scholarly journals Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions

2000 ◽  
Vol 347 (1) ◽  
pp. 37-44 ◽  
Author(s):  
John K. SHEEHAN ◽  
Caroline BRAZEAU ◽  
Saduman KUTAY ◽  
Helena PIGEON ◽  
Sara KIRKHAM ◽  
...  
Keyword(s):  
Cell Culture ◽  
Gel Electrophoresis ◽  
Early Stage ◽  
Average Length ◽  
Buoyant Density ◽  
Gel Chromatography ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Major Band

We have isolated the high-Mr mucins from growth medium of the early stage of an HT-29 cell culture by gel chromatography and isopycnic density gradient centrifugation. The mucins (buoyant density 1.34-1.44 g/ml) were reactive with an anti-peptide antiserum (MAN-5ACI) raised against a sequence from within the MUC5AC mucin. Similar antisera raised against the MUC2 and MUC5B mucins were not reactive. The MUC5AC reduced-mucin subunits exhibited a homogeneous charge distribution on anion-exchange chromatography, but appeared as two bands, one major and one more minor, after agarose gel electrophoresis. The unreduced mucins had an average Mr in excess of 40 MDa and were visualized in the electron microscope as large, fine filamentous threads (many microns in length) that after reduction were greatly reduced in size (number average length 570 nm). Agarose gel electrophoresis of unreduced MUC5AC mucins identified a major band just entering the gel with evidence of a ‘ladder’ of faster-migrating minor bands. Partial reduction of the mucins increased the proportion of the faster bands and at least 16 could be discriminated. Mr measurements showed that these bands differed by single monomer units. The mucins behaved as very stiff extended structures in solution and this characteristic might explain the poor separation of different-sized oligomers in sedimentation-rate experiments. The cell-culture mucin preparation had similar characteristics of charge and buoyant density to MUC5AC mucins from respiratory secretions in vivo. In addition the MUC5AC mucin from respiratory tract secretions exhibited similar behaviour, reduced and unreduced on agarose gel electrophoresis, indicating that the mucin has a similar molecular phenotype in vivo and in vitro.

Characterisation Of Active Components Of Fix Concentrates Used In FVIII Inhibitor Patients

1981 ◽  
Author(s):  
M J Seghatchian ◽  
I J Mackie
Keyword(s):  
Gel Electrophoresis ◽  
Molecular Form ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Active Components ◽  
Fviii Inhibitor ◽  
Antigenic Sites ◽  
Twin Peak

It is well recognised that haemostasis may be achieved in patients with FVIII inhibitors using FIX concentrates (PTX). However, it has not been possible to correlate the clinical response with enzymicity, FVIII levels and other in vitro results. This is partially due to the presence of variable amounts of inhibitors and stabilizers such as: citrate, heparin, and anti proteases in different preparations. Agarose gel electrophoresis was performed in an attempt to separate these inhibitors so that the various activities may be measured relative to one another. PTX from 4 sources were used in this study,(Kabi Vitrum, Immuno, Oxford and Edinburgh fractionation centres); FVIII was measured by two stage clotting(FVIII:C)and amidolytic (CAM) assays, and its electrophoretic distribution was followed by preincubation with 125I-anti VIII:C (VIII:CAg). Enzymicity was measured with various substrates (S2160, S2222, S2302, S2238, S2288, S2251). All concentrates showed a fast moving peak of FVIII:C and VIII:CAm activity, in amounts in excess of that detected before electrophoresis. This peak correlated well with the distribution of bound radiolabel (VIII:CAg). Preincubation of PTX with FVIII concentrate caused the VIII:CAg peak to migrate more slowly, in its normal α 2 position. FVIII:RAg was present in much lower amounts than FVIII:C. When a twin- peak of VIII:CAm activity was sometimes seen, the slower peak was associated with S2288 activity, which may represent the action of activated factors VII or IX: other substrates showed variable patterns of enzymicity but the strongest activity was with S2160 and S2302 from the well to the β region, and this could be completely inhibited by addition of anti Kallikrein. The presence of an abnormal molecular form of FVIII, and the discrepancy between pre and post electrophoresis FVIII levels, indicate that FVIII could play a major role in the action of PTX. This may involve 1) a complex with phospholipid, FIX and FX making it potentially more effective; 2) enhancement of VIII:C activity in vivo by binding to FVIII:RAg; 3) altered antigenic sites on the FVIII, making it unrecognisable to inhibitor.

