bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved electrophoretic separation and immunoblotting of beta‐amyloid (Aβ) peptides 1–40, 1–42, and 1–43
Authors: Wiltfang Jens , Smirnov Alexandr , Schnierstein Beate , Kelemen Georg , Matthies Uta , Klafki Hans‐Wolfgang , Staufenbiel Matthias , Hüther Gerald , Rüther Eckhardt , Kornhuber Johannes
Published: May 2005

Beta‐amyloid peptides (Aβ peptides) form the main protein component of the amyloid deposits found in the brains of Alzheimer's disease (AD) patients. Soluble Aβ peptides, which are proteolytic fragments of the amyloid‐precursor protein (APP) are constitutively secreted by cells expressing APP during normal metabolism [1] and are also present in human plasma and cerebrospinal fluid [2]. Missense mutations in Codon 717 of the APP gene are reponsible for a small percentage of inherited AD cases (FAD) and increase the amount of Aβ peptides containing additional carboxy terminal amino acids (Aβ1–42, Aβ1–43) [3, 4]. Recent findings indicate that FAD mutations in the presenilin 1 and 2 genes also increase the amount of these longer Aβ peptides [5]. Aβ1–42 polymerizes more rapidly in vitro [6] than Aβ1–40 and has been identified as the major component of the brain amyloid deposits [7–9]. We recently developed a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) system [10] for the separation of these two peptides. Here we describe a modified version of the original SDS‐PAGE procedure, which allows the separation of Aβ1–40, Aβ1–42, and Aβ1–43 for the first time. Detection of the three Aβ peptides in the lower ng and pg range is realized by optimized silver staining or immunoblot procedures. These nonradioactive methods may validate results obtained by ELISA procedures used to study the metabolic fate of APP. They may help to define the neurotoxic potential of the longer Aβ peptides in relation to their aggregation state.
Identification of ADP‐ribosylation factor‐6 in brush‐border membrane and early endosomes of human kidney proximal tubules
Authors: Marshansky Vladimir , Bourgoin Sylvain , Londoño Irene , Bendayan Moïse , Vinay Patrick
Published: May 2005

The expression and distribution of ADP‐ribosylation factor (ARF) small GTP‐binding proteins in kidney tissue was examined. Various anti‐ARF antibodies were raised against purified rec‐ARF 1 and rec‐ARF 6 and their specificity was determined. Using indirect immunofluorescence analysis of intact kidney, ARF proteins were found to be predominantly expressed in kidney tubules as compared to glomeruli. This result was further supported by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot analysis of purified human kidney glomeruli and proximal tubules. Both ARF 1 and ARF 6 were detected in purified human glomeruli and proximal tubules; however, ARF 1 was more abundant than ARF 6 in these kidney structures. Brush‐border membrane vesicles (BBMV) and early endosomes (EE) derived from the receptor‐mediated endocytosis pathway were isolated from purified proximal tubules of rat, dog and human kidney using a combination of magnesium precipitation and wheat‐germ agglutinin negative selection techniques. We demonstrated that ARF 6 is associated with BBMV and with EE derived from receptor‐mediated endocytosis pathway of human kidney proximal tubules. Using a combination of SDS‐PAGE and quantitative enhanced chemiluminescence Western blot analysis, the quantification of the ARF 6 distribution in membrane and cytoplasmic fractions of proximal tubules was made and its predominance in membrane fractions was demonstrated. By analogy with the functional role of ARF 1 in Golgi protein transport, we suggest that ARF 6 may play an important role in the regulation of receptor‐mediated endocytosis and protein reabsorption by kidney proximal tubules.
Spermatocytes and round spermatids of rat testis: The difference betweenin vivoandin vitroprotein patterns
Authors: Cossio Gabriela , Sanchez Jean‐Charles , Wettstein Rodolfo , Hochstrasser Denis F.
Published: May 2005

During mammalian spermatogenesis meiotic cell division and spermiogenesis occurs. Gene expression during this process is temporally regulated at the transcriptional and translational levels but the mechanisms are not well understood. In this publication we have investigated the synthesis of proteinsin vitroto detect the proteins with a high metabolic turnover and to compare them with thein vivoprotein map. RNA of spermatocytes and round spermatid cell populations, purified by centrifugal elutriation, and total testis was isolated. The poly A+ mRNA fraction was translated using a rabbit reticulocyte lysate. The translation products were separated by two‐dimensional (2‐D) gel electrophoresis using nonlinear 3.5–10 immobilized pH gradients for the first‐dimensional separation. The gels with35S‐translated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and scanned using a phosphorimager. A highly reproducible and complex protein pattern was obtained using this methodology. Only rat testis messages were translated. Using Melanie 2 software we could compare and detect more than 1000 proteins on 2‐D radioactive images. Some changes could be observed in protein expression between the different cell types but they were not statistically significant. The comparison between the 2‐D rat testis map and thein vitrotranslated patterns show no matching between any spots. This result suggests that the post‐transcriptional modifications occurring in the reticulocyte system are not the same as those that occurin vivoin the testis. Rabbit reticulocyte proteins were detected by staining PVDF membranes with colloidal gold. Rat testis and reticulocyte patterns were completely different.
Electrophoretic analysis of the novel antigen for the gastrointestinal‐specific monoclonal antibody, A33
Authors: Ji Hong , Moritz Robert L. , Reid Gavin E. , Ritter Gerd , Catimel Bruno , Nice Ed , Heath Joan K. , White Sara J. , Welt Sydney , Old Lloyd J. , Burgess Antony W. , Simpson Richard J.
Published: May 2005

