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1.
Biomed Chromatogr ; 34(2): e4735, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31691999

ABSTRACT

The biosynthesis of sialic acid (Neu5Ac) leads to the intracellular production of cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), the active sialic acid donor to nascent glycans (glycoproteins and glycolipids) in the Golgi. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase myopathy is a rare autosomal recessive muscular disease characterized by progressive muscle weakness and atrophy. To quantify the intracellular levels of CMP-Neu5Ac as well as N-acetylmannosamine (ManNAc) and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography-tandem mass spectrometry methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography was used to retain three hydrophilic analytes. The human leukocyte pellets were lysed and extracted in a methanol-water mixture and the leukocyte extract was used for LC-MS/MS analysis. The lower limits of quantitation for ManNAc, Neu5Ac and CMP-Neu5Ac were 25.0, 25.0 and 10.0 ng/ml, respectively. These validated methods were applied to a clinical study.


Subject(s)
Chromatography, Liquid/methods , Cytidine Monophosphate/analogs & derivatives , Leukocytes/chemistry , Sialic Acids/blood , Tandem Mass Spectrometry/methods , Cytidine Monophosphate/blood , Drug Stability , Humans , Limit of Detection , Linear Models , Reproducibility of Results
2.
Talanta ; 201: 358-363, 2019 Aug 15.
Article in English | MEDLINE | ID: mdl-31122435

ABSTRACT

Single base mismatch can always connect with various gene-related diseases, whose determination has aroused widespread interest. So far, various methods have been developed to determine the common base mismatch. However most of them are complex, time-consuming. Herein, we report a novel method, which only need one conventional endonuclease (NEase) and achieve site-specific cleavage in a programmable way, to detect single base mismatch, termed aligner-mediated cleavage-based single base mismatch discrimination (AMCMD). The DNA aligner (DA) is in a stem-loop structure, consistent with an incomplete recognition site of NEase on its stem and a 5'-side arm complementary to the target sequence (TS). Once TS contains matched base and hybridizes with DA, the complete recognition site of NEase is formed, and the TS will be cleavaged with fast speed, while converse is not. Based on it, the method can clearly distinguish mismatched and complementary bases. Without sample pre-processing, we were able to obtain and verify all the test result in about 30 min through the polyacrylamide gel electrophoresis analysis. This endows the proposed method with a simpler advantage. Then we combined AMCMD and EXPAR to create a new method for single base mismatch discrimination, the short sequence obtained by AMCMD as a target to trigger EXPAR, with a detection limit at 1pM level. Another process with human serum underlines that AMCMD is compatible with the complex biological sample, thus it has the potentials for practical applications.


Subject(s)
Base Pair Mismatch , Biosensing Techniques/methods , Cytidine Monophosphate/blood , DNA Probes/chemistry , DNA/chemistry , Base Sequence , Cytidine Monophosphate/genetics , DNA/genetics , DNA Probes/genetics , Deoxyribonucleases, Type II Site-Specific/chemistry , Humans , Inverted Repeat Sequences , Limit of Detection , Nucleic Acid Hybridization
3.
Int J Hematol ; 87(2): 118-125, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18228114

ABSTRACT

Twenty-one acute myeloid leukemia (AML) patients were enrolled and received oral induction therapy with cytarabine ocfosfate (SPAC) and etoposide (EP). The median age was 69 years (range: 33-86). There were 11 patients with de novo AML and 10 AML cases that had evolved from myelodysplastic syndromes. Seventeen patients had abnormal karyotypes including eight complex abnormalities, various complications, and 7 of 21 had a poor performance status (PS) with Eastern Cooperative Oncology Group (ECOG) scores of 3-4. All patients completed induction therapy without severe adverse events. Seven achieved complete remission (CR), and two achieved partial remission (PR). Uni- and multivariate analyses demonstrated a positive and significant correlation between the results of therapy (CR +/- PR) and overall survival. The plasma concentrations of cytosine arabinoside (ara-C) in some cases were higher than those previously reported, indicating the accumulation of ara-C with increasing numbers of days of SPAC administration. We conclude that this therapy is well tolerated and useful for refractory and elderly AML patients.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Leukemia, Myeloid, Acute/drug therapy , Neoplasm Recurrence, Local/drug therapy , Administration, Oral , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/blood , Arabinonucleotides/administration & dosage , Arabinonucleotides/adverse effects , Arabinonucleotides/blood , Cytidine Monophosphate/administration & dosage , Cytidine Monophosphate/adverse effects , Cytidine Monophosphate/analogs & derivatives , Cytidine Monophosphate/blood , Etoposide/administration & dosage , Etoposide/adverse effects , Etoposide/blood , Female , Humans , Kaplan-Meier Estimate , Male , Middle Aged
4.
Pol Arch Med Wewn ; 93(4): 283-7, 1995 Apr.
Article in Polish | MEDLINE | ID: mdl-7479251

