scholarly journals Comparison of Organosulfur and Amino Acid Composition between Triploid Onion Allium cornutum Clementi ex Visiani, 1842, and Common Onion Allium cepa L., and Evidences for Antiproliferative Activity of Their Extracts

Plants ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 98 ◽  
Author(s):  
Željana Fredotović ◽  
Barbara Soldo ◽  
Matilda Šprung ◽  
Zvonimir Marijanović ◽  
Igor Jerković ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Hela Cells ◽  
Antiproliferative Activity ◽  
Dapi Staining ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Allium Species ◽  
Allium Cepa L ◽  
Common Onion

Species that belong to the genus Allium have been widely used for human food and traditional medicine. Their beneficial health effects, as well as the specific aroma, are associated with their bioactive chemical compounds, such as sulfur compounds and flavonoids. Gas chromatography and mass spectrometry (GC–MS) and reverse-phase high-performance liquid chromatography (reverse-phase HPLC) were used to identify organosulfur and amino acid content of triploid hybrid onion, Allium cornutum Clement ex Visiani, 1842, and common onion, Allium cepa L. Allium extracts were tested for their antiproliferative activity in three human cancer cell lines (HeLa, HCT116, and U2OS). DNA fragmentation and DAPI staining analysis were performed on HeLa cells to evaluate the effect of extracts on DNA damage and cell morphology. The mRNA expression of p53, Bax, and Caspase-3 genes involved in apoptosis were analyzed by real-time PCR. Using GC–MS, 27 compounds were found in two Allium species headspaces. Differences were noted among the main compound abundance in the headspace (although the major thiols and disulfides were qualitatively identic in both Allium species) and dipropyl disulfide, diisopropyl trisulfide, and (Z)-prop-1-enyl propyl trisulfide were predominant sulfides. Identification of amino acids and their quantities were determined by reverse-phase HPLC. Most abundant amino acids in both onions were arginine (Arg) and glutamic acid (Glu). The results of cytotoxicity testing confirmed antiproliferative effects of both species. The DNA fragmentation assay, DAPI staining and real time PCR analysis confirmed that A. cornutum and A. cepa extracts induced apoptosis in HeLa cells. This study presents the evidence for possible therapeutic use of A. cornutum and A. cepa extracts against human cervical carcinoma cell line.

scholarly journals Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

2007 ◽  
Vol 81 (16) ◽  
pp. 8648-8655 ◽  
Author(s):  
Melissa Stewart Kim ◽  
Vincent R. Racaniello
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rhesus Monkey ◽  
Host Range ◽  
Hela Cells ◽  
Lytic Replication ◽  
Monkey Kidney ◽  
Kidney Cells ◽  
Cytopathic Effects ◽  
Axis Of Symmetry

ABSTRACT Enterovirus type 70, an etiologic agent of acute hemorrhagic conjunctivitis, may bind different cellular receptors depending on cell type. To understand how EV70-receptor interaction is controlled, we studied two variants of the virus with distinct receptor utilization. EV70-Rmk, derived by passage in rhesus monkey kidney cells, replicates poorly in HeLa cells and does not cause cytopathic effects. Decay accelerating factor (DAF) is not a cell receptor for EV70-Rmk. Passage of EV70-Rmk in HeLa cells lead to isolation of EV70-Dne, which does not replicate in rhesus monkey kidney cells but grows to high titers in HeLa cells and causes cytopathic effects. DAF is sufficient for cell entry of EV70-Dne. EV70-Rmk replicates in human eye and brain-derived cell lines, whereas the Dne strain replicates only in HeLa cells and in conjunctiva-derived 15C4 cells. The two EV70 strains differ by five amino acid changes in the viral capsid. Single substitution of four of the five EV70-Rmk amino acids with the residue from EV70-Dne leads to lytic replication in HeLa cells. Conversely, substitution of any of the five EV70-Dne amino acids with the EV70-Rmk amino acid does not alter replication in HeLa cells. Three of these capsid amino acids are predicted to be located in the canyon encircling the fivefold axis of symmetry, one amino acid is found at the fivefold axis of symmetry, and one is located the interior of the capsid. The five EV70 residues define a region of the capsid that controls viral host range, DAF utilization, and cytopathogenicity.