The murine monoclonal antibody A33 (mAbA33) recognises a human cell membrane‐associated antigen selectively expressed in epithelial cells of the lower gastrointestinal tract and > 90% of colonic cancers, but is not detected in a wide range of other normal tissues by immunohistochemical analysis. In phase I/II clinical triasl, mAbA33 has been shown to target advanced colon cancers and the humanised version is currently being evaluated in therapy studies. Although the mAbA33 has been well characterised by immunohistochemical and clinical studies, until recently, the target antigen has remained poorly defined. This was largely attributable to the antigenic determinant recognised by mAbA33 being dependent on the native spatial conformation of the A33 antigen which impeded its identification by conventional two‐dimensional electrophoresis (2‐DE) and immunoblot analysis. We have developed an immunoblot method, based on nonreducing/non‐urea precast 2‐DE gels, that has facilitated the purification of the detergent (0.3% Triton X‐100) solubilised A33 antigen from the human colon cancer cell lines LIM1215 and SW1222. Under these 2‐DE conditions, the A33 antigen electrophoreses with an apparent Mr ˜ 41000 and pI 5.0–6.0. Attempts to isolate the A33 antigen from 2‐DE gels for direct structural analysis were unsuccessful, due to its co‐electrophoresis with actin and cytokeratin proteins. However, using Western blot and biosensor detection the A33 antigen has been purified chromatographically and N‐terminal sequence analysis was possible. Using polyclonal antibodies raised against a synthetic peptide corresponding to the N‐terminal region of the A33 antigen we have used Western blot analysis to localise the molecule in our master 2‐DE protein database for normal human colon crypts and several colon carcinoma cell lines (URL address: www.ludwig.edu.au). Under reducing 2‐DE conditions, the A33 antigen electrophoresis as 6 differentially charged isoforms (pI 4.6–4.8) with a single molecular weight species at Mr ˜ 55000.
Two‐dimensional electrophoresis of precision‐cut testis slices: Toxicologic application
Authors: Witzmann Frank A. , Fultz Carla D. , Wyman John F.
Published: May 2005

Advances in tissue slice technology and a recent novel application of this technique to reproductive toxicology using bovine testis have demonstrated the remarkable utility of this approach. The objective of the present study was to combine this in vitro toxicity test system with large‐scale two‐dimensional polyacrylamide gel electrophoresis (2‐DE) to detect and study alterations in testicular‐slice protein patterns as molecular correlates of 1,3,5‐trinitrobenzene (TNB) and 1,3‐dinitrobenzene (DNB) toxicity. Previous studies have shown that testicular slices remain viable for > 24 h and, as measured by protein synthesis inhibition, TNB causes dose‐related injury. Tissue‐slices were prepared from bovine testicles incubated for 2, 4 or 6 h and exposed to either 100 μM, 500 μM or 1 mM DNB or TNB in the incubation medium. Slices were collected, solubilized, and separated by large scale 2‐DE. Resulting protein patterns were then examined by image analysis, which revealed coefficients of variation in protein spot abundance comparable to patterns from fresh rodent tissue samples. Furthermore, specific protein alterations indicated dose‐related inductions and declines in protein abundance, some progressive over time. The results of this investigation demonstrate the potential toxicologic utility of combining in vitro tissue‐slice technology with high‐resolution 2‐DE protein mapping. The consolidation of these methods offers a novel approach for toxicity screening and testing, reduces experimental cost, and reduces the use of laboratory animals.
Effects of local changes in the helix flexibility on electrophoretic migration of DNA in agarose gel
Authors: Gisselfält Katrin , Åkerman Björn , Jonsson Mats
Published: May 2005

We present a study of how kinks, flexible bends, and flexible joints in the DNA helix, induced by binding cis‐diamminedichloroplatinum(II) (cis‐DDP), transdiamminedichloroplatinum(II) (trans‐DDP), and chlorodiethylenetriammineplatinum(II) (dien‐Pt) to the DNA, affect the electrophoretic migration of DNA in agarose gels. For long DNA the conformation oscillates between extended and compact states during the migration, as for native DNA. The presence of flexible joints decreases both the length of time and the step length of the cycles, but in a compensatory manner so that there is no net effect on the mobility. This demonstrates that in some cases mobility alone cannot detect pertubations in the DNA helix. Kinks and flexible bends reduce the mobility because they both lead to longer time periods of the cycles. With kinks the reduction is strongest at low fields because at high fields the kinks are straightened out; the steps thus become even longer than for native DNA. The results suggest that a combination of mobility and orientation measurements on reptating DNA can be used for distinguishing different kinds of structural alterations in the DNA.
Detection of somatic mutations in the mitochondrial DNA control region of colorectal and gastric tumors by heteroduplex and single‐strand conformation analysis
Authors: Alonso Antonio , Martin Pablo , Albarran Cristina , Aguilera Beatriz , Garcia Oscar , Guzman Ana , Oliva Horacio , Sancho Manuel
Published: May 2005