ABSTRACT

Activity of pyrimidine 5'nucleotidase was measured in hemolysate of erythrocytes of healthy persons and patients with essential hypertension. Cytidine 5'monophosphate (CMP) and uridine 5'monophosphate (UMP) were used as the substrates for evaluation of activity of so called I-isoenzyme and uridine 3'monophosphate (U3'MP) was used as a substrate for the II isoenzyme of Py5'Nd. It was found that the activity of Py5'Nd I was lower in hypertensives (26.8 mU/gHb (CMP)) and 69.3 mU/gHb (UMP) in comparison with normotensives (62.3 and 117.4 mU/gHb respectively) (p < 0.05). The activity of Py5'Nd II did not differ between studied groups. Possible metabolic consequences of decreased activity of Py5'Nd are discussed.


Subject(s)
5'-Nucleotidase/blood , Erythrocytes/enzymology , Hypertension/enzymology , Adult , Case-Control Studies , Cytidine Monophosphate/blood , Humans , Isoenzymes/blood , Middle Aged , Substrate Specificity , Uridine Monophosphate/blood
5.
J Chromatogr B Biomed Appl ; 665(1): 183-92, 1995 Mar 10.
Article in English | MEDLINE | ID: mdl-7795790

ABSTRACT

An ion-pair HPLC method for the determination of 1-beta-D-arabinofuranosylcytosine-5'-stearyl phosphate (cytarabine-ocfosfate I) was developed, using a phenyl-bonded column under reversed-phase conditions with a mobile phase of acetonitrile-buffered water (pH 6.8) (50:50) for isocratic elution. A reproducible sample clean-up was achieved by solid-phase extraction. In order to reach the low limit of detection of 2 ng/ml, an enrichment switching system was used. The present validation leads to a limit of quantification of 5 ng/ml with a coefficient of variation (C.V.) of 10%. The total time of measurement was shortened by a back-flush procedure to restore the conditions after each run. UV detection at 275 nm was applied. The recoveries for plasma samples ranged from 56.4 to 64.1%, regardless of drug concentrations. The intra-assay C.V. was about 4% (40 measurements at four different concentrations). The inter-assay recovery (ten measurements over ten days) at a plasma concentration of 50 ng/ml was 57% with a C.V. of 8.25%. Based on this HPLC method, the pharmacokinetics of I were measured during a clinical phase I/II study.


Subject(s)
Antineoplastic Agents/analysis , Arabinonucleotides/analysis , Chromatography, High Pressure Liquid/methods , Cytidine Monophosphate/analogs & derivatives , Antineoplastic Agents/blood , Antineoplastic Agents/urine , Arabinonucleotides/blood , Arabinonucleotides/urine , Cytidine Monophosphate/analysis , Cytidine Monophosphate/blood , Cytidine Monophosphate/urine , Drug Stability , Humans , Reproducibility of Results
6.
Biochem J ; 275 ( Pt 1): 187-92, 1991 Apr 01.
Article in English | MEDLINE | ID: mdl-1850237

ABSTRACT

Unlike human erythrocytes, those from avian species, such as turkeys and chicks, rapidly incorporate myo-[3H]inositol into membrane phospholipids. The mechanisms regulating [3H]Ins labelling of phosphatidylinositol have been investigated using turkey erythrocyte membranes. In the absence of added nucleotides, [3H]inositol incorporation appears to proceed via phosphatidylinositol/inositol exchange, with a Km for inositol of 0.01 mM. The reaction was dependent upon divalent cations, either Mg2+ or Mn2+, with the latter metal ion being the more effective. [3H]Inositol incorporation was accelerated by CMP, especially when the concentration of Ins was greater than the Km for the exchange reaction. CMP-dependent labelling of PtdIns had a Km for inositol of 0.3 mM and for CMP of 0.015 mM. Divalent cations were also required for this reaction: activity peaked at 0.5 mM-Mn2+ and declined at higher concentrations. At relatively high concentrations, Mg2+ was more effective than Mn2+, with peak activity being achieved above 10 mM. CMP-dependent incorporation of [3H]inositol appears to reflect an exchange reaction catalysed by PtdIns synthase. Definitive evidence for the occurrence of PtdIns synthase in turkey erythrocyte membranes was obtained by demonstrating the formation of [14C]CMP-phosphatidate from [14C]CMP. The radioactivity could be efficiently chased from [14C]CMP-phosphatidate in the presence of unlabelled inositol. The detection of PtdIns synthase activity in morphologically simple turkey erythrocytes should help to clarify the subcellular distribution of this important component of the phosphatidylinositol cycle.


