bluuebunny/crossref_metadata_2025 huggingface dataset:
Childhood cancer patients' access to cooperative group cancer programs
Authors: Liu Lihua , Krailo Mark , Reaman Gregory H. , Bernstein Leslie
Published: February 2003

BACKGROUND

The Children's Oncology Group (COG), a merger of the Children's Cancer Group (CCG) and the Pediatric Oncology Group (POG), conducts clinical trials for the treatment of childhood cancer. To assess the feasibility of developing a nationwide childhood cancer registry, the authors attempted to determine whether COG could serve as a resource for identifying all children with cancer.

METHODS

A consolidated file of children age < 20 years who were diagnosed with cancer between 1992–1997 and registered with either CCG or POG was linked with records from the National Cancer Institute's Surveillance, Epidemiology and End Results (SEER) Program. Age‐specific registration rates and age‐adjusted registration rates (AARR) were calculated overall and by year of diagnosis, gender, race/ethnicity, stage of disease at diagnosis, and type of cancer.

RESULTS

Of 10,108 children age < 20 years with cancer who were identified by the 11 SEER registries between 1992–1997, 5796 were registered with CCG or POG. The AARR was 71% for children age < 15 years, 24% for adolescents ages 15–19 years, and 57% for children age < 20 years. Registration rates were stable over the years studied, varied by geographic region, and were found to be higher among children with more advanced disease. Registration rates were highest for children with leukemia, hepatic tumors, and renal tumors, and were lowest for carcinoma and retinoblastoma.

CONCLUSIONS

The results of the current study demonstrate that not all children with cancer are registered by the cooperative groups; however, a national registry program can be achieved by supplementing cases identified through COG with data collected by statewide population‐based cancer registries. Such a partnership would be mutually beneficial, allowing COG to achieve 100% registration of children with cancer and, for the statewide cancer registries, improving the timeliness of case‐finding and follow‐up information for cancer outcomes. Cancer 2003;97:1339–45. © 2003 American Cancer Society.

DOI 10.1002/cncr.11192
Caveolin‐1 expression is a predictor of recurrence‐free survival in pT2N0 prostate carcinoma diagnosed in Japanese patients
Authors: Satoh Takefumi , Yang Guang , Egawa Shin , Addai Josephine , Frolov Anna , Kuwao Sadahito , Timme Terry L. , Baba Shiro , Thompson Timothy C.
Published: February 2003

BACKGROUND

The authors previously identified elevated caveolin‐1 expression in human prostate carcinoma and determined that caveolin‐1 levels as detected by immunohistochemistry of radical prostatectomy specimens offered novel prognostic information. A higher incidence of caveolin‐1 expression also was reported in African‐American men compared with white men in the U.S. To explore these ethnic/racial differences in caveolin‐1 expression further, the authors evaluated caveolin‐1 expression as a predictive marker in Japanese men with prostate carcinoma.

METHODS

Immunohistochemical staining with a caveolin‐1 specific antibody was performed on routinely processed paraffin sections from 152 consecutively collected radical prostatectomy specimens. The mean patient age was 64.3 years (range, 49–74 years; median, 64.5 years) and the mean follow‐up period was 49.5 months (range, 1.3–103.3 months; median, 48.2 months). Caveolin‐1 immunoreactivity was evaluated in association with patient's age; preoperative prostate specific antigen level; clinical stage; and pathologic features including Gleason score, extraprostatic extension, status of surgical margins, seminal vesicle involvement, lymph node involvement, and time to disease progression after surgery.

RESULTS

Positive caveolin‐1 immunostaining was detected in 46 of the 152 tumors (30.3%) and was found to be associated significantly with a positive surgical margin (P= 0.022). A higher incidence of caveolin‐1 expression tended to be found in patients with poorly differentiated tumors (Gleason score > 7, 6–7, and < 6, 35.0% vs. 34.9% vs. 20.4%, respectively) or in patients with extraprostatic extension versus those without extraprostatic extension (35.4% vs. 24.7%) or patients with lymph node involvement compared with those without lymph node involvement (50% vs. 29.5%), although these differences did not reach statistical significance (P= 0.100,P= 0.150, andP= 0.178, respectively, by the Spearman correlation test). Kaplan–Meier analysis revealed that increased caveolin‐1 expression was associated with an increased risk of disease progression at 5 years (P= 0.0122 by the log‐rank test). In patients with organ‐confined (pT2N0) disease, univariate Cox proportional hazards regression analysis revealed that positive caveolin‐1 expression was the only significant predictor of disease recurrence after radical prostatectomy (P= 0.011; hazards ratio = 4.75; and 95% confidence interval, 1.43–15.76).