Each entire hypervariable region of the mitochondrial DNA control region was screened for mutations from paired normal and tumor DNA corresponding to a group of 21 patients (13 colorectal and 8 gastric adenocarcinomas) using both heteroduplex analysis and single‐strand conformation analysis. These two mutation scanning strategies allowed the identification of sequence alterations in 3/13 (23%) colorectal tumors and in 3/8 (37%) gastric tumors. Heteroduplex analysis showed the heteroplasmic state of the majority of these tumor mutations. Sequence analysis revealed two A:T/G:C transitions (nucleotide positions: 16241 and 16166) in hypervariable region 1 (HV1) and two C:G/T:A transitions (nucleotide positions: 76 and 312), one A:T/G:C transition (nucleotide position: 93), a 1‐basepair C:G deletion (nucleotide position: 309), and a 2‐base‐pair CC:GG insertion (nucleotide position: 309) in the HV 2 region. A considerable proportion of these mutations was found in homopolymeric regions which are highly polymorphic among humans. Different mechanisms (clonal expansion, increased oxidative damage, and nuclear mutator mutations) were suggested to explain the increased mitochondrial DNA mutation rate observed in cancer.
Quantitative description of analyte migration behavior based on dynamic complexation in capillary electrophoresis with one or more additives
Authors: Peng Xuejun , Bowser Michael T. , Britz‐McKibbin Philip , Bebault Gwendolyn M. , Morris Jamie R. , Chen David D. Y.
Published: May 2005

A comprehensive theory is proposed to describe the migration behavior of analytes in capillary electrophoresis (CE) when one or more additives are present in the buffer solution. This theory amalgamates and extends the previous work done by others. The capacity factor (ḱ) in this theory is defined as the product of the equilibrium constant and the additive concentration, thus, ḱ changes linearly with additive concentration. The net electrophoretic mobility of an analyte is a function of ḱ, therefore, it can be changed by varying the additive concentration. Three parameters are needed to predict the mobility of an analyte in a one‐additive CE system: the mobility of the free analyte, the mobility of the complex, and the equilibrium constant for the analyte‐additive interaction (which determines the fraction of the free analyte at different additive concentrations). When additives are used, the change in viscosity obscures this relationship, therefore, a viscosity correction factor is required to convert all mobilities to an ideal state where the viscosity remains constant. The migration behavior of an analyte in a solution with multiple additives can be predicted and controlled, once the equilibrium constants of the interactions between the analyte and each of the additives are obtained separately. β‐Cyclodextrin and hydroxypropyl‐β‐cyclodextrin are used as additives and the migration behavior of phenol, p‐nitrophenol, and benzoic acid are studied as a model system to verify this theory. When the necessary viscosity correction factor is included, the net electrophoretic mobilities of the analytes obtained from experimental results agree with the values predicted by the theory based on dynamic complexation. Although only experiments with one and two additives were carried out to verify the theory, the equations apply to situations when more than two additives are used. The relationship between the theories of electrophoresis and chromatography is clarified.
Density gradient isoelectric focusing of proteins in artificial pH gradients made up of binary mixtures of amphoteric buffers
Authors: Tulp Abraham , Verwoerd Desirée , Hart Augustinus A. M.
Published: May 2005

A density gradient electrophoresis apparatus made of Perspex (7 cm, Ø 2.2 cm) with a circular platinum anode and a palladium cathode was used for the separation of proteins in free liquid. Following a concept developed by M. Bieret al.(Electrophoresis1993,14, 1011–1018), mixtures of two suitable amphoteric buffers I and II provide for media with a fixed and electrophoretically stable pH or were used for the generation of preformed (electrophoretically stable) pH gradients covering about 1 pH unit. Amphoters I and II are considered suitable if there is overlap between (pK1,1–1–2) and the pK2,II+1+2) region. 3‐(N‐Morpholino)propanesulfonic acid (MOPS) and γ‐amino‐n‐butyric acid (GABA) were used as an example. Two approaches were followed: (i) rate‐zonal separation of test proteins in a pH window, formed by a fixed ratio of MOPS/GABA. (ii) Isoelectric focusing in a shallow preformed pH gradient, made up of inverse reciprocal linear gradients of MOPS and GABA. At isopH, test proteins (bovine serum albumin, cytochromec, ferritin, hemoglobin, lactoglobulin, myoglobin, and transferrin) were rate‐zonally separated within a short time. Even the separation of the A and B forms of lactoglobulin was feasible at isopH. The glycoforms of transferrin were separated and enriched on a pH 5.2–6.1 pH gradient, indicating that pH differences of about 0.01 still permit resolution. Contrary to the ill‐defined Ampholines, the cost of these well‐defined amphoters is low

Presented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996

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Changes in nuclear protein composition in response to chronic electrical stimulation of skeletal muscle
Authors: Gilroy Stephen J. , Salmons Stanley , Pennington Stephen R.
Published: May 2005

The adaptive response of skeletal muscle to increased functional demand involves phenotypic changes that affect contractile properties, energy metabolism and calcium kinetics. Some of these changes are known to be initiated at a pre‐translational level, but the underlying regulatory mechanisms have not yet been identified. In this study we used chronic electrical stimulation (10 Hz, continuous) to initiate fast‐to‐slow muscle fibre‐type transformation, and two‐dimensional electrophoresis (2‐DE) to assess changes in nuclear protein composition after 24 and 72 h. We report an early and sustained increase in the level of a 3 kDa protein in stimulated fast muscle (n = 6). The presence of the same protein in control slow muscle is consistent with a possible functional role in the determination of the slow phenotype.
Determination of association constants of dansyl‐amino acids and β‐cyclodextrin in N‐methylformamide by capillary electrophoresis
Authors: Valkó István E. , Sirén Heli , Riekkola Marja‐Liisa
Published: May 2005