Subject(s)
Erythrocyte Membrane/metabolism , Inositol/blood , Phosphatidylinositols/blood , Phosphotransferases/blood , Transferases (Other Substituted Phosphate Groups) , Turkeys/blood , Animals , CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase , Cations, Divalent , Cytidine Monophosphate/blood , Cytidine Monophosphate/pharmacology , Kinetics , Magnesium/pharmacology , Manganese/pharmacology , Phosphatidic Acids/blood
7.
Circulation ; 77(2): 398-406, 1988 Feb.
Article in English | MEDLINE | ID: mdl-2962788

ABSTRACT

In an animal preparation of congestive heart failure in the dog, during the development of cardiac failure due to rapid right ventricular pacing we observed significant decreases in cardiac output and arterial pressure and increases in pulmonary arterial and right atrial pressure. We also observed a related increase in right atrial pressure and increases in plasma levels of atrial natriuretic peptide (ANP) and cyclic guanosine monophosphate (c-GMP). Ultrastructure changes in the atrial myoendocrine cells indicated extreme stimulation of the secretory apparatus of ANP. The response of hemodynamic, renal, and hormonal variables was investigated after incremental infusions (0.01, 0.03, 0.1, 0.3, and 0.06 microgram/kg/min) of exogenous ANP. In healthy animals ANP significantly decreased mean arterial pressure, cardiac output, stroke volume, and right atrial pressure without changing heart rate or peripheral vascular resistance. As expected, we found a striking increase in urine flow and urinary excretion of sodium, chloride, magnesium and calcium and a smaller increase in potassium excretion. ANP suppressed renin secretion, and increased renal plasma flow, glomerular filtration rate, and filtration fraction. In dogs with heart failure ANP caused a small reduction in mean arterial pressure. No effect was seen on other hemodynamic variables or plasma renin concentration. The excretory effects on the kidneys were completely absent, and smaller increases in glomerular filtration rate and filtration fraction were observed. We found no difference between healthy dogs and animals with heart failure with respect to the secretion of c-GMP during ANP infusions in relation to the plasma levels of ANP. This suggests an intracellular defect that prevents the mediation of the hormonal signal into biological action in the presence of heart failure.


Subject(s)
Atrial Natriuretic Factor/blood , Cytidine Monophosphate/blood , Cytosine Nucleotides/blood , Heart Failure/blood , Hemodynamics/drug effects , Kidney/drug effects , Myocardium/ultrastructure , Animals , Atrial Natriuretic Factor/administration & dosage , Dogs , Dose-Response Relationship, Drug , Female , Heart Atria/ultrastructure , Heart Failure/pathology , Heart Failure/physiopathology , Infusions, Intravenous , Renin/blood
8.
Biomed Biochim Acta ; 46(2-3): S280-4, 1987.
Article in English | MEDLINE | ID: mdl-2439075

ABSTRACT

The changes in inosine 5'-monophosphate (IMP) and adenine nucleotides were studied in human erythrocytes incubated with glucose alone, or with glucose plus inosine in the presence or absence of monoiodoacetate, a strong inhibitor of the glycolytic pathways of the cells. HPLC was useful in analyzing the changes in these nucleotides. When the cells were incubated with glucose and inosine for several hours at pH 7.0, 37 degrees C, they produced much IMP, while the cells incubated with glucose and monoiodoacetate accumulated AMP rapidly within 2 hours, accompanied with the decomposition of ADP and ATP; but IMP was not accumulated. When the cells were incubated with glucose and inosine in the presence of monoiodoacetate, AMP increased more rapidly to about 1400 mu mols/ml of cells within 1 hour and then gradually decreased. ATP was consumed completely and ADP decreased correspondingly. However, IMP was produced in this case, though its accumulation was not so high as in the case with glucose plus inosine without monoiodoacetate. The production of IMP depended on the pH of the cell suspension, i.e., more IMP was produced at higher pHs.