The extraction, characterization andin vitrotranslation of RNA from adultSchistosoma mansoni

Parasitology ◽  
1982 ◽  
Vol 84 (2) ◽  
pp. 253-261 ◽  
Author(s):  
M. P. R. Tenniswood ◽  
A. J. G. Simpson
Keyword(s):  
Schistosoma Mansoni ◽  
Lithium Chloride ◽  
Gel Electrophoresis ◽  
Rabbit Reticulocyte Lysate ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Molecular Weights ◽  
Reticulocyte Lysate ◽  
Urea Method

SUMMARYWe have extracted RNA fromSchistosoma mansoniusing the lithium chloride–urea method which gives good yields of undegraded RNA. The results of agarose gel electrophoresis of RNA extracted by this procedure suggest thatS. mansonihas anin vivonick in the large rRNA sub-unit. Translation of the RNA in a rabbit reticulocyte lysate gave significant incorporation of [35S]methionine into synthesized proteins. Immuno-precipitation of these translation products using a hyperimmune monkey serum sedimented between 5 and 8% of the radioactivity, which appeared to be present in approximately 13 proteins of molecular weights 18, 20, 21, 22, 23, 35, 40, 54, 60, 70, 74, 78 and 105 K Daltons.

L-Carnitine and Apoptosis in Thalassemia Major Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3767-3767
Author(s):  
Ali Taher ◽  
H. Seoud ◽  
A. Ibrahim ◽  
H. Gabre ◽  
I. Youssry Ibrahim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Enzyme Immunoassay ◽  
Gel Electrophoresis ◽  
Large Scale ◽  
Early Stage ◽  
Thalassemia Major ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Erythroid Progenitors ◽  
Erythroid Precursors

Abstract Background and Objectives: Thalassemia major patients suffer from severe anemia due to intramedullary hemolysis of erythroid progenitors. Depending on the severity of their disease, thalassemic patients exhibit different patterns of apoptosis. In cases of β-thalassemia major, apoptosis appears to be greatly enhanced in the early-stage erythroid precursors in the bone marrow and it is considered to be the machinery via which ineffective erythropoiesis develops. L-Carnitine is a butyrate derivative found to strongly reduce apoptosis in different diseases. We investigated the effect of oral L-carnitine therapy on apoptosis in thalassemia major patients. Methods: eighteen Thalassemia major patients were included. Detection of apoptosis was done by Photometric enzyme immunoassay (Elisa) and agarose gel electrophoresis before and after 6 months oral therapy with L-Carnitine (50mg/kg/day). Results: The mean age was 12.2±6.6 years. All patients tolerated L-carnitine with no side effects. A significant decrease of apoptosis rates in the erythroid precursors in the bone marrow of studied cases was noted after therapy by. The enrichment factor measured by the photometric enzyme immunoassay dropped from 3.65±1.338 before therapy to 1.60±0.65 after therapy (p=0.005). A positive ladder pattern reflecting apoptosis on agarose gel electrophoresis was detected in 88.9% of cases prior to treatment (figure 1) versus 11.1% after therapy (figure 2) (p=0.006). Patients also had significant decrease in the frequency of transfusions and increase in the pre-transfusion hemoglobin levels after therapy. These results were found to significantly correlate with the reduction in apoptosis rates (p=0.006, r = 0.543 and p=0.027 &r = −0.7762 respectively). Conclusion: Apoptosis plays a major role in the underlying pathophysiology of anemia in thalassemic patients. L-Carnitine seems to be a good modulator of apoptotic processes in such patients leading to decreased rates of programmed erythroblast death and general improvement of the disease condition. Further large-scale studies are needed to evaluate the overall role of L-Carnitine in the management of these patients. Figure Figure

scholarly journals Curing of Escherichia coli Antibiotic Resistance Plasmids by Aspirin