CONCLUSIONS

The results of the current study confirm that positive caveolin‐1 expression is associated with clinical markers of disease progression and is predictive of poor clinical outcome after surgery in Japanese patients with pT2N0 prostate carcinoma. Cancer 2003;97:1225–33. © 2003 American Cancer Society.

DOI 10.1002/cncr.11198
Capecitabine in combination with docetaxel and epirubicin in patients with previously untreated, advanced breast carcinoma
Authors: Venturini Marco , Durando Antonio , Garrone Ornella , Colozza Maria Antonietta , Contu Antonio , Stevani Ilaria , Genta Franco , Bighin Claudia , Lambiase Antonio , Del Mastro Lucia
Published: February 2003

BACKGROUND

The objective of this study was to evaluate the activity and safety of oral capecitabine in combination with docetaxel and epirubicin (TEX) as first‐line treatment for patients with locally advanced/metastatic breast carcinoma.

METHODS

This open‐label, Phase II study was conducted at six Italian centers. Treatment consisted of epirubicin, 75 mg/m2 (intravenous bolus), and docetaxel, 75 mg/m2 (1‐hour infusion), both administered on Day 1, plus oral capecitabine, 1000 mg/m2 twice daily, on Days 1–14 of each 3‐week treatment cycle.

RESULTS

A total of 67 patients received 392 cycles of treatment, with a median of 6 cycles in patients with Stage III disease (n = 34 patients) and a median of 8 cycles in patients with Stage IV disease (n = 33 patients). The objective response rate was 82%, including complete responses in 21% of patients. A greater proportion of patients with Stage III disease achieved tumor responses compared with patients who had Stage IV disease (97% vs. 67%, respectively). Among 34 patients with Stage III disease, pathologic complete responses were confirmed in 10 patients (29%). TEX chemotherapy demonstrated an acceptable safety profile. There was a low incidence of Grade 3 adverse events, and Grade 4 adverse events were particularly rare (4%). The most common Grade 3–4 adverse event was febrile neutropenia, which occurred in 16% of patients.

CONCLUSIONS

TEX combination therapy has important antitumor activity and an acceptable safety profile in this setting. A large, randomized, Phase III trial is ongoing to compare TEX chemotherapy with an epirubicin plus docetaxel regimen in patients with untreated, advanced breast carcinoma. Cancer 2003;97:1174–80. © 2003 American Cancer Society.

DOI 10.1002/cncr.11203
Increased telomerase activity is associated with shorter survival in patients with chronic phase chronic myeloid leukemia
Authors: Verstovsek Srdan , Kantarjian Hagop , Manshouri Taghi , Cortes Jorge , Faderl Stefan , Giles Francis J. , Keating Michael , Albitar Maher
Published: February 2003

BACKGROUND

Significantly elevated telomerase activity (TA) has been found in samples from patients with many malignant hematologic diseases. However, the impact of elevated TA on the course of patients with chronic phase chronic myeloid leukemia (CP‐CML) is unknown.

METHODS

Using a modified polymerase chain reaction‐based telomeric repeat amplification protocol assay, the authors measured TA in bone marrow samples from 93 patients with CP‐CML and correlated it with patient characteristics and survival. TA also was measured in bone marrow samples from 29 patients with accelerated/blastic phase CML.

RESULTS

Patients with accelerated/blastic phase CML were found to have somewhat higher levels of TA compared with patients with CP‐CML (P = 0.07). Among patients with CP‐CML, those with high TA progressed to advanced stages of disease sooner (P = 0.05) and had a significantly shorter survival (P = 0.04) than patients with low TA. No correlation was found between TA and patient age, hemoglobin, platelet and leukocyte counts, percentage of peripheral or bone marrow blasts or basophils, or bone marrow cellularity. On multivariate analysis, high TA retained its significance as a factor associated with shorter patient survival (P = 0.02).