Molecular Characterization and Pharmacology of Epoetin Delta − The Only Erythropoietin Produced in a Human Cell Line.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1299-1299
Author(s):  
Raymond D. Pratt ◽  
J. Dowell ◽  
Z. Shahrokh ◽  
S. Flatman ◽  
M. Davies ◽  
...  
Keyword(s):  
Amino Acid ◽  
Adverse Events ◽  
Human Cell Line ◽  
Pharmacokinetic Parameters ◽  
Epoetin Alfa ◽  
Healthy Individuals ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Dose Dependent ◽  
Serum Erythropoietin

Abstract Recombinant erythropoietins are used extensively in the management of anemia associated with chronic kidney disease (CKD). All the currently available erythropoietins are synthesized in Chinese hamster ovary (CHO) cell lines resulting in differences in glycosylation compared with human serum erythropoietin. We report the molecular characterization and pharmacokinetic properties of epoetin delta (Dynepo®, Shire plc), the only erythropoietin produced in a human cell line. A variety of techniques have been used to characterize epoetin delta, including amino acid sequencing, peptide mapping with reverse-phase HPLC/mass spectrometry, oligosaccharide profiling and MALDI-TOF of released glycans. Sialic acid and N-glycolylneuraminic acid (Neu5Gc) residues were quantified, following labeling of the released glycans, by reverse-phase HPLC analysis with fluorescence detection. Epoetin delta was produced as a highly pure and stable protein with the full human primary amino acid sequence. Neu5Gc residues were not detectable in epoetin delta within the validated limits of the assay but became readily detectable in CHO derived epoetins. Following characterization of the molecule early studies were done to determine the pharmacokinetic and pharmacodynamic properties of epoetin delta. Two studies investigated responses to epoetin delta in healthy individuals. The first study involved randomization of 21 men to either intravenous (i.v.) epoetin delta (15, 40 or 100 IU/kg) or placebo. The second was an open-label, crossover study, involving 32 volunteers randomized to receive single doses of epoetin delta 75 IU/kg given i.v. or subcutaneous (s.c.) by injection. Two further studies were carried out with CKD patients requiring hemodialysis who had previously received epoetin alfa. The first involved 40 patients randomized to epoetin delta or epoetin alfa (50 or 100 IU/kg), three-times per week for 4 weeks. The second was a single-dose study in 28 patients comparing epoetin delta 150 and 300 IU/kg given i.v. or s.c. Pharmacokinetic parameters were calculated from serum erythropoietin concentrations (determined by ELISA) by validated non-compartmental techniques using WinNonlin Professional v3.0A. Intravenous epoetin delta in healthy individuals displayed non-linear and dose-dependent pharmacokinetics, with a dose-dependent effect on serum hemoglobin. The bioavailability of s.c. epoetin delta is around 30%, and concentrations peak later and decline more slowly than with i.v. injection. In hemodialysis patients pharmacokinetic parameters were similar to those in healthy individuals, although AUC and half-life were increased. Compared with the 50 IU/kg dose, treatment with epoetin delta 100 IU/kg was associated with a trend to increased hemoglobin and hematocrit levels. Epoetin delta was well tolerated in all participants with the frequency of adverse events occurring in dialysis patients as expected for that population. Adverse events were similar with epoetin alfa and epoetin delta. No neutralizing anti-erythropoietin antibodies were detected in any patient. Epoetin delta was characterized at the molecular level and levels of Neu5Gc residues were undetectable within the validated limits of the assay. The pharmacokinetic profile of epoetin delta and effects on hemoglobin and hematocrit levels were suitable for the treatment of anemia in CKD patients by either s.c or i.v. administration.