The use of nonaqueous background electrolytes in capillary electrophoresis (CE) is a promising new trend which should widen the scope of this technique. We demonstrate the chiral separation of dansyl‐amino acids (Dns‐AAs) in N‐methylformamide (NMF) using β‐cyclodextrin (β‐CD) as chiral selector. The solubility of β‐CD is much better in NMF than in water, allowing high concentration of the chiral selector and successful enantioseparation despite the weak host‐guest interaction between the Dns‐AAs and β‐CD. The association constants for the complexation between Dns‐AAs and β‐CD could be calculated from the electrophoretic mobilities, with attention paid to the change in viscosity of the electrolyte upon addition of β‐CD. The association constants ranged between 2 and 13 M−1.
Chiral o‐phthaldialdehyde reagents for fluorogenic on‐column labeling of D‐ and L‐amino acids in micellar electrokinetic chromatography
Authors: Tivesten Anna , Folestad Staffan
Published: May 2005

Following a recent communication from this laboratory (A. Tivesten et al., J. High Resol. Chromatogr. 1996, 19, 229–233) where on‐column chiral derivatization of D‐ and L‐amino acids in micellar electrokinetic chromatography (MEKC) was demonstrated for the first time, we now present further details of the labeling procedure. The basis of the method is the consecutive injection of a sample and the reagent onto the capillary as two discrete plugs. By utilizing their difference in mobility, the zones are mixed by the electrophoretic process in a controllable way. In this way the amino acids are both derivatized within a few seconds and subsequently separated in a single step. Compared with precolumn derivatization, dilution of the original sample is minimized, which is why the method is highly useful for microchemical analytical work, i.e., labeling of nano‐ to picoliter samples. Four different chiral thiols were compared in this study, 2,3,4,6‐tetra‐O‐acetyl‐l‐thio‐β‐D‐glucopyranose (TATG), N‐acetyl‐L‐cysteine (AC), N‐acetyl‐D‐penicillamine (AP), and N‐acetyl‐L‐cysteine (IBC). Together with o‐phthaldialdehyde (OPA) these constitute the chiral reagent. The reaction rate as well as the spectroscopic and chromatographic properties of the formed derivatives were examined. It was found that the fastest reaction is obtained with OPA/TATG, as was the case with L‐alanine (L‐ala), and that the rate is greatly affected by the presence and concentration of acetonitrile or methanol. Moreover, OPA/TATG yields superior resolution of D‐ and L‐amino acids over the other OPA/thiol combinations in a sodium dodecyl sulfate (SDS) micellar buffer, whereas the OPA/AC and OPA/IBC‐amino acid derivatives have a higher fluorescence quantum yield. With laser‐induced fluorescence detection (He‐Cd, 325 nm) the mass limit of detection is at the low amol level.
Identification of wallaby milk whey proteins separated by two‐dimensional electrophoresis, using amino acid analysis and sequence tagging
Authors: Molloy Mark P. , Herbert Ben R. , Yan Jun X. , Williams Keith L. , Gooley Andrew A.
Published: May 2005

Micropreparative two‐dimensional polyacrylamide gel electrophoresis has been used to separate milk whey proteins from the Tammar wallaby (Macropus eugenii). We have used a combination of amino acid analysis and N‐terminal sequence tagging as a rapid and sensitive method to identify the major whey proteins. Using these techniques, we confidently identified α‐lactalbumin and late lactation protein. While these are the only two M. eugenii whey proteins with a corresponding SWISS‐PROT entry, we demonstrate that by using amino acid analysis and matching across species boundaries, we can identify previously unsequenced conserved wallaby whey proteins including β‐lactoglobulin and serum albumin.
Strategies for whole microbial genome sequencing and analysis
Authors: Fraser Claire M. , Fleischmann Robert D.
Published: May 2005

The introduction of methods for automated DNA sequence analysis nearly a decade ago, together with more recent advances in the field of bioinformatics, have revolutionized biology and medicine and have ushered in a new era of genomic science, the study of genes and genomes. These new technologies have had an impact on many areas of research, including the association between genes and disease, in DNA‐based diagnostics, and in the sequencing of genomes from human and other model organisms. The demonstration in 1995, that automated DNA sequencing methods could be used to decipher the entire genome sequence of a free‐living organism, Haemophilus influenzae, was a milestone in both the genomics and microbial fields [1]. Since the first report of the complete sequence of H. influenzae, these methodologies have been adopted by laboratories around the world. The complete genomic sequence of five eubacterial species [1–5], one archaea [6], and the eukaryote, Saccharomyces cerevisiae [7], have been reported in the last 18 months. At the beginning of 1997 more than a dozen microbial genome projects are at or near completion, with many others in progress. It is likely that in the next few years we will see the complete sequence of perhaps as many as 30–40 microbial genomes. In this article, we will review methods for whole genome sequencing and analysis and examine how this information can be exploited to better understand microbial physiology and evolution.
Proteome analysis of Saccharomyces cerevisiae: A methodological outline
Authors: Fey Stephen J. , Nawrocki Arkadiusz , Larsen Martin R. , Görg Angelika , Roepstorff Peter , Skews Gerard N. , Williams Richard , Larsen Peter Mose
Published: May 2005