CONCLUSIONS

The current study data suggest that TA plays a role in the propagation of CP‐CML and that the potential of telomerase inhibitors in patients with CML should be explored, even in those with early phase disease. Cancer 2003;97:1248–52. © 2003 American Cancer Society.

DOI 10.1002/cncr.11217
Attractors of reaction‐diffusion systems in unbounded domains and their spatial complexity
Authors: Zelik Sergey V.
Published: February 2003

The nonlinear reaction‐diffusion system in an unbounded domain is studied. It is proven that, under some natural assumptions on the nonlinear term and on the diffusion matrix, this system possesses a global attractor 𝒜 in the corresponding phase space. Since the dimension of the attractor happens to be infinite, we study its Kolmogorov's ϵ‐entropy. Upper and lower bounds of this entropy are obtained.

Moreover, we give a more detailed study of the attractor for the spatially homogeneous RDE in ℝn. In this case, a group of spatial shifts acts on the attractor. In order to study the spatial complexity of the attractor, we interpret this group as a dynamical system (with multidimensional “time” if n > 1) acting on a phase space 𝒜. It is proven that the dynamical system thus obtained is chaotic and has infinite topological entropy.

In order to clarify the nature of this chaos, we suggest a new model dynamical system that generalizes the symbolic dynamics to the case of the infinite entropy and construct the homeomorphic (and even Lipschitz‐continuous) embedding of this system into the spatial shifts on the attractor.

Finally, we consider also the temporal evolution of the spatially chaotic structures in the attractor and prove that the spatial chaos is preserved under this evolution. © 2003 Wiley Periodicals, Inc.
Vortex disaggregation for flow cytometry allows direct histologic correlation: A novel approach for small biopsies and inaspirable bone marrows
Authors: Vos Jeffrey A. , Simurdak Jerry H. , Davis Brad J. , Myers Jerome B. , Brissette Mark D.
Published: February 2003

Background

Many approaches to obtaining single cells from tissue for flow cytometric immunophenotyping are used; however, these methods result in tissue that is too disrupted for subsequent histologic examination. We introduce a new technique for cell dissociation of hematopoietic malignancies that preserves tissue for histology. This is especially important with small specimens for which this type of correlation is critical.

Methods

Fresh tissue from lymph node, gastrointestinal (GI) tract, skin, and other soft tissue biopsies, in addition to cores of inaspirable bone marrows, were briefly vortexed until the RPMI cell culture medium became cloudy. Larger specimens such as lymph nodes were sectioned before disaggregating, whereas smaller ones were vortexed in toto. Resultant flow cytometric analyses were compared with the histology and, in some cases, the immunohistochemistry (IHC) to determine whether the data were concordant. Cell suspensions of 104 specimens–composed of 48 lymph nodes, 19 bone marrow cores (BMCs), 11 GI biopsies, 11 skin/soft tissue biopsies, and 15 miscellaneous specimens–were prepared via vortex disaggregation.

Results

Flow cytometric analysis of 96 specimens (92.3%) showed adequacy of material and diagnostic correlation with the histology and IHC. Of the eight cases (7.7%) that were discordant, seven were attributable to significant specimen fibrosis or necrosis. With respect to tissue type, this method produced diagnostic cell suspensions for most lymph nodes (95.8%), GI biopsies (90.9%), and BMCs (89.5%); however, it was less useful for skin/soft tissue samples (81.8%).

Conclusions

Disaggregation of tissue for flow cytometric analysis by vortexing appears to provide adequate and representative cellular material. This technique is ideal for inaspirable bone marrows and small biopsies where tissue preservation for histology is paramount. Cytometry Part B (Clin. Cytometry) 52B:20–31, 2003. Published 2003 Wiley‐Liss, Inc.
Importance of CD117 in the evaluation of acute leukemias by flow cytometry
Authors: Newell James O. , Cessna Melissa H. , Greenwood Jay , Hartung Leah , Bahler David W.
Published: February 2003

Background

The issue of which specific antibodies need to be used when evaluating acute leukemias by flow cytometry is controversial.

Methods

Recent studies have suggested that antibodies against CD117 or c‐kit are not essential for the assignment of blast lineage by flow cytometry, even though CD117 appears to be a very specific marker for myeloid lineage acute leukemias. We report a case of acute myeloid leukemia M2 subtype with an 8:21 translocation, where the leukemic blasts expressed CD117, CD19, and CD15 but did not show definitive expression of the myeloid markers CD13 or CD33.

