Discontinuous Buffer System Research Articles

Molecular and Functional Analyses of a Novel Class I Secretory Nuclease from the Human Pathogen, Leishmania donovani

The primitive protozoan pathogen of humans, Leishmania donovani, resides and multiplies in highly restricted micro-environments within their hosts (i.e. as promastigotes in the gut lumen of their sandfly vectors and as amastigotes in the phagolysosomal compartments of infected mammalian macrophages). Like other trypanosomatid parasites, they are purine auxotrophs (i.e. lack the ability to synthesize purines de novo) and therefore are totally dependent upon salvaging these essential nutrients from their hosts. In that context, in this study we identified a unique 35-kDa, dithiothreitol-sensitive nuclease and showed that it was constitutively released/secreted by both promastigote and amastigote developmental forms of this parasite. By using several different molecular approaches, we identified and characterized the structure of LdNuc(s), a gene that encodes this new 35-kDa class I nuclease family member in these organisms. Homologous episomal expression of an epitope-tagged LdNuc(s) chimeric construct was used in conjunction with an anti-LdNuc(s) peptide antibody to delineate the functional and biochemical properties of this unique 35-kDa parasite released/secreted enzyme. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that this "secretory" enzyme could hydrolyze a variety of synthetic polynucleotides as well as several natural nucleic acid substrates, including RNA and single- and double-stranded DNA. Based on these cumulative observations, we hypothesize that within the micro-environments of its host, this leishmanial "secretory" nuclease could function at a distance away from the parasite to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, this enzyme might play an important role(s) in facilitating the survival, growth, and development of this important human pathogen.

Group VIA Calcium-independent Phospholipase A2 Mediates Endothelial Cell S Phase Progression

Arachidonic acid and its metabolites have been previously implicated in the regulation of endothelial cell proliferation. Arachidonic acid may be liberated from cellular phospholipids by the action of group VIA calcium-independent phospholipase A2 (iPLA2-VIA). Consequently, we tested the hypothesis that iPLA2-VIA activity is linked to the regulation of endothelial cell proliferation. Inhibition of iPLA2 activity by bromoenol lactone (BEL) was sufficient to entirely block endothelial cell growth. BEL dose-dependently inhibited endothelial cell DNA synthesis in a manner that was reversed upon the exogenous addition of arachidonic acid. DNA synthesis was inhibited by the S-isomer and not by the R-isomer of BEL, demonstrating that endothelial cell proliferation is mediated specifically by iPLA2-VIA. iPLA2-VIA activity was critical to the progression of endothelial cells through S phase and is required for the expression of the cyclin A/cdk2 complex. Thus, inhibition of iPLA2-VIA blocks S phase progression and results in exit from the cell cycle. Inhibition of iPLA2-VIA-mediated endothelial cell proliferation is sufficient to block angiogenic tubule formation in co-culture assays. Consequently, iPLA2-VIA is a novel regulator of endothelial cell S phase progression, cell cycle residence, and angiogenesis.

Discontinuous native protein gel electrophoresis

Analysis of the oligomeric state of a native protein usually requires analytical ultracentrifugation or repeated gel filtration to calculate the protein's size. We have developed a discontinuous native protein gel electrophoresis system that allows the separation of even basic proteins according to their size, oligomeric state, and shape. This gel system combines the addition of negative charges to the proteins by Serva Blue G with a discontinuous buffer system and gradient gels. As in SDS-PAGE, chloride constitutes the high mobility anion in the gel and anode buffer. However, for sample focusing this system employs histidine instead of glycine as the slow dipolar ion following from the cathode buffer to improve migration of basic proteins. In addition, proteins run into gel pores corresponding to their size and shape in the gradient gel. Using this gel system, we show that the polypyrimidine tract-binding protein (PTB) is a monomer.