Proteome analysis offers a unique means of identifying important proteins, characterizing their modifications and beginning to describe their function. This is achieved through the combination of two technologies: protein separation and selection by two‐dimensional gel electrophoresis, and protein identification and characterization by mass spectrometry. This methodological outline sketches the strengths and weaknesses of the two central technologies used, and provides both practical tips and the theoretical background for their utilization. One application of these technologies is illustrated by the characterization of genes, revealed by sequencing, but which have no — or only weak homology — to any other known genes. Other applications, for example the identification of protein markers for particular human diseases, are only referred to. The aim of the article is thus to provide the basis for a sound understanding of the full potential and limitations of proteome analysis.
Stress proteins in Listeria monocytogenes
Authors: Phan‐Thanh Luu , Gormon Thierry
Published: May 2005

The proteins induced by the different stress conditions in Listeria monocytogenes were analyzed by two‐dimensional (2‐D) electrophoresis with the aid of a computerized 2‐D gel analysis system. The stress conditions imposed were pH 4, pH 10, 0.015% sodium dodecyl sulfate (SDS), 0.03% sodium deoxycholate and 4% ethanol. As previously seen for heat shock and cold shock, more than half of the proteins normally synthesized by Listeria cells were repressed under these stress conditions. Conversely, the synthesis of a great number of proteins was increased and novel proteins appeared upon stress. Each stress factor induced a specific set of proteins. These stress proteins were characterized by their apparent molecular mass and isoelectric point. No universal stress proteins were found to be common to all the stresses studied, while some proteins were commonly induced by two or three stress conditions. The degree of dissimilarity in stress responses was best illustrated by the induction of only two proteins common to exposure to the two detergents SDS and sodium deoxycholate. This work, together with that on heat and cold shock, constitutes the basic step for the identification of stress proteins in Listeria.
Identification and characterization of Brucella ovis immunogenic proteins using two‐dimensional electrophoresis and immunoblotting
Authors: Teixeira‐Gomes Ana P. , Cloeckaert Axel , Bézard Guy , Bowden Raúl A. , Dubray Gérard , Zygmunt Michel S.
Published: May 2005

In a previous report, proteins from Brucella melitensis were characterized by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) and N‐terminal microsequencing. In the present report, we have extended this study to the second etiologic agent in ovine brucellosis, B. ovis, responsible for ram epididymitis and infertility. The combination of 2‐D gel electrophoresis and protein microsequencing facilitated the location and identification of the major proteins of B. ovis on the 2‐D pattern. These proteins comprised cytoplasmic, periplasmic and some membrane proteins except the major outer membrane proteins. By comparing 2‐D gel profiles of B. ovis with that of B. melitensis described previously, a few proteins with different expression levels were readily identified. Serum from a ram naturally infected with B. ovis was used in immunoblotting studies to identify immunogenic proteins recognized during the course of infection. This serum showed antibody reactivity against approximately 82 protein spots. Twenty‐one of these proteins were identified either by use of monoclonal antibodies or by N‐terminal microsequencing. Several proteins previously described in earlier Brucella works were identified: the 89 kDa outer membrane protein, DnaK, GroEL, BP26, and Cu‐Zn superoxide dismutase. Eight proteins had amino acid sequences homologous to those of various proteins from other bacteria found in protein databases: NikA, dihydrolipoamide succinyltransferase, a hypothetical 31 kDa protein, malate dehydrogenase, succinyl‐CoA synthetase alpha subunit, an amino acid ABC type transporter, Leu/ Ile/ Val‐binding protein precursor, and ClpP. The remaining eight proteins had N‐terminal sequences lacking similarity to existing database entries. Thus, the 2‐D PAGE analysis provided a convenient first approach in the characterization of immunogenic proteins.
Ancient DNA from polynesian rats: Extraction, amplification and sequence from single small bones
Authors: Matisoo‐Smith Elizabeth , Allen John S. , Ladefoged Thegn N. , Roberts R. Mere , Lambert David M.
Published: May 2005

The human colonisation of Polynesia was a major event in world prehistory. It represents one of the last human population migrations, and one which required crossing major water barriers. Though the subject of Pacific population origins has been approached by scholars from numerous fields for nearly a century, recent years have seen the problem addressed by human geneticists [1–5]. Since the initial report describing the recovery of DNA from skeletal remains [6], ancient DNA studies have also focused on the Pacific region [7, 8]. In this paper we present the results of ancient DNA analyses of Rattus exulans, an animal that was transported by ancestral Polynesians through the Pacific to the far reaches of the Polynesian triangle. Analysis of DNA of R. exulans skeletal remains has many advantages over studies of ancient human remains, yet the one drawback has been the recovery of ancient DNA from single bones of these very small rodents. We have successfully modified standard extraction protocols for ancient DNA [9] and have consistently extracted, amplified and sequenced mitochondrial DNA (mtDNA) from less than 0.1 g of R. exulans bone and tooth samples recovered from archaeological sites throughout the Pacific, ranging from 400 to 2000 years old.
A study of the genetic diversity of Mycobacterium tuberculosis isolated from patients in the eastern province of South Africa using random amplified polymorphic DNA profiling
Authors: Richner Sharon M. , Meiring Jill , Kirby Ralph
Published: May 2005

