Agarose Gel Electrophoresis Patterns Research Articles

Agarose gel electrophoresis pattern of serum creatine kinase and lactate dehydrogenase isoenzymes in zoo-managed Asian elephants (Elephas maximus).

Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH) isoenzymes were evaluated in nine zoo-managed Asian elephants (Elephas maximus) using a commercial agarose gel electrophoresis (AGE) kit. CK was separated into two major fractions, CK-BB and CK-MM, along with a small fraction of macroenzyme-CK type 2 (mCK2); CK-MM was the largest fraction. LDH was separated into five fractions (LDH1-5); LDH3 was the largest fraction. Age was negatively and positively correlated with the percentages of CK-BB and CK-MM, respectively, and negatively correlated with CK-BB and mCK2 activities. These results indicate that an AGE kit can be used to evaluate CK and LDH isoenzymes. Routine isoenzyme testing may enable early detection of disease and physiological changes.

Agarose gel electrophoresis pattern of serum alkaline phosphatase isoenzymes in Holstein cows during lactation.

A recent study found that an agarose gel electrophoresis (AGE) method yielded two distinct major bands corresponding to the hepatic and bone ALP isoenzymes (ALP2 and ALP3, respec-tively) in bovine serum treated with protease and neuraminidase (PN-treatment), although there were concerns that the intestinal ALP isoenzyme (ALP5) often overlapped with ALP3 in human serum treated with neuraminidase. Because ALP5 was separated from ALP3 in bovine serum treated with protease alone (P-treatment), we used a modified method employing both P- and PN-treated bovine sera to measure the activities of the three ALP isoenzymes in 53 lacta-ting Holstein cows: 24 primiparous and 29 multiparous. Upon electrophoresis, 51 of 53 samples (96.2%) subjected to P-treatment yielded a distinct fraction corresponding to ALP5, as did the control serum. All PN-treated sera yielded a definite ALP2 fraction. The ALP3 fraction was calculated as the remainder after excluding ALP2 and ALP5. The activities of total ALP (t-ALP) and ALP3 in primiparous cows were higher than those in multiparous cows (p ⟨ 0.001) at early-to-peak [10-110 days in milk (DIM)] and mid (111-220 DIM) lactation. In the multi-parous cows, the ALP3 activity at late lactation (221-477 DIM) was significantly higher than that at early-to-peak lactation. Thus, the modified AGE method described here is able to discrimi-nate three fractions of ALP isoenzymes in the sera of lactating cows. The AGE pattern of circu-lating ALP isoenzymes will contribute to the understanding of the physiological bone metabolism status in lactating cows.

Evidence of altered DNA integrity in the brain regions of suicidal victims of Bipolar Depression

Deoxyribonucleic acid (DNA) integrity plays a significant role in cell function. There are limited studies with regard to the role of DNA damage in bipolar affective disorder (BP). In the present study, we have assessed DNA integrity, conformation, and stability in the brain region of bipolar depression (BD) patients (n=10) compared to age-matched controls (n=8). Genomic DNA was isolated from 10 postmortem BD patients’ brain regions (frontal cortex, Pons, medulla, thalamus, cerebellum, hypothalamus, Parietal, temporal, occipital lobe, and hippocampus) and from the age-matched control subjects. DNA from the frontal cortex, pons, medulla, and thalamus showed significantly higher number of strand breaks in BD (P<0.01) compared to the age-matched controls. However, DNA from the hippocampus region was intact and did not show any strand breaks. The stability studies also indicated that the melting temperature and ethidium bromide binding pattern were altered in the DNA of BD patients’ brain regions, except in the hippocampus. The conformation studies showed B-A or secondary B-DNA conformation (instead of the normal B-DNA) in BD patients’ brain regions, with the exception of the hippocampus. The levels of redox metals such as Copper (Cu) and Iron (Fe) were significantly elevated in the brain regions of the sufferers of BD, while the Zinc (Zn) level was decreased. In the hippocampus, there was no change in the Fe or Cu levels, whereas, the Zn level was elevated. There was a clear correlation between Cu and Fe levels versus strand breaks in the brain regions of the BD. To date, as far as we are aware, this is a new comprehensive database on stability and conformations of DNA in different brain regions of patients affected with BD. The biological significance of these findings is discussed here.