Phosphorylation of Ser-129 Is the Dominant Pathological Modification of α-Synuclein in Familial and Sporadic Lewy Body Disease

A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two-dimensional immunoblot analysis, novel sandwich enzyme-linked immunosorbent assays with modification-specific synuclein antibodies, and mass spectroscopy. The predominant modification of alpha-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N-terminal acetylation. Small amounts of Ser-129 phosphorylated and Asp-119-truncated alpha-synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of alpha-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent-insoluble fraction of brain from patients with familial Parkinson's disease (synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser-129 phosphorylated alpha-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.

Reversed‐phase high‐performance liquid chromatographic, gel electrophoretic and size exclusion chromatographic studies of subunit structure of arachin and its molecular species

Arachin and its molecular species (arachin I and arachin II) were separated and isolated. The number and kind of subunits of arachin, arachin I and arachin II were determined. Studies were carried out under different experimental conditions using slab gel electrophoresis, size-exclusion chromatography and reversed-phase high-performance liquid chromatography. Gel electrophoresis was done under varying concentrations of resolving gel. Tube gel as well as slab gel electrophoresis were used and continuous as well as discontinuous buffer systems were used for both types of electrophoresis. In addition, the subunits were separated by reversed-phase HPLC using a gradient program. Arachin and arachin II were found to have 12 subunits each while arachin I showed six subunits. The subunits of arachin I were allowed to reconstitute by removing SDS. Eight combinations were tried for studying the reconstitution pattern. Molecular weight and weight ratio in each case were also determined.

A Unique ATP Hydrolysis Mechanism of Single-headed Processive Myosin, Myosin IX

Recent studies have revealed that myosin IX is a single-headed processive myosin, yet it is unclear how myosin IX can achieve the processive movement. Here we studied the mechanism of ATP hydrolysis cycle of actomyosin IXb. We found that myosin IXb has a rate-limiting ATP hydrolysis step unlike other known myosins, thus populating the prehydrolysis intermediate (M.ATP). M.ATP has a high affinity for actin, and, unlike other myosins, the dissociation of M.ATP from actin was extremely slow, thus preventing myosin from dissociating away from actin. The ADP dissociation step was 10-fold faster than the overall ATP hydrolysis cycle rate and thus not rate-limiting. We propose the following model for single-headed processive myosin. Upon the formation of the M.ATP intermediate, the tight binding of actomyosin IX at the interface is weakened. However, the head is kept in close proximity to actin due to the tethering role of loop 2/large unique insertion of myosin IX. There is enough freedom for the myosin head to find the next location of the binding site along with the actin filament before complete dissociation from the filament. After ATP hydrolysis, Pi is quickly released to form a strong actin binding form, and a power stroke takes place.

Allele Frequencies for Two STR Loci GATA31E08 and DXS6809 in Chinese Population

Abstract Blood samples were collected from unrelated individuals of Chinese Han ethnic group in Chengdu of China. DNAwas extracted using Chelex method (1). PCR amplification conditions can be accessed at http://www.legalmed.org/dna/DXS6809.htm (2). The volume of PCR reaction for each locus was 37.5 µL. The PCR products were analyzed by horizontal non-denaturing polyacrylamide gel electrophoresis with discontinuous buffer system and visualized by sliver staining (2,3). Data were analyzed using POWERSTATS program (4).

Concentration and Isolation of DNA from Biological Fluids by Agarose Gel Isotachophoresis

Isotachophoresis is an electrophoretic method of separation of charged substances. The method is characterized by a discontinuous buffer system, constant velocity of separated molecules, and the distribution of separated components in the form of narrow concentrated bands located one right after another. As a rule, isotachophoresis is not used for the separation of nucleic acids because the mobility of polynucleotides in this system does not depend on their size. However, this circumstance proved to be very useful for the quantitative isolation of heterogeneous DNA fragments from biological fluids, for gene diagnostics of cancer in particular. The proposed method of agarose gel isotachophoresis of DNA has been used for the isolation of blood DNA and its successful PCR analysis.