An Improved Reverse Phase HPLC Separation and Postcolumn Detection of Amino Acids

Proteins ◽  
1987 ◽  
pp. 215-223
Author(s):  
Stephen Gruber ◽  
Noel M. Meltzer ◽  
Stanley Stein ◽  
Guillermo I. Tous
Keyword(s):  
Amino Acids ◽  
Hplc Separation ◽  
Reverse Phase Hplc ◽  
Reverse Phase

Separation of unmodified α-amino acid enantiomers by reverse phase HPLC

1980 ◽  
Vol 13 (11) ◽  
pp. 677-685 ◽  
Author(s):  
V. A. Davankov ◽  
A. S. Bochkov ◽  
A. A. Kurganov ◽  
P. Roumeliotis ◽  
K. K. Unger
Keyword(s):  
Amino Acid ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Amino Acid Enantiomers

scholarly journals T-kininogenase activity of the rat submandibular gland is predominantly due to the kallikrein-like serine protease antigen γ

1991 ◽  
Vol 280 (1) ◽  
pp. 19-25 ◽  
Author(s):  
T Berg ◽  
I Wassdal ◽  
T Mindroiu ◽  
K Sletten ◽  
G Scicli ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mass ◽  
Submandibular Gland ◽  
Sequence Similarity ◽  
Rat Plasma ◽  
Amino Acid Sequence Similarity ◽  
Tissue Kallikrein ◽  
Reverse Phase ◽  
Rat Submandibular Gland

T-kininogen, the major kininogen in rat plasma, releases Ile-Ser-bradykinin (T-kinin) when incubated with trypsin, but is not a substrate for tissue kallikrein. Enzymes able to release T-kinins from T-kininogen have been found in the rat submandibular gland, but precise identification of these enzymes and their possible relationship to kallikrein-like enzymes has not been established. We studied T-kininogenase activity in fractionated submandibular gland homogenate. The main T-kininogen catalytic enzyme was purified and characterized, and found to be identical to antigen gamma, a kallikrein-like enzyme which we have previously characterized. Of other identified kallikrein-like enzymes only tonin showed weak T-kininogenase activity, which was about 0.25% of that of antigen gamma. No other T-kininogen catalytic enzymes were observed. Antigen gamma released a kinin which was identified as T-kinin by reverse-phase h.p.l.c. The T-kininogenase activity of antigen gamma had a Km of 29 +/- 4 microM and a kcat/Km of 140 M-1.s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10 micrograms of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42,200 micrograms of kinin/h per mg respectively. Thus antigen gamma is a weak kininogenase. The isoelectric point of antigen gamma, but not its molecular mass, differed from that of other kallikrein-like enzymes. Isoelectrofocusing in flat-bed gels combined with immunostaining was therefore a convenient method for identification. The kallikrein-like nature of antigen gamma was demonstrated by its immunological similarity to tissue kallikrein and tonin and by 91% and 87% amino acid sequence similarity with tonin and kallikrein respectively (67 amino acids sequenced). Complete identity was also not observed with other sequenced kallikrein genes, mRNAs or proteins.

scholarly journals Isolation and Characterisation of a Reserve Protein from the Seeds of Cereus jamacaru (Cactaceae)

2001 ◽  
Vol 44 (4) ◽  
pp. 331-335 ◽  
Author(s):  
Itayguara Ribeiro da Costa ◽  
Petrônio Augusto Simão de Souza ◽  
Carlos Bloch Jr. ◽  
Romulo Marino Llamoca-Zárate ◽  
Francisco A. P. Campos
Keyword(s):  
Amino Acid ◽  
Molecular Marker ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Storage Protein ◽  
Gel Filtration ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
2S Albumin ◽  
Reserve Protein

We describe here the isolation and characterisation of a major reserve protein from the seeds of Cereus jamacaru. (Cactaceae). This protein has a molecular mass of 5319 kDa and was isolated by a combination of gel filtration chromatography and reverse phase HPLC. The amino acid composition of the protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The usefulness of this protein as a molecular marker in the Cactaceae is also discussed.

scholarly journals Functional Regions of the Pseudomonas aeruginosa Cytotoxin ExoU

2005 ◽  
Vol 73 (1) ◽  
pp. 573-582 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Alan R. Hauser
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Phospholipase Activity ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.

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