Results and Conclusions

This study highlights the importance of CD117 when evaluating acute leukemias by flow cytometry, which was necessary in this case to suggest that the blasts were phenotypically abnormal myeloblasts. In addition, this case presented an unusual acute myeloid leukemia phenotype that will likely be encountered by others and could be difficult to interpret. Cytometry Part B (Clin. Cytometry) 52B:40–43, 2003. © 2003 Wiley‐Liss, Inc.
Normal values of CD4 and CD8 lymphocyte subsets in healthy indian adults and the effects of sex, age, ethnicity, and smoking
Authors: Uppal S. S. , Verma Shashi , Dhot P. S.
Published: February 2003

Background

Information on lymphocyte populations (T, B, and natural killer cells) and subpopulations (CD4 and CD8) in India is generally lacking. Measurement of T‐cell subsets is important in India for evaluating disease stage and progression in individuals with the human immunodeficiency virus (HIV). Hence, this study was conducted to provide normal ranges of absolute and percentage values of CD4 and CD8 T‐lymphocyte subsets and the ratio of CD4 to CD8 in normal Indian adults.

Methods

Flow cytometric analysis (EPICS‐XL) was used to determine the range of T‐lymphocyte subpopulations in normal Indian blood donors at Command Hospital and the Armed Forces Medical College, Pune, India. The reference population consisted of 94 healthy HIV‐seronegative blood donors. T‐lymphocyte subsets were analyzed with two‐color immunophenotyping of peripheral blood lymphocytes with the use of a lysed whole‐blood technique and enumerated.

Results

For normal values of various blood components, we found mean values of 2114 cells/μl for total lymphocytes, 865 cells/μl (40.2%) for CD4+ lymphocytes, 552 cells/μl (31.3%) for CD8+ lymphocytes, and 1.7 for the CD4:CD8 ratio. The 95% confidence intervals for the same parameters were 1115–4009 cells/μl, 430–1740 cells/μl (30.75–49.60%), 218–1396 cells/μl (20.06–42.52%), and 0.39–3.02 respectively. Females had significantly higher CD4 counts (P < 0.05), percentage of CD4 lymphocytes (P < 0.01), and CD4:CD8 ratio (P < 0.01). Males had a significantly higher percentage of CD8 lymphocytes (P < 0.01). They also had higher CD8 counts that did not reach significance. Age, ethnicity (Dravidian versus Aryan), smoking, alcohol consumption, and the interval between drawing the blood sample and its analysis were factors that did not produce statistically significant differences in the T‐cell subsets studied.

Conclusions

When compared with other published series, the CD4 and CD8 values in healthy Indians were no different from those reported in the West. These observations have important clinical implications for the use of T‐lymphocyte subset measurements in India, especially in the management of HIV infection. The normal ranges established by this study can be used as a reference for decisions made in clinical practice. Cytometry Part B (Clin. Cytometry) 52B:32–36, 2003. © 2003 Wiley‐Liss, Inc.
Flow cytometry versus histamine release analysis of in vitro basophil degranulation in allergy to Hymenoptera venom
Authors: Lambert C. , Guilloux L. , Dzviga C. , Gourgaud‐Massias C. , Genin C.
Published: February 2003

Background

Flow cytometry (FCM) has been proposed for specific allergy in vitro testing. We investigated its biological significance for allergy to Hymenoptera venoms and compared it with the routinely performed basophil histamine release test (HRT).

Methods

Blood samples from 26 allergic and 8 nonallergic donors were incubated with venom at serial concentrations. Basophils were analyzed with anti‐CD45‐PE‐Cyanin 5, Anti‐IgE‐FITC, and Anti‐CD63‐Phycoerythrine. HRT was measured by radioimmunoassay.

Results

FCM was as convenient as HRT for measuring basophil reactivity in at least 87% of allergic and 75% of nonallergic subjects. CD63 outer expression was specifically induced in 91% of releaser subjects (86% on HRT) and in 1 of 10 tests in nonallergic donors, or one of six tests (16% on HRT) in allergic patients tested with an irrelevant allergen. Both methods were concordant in 85.7% of the tests. The three discordant patients had low‐grade reactions and borderline biological responses on FCM (n = 2) or HRT (n = 1).