A case of Benign familial hyperphosphatasemia of intestinal origin

We recently encountered a case of hyperphosphatasemia, in which >90% of serum alkaline phosphatase (ALP) was of intestinal origin. The patient, a 51-year old man, was found to have hyperphosphotasemia (2,3421 U/L) during a routine medical check-up. All other laboratory tests and physical findings were normal. The agarose gel electrophoresis pattern of the patient's serum ALP was identical to that of common intestinal ALP from healthy adults, and only a single band of intestinal ALP was detected by immunoaffinity electrophoresis. In addition, 89% of total ALP was defined as intestinal ALP by an immunoprecipitation method. The molecular mass of the ALP was 154 kDa, almost identical with that of adult duodenal ALP. Analysis of the sugar chain structure showed an increased la fraction (74%) compared with adult duodenal ALP. Genealogical study revealed that two persons in the 5 members of the proband's family had hyperphosphatasemia of intestinal origin, indicating possible autosomal dominant inheritance.

Comparative studies on the action of 7-N-[2-[[2-(γ-l-glutamylamino)ethyl]dithio]ethyl]mitomycin C and of mitomycin C on cultured HL-60 cells and isolated phage and plasmid DNA

The mechanism of action of a new mitomycin C (MMC) derivative, KT6149, was studied in human leukemia HL-60 cells and in isolated phage and plasmid DNA, and its effects were compared with those of MMC. Cell growth was markedly inhibited by KT6149, with an IC50 of 2 x 10(-9) M, that for MMC being 2 x 10(-8) M. DNA synthesis of HL-60 cells as determined by incorporation of [3H]-thymidine was also inhibited by KT6149, with an IC50 of 2 x 10(-7) M as compared with 2 x 10(-6) M for MMC. RNA and protein synthesis were less markedly inhibited at low concentrations. Alkaline sucrose density-gradient centrifugation revealed a significant decrease in sedimentation velocity for cellular DNA of the cells after 1 h treatment with KT6149 at concentrations higher than 10(-7) M. In contrast, no such change was observed for DNA of cells treated with MMC, even at a concentration of 10(-5) M. In a cell-free system, analysis by agarose gel electrophoresis patterns showed that the drug induced a decrease in the amount of covalently closed circular DNA of phage PM2 and an increase in that of open circular DNA in the presence of dithiothreitol (DTT), whereas MMC did not cause any change in DNA subfraction amounts. Furthermore, the electrophoretic mobility of linearized pBR322 DNA in alkaline agarose gel was significantly decreased by KT6149 in the presence of DTT and FeSO4, no such change being observed in the case of MMC. The results clearly indicate that the inhibitory effects of KT6149 on the growth and DNA synthesis of HL-60 cells are more potent than those of MMC and that KT6149-induced DNA damage is due to single-strand scission and to cross-linking of DNA, suggesting a mode of activation different from that of MMC.

Nick translation studies on DNA strand breaks in pBR322 plasmid induced by different chromium species

Single strand breaks are DNA defects caused by various chemicals. DNA strand breaks induced in vitro by chromium(VI) during the reduction of this chromium species with glutathione or hydrogen peroxide were examined. Using DNA agarose gel electrophoresis and a nick translation assay, strand breaks were detected only when chromium(VI) was reduced by hydrogen peroxide. The reduction of chromium(VI) by an excess of glutathione led to no alteration in the DNA agarose gel electrophoresis pattern of the double-stranded plasmid pBR322 DNA and in the nick translation assay, indicating that no strand breaks had occurred under these conditions. No strand breaks could be detected during the reduction by hydrogen peroxide after the addition of superoxide dismutase. This indicates that hydroxyl radicals from peroxochromium complexes may be a relevant reactive species involved in chromate genotoxicity.