2014 ◽  
Vol 8 (1) ◽  
pp. 30-35
Author(s):  
Ibtesam H. Al Musawi ◽  
Ali A. Al Zaag
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Agarose Gel ◽  
Large Plasmid ◽  
Agarose Gel Electrophoresis ◽  
E Coli ◽  
Resistance Plasmids ◽  
Minimum Number

An isolate of Escherichia coli (E. coli) was isolated from urine sample due to person infected with urinary tract infection (UTI).The isolate was resistant to the following antibiotics: Ampicillin, Cotrimoxazole, Chloramphenicol and Tetracycline. Agarose gel electrophoresis of its plasmids content has revealed the presence of single large plasmid and two small plasmids bands. The large plasmid was conjugative and contained the resistance genes for four antibiotics. In vitro curing of this plasmid was achieved by treatment with salicylic acid (aspirin) with 150,200,250 and 300 µg/ml as indicated by the elimination of resistance, also by absence of large plasmid band following agarose gel electrophoresis. In vivo curing was conducted using New Zealand rabbits. UTI was induced by bacterial inoculation via urethral catheterization. The E. coli from urine samples of the rabbits, the count of which was proportional to the type of treatment. Minimum number of colonies was associated with group treated with metheprim was aspirin 300mg/kg daily dosage. This result may indicate that the side effect of metheprim was in its maximum with aspirin. Survivor bacteria may indicate incomplete exposure to the drug.

scholarly journals A Functional Single Nucleotide Polymorphism in the Aγ Globin Gene Promoter Affects Globin Chain Synthesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-20
Author(s):  
Elena Spiteri ◽  
Laura Grech ◽  
Alexander E. Felice ◽  
Joseph Borg
Keyword(s):  
Restriction Enzyme ◽  
Gel Electrophoresis ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Total Study Population ◽  
Study Population ◽  
The Mean

Beta Thalassemia and related hemoglobinopathies (such as sickle cell disease), are usually characterized by high levels of HbF since this compensates for the lack of, or abnormal HbA that is expressed in vivo. Further insight on how the hemoglobin switch is controlled and regulated can become crucial in the treatment of hemoglobin disorders. The present treatment options currently include routine blood transfusions with the occasional complications attributed to iron overload in major organs. Human stem cell therapy and bone marrow transplantation are some other options that are very risky and not without complications. Previous studies have shown that HbF levels differ in adults and that at least three loci independent of the β globin locus are responsible including XMN1, the HBS1L-MYB intergenic interval, and BCL11A. In another study conducted on an extended Maltese family, revealed that KLF1 also controls HbF levels in vivo. This study also suggested that other genes in conjunction with KLF1, play a part in activating the HbF switch upon birth. To date, the complete mechanism of how globin gene switching works is still elusive. A total of 355 DNA samples were tested. The majority of the DNA samples were obtained from an existing collection of normal to borderline HbA2 and HbF cases, while other samples were collected along the course of the study. Data from anonymous and randomized cord blood DNA samples were also used and obtained from the Laboratory of Molecular Genetics, University of Malta. These were tested for the rs368698783 polymorphism under study and were used for reference purposes. The SNP rs368698783 was amplified by PCR and checked by agarose gel electrophoresis using a 1.5% agarose gel. Restriction enzyme fragment length polymorphism analysis was then performed using Mnl1 restriction enzyme. Agarose gel electrophoresis was performed again in order to separate the Mnl1- incubated products according to size and thus determine the frequency of the mutation identified. Some samples were chosen at random and were genotyped by DNA sequencing. Amongst the total study population (~355 individuals), the frequency of the mutant allele was 0.14 and close to 0.25 for the random newborn cord blood DNA analyzed in a previous study. The total study population was divided into 125 healthy adults and 193 into borderline HbF/HbA2 The mean fetal hemoglobin and HbA2 for the normal sub-population were 0.39± 0.034 and 2.68± 0.02 respectively. The mean levels in the borderline population were slightly higher with a mean of 0.6± 0.04 for fetal hemoglobin and 3.7±0.04 for HbA2 The effects of rs368698783 A allele on HbF levels examined in the total population and furthermore in the two sub populations of (i) normal healthy adults and (ii) adults with borderline levels of HbF/HbA2 showed that the mutant A allele of HBG1-rs368698783 exhibited a significantly elevated level of HbF (p < 0.05) compared with the wildtype G allele in each of the cohorts from Malta. Intriguingly no association was determined between this point mutation and HbA2 levels. The data is suggestive that the variant HBG1-rs368698783 could act as a modifier associated with elevated levels of HbF in human adults. The SNP is located within the LYAR binding motif. The LYAR is a transcriptive factor which is said to silence the expression of fetal hemoglobin. thus, the G to A polymorphism reduces the DNA binding affinity increasing HbF levels invivo. Figure Disclosures No relevant conflicts of interest to declare.