Extracellular Cleavage of E-Cadherin by Leukocyte Elastase During Acute Experimental Pancreatitis in Rats

Cadherins play an important role in cell-cell contact formation at adherens junctions. During the course of acute pancreatitis, adherens junctions are known to dissociate-a requirement for the interstitial accumulation of fluid and inflammatory cells-but the underlying mechanism is unknown. Acute pancreatitis was induced in rats by supramaximal cerulein infusion. The pancreas and lungs were either homogenized for protein analysis or fixed for morphology. Protein sequencing was used to identify proteolytic cleavage sites and freshly prepared acini for ex vivo studies with recombinant proteases. Results were confirmed in vivo by treating experimental pancreatitis animals with specific protease inhibitors. A 15-kilodalton smaller variant of E-cadherin was detected in the pancreas within 60 minutes of pancreatitis, was found to be the product of E-cadherin cleavage at amino acid 394 in the extracellular domain that controls cell-contact formation, and was consistent with E-cadherin cleavage by leukocyte elastase. Employing cell culture and ex vivo acini leukocyte elastase was confirmed to cleave E-cadherin at the identified position, followed by dissociation of cell contacts and the internalization of cleaved E-cadherin to the cytosol. Inhibition of leukocyte elastase in vivo prevented E-cadherin cleavage during pancreatitis and reduced leukocyte transmigration into the pancreas. These data provide evidence that polymorphonuclear leukocyte elastase is involved in, and required for, the dissociation of cell-cell contacts at adherens junctions, the extracellular cleavage of E-cadherin, and, ultimately, the transmigration of leukocytes into the epithelial tissue during the initial phase of experimental pancreatitis.

Population Genetics of three STR Loci D19S591, D5S1462 and D3S2432 in a Chinese Han Population

Blood samples were collected from unrelated healthy individuals of Chinese Han ethnic group in Chengdu of Sichuan, China after informed consent. Genomic DNA samples were extracted using Chelex-100 method (1). PCR amplification conditions can be accessed at http://www.legalmed.org/dna/D19S591.htm., http:// www. legalmed.org/dna/D5S1462.htm. and http://www.legalmed. org/ dna/D3S2432.htm. The volume of PCR reaction for each locus was 37.5μL. The PCR products were analyzed by horizontal nondenaturing polyacrylamide gel electrophoresis with discontinuous buffer system and visualized by silver staining (2). Data of population genetics and forensic science of the locus D19S591, D5S1462, D3S2432 was analyzed using POWERSTATS program (3). The

An efficient proteomics-based approach for the screening of autoantibodies

This study presents an improved method for the complete transfer of proteins separated by two-dimensional gel electrophoresis to a membrane, specifically designed for the screening and identification of antigens recognized by autoantibodies in patients with breast cancer (BCP) and healthy volunteers. This paper reports the evaluation of this technique using proteins from MCF7 as a source of antigens following 2-DE separation. The appropriate quantity of protein to be loaded on gels (150 μg) has been determined, the aim being a complete and reproducible recovery of all separated proteins onto the polyvinylidene fluoride membrane (2D-blot) after a semi-dry electrotransfer. Several different transfer methods were tested in parallel, resulting in the selection and optimisation of one using a discontinuous buffer system, based on the isotachophoresis theory. To facilitate the comparative analysis of the different sets of 2D-blots probed with individual sera from BCP and healthy volunteers, the 2D-blots were stained with colloidal gold following the immunodetection step. The gels and 2D-blots were scanned and analysed by imaging software. The matching permitted exact localisation of particular relevant protein spots hybridised by antibodies on the 2D-blots. These spots were subsequently located on preparative gels for identification by mass spectrometry. A set of 40 2D-blots was probed with 20 sera from patients with breast cancer and 20 sera from healthy volunteers. In the protein profiles submitted to immunodetection, 15 proteins were repeatedly immunodetected by both BCP and sera from healthy people. Those proteins were identified by mass spectrometry. Conversely, some protein isoforms were preferentially immunodetected by BCP sera and may reflect the presence of this cancer. The improved isotachophoretic method described in this study is suitable for comparing the overall profile of autoimmunity between different populations and for subsequent identification of relevant antigens.