Conclusions

The dynamic, physiologic significance of CD63, the dose–response curve, and dependency on ethylene‐diaminetetra acetic acid suggested that both tests reflect the same mechanism. Cytometry Part B (Clin. Cytometry) 52B:13–19, 2003. © 2003 Wiley‐Liss, Inc.
B‐cell monoclonal lymphocytosis and B‐cell abnormalities in the setting of familial B‐cell chronic lymphocytic leukemia
Authors: Marti Gerald E. , Carter Patricia , Abbasi Fatima , Washington Glennelle C. , Jain Nisha , Zenger Vincent E. , Ishibe Naoko , Goldin Lynn , Fontaine Laura , Weissman Nancy , Sgambati Maria , Fauget Guy , Bertin Pablo , Vogt Robert F. , Slade Barbara , Noguchi Philip D. , Stetler‐Stevenson M. A. , Caporaso Neil
Published: February 2003

Background

Among all hematologic malignancies, B‐cell chronic lymphocytic leukemia (BCLL) has the highest familial clustering (three‐ to sevenfold increase), strongly suggesting a genetic component to its etiology. Familial BCLL can be used as a model to study the early pathogenesis of this disease.

Methods

We examined nine kindreds from the National Cancer Institute's Familial BCLL Registry, consisting of 19 affected members with BCLL and 33 clinically unaffected first‐degree relatives. Flow cytometric immunophenotyping to detect a B‐cell monoclonal lymphocytosis (BCML) was performed. Monoclonality was confirmed by polymerase chain reaction analysis of whole blood DNA. Cell cycle analysis for aneuploidy was conducted.

Results

In all affected individuals, we observed the classic BCLL CD5/CD19/CD20/CD23 immunophenotypic patterns. Six of the 33 unaffected individuals (18%) had evidence of BCML. Additional individuals (13/33, 39%) showed some other abnormality, whereas 14 individuals (42%) were normal. Based on an estimated prevalence of 0.7% for BCML in the general population, the finding of six subjects (18%) with clonal abnormalities in this relatively modest sample was significantly greater than expected (i.e., 18% vs. 0.7%, P < 5.7 × 10−9).

Conclusions

Individual components of BCML and other B‐cell abnormalities were observed in almost half of the apparently unaffected individuals. Our findings suggested that BCML may be an early detectable abnormality in BCLL. The spectrum of some of these observed abnormalities suggested the involvement of different B‐cell subpopulations or different pathways in clonal evolution. Population‐based, longitudinal studies will be required to determine the incidence of BCML and other B‐cell abnormalities and their relation to disease progression in BCLL and other closely related B‐cell lymphoproliferative disorders. Cytometry Part B (Clin. Cytometry) 52B:1–12, 2003. Published 2003 Wiley‐Liss, Inc.
Separation of proteins in a multicompartment electrolyzer with chambers defined by a bed of gel beads
Authors: Cretich Marina , Pirri Giovanna , Carrea Giacomo , Chiari Marcella
Published: February 2003

Multicompartment electrolyzers (MEs) with isoelectric membranes were introduced in 1989 for purifying proteins in an electric field. At the basis of ME technology there are membranes consisting of cross‐linked copolymers of acrylamide and acrylamido monomers bearing protolytic groups. The technology employed for casting the membranes is an extension of the isoelectric focusing in immobilized pH gradient technique for which specific acrylamido monomers, known with the trade name of Immobiline, have been developed. However, the use of continuous membranes presents several disadvantages. Due to the mechanical characteristics of polyacrylamide, the gel must physically adhere onto a rigid support, which prevents it from collapsing. The support must have a highly porous structure in order to be permeable to proteins. The mechanical fragility of the membranes is one of the main problems that hinders the industrial scale application of ME separators. In order to overcome this problem, we propose to substitute the continuous membranes with a bed of gel beads of identical comonomer composition, obtained by an inverse emulsion polymerization process.
Rapid high‐resolution electrophoresis of multimeric von Willebrand Factor using a thermopiloted gel apparatus
Authors: Smejkal Gary B. , Shainoff John R. , Kottke‐Marchant Kandice M.
Published: February 2003