This population genetics study was done to determine the degree of genetic diversity amongst clinical isolates of Mycobacterium tuberculosis in one of the largest provinces of South Africa. Three hundred and fifty‐nine individual cultures were obtained from single patients over a nine‐month period. Bacterial DNA was extracted and amplified with two arbitrary ten‐mer primers, using the random amplified polymorphic DNA‐polymerase chain reaction (RAPD‐PCR) technique. RAPD markers were separated by agarose gel electrophoresis, visualised with ethidium bromide, and then analysed for similarity using the GelCompar programme. The isolates were seen to fall into two major population groups. A high degree of genetic diversity was seen, with a total of 350 unique strains occurring amongst the 359 isolates. The majority of drug‐resistant isolates grouped together in the smaller population group. The genetic data were also correlated with geographical locality. It was found that a slight preponderance of isolates that grouped together closely, vis. that were either genetically identical or very similar (similarity index of approximately 97%), were from areas that were considered to be geographically distant. Antibiotic resistance patterns were also examined, revealing a total of 16 different resistance profiles among the 68 drug‐resistant isolates. RAPD‐PCR has proved to be a cost‐ and time‐effective technique, suitable for such a large‐scale population genetics study. It has provided useful information on the genetic diversity of an organism which is responsible for an increasing amount of morbidity and mortality in South Africa's second‐largest province.
Tetra‐and pentanucleotide short tandem repeat instability in gastric cancer
Authors: Silva Filipe , Gusmão Leonor , Alves Cíntia , Seruca Raquel , David Leonor , Amorim António
Published: May 2005

The association between genetic instability in repetitive DNA domains and cancer has been reported in different types of malignancies. In this work we perform a comparative study of 29 gastric tumors with paired normal tissue using seven tetra‐(FES/FPS, VWA31/A, HTPO, TH01, MBPB) and pentanucleotide (CD4, TP53) STR polymorphic markers regarding loss of heterozygosity and replication error status. Furthermore, we compare the gene frequencies obtained in normal tissue from patients with those of a normal control population from the same area, looking for allele associations between any of these polymorphic loci and gastric cancer risk. The results have shown that FES/FPS and TP53 present the higher rates of somatic instability. The observed results for TP53 are in accordance with those previously reported in gastric carcinogenesis, while instability of FES/FPS is for the first time reported in this tumor type. Our data suggest that different loci show different rates of instability and/or loss of heterozygosity and do not seem to consist of a result of an RER+ phenotype affecting several genomic repetitive domains. Furthermore, the instability in markers TH01, MBPB, TP53, and FES was generally detected in genotypes involving alleles with a high number of repeats. Comparing gene frequencies in patients and normal controls, no significant differences were found, although longer alleles are consistently more frequent in patients for the markers MBPB, TH01, and CD4.
United Arab Emirates population data on three DNA tetrameric short tandem repeat loci: HUMTHO1, TPOX and CSF1PO — derived using multiplex polymerase chain reaction and manual typing
Authors: Bayoumi Riad A. , Al‐Gazali Lihadh I. , Jaffer Uzma , Nur‐E‐kamal Mohamed S. A. , Dawodu Adekunle , Bener Abdulbari , Eapen Valsamma , Budowle Bruce
Published: May 2005

Allele and genotype frequencies for three tetrameric short tandem repeat (STR) loci: HUMTHO1, TPOX, and CSF1PO, were determined in a United Arab Emirates (UAE) national population sample (n= 119). The loci were amplified simultaneously and the PCR products were separated in polyacrylamide DNA sequencing gels. Detection of the DNA fragments was accomplished using silver staining. Allele designations were determined by comparison to an allelic ladder. One allele at each locus was sequenced to confirm the nature of the repeats and their number. Alleles at the HUMTHO1 and TPOX loci were distributed bimodally, while CSF1PO showed unimodal distribution. The observed heterozygosities were 76% for HUMTHO1, 64% for TPOX, and 71% for CSF1PO. No deviation from the Hardy‐Weinberg expectations was observed in the genotype distribution for the loci TPOX and CSF1PO, but the HUMTHO1 locus did depart from HWE based on the likelihood ratio and the exact test. No correlation was detected between the alleles at any of the three pairs of loci. The allelic frequency data of these three loci in the UAE population sample can thus be used in human identity testing.
Genetic variation in Asiatic lions and Indian tigers
Authors: Shankaranarayanan Pattabhiraman , Banerjee Moinak , Kacker Raj Kumar , Aggarwal Ramesh Kumar , Singh Lalji
Published: May 2005

Previous reports have suggested that Asiatic lions and tigers are highly inbred and exhibit very low levels of genetic variation. Our analyses on these species have shown much higher degrees of polymorphism than reported. Randomly amplified polymorphic DNA (RAPD) analysis of 38 Asiatic lions, which exist as a single population in the Gir Forest Sanctuary in India, shows an average heterozygosity of 25.82% with four primers. Sperm motility studies by our colleagues corroborate this data. In Indian tigers, microsatellite analysis of five CA repeat loci and multilocus fingerprinting using Bkm 2(8) probe on a population of 22 individuals revealed a heterozygosity of 22.65%. Microsatellite analysis at loci Fca 77 and Fca 126 revealed polymorphism amongst the Asiatic X African lion hybrids, which has enabled us to use these as markers to discriminate the pure Asiatic lions from the hybrids. A similar analysis was used to identify hybrids of Indian and Siberian tigers through polymerase chain reaction (PCR) amplification of hair samples. To ascertain the variation which existed before the population bottleneck at the turn of the present century, microsatellite analysis was performed on 50‐ to 125‐year‐old skin samples from museum specimens. Our results show similar levels of genetic variability as in the present population (21.01%). This suggests that low genetic variability may be the characteristic feature of these species and not the result of intensive inbreeding. DNA fingerprinting studies of Asiatic lions and tigers have helped in identifying individuals with high genetic variability which can be used for conservation breeding programs.
Measurements of catecholamine‐mediated apoptosis of immunocompetent cells by capillary electrophoresis
Authors: Bergquist Jonas , Josefsson Elisabet , Tarkowski Andrej , Ekman Rolf , Ewing Andrew
Published: May 2005