Population Genetics of Two STR Loci D13S796 and DYS439 in a Chinese Han Population

Blood samples were collected from unrelated healthy individuals ofChineseHan ethnic group inChengdu of Sichuan.GenomicDNA samples were extracted using Chelex-100method (1). PCR amplification conditions can be accessed at http://www.legalmed.org/dna/ D13S796 and DYS439.htm. The volume of PCR reaction for each locus was 37.5 μL. The PCR products were analyzed by horizontal non-denaturing polyacrylamide gel electrophoresis with discontinuous buffer system and visualized by sliver staining (2). Data of population genetics and forensic science of the locus D13S796 was analyzed using POWERSTATS program (3). Data of population

Polymorphism of Three STR Loci in Chinese Population

Blood samples were collected from unrelated individuals of Chinese Han ethnic group in Chengdu of China. DNA was extracted using Chelex method (1). PCR amplification conditions can be accessed at http://www.legalmed.org/dna/D12S1064.htm. The volume of PCR reaction for each locus was 37.5 μL. The PCR products were analyzed by horizontal non-denaturing polyacrylamide gel electrophoresis with discontinuous buffer system and visualized by silver staining (2). Data of population genetics and

325 ACTIVITY OF MATURATION-PROMOTING FACTOR (MPF) AND MITOGEN-ACTIVATED PROTEIN KINASES DURING IN VITRO MATURATION OF BUFFALO OOCYTES (BUBALUS BUBALIS)

The maturation promoting factor (MPF) and mitogen-activated protein kinases (MAPK) are the key regulators of both meiotic and mitotic cell cycles. The absence of data on the activity of the major cell cycle kinases in buffalo oocytes during meiotic progression provided the bases for this study. More specifically we assayed the MPF and MAP kinase activity of buffalo oocytes during meiosis. Abattoir-derived cumulus-oocyte complexes (COCs) with a compact, non-atretic cumulus and a homogeneous cytoplasm were utilized for the study. The COCs (n = 293, over four replicates) were matured in vitro in TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 0.5 �g/mL FSH, 5 �g/mL LH, 1 �g/mL 17�-estradiol, 50 �M of cysteamine, and 50 �g/mL kanamycin (B199). In vitro maturation (IVM) was carried out at 38.5�C under a controlled gas atmosphere of 5% CO2 in humidified air. At scale times during the culture (0, 3, 6, 9, 12, 15, 18, 21, 24 h) groups of oocytes were stained with Hoechst 33342 to assess chromatin configuration and stored according to the maturation stage (GV, GVDB, MI, and MII) at -80�C pending protein analysis. SDS-polyacrylamide gel electrophoresis wase performed using Laemmli discontinuous buffer system (Laemmli 1970 Nature 227, 680) with a 12% running gel. Groups of oocytes were analyzed for MPF activity (n = 65) by histone H1 kinase activity (Naito and Toyoda 1991 J. Reprod. Fertil. 93, 467-473) and for MAPK activity (n = 48) by myelin basic protein assays (Chesnel et al. 1995 Biol. Reprod. 52, 895-902). The activity of both MPF and MAP kinases was quantified by measuring the density of the bands on the autoradiographic film with a densitometer. Differences in the levels of the kinases among groups were analyzed by ANOVA. It was assumed that the value of MPF and MAPK was 100% in metaphase II (MII) stage oocytes. The lowest levels of MPF and MAPK activities were found in the oocytes at the GV (0-6 h post-IVM: 40% and 17.2%, respectively) and at the GVBD (6-9 h post-IVM: 41.2% and 18%) stages. The activities increased at metaphase I (MI) stage (9-15 h post-IVM) and at MII (21-24 post-IVM). Interestingly, although similar levels of MAP kinases were found at MI and MII stages (95.1% vs. 100%), MPF levels were significantly lower (P < 0.01) at the MI stage compared to those detected at MII (82.8% vs. 100%). The fluctuations of the MPF levels in buffalo appear different compared to those observed in other species; in particular, no differences were recorded between the GV and the GVBD stages whereas a significant increase of the MPF levels was found at MII compared to the MI stage. It seems that MPF and MAPK could differently guide meiotic resumption and progression to the MII arrest in this species. To our knowledge, this is the first report on biochemical analysis of the cell cycle regulation in buffalo oocytes.