Subject(s)
Erythrocytes/metabolism , Inosine Monophosphate/blood , Inosine Nucleotides/blood , Inosine/blood , Adenine Nucleotides/blood , Blood Glucose/metabolism , Cytidine Monophosphate/blood , Humans , Hydrogen-Ion Concentration , In Vitro Techniques , Phosphoribosyl Pyrophosphate/blood , Ribose-Phosphate Pyrophosphokinase/blood
9.
J Biochem Toxicol ; 1(3): 51-9, 1986 Sep.
Article in English | MEDLINE | ID: mdl-2856073

ABSTRACT

Inhibition by lead of erythrocyte pyrimidine 5'-nucleotidase (P5N) is thought to contribute to morphological abnormalities observed in red blood cells (RBC) of lead-exposed subjects. However, neither the mechanism of lead inhibition of P5N nor the relationship of this inhibition to blood lead levels attained in exposed subjects is known. In the present investigation, acute in vivo and in vitro lead acetate effects on erythrocyte P5N from 21-day-old rat pups were determined and were related to blood lead concentrations ascertained by atomic absorption spectrophotometry. Acute lead administration to rat pups resulted in a 16% to 21% reduction in erythrocyte P5N, with mean blood lead levels ranging from 77 to 108 micrograms/dl 24 hours later. Inhibition of erythrocyte P5N was linearly related to blood lead level (r = -0.67, P less than 0.05) following acute lead administration. Lead acetate addition to RBC preparations from 21-day-old rats resulted in concentration-dependent P5N inhibition which was comparable to that produced following acute in vivo exposure. The results indicate that acute P5N inhibition in lead-treated neonatal rats is due to noncompetitive P5N inhibition by lead. The inhibition of P5N produced by acute lead treatment is linearly related to blood lead concentrations.


Subject(s)
5'-Nucleotidase/blood , Erythrocytes/enzymology , Organometallic Compounds/toxicity , 5'-Nucleotidase/antagonists & inhibitors , Animals , Animals, Newborn , Cytidine Monophosphate/blood , Lead/blood , Organometallic Compounds/pharmacology , Rats
10.
Br J Haematol ; 43(3): 423-34, 1979 Nov.
Article in English | MEDLINE | ID: mdl-497119

ABSTRACT

In pyrimidine 5'-nucleotidase deficiency, erythrocytes contain elevated levels of pyrimidine nucleotides. The composition of this nucleotide pool was examined by ion exchange chromatography on Dowex formate columns using a linear ammonium formate elution gradient. In contradistinction to normal erythrocytes, adenine nucleotides accounted for only 32% of the nucleotide pool. The remainder consisted of 50% cytidine and 16% uridine nucleotides. The remaining 2% was not identified. The most abundant compound appeared to be UDP glucose whilst high levels of CTP, CMP and an unidentified cytidine compound less polar than CMP accounted for most of the cytidine nucleotide pool. The possibility that the abnormal nucleotides were due to an elevated reticulocyte count was excluded and it was also shown that erythrocytes from subjects heterozygous for pyrimidine 5'-nucleotidase deficiency did not have detectable levels of the abnormal nucleotides.


Subject(s)
Erythrocytes/enzymology , Nucleotidases/deficiency , Pyrimidine Nucleotides/blood , Adenosine Triphosphate/blood , Anemia, Hemolytic, Congenital Nonspherocytic/enzymology , Anemia, Pernicious/enzymology , Cytidine Diphosphate/blood , Cytidine Monophosphate/blood , Erythrocytes/analysis , Humans , Infant , Uridine Diphosphate/blood , Uridine Monophosphate/blood , Uridine Triphosphate/blood
11.
Blut ; 30(1): 39-46, 1975 Jan.
Article in German | MEDLINE | ID: mdl-1172767

ABSTRACT

In a case with leukemic lymphosarcomatosis huge nucleoli developed at a final stage refractory to chemotherapy. The lymphosarcoma cells were characterized before and after their macronucleolar transformation by cell fractionation and by biochemical characterization of their nuclear 45S RNA fraction after high specific labelling with 32P-orthophosphate. The analyses provided evidence for a marked increase in nucleolar 45S RNA production. Processing of preribosomal RNA did not seem to be blocked. The biochemical alterations are discussed especially with respect to a possible significance of biological changes during the development of drug resistance against cytostatic agents.


Subject(s)
Cell Nucleolus , Lymphoma, Non-Hodgkin/blood , Adult , Blood Cell Count , Cell Fractionation , Centrifugation, Density Gradient , Cytidine Monophosphate/blood , Female , Guanine Nucleotides/blood , Humans , Lymph Nodes/pathology , Lymphoma, Non-Hodgkin/pathology , RNA, Neoplasm/blood , RNA, Neoplasm/metabolism , Ribosomes/metabolism
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