Rapid and highly reproducible nonreducing agarose gel electrophoresis (NRAGE) of von Willebrand Factor (vWF) multimers was performed using a thermostated minigel apparatus that monitors and precisely controls internal gel temperature. The substitution of lithium dodecyl sulfate (LiDS) for sodium dodecyl sulfate (SDS) allowed electrophoresis to be performed below the 16°C Krafft point of SDS and facilitated NRAGE of vWF over the entire range of 0–35°C. Internal gel temperature was regulated by a thermocouple probe inserted directly into the gel during electrophoresis which interfaced with a thermopilot that continually measures and adjusts temperature to within ± 0.5°C. At 10°C operative temperature, NRAGE at 1.5% agarose concentration was completed in 20 min at 250 V. Electrophoresis could be performed in only 10 min at 500 V, but at such high voltages, localized temperature fluctuations as much as 6°C resulted in perturbation of banding patterns in those vicinities. In the optimized method, both high molecular weight multimers and proteolytic fragments of vWF were separable suggesting clinical applicability of this system for the diagnosis of von Willebrand Disease and thrombotic thrombocytopenic purpura.
Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro‐Q Emerald 488 dye, a fluorescent periodate Schiff‐base stain
Authors: Hart Courtenay , Schulenberg Birte , Steinberg Thomas H. , Leung Wai‐Yee , Patton Wayne F.
Published: February 2003

Pro‐Q Emerald 488 glycoprotein stain reacts with periodic acid‐oxidized carbohydrate groups, generating a bright green‐fluorescent signal on glycoproteins. The stain permits detection of less than 5–18 ng of glycoprotein per band, depending upon the nature and the degree of protein glycosylation, making it roughly 8–16‐fold more sensitive than the standard colorimetric periodic acid‐Schiff base method using acidic fuchsin dye (pararosaniline). The green‐fluorescent signal from Pro‐Q Emerald 488 stain may optimally be visualized using charge‐coupled device/xenon arc lamp‐based imaging systems or 470–488 nm laser‐based gel scanners. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and the specificity of staining is better in gels. After detecting glycoproteins with Pro‐Q Emerald 488 dye, total protein profiles may subsequently be detected using SYPRO Ruby protein gel stain. Using computer‐assisted registration techniques, images may then be merged to generate differential display maps.
Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate‐polyacrylamide gels
Authors: Hart Courtenay , Schulenberg Birte , Diwu Zhenjun , Leung Wai‐Yee , Patton Wayne F.
Published: February 2003

Two fluorophore‐nitrilotriacetic acid conjugates, Pro‐Q Sapphire 365 and Pro‐Q Sapphire 488 oligohistidine gel stains, have been developed for the fluorescence detection of fusion proteins containing oligohistidine tags directly in sodium dodecyl sulfate polyacrylamide gels, without the requirement for electroblotting, reporter enzymes or secondary detection reagents. Pro‐Q Sapphire 365 oligohistidine gel stain exhibits bright‐blue fluorescence (emission maximum = 450 nm) when illuminated with UV‐A or UV‐B light from a standard ultraviolet transilluminator. Pro‐Q Sapphire 488 oligohistidine gel stain exhibits bright‐green fluorescence (emission maximum = 515 nm) when illuminated with visible light from a laser‐based gel scanner equipped with a 470 nm second‐harmonic generation (SHG) or 488 nm argon‐ion laser source. Typically, 25–65 ng of oligohistidine‐tagged fusion protein in whole cell lysates is detectable using either stain. After documenting the fluorescence signal from the Pro‐Q Sapphire dyes, gels may be post‐stained with the red‐fluorescent SYPRO Ruby protein gel stain in order to reveal the total protein pattern.
Microscale analysis of mucin‐type O‐glycans by a coordinated fluorophore‐assisted carbohydrate electrophoresis and mass spectrometry approach
Authors: Robbe Catherine , Capon Calliope , Flahaut Christophe , Michalski Jean‐Claude
Published: February 2003

