene Encoding ◽

Target Dna ◽

Oflisteria Monocytogenes ◽

Function Relationship

ABSTRACT Transcription factor PrfA controls the expression of virulence genes essential for Listeria monocytogenes pathogenesis. To gain insight into the structure-function relationship of PrfA, we devised a positive-selection system to isolate mutations reducing or abolishing transcriptional activity. The system is based on the observation that the listerial iap gene, encoding the p60 protein, is lethal if overexpressed in Bacillus subtilis. A plasmid in which the iap gene is placed under the control of the PrfA-dependent hlypromoter was constructed and introduced into B. subtilis. This strain was rapidly killed when expression ofiap was induced by introduction of a second plasmid carrying prfA. Two classes of B. subtilissurvivor mutants were identified: one carried mutations iniap, and the second carried mutations inprfA. Sequence analysis of the defectiveprfA genes identified mutations in three regions of the PrfA protein: region A, between amino acids 58 and 67 in the β-roll domain of PrfA; region B, between amino acids 169 and 193, which corresponds to the DNA-binding helix-turn-helix motif; and region C, comprising the 38 C-terminal amino acids of PrfA, which form a leucine zipper-like structure. PrfA proteins with mutations in regions B and C were unable to bind to the PrfA-binding site in the target DNA, while mutations in region A resulted in a protein still binding the target DNA but unable to form a stable complex with RNA polymerase and initiate transcription in vitro.

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In Vitro Knock-Down of Myh9 Leads to a Myh9-Related Disorder Phenotype in Human Megakaryocytes.

Blood ◽

10.1182/blood.v114.22.4596.4596 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4596-4596

Author(s):

Yolande Chen ◽

Siham Boukour ◽

Monia Romdhane ◽

Ababacar Seye ◽

Olivier Bluteau ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myosin Ii ◽

In Vitro Study ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Gene Encoding ◽

Related Disorder ◽

Normal Platelet ◽

Negative Effect

Abstract Abstract 4596 Normal platelet production is dependent on the formation of branched long cytoplasmic extensions, called proplatelets (PPT). Mutations of the Myh9 gene (encoding for the nonmuscle myosin heavy chain IIA) result in autosomal dominant disorders, where patients develop various degrees of macrothrombocytopenia, with sometimes glomerular impairment, hearing loss and cataracts. There has been questioning as to whether the mechanism for the macrothrombocytopenia is haploinsufficiency, or a dominant negative effect of the mutated gene. We performed an in vitro study to investigate PPF from patient megakaryocytes (MK). By this approach, a decrease in PPF from patient CD34 derived MKs was observed in comparison to normal cultured MK. Surprisingly this defect of PPF observed in patients was rescued by blebbistatin, an inhibitor of class II myosin. Immunofluorescence studies performed showed that besides clusterization of GPIb in patient's platelets, no major repartition abnormalities were seen in cultured MKs derived from patient's CD34 for myosin, actin, tubulin, vWF, and Rac (except in one patient where actin and Rac formed aggregates to some extend, in a small number of MKs). In order to better understand the role of myosin during normal and abnormal PPF, we used a shRNA strategy to disrupt the Myh9 expression during normal MK differentiation and compared shRNA-treated MKs with MKs derived from patient CD34. Megakaryocytes treated with a shRNA that knocks down the protein of about 50%, did not alter MK ploidization, but decreased in vitro PPF, as previously observed for cells issued from patients. Moreover, shRNA-treated MKs exhibited the same ultrastructural abnormalities as patient MKs. Addition of Blebbistatin to shRNA treated MKs led to an increase of PPF, suggesting that the remaining myosin II might be hyperactivated and inhibit PPF. Altogether this study strongly suggests that the thrombocytopenia of the Myh9 syndrome is essentially related to haploinsufficiency in myosin II. Disclosures: No relevant conflicts of interest to declare.

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