DNA Breaks Induction by Mimosine

2003 ◽  
Vol 58 (9-10) ◽  
pp. 732-735 ◽  
Author(s):  
Emil Mladenov ◽  
Boyka Anachkova
Keyword(s):  
Nuclear Matrix ◽  
Gel Electrophoresis ◽  
Average Length ◽  
Agarose Gel ◽  
Dna Fragments ◽  
Agarose Gel Electrophoresis ◽  
Chromosomal Dna ◽  
Dna Breaks ◽  
Random Size ◽  
Random Fashion

AbstractMouse erythroleukemic F4 N cells were treated with mimosine, etoposide, Fe(II)-EDTA, and Cu(II) in the presence of ascorbate. DNA was isolated and subjected to agarose gel electrophoresis and the size and distribution of the DNA fragments produced by the agents were compared. With increasing concentration of Cu(II) the production of DNA fragments was increased without decrease of the average length of the fragments, and their sizes were similar to those produced by etoposide as expected for cleavage of DNA at the nuclear matrix attachments sites. In contrast, mimosine and Fe(II) produced fragments of random size and with the progression of the reaction the average length of the fragments decreased. These results indicate that mimosine cuts DNA in a random fashion, regardless of its higher order chromatin organization. A conclusion is drawn that the DNA fragments obtained after mimosine treatment are a result of mimosine-assisted, Fe(II) dependent Fenton-like reactions randomly cutting chromosomal DNA.

Luminography – an Alternative Assay for Detection of von Willebrand Factor Multimers

1988 ◽  
Vol 60 (02) ◽  
pp. 133-136 ◽  
Author(s):  
R Schneppenheim ◽  
H Plendl ◽  
U Budde
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Detection Methods ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Luminescence Assay ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA luminescence assay was adapted for detection of von Willebrand factor multimers subsequent to SDS-agarose gel electrophoresis and electroblotting onto nitrocellulose. The method is as fast as chromogenic detection methods and appears to be as sensitive as autoradiography without the disadvantages of the latter.

Identification of Fibrinogen Derivatives in Plasma Samples

1972 ◽  
Vol 27 (03) ◽  
pp. 610-618 ◽  
Author(s):  
H Graeff ◽  
R von Hugo
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Gel Chromatography ◽  
Agarose Gel ◽  
Plasma Samples ◽  
Methodological Study ◽  
Parent Molecule ◽  
Electrophoretic Mobilities ◽  
Fibrin Monomer

SummaryThe observation of fibrinogen derivatives with a molecular weight higher than the parent molecule in human cases of DIC initiated the present methodological study. These derivatives were identified by the following methods : 2.5 M β-alanine precipitation of the plasma samples, PAA gel electrophoresis, intra gel immunoprecipitation and agarose gel chromatography. In the plasma of a patient with severe eclampsia and laboratory signs of DIC two derivatives with a molecular weight higher than that of fibrinogen were identified according to their relative electrophoretic mobilities: 0.18 and 0.28 × 10−5 cm2/V × sec (fibrinogen: 0.43 × 10−5 cm2/V × sec). Electrophoretic studies in the presence of 5 M urea indicated that the 0.28 derivative is a complex probably formed by fibrinogen and a fibrin monomer.

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