The total glycan moiety was released in a single step from native glycoproteins by a nonreductive β‐elimination procedure. The generated oligosaccharides were further derivatized either with the hydrophobic fluorophore 2‐aminoacridone (AMAC) or the charged 8‐aminonaphthalene‐1,3,6‐trisulfonic acid (ANTS) fluorophore, and the resulting fluorescent derivatives were separated according to their hydrodynamic size or charge with high‐resolution gel electrophoresis. Both N‐ and O‐glycans released by this β‐elimination procedure might be analyzed simultaneously. AMAC derivatization allows a rapid separation of neutral and charged oligosaccharides without prior fractionation. Derivatized oligosaccharide species were then eluted from the gel slices and analyzed by mass spectrometry. This methodology allowed the rapid structural characterization of each glycan in term of monosaccharide composition and sequence. Using this technique we were able to screen several heterogeneous O‐glycan mixtures isolated at the picomolar range from reference glycoproteins or mucins.
Congruence between starch gel and polyacrylamide gel electrophoresis in detecting allozyme variation in pulmonate land slugs
Authors: Geenen Sofie , Jordaens Kurt , Castilho Rita , Backeljau Thierry
Published: February 2003

The predominantly selfing slug species Arion (Carinarion) fasciatus, A. (C.) silvaticus and A. (C.) circumscriptus are native in Europe and have been introduced into North America, where each species consists of a single, homozygous multilocus genotype (strain), as defined by starch gel electrophoresis (SGE) of allozymes. In Europe, the “one strain per species” hypothesis does not hold since polyacrylamide gel electrophoresis (PAGE) of allozymes uncovered 46 strains divided over the three species. However, electrophoretic techniques may differ in their ability to detect allozyme variation. Therefore, several Carinarion populations from both continents were screened by applying the two techniques simultaneously on the same individual slugs and enzyme loci. SGE and PAGE yielded exactly the same results, so that the different degree of variation in North American and European populations cannot be attributed to differences in resolving power between SGE and PAGE. We found four A. (C.) silvaticus strains in North America indicating that in this region the “one strain per species” hypothesis also cannot be maintained. Hence, the discrepancies between previous electrophoretic studies on Carinarion are most likely due to sampling artefacts and possible founder effects.
Sequence‐specific and nonspecific mobilities of single‐stranded oligonucleotides observed by changing the borate buffer concentration
Authors: Biyani Manish , Nishigaki Koichi
Published: February 2003

The suitability of gel electrophoresis to structural analysis of nucleic acids has been examined, using from borate buffer concentrations (in the range from 4.5 to 450 mM. The gel electrophoretic mobility of single‐stranded oligonucleotides was shown to be sequence‐dependent at higher concentrations than 27 mM of borate buffer and nondependent at lower one (less than 9 mM). As a result, each dodecamer had a sequence‐specific critical concentration of Tris‐borate‐EDTA (TBE) buffer at which each seems to change its structural state. At the lower concentration than the critical one, all the dodecamers turned to the state of a finite mobility and migrated in a sharp band. This finding is discussed and rationalized by the assumption that the difference in conformational dynamics of oligonucleotides, due to the difference in their sequence, is mainly responsible for the observed difference in their mobility.
Detection of single‐nucleotide polymorphisms coding for three ovine prion protein variants by primer extension assay and capillary electrophoresis
Authors: Zsolnai Attila , Anton István , Kühn Christa , Fésüs László
Published: February 2003

An alternative method is described for the determination of ovine prion protein allelic variants at codon 136, 154, and 171. The four mutations responsible for amino acid changes are typed simultaneously. The technique utilizes dideoxy chain termination reaction using fluorescently labeled dideoxy nucleotides. The single‐base extended primers are resolved on a capillary electrophoresis instrument. Data obtained by our approach are presented according to genotype distribution in some breeds as a part of the validation procedure.
Application of high‐resolution capillary array electrophoresis with automated fraction collection for GeneCalling™ analysis of the yeast genomic DNA
Authors: Berka Jan , Ruiz‐Martinez Marie C. , Hammond Richard , Minarik Marek , Foret Frantisek , Sosic Zoran , Kleparnik Karel , Karger Barry L.
Published: February 2003

Capillary array instrument was applied to transcript profiling of the yeast genomic DNA using GeneCalling™ chemistry. The instrument integrated a 12‐capillary array for DNA separation with a replaceable sieving matrix, laser‐induced fluorescence detection and an automated microfraction collector. The DNA fractions, exiting the separation capillaries, were continuously deposited in a 1536‐well collection plate made of agarose gel. DNA fragments recovered from selected fractions were cloned and then sequenced. Over 80% of theoretically predicted fragments could be recovered in the collected fractions, cloned and sequenced with an average redundancy of threefold. Excellent correlation of the experimentally obtained sequences with the theoretically predicted gene fragments demonstrated the suitability of capillary array electrophoresis for micropreparative recovery of DNA fragments. This approach, useful especially for rapid DNA expression profiling of unknown genes for nonsequenced organisms, demonstrates the practical capability of the prototype multicapillary fraction collector.
Simplex optimization of electrokinetic injection of DNA in capillary electrophoresis using dilute polymer solution
Authors: Catai Jonatan Ricardo , Carrilho Emanuel
Published: February 2003