Single cell analysis with capillary electrophoresis, a technique capable of detecting zeptomole quantities (10−21 mole) of neurochemical species, has been used to demonstrate that lymphocytes are capable of active synthesis of dopamine and norepinephrine. Exposure of lymphocytes to catecholamines at concentrations as low as 10 nM leads to decreased proliferation and differentiation, e.g. interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4) and immunoglobulin (Ig). In addition, both inhibition of dopamine uptake with nomifensine and inhibition of packing of catecholamines into vesicles with tetrabenazine, results in sinificantly lower levels of dopamine and norepinephrine (p < 0.01 and p < 0.05, respectively). The catecholamine‐dependent inhibition of T‐ and B‐lymphocyte activity is mediated via an induction of a Bcl‐2/Bax and Fas/FasL involved apoptosis. These findings indicate a novel mechanism for regulation of lymphocyte activity in the central nervous system, whereby elevated regional levels of catecholamines might lead to the immunoprivilege of the brain.
Hydrophobic peptide mapping of clinically relevant heptathelical membrane proteins by capillary electrophoresis
Authors: Dong Maoqing , Oda Robert P. , Strausbauch Michael A. , Wettstein Peter J. , Landers James P. , Miller Laurence J.
Published: May 2005

The structural investigation of G protein‐coupled receptors has been hindered by the lack of techniques to effectively resolve the hydrophobic peptides obtained by chemical or proteolytic cleavage, as well as the minute amounts of protein typically isolated. We have developed a capillary electrophoresis method for efficient separation of hydrophobic peptides using a cyanogen bromide digest of bacteriorhodopsin as a model for these clinically important membrane proteins. This procedure includes (i) solubilization of the protein digest in acetic acid; and (ii) electrophoresis using an acetic acid‐based buffer system augmented by acetonitrile and hexane sulfonic acid, in a Polybrenecoated fused silica capillary. The potential for detection sensitivity to be increased at least 100‐fold by use of on‐line solid‐phase extraction on C18‐silica is shown. This approach is potentially useful for peptide fingerprinting of sparse and extremely hydrophobic membrane receptors.
Identification of monoclonal proteins in serum: A quantitative comparison of acetate, agarose gel, and capillary electrophoresis
Authors: Katzmann Jerry A. , Clark Raynell , Wiegert Elaine , Sanders Elisabeth , Oda Robert P. , Kyle Robert A. , Namyst‐Goldberg Colette , Landers James P.
Published: May 2005

A selected group of 308 sera were analyzed by capillary electrophoresis (CE), agarose gel electrophoresis (AGE), and cellulose acetate electrophoresis (CAE) and evaluated for abnormalities that would suggest the presence of a monoclonal protein. The sensitivity (an electrophoretic abnormality in sera that contained a monoclonal protein) and specificity (a normal electrophoretic pattern in sera that did not contain a monoclonal protein) was determined for each electrophoretic procedure. CAE was the most specific procedure and CE was the most sensitive. The increase in sensitivity of CE was primarily due to increased detection of cryoglobulins and free light chains. The quantitation of the gamma region and/or monoclonal antibody peaks by CE was similar to results obtained by AGE. Quantitation of very large monoclonal protein peaks (> 3.0 g/dL) by on‐line absorption detection (CE) yielded higher results than quantitation by dye‐binding (AGE).
Development of a lipoprotein profile using capillary electrophoresis and mass spectrometry
Authors: Macfarlane Ronald D. , Bondarenko Pavel V. , Cockrill Steven L. , Cruzado Ingrid D. , Koss William , McNeal Catherine J. , Spiekerman A. Michael , Watkins Layle K.
Published: May 2005

A new program for lipoprotein characterization is outlined where capillary electrophoresis (CE) plays a central role in the analysis of intact lipoprotein serum components and the apoprotein domains. The first characterization step involves separation and particle density analysis of very low‐, low‐, and high‐density lipoprotein fractions (VLDL, LDL, HDL) by ultracentrifugation and image analysis. VLDL, HDL, and LDL fractions are analyzed by capillary electrophoresis. Sodium dodecyl sulfate (SDS) at low concentrations in the background electrolyte used in the CE analysis is incorporated into the lipoprotein particle without appreciable delipidation, as determined by ultracentrifuge particle density analysis. Increasing the concentration of SDS results in extensive delipidation, resulting in the release of apoproteins (apo) which are detected as components of the electropherogram. Apo B‐100 is detected in the delipidated VLDL and LDL fractions along with micelles of the lipids. Micelles from LDL delipidation have uniform charge densities. Apo A‐I and A‐II are detected in the HDL fraction. A new method for lipoprotein delipidation is introduced where the lipoprotein fraction is adsorbed on a reversed‐phase hydrophobic cartridge. Delipidation and recovery of the apoprotein fractions is made by serial clutions with acetonitrile. CE of the lipid‐free apoprotein mixture shows the presence of apoC‐I,II,III and apoE in the VLDL fraction, and apoA‐I,II apoC‐I and apoE in the HDL fraction. Electrospray ionization mass spectrometry analysis gives the isoform distribution for each apoprotein. The identification of the apoproteins in the electropherograms is the first step in developing a CE‐based quantitation method for measuring serum levels of these apoproteins and their distribution between the lipoprotein fractions. The assay described in this paper is being used as a level 2 and 3 cardiac risk profile analysis for individuals with normal lipid profiles who have a documented or family history of cardiovascular disease.
Capillary electrophoresis‐based separation of transferrin sialoforms in patients with carbohydrate‐deficient glycoprotein syndrome
Authors: Oda Robert P. , Prasad Rajani , Stout Robert L. , Coffin David , Patton Wendy P. , Kraft Daniel L. , O'Brien John F. , Landers James P.
Published: May 2005