Over 1000-fold protein preconcentration for microliter-volume samples at a pH junction using capillary electrophoresis

An effective protein preconcentration technique specifically designed for microliter-volume samples is presented. The preconcentration is based on the capturing of protein in its isoelectric point (p I) within an applied electric field, using a pH junction created by a discontinuous buffer system. The buffers were chosen to selectively preconcentrate proteins of neutral p I, myoglobin in this case, while removing other proteins with acidic or basic p Is. For the suppression of electro-osmotic flow (EOF) and protein adsorption, the capillary inner wall was modified with a zwitterionic phospholipid bilayer coating. A preconcentration factor of up to 1700 was obtained for a 1 μg/mL solution of myoglobin. The preconcentration was completed in approximately 20 min. Several sample introduction conditions were presented to accommodate sample volume from one to a few hundreds of microliters. The final volume of the preconcentrated sample band was estimated to be approximately 5 nL.

Polymorphism Data of Two STR Loci DYS632 and DYS634 in a Chinese Han Population

Blood samples were collected from 100 unrelated healthy males of the Chinese Han ethnic group in Chengdu of China. DNA was extracted using the Chelex method (1). The allelic variation at the two Y-STR loci named as DYS632 and DYS634 were analyzed by PCR amplification system. PCR amplification conditions can be accessed at: http://www.legalmed.org/dna/DYS632.htm. The volume of PCR reaction for each locus was 37.5 μL. The PCR products were analyzed by horizontal non-denaturing polyacrylamide gel electrophoresis with discontinuous buffer system and visualized by silver staining (2). Alleles were designated according to the recommendation of the International Society of Forensic Genetics (3). The gene diversity, the haplotype diversity, and the standard errors of diversity were calculated in accordance with Hou’s method (4). The complete data can be accessed at: http://www.legalmed. org/dna/DYS632.htm.

Phytophthora cinnamomi

<i>Phytophthora cinnamomi</i>

Characterization and haplotype analysis of 10 novel Y-STR loci in Chinese Han population

In this study, we analyzed allelic sequences of 10 novel Y-specific STR loci, DYS454, DYS510, DYS513, DYS520, DYS542, DYS544, DYS552, DYS561, DYS587 and DYS593, surveyed the distribution of haplotypes in a Chinese Han population. Extracted DNA was amplified with PCR, followed by a horizontal non-denaturing polyacrylamide gel electrophoresis with discontinuous buffer system. Purified alleles were sequenced on DNA sequencer (ABI Model 377) to verify the number of motif repeats. The number of alleles observed at each locus ranged from 3 to 8, yielding 102 haplotypes in 103 unrelated males samples. The allele diversity values for each locus ranged from 0.2099 (DYS544) to 0.7523 (DYS552). The haplotype diversity using all these loci was 0.9998. Our study revealed that they were valuable Y-specific markers for forensic applications.

High-salt stacking principles and sweeping: comments and contrasts on mechanisms for high-sensitivity analysis in capillary electrophoresis

High-salt stacking in electrokinetic chromatography (EKC) is defined and contrasted to the sweeping method. A recent paper argued the two methods are identical, where high concentrations of micelle in the sample were intended to mimic the effect of high-salt stacking. However, high micelle concentration in the sample matrix in EKC is analogous to using a high-conductivity sample instead of a low-conductivity sample in field amplified stacking. High-salt stacking does not require a sample free of pseuostationary phase, only a sample with a high-mobility co-ion compared to the separation buffer electrokinetic vector. High-salt stacking uses a discontinuous buffer system and should not be confused with continuous buffer stacking systems such as sweeping.

Discontinuous buffer systems operative at pH 2.5 - 11.0, 0 degrees C and 25 degrees C, available on the Internet.

This communication is an announcement of the availability on the Internet of a computer output of discontinuous buffer systems operative at pH 2.5-11.0, 0 degrees C and 25 degrees C, generated by the theory and program of T. M. Jovin. The output and instructions for its use can be accessed under http://www.buffers.nichd.nih.gov.