KM1, a Bacteriophage of Clostridium butyricum

Summary: Characteristics of bacteriophage KM1, which infects Clostridium butyricum, are described, and several important properties are compared with those of phage 5, which infects the same species. Phage KM1 has a hexagonal head and a tail with a contractile sheath. Agarose gel electrophoresis patterns of BglII, EcoRI and HindIII fragments of the two phage DNAs differed greatly. The G+C contents were 30·5 mol% for phage KM1 and 35·4 mol% for phage 5. Among the 32 strains of clostridia tested, phage KM1 infected only C. butyricum MII588. Other properties of phage KM1 are also discussed.

Studies of the pathophysiology of acquired von Willebrand's disease in seven patients with lymphoproliferative disorders or benign monoclonal gammopathies

In seven patients with acquired von Willebrand's disease (AvWD) associated with lymphoproliferative disorders or benign monoclonal gammopathies, the platelet contents of von Willebrand factor antigen and ristocetin cofactor (vWF:Ag and vWF:RiCof, respectively) were normal. All the multimers of vWF:Ag could be seen in the 1.6% SDS- agarose gel electrophoresis patterns of plasma and platelet lysates. Infusion of 1-deamino-8-D-arginine vasopressin (DDAVP) augmented plasma levels of vWF:Ag and vWF:RiCof of all patients and corrected prolonged bleeding times (BT). However, compared with patients with congenital vWD type I and comparable degrees of baseline abnormalities treated in the same way, vWF:Ag and vWF:RiCof were increased less and cleared more rapidly from plasma and the BT remained normal for a shorter period of time. These studies provide evidence that these AvWD patients have qualitatively normal vWF in plasma, but at lower concentrations, that vWF in platelets is normal both qualitatively and quantitatively, and that cellular vWF can be rapidly released into plasma by DDAVP to correct the hemostatic abnormalities. However, vWF is removed rapidly from plasma, making the correction more transient than in congenital vWD type I.

An aberrant adenylate kinase isoenzyme from the serum of patients with Duchenne muscular dystrophy

The sera from patients with human Duchenne (X-linked) progressive muscular dystrophy contain elevated adenylate kinase (ATP: AMP phosphotransferase, EC 2.7.4.3) activities, in addition to their characteristically high creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) activities. By agarose gel electrophoresis of human Duchenne dystrophic serum, the presence of an apparently normal human serum adenylate kinase together with a variant species of adenylate kinase was detected. The latter enzyme species appeared, in its mobility, to be similar to that of the normal human liver-type adenylate kinase. The presence of this aberrant liver-type adenylate kinase could also be demonstrated by characteristic (for the liver type) inhibition patterns with P 1, P 5-di-(denosine)-5′)pentaphosphate, 5,5′-dithiobis(2-nitrobenzoate) and phospho enolpyruvate. On the other hand, by inhibition titrations with an anti-muscle-type adenylate kinase, hemolysates from the erythrocytes of several Duchenne and Becker's dystrophics were found to contain approx. 96% muscle-type adenylate kinase and their serum approx. 97% muscle-type adenylate kinase. These same patients contained approx. 89% M-M type creatine kinase in their serum (by inhibition against anti-human muscle-type creatine kinase) indicative of the presence also of M-B plus B-B type active isoenzymes. All of these data can best be explained by the presence of a variant or mutant adenylate kinase isoenzyme in the dystrophic serum. This isoenzyme appears to resemble the liver type in its inhibition patterns with P 1, P 5-di(adenosine-5′)pentaphosphate, 5,5′dithiobis(2-nitrobenzoate) and phospho enolpyruvate, and in its heat stability (compare also the agarose gel electrophoresis pattern); but structurally, it is a muscle type, or derived from a muscle type, as shown immunologically by inhibition reactions with anti-muscle-type adenylate kinase. Whether this is a fetal-type isoenzyme of adenylate kinase will require further investigation.