The heterogeneity associated with protein glycoforms has been a challenge to analytical chemists and the subject of structure‐function studies for biochemists since their presence in biological systems had been confirmed some three decades ago. Initial investigations led to discoveries of synthetic and degradative pathways, and brief forays into functional determination of the “glyco” portion on the protein activity in glycoproteins. Only recently has it come to our understanding that variations from the “normal” glycosylation patterns might be indicative of pathological states. The presence of certain transferrin (Tf) glycoforms in human serum has been shown to correlate with certain clinical syndromes. Hence, the ability to separate and quantitatively measure the various forms of human Tf has become increasingly important. It this study, we demonstrate that a simple method utilizing a DB‐17‐coated capillary to slow endoosmotic flow and a sieving buffer containing hydroxyethyl cellulose allows for the resolution of sialoforms of transferrin. An analysis time of less than eight minutes allows for baseline resolution of the lower sialoforms of Tf, presenting a simple, rapid test for carbohydrate‐deficient transferrin (CDT). We demonstrate the utility of this methodology for the facile diagnosis of carbohydrate‐deficient glycoprotein syndrome, and postulate that it may allow for the detection of other carbohydrate‐deficient protein‐related disease states.
Micellar electrokinetic capillary chromatography (MECC) of UV‐absorbing constituents in normal urine: A chemometric optimisation of the separation
Authors: Alfazema Luwiza N. , Hows Mark E. P. , Howells Sian , Perrett David
Published: May 2005

A micellar electrokinetic capillary chromatography (MECC) method when compared to free solution capillary electrophoresis (CZE) was shown to offer improved selectivity and resolution for the separation of UV‐absorbing components of human urine. Some of the factors affecting MECC separation e.g. methanol concentration, sodium dodecyl sulphate (SDS) concentration, β‐cyclodextrin (β‐CD) concentration, voltage, pH, temperature and electrolyte additives (urea, β‐CD and Brij 35) were optimised using chemometric techniques. Three‐level three‐factor (33) factorial designs and simplex optimisation were used to achieve optimised conditions with the goal of obtaining the maximum number of peaks in the shortest possible analysis time. Using a TSP CE2000 instrument with detection from 195–300 nm and fitted with a 75 μm × 44 cm (37 cm effective length) fused silica capillary the final optimum conditions were found to be, an electrolyte consisting of 30 mM sodium tetraborate, pH 10, containing 75 mM SDS and 10 mM β‐CD, 15°C, 20 kV, 4 s hydrodynamic injection of filtered urine. These conditions were capable of separating 70 peaks from a normal human urine pool in less than 12 min. The separation of components in urine using the optimised MECC was simpler, more reproducible, faster and gave better resolution than gradient reversed‐phase high performance liquid chromatography

This work was first presented at the 4th International Symposium on Capillary Electrophoresis, University of York, UK, August 1996

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A biostatistical study into the efficiency of individualisation using nonisotopic chemiluminescent‐enhanced NICE™ multilocus DNA probes
Authors: Hau Peter P. C. , Watt Elizabeth H. , Hau Cathyrn M.
Published: May 2005

The efficiency of individualisation using nonisotopic chemiluminescent‐ enhanced probes (NICE™) was investigated by analysing DNA fingerprints obtained from 190 unrelated Caucasians. Novel analysis of the scoring procedure enabled us to include the comparison of 585 pairs of samples for each of two probes. When the results of NICE probes 33.6 and 33.15 were combined, the mean percentage band share between two unrealated individuals was 16.8% and the mean number of bands identified in an individual DNA fingerprint was 54.8. Results were compared with those obtained using isotopically labelled probes and suggest that the two labelling systems gave similar efficiencies for differentiating between individuals. Analysis of DNA fingerprints from 37 family trios (mother, child and father groups) gave a mutation rate of 0.10% when using NICE probes. The two labelling systems compared were equally efficient in establishing family relationships.
Improved sensitivity of detection with the commercial automated gel electrohoresis (HPGE‐1000) apparatus through modification of its optical system
Authors: Yarmola Elena , Chrambach Andreas
Published: May 2005

In a representative application to a fluorescently detectable protein of commercial automated gel electrophoresis apparatus (HPGE‐1000, LabIntelligence, Belmont, CA) the sensitivity of detection by fluorescence was significantly increased by elimination of the mirror below the gel tray. That increase in detection sensitivity is due to a decrease of fluorescent background noise by nearly one order of magnitude, overcompensating a decrease in signal by a factor of two. The resulting increase in signal/noise ratio, i.e., detection sensitivity, should allow for lowered sample loads by which the band width is reduced with benefits to resolution.