Characterization of the Serum High Density Lipoproteins and Apolipoproteins of Pink Salmon

Abstract High density lipoproteins (HDL) were isolated by centrifugation from the serum of salmon (Oncorhynchus gorbuscha) captured just before spawning. There were no detectable low density lipoproteins in their serum. Analytical ultracentrifugation of the lipoprotein fraction yielded only one band with a hydrated density of 1.103 g per ml and a peak F1.20 rate of 3.21 S. If the molecule is spherical, it would have minimum molecular weight of 180,000 and an average diameter of 80 A. The agarose gel electrophoresis pattern had one major and one minor band with the approximate mobility of human α-lipoprotein. The lipid class composition was very similar to that reported for human HDL. The phospholipids were mainly phosphatidylcholine (80%), its lysoderivative, and sphingomyelin. The major fatty acids were 16:0, 18:0, 18:1 (n-9), 20:5 (n-3), and 22:6 (n-3). Lipids accounted for 60% of the mass of the molecule. Disc electrophoresis in polyacrylamide gels (in 8 m urea at pH 8.6) produced a pattern with two major and several minor bands for both the intact lipoproteins and the lipid-free protein moiety. The over-all pattern was very similar to that of human HDL. The apoproteins from the salmon HDL were eluted from diethylaminoethylcellulose columns under conditions similar to those used for elution of apoproteins from human HDL; they also gave similar elution patterns. Their circular dichroism spectra resembled those of the corresponding human apolipoproteins. Despite all of these physical similarities, there were distinct and major differences between the amino acid compositions of salmon apoproteins and their human counterparts. These data suggest that general physical properties of the HDL are not specifically dependent on the total amino acid composition of the molecule but rather on its general secondary or tertiary structure and these same features must govern the binding of lipids to proteins in soluble lipoproteins.

The lipid composition of plasma lipoprotein density classes of sheep Ovis aries

1.1. The lipoproteins of sheep plasma were separated by preparative ultracentrifugation into five different classes based on hydrated density.2.2. Agarose gel electrophoresis patterns of the sheep lipoprotein classes and whole plasma were obtained.3.3. No pre-beta band (VLDL) was found in sheep plasma; beta (LDL) was present along with two alpha (HDL) bands.4.4. The lipids associated with the various lipoprotein classes did not vary appreciably with density except that the fraction with ρ>1·20 g/ml contained most of the free fatty acids and lysophosphatidyl choline present in sheep plasma.5.5. Cholesterol ester was the major neutral lipid in the LDL and HDL fractions, while phosphatidyl choline was the major phospholipid in the HDL, and equal amounts of sphingomyelin and phosphatidyl choline were present in the LDL fraction.6.6. The absence of VLDL and chylomicrons in sheep plasma suggest that sheep absorb dietary fat differently than do humans.

Biochemical Profile of Multiple Myeloma in the Patients Visiting BPKIHS as Diagnosed by Agarose Gel Electrophoresis

Multiple myeloma is a proliferative disease of plasma cells. The incidence of the disease increases with age.Objectives : To study different biochemical parameters and serum agarose gel electrophoresis patterns of patients consistent with clinical symptoms of multiple myeloma.Subject and Methods : A retrospective study was carried out in Department of Biochemistry, B.P. Koirala Institute of Health Sciences, from October 2008 to September 2009. Patients consistent with the symptoms of multiple myeloma were selected for the study. Alkaline phosphatase, total calcium, albumin and globulin levels were estimated. Bence Jones Protein, cell counts of bone marrow and x-ray of skull was carried out. Agarose gel electrophoresis was performed at buffer pH 8.6, 5 mili ampere current and the constant voltage of 200 volts was applied.Results : Serum electrophoresis patterns in all the patients showed M-Protein band (M-band) at gamma-region. Serum alkaline phosphatase levels were 216.0 ± 35.7 . Serum total calcium levels were 10.0± 1.5, serum albumin levels were 4.1 ± 0.5 and serum globulin levels were 3.7 ± 0.9 respectively. Bence Jones Protein was present in urine of the 7 patients out of 14. Plasma cell counts were more than 10% and lytic lesions on skull were present.Conclusion : Patients with multiple myeloma showed consistently increased total calcium, decreased serum albumin and normal alkalline phosphatase levels. Electrophoretic patterns showed M-band in all the patients, giving the confirmation of the diagnosis. Though a conventional technique, electrophoresis remains as the gold standard for the diagnosis of multiple myeloma.Health Renaissance, May-Aug 2010; Vol 8 (No.2):122-125