In DNA analysis by capillary electrophoresis with polymer solutions there are many variables that can be optimized. However, electric field strength, polymer solution concentration and temperature of analysis are the most relevant ones. These are the variables most responsible for the fragment resolution and analysis time. Optimization of such parameters can be obtained simultaneously using chemometric techniques, reaching the optimum working conditions with few experiments. In this work, we have studied the influence of the sample composition and electrokinetic injection conditions in the reproducibility and the quality of the DNA separation results. A simplex optimization has been carried out and the optimum condition was reached with nine experiments.
Detection of Escherichia coli O157:H7 bacteria by a combination of immunofluorescent staining and capillary electrophoresis
Authors: Kourkine Igor V. , Ristic‐Petrovic Mirjana , Davis Edward , Ruffolo Carmel G. , Kapsalis Andreas , Barron Annelise E.
Published: February 2003

As the number of incidents of bacterial infections continues to rise around the globe, simpler, faster, and more sensitive diagnostic techniques are required to improve the safety of the food supply and to screen for potential bacterial infections in humans. We present here direct and indirect approaches for the detection of bacteria, which are based upon a combination of immunofluorescent staining and capillary electrophoresis. In the direct approach, Escherichia coli O157:H7 bacteria stained with fluorescein‐tagged specific antibodies are detected by CE, while in the indirect approach fluorescein‐tagged specific antibodies to E. coli are first captured by E. coli O157:H7 bacteria and then released and detected by CE. We have identified suitable bacteria staining and CE protocols, which involved a 10 mM Tris‐borate‐EDTA (TBE) buffer, 0.25 μg antibody/1 million bacteria, and capillaries dynamically coated with poly‐N‐hydroxyethylacrylamide (polyDuramide). We have also successfully detected the presence of E. coli O157:H7 in contaminated meat. The total time required for analysis was 6–8 h, which is less than that realized in most commercial assays presently available.
Simultaneous separation of fifteen approved protease and reverse transcriptase inhibitors for human immunodeficiency virus therapy by capillary electrophoresis
Authors: Tuan Nguyen Duc , Gutleben Wolfgang , Scherer Karin , Stoiber Heribert , Falkensammer Barbara , Dierich Manfred P. , Zemann Andreas
Published: February 2003

In the present investigation, a novel approach towards a complete separation of all 15 protease and reverse transcriptase inhibitors which are currently approved for use in highly active antiretroviral therapy in a single analytical run is presented. The developed method employs an acidic background electrolyte with sodium polyanethol sulfonate (SPAS) as polyanionic electroosmotic flow (EOF) modifier to establish a strong cathodic EOF, sodium dodecyl sulfate (SDS) as pseudostationary selector, and acetonitrile and ethanol as organic modifiers. Separation of the analytes is based on two different mechanisms. The more basic analytes are protonated at the prevailing pH conditions and thus migrate in front of the cathodic EOF, whereas the less basic and neutral analytes interact with the SDS and are retained after the EOF. By optimizing electrolyte pH, the amount of solvents and SDS concentrations in the background electrolyte it is possible to completely separate all compounds of interest.
Separation of twenty underivatized essential amino acids by capillary zone electrophoresis with contactless conductivity detection
Authors: Coufal Pavel , Zuska Jiří , van de Goor Tom , Smith Vonda , Gaš Bohuslav
Published: February 2003

Twenty underivatized essential amino acids were separated using capillary zone electrophoresis and consequently detected with contactless conductivity detection (CCD). A simple acidic background electrolyte (BGE) containing 2.3 M acetic acid and 0.1% w/w hydroxyethylcellulose (HEC) allowed the electrophoretic separation and sensitive detection of all 20 essential amino acids in their underivatized cationic form. The addition of HEC to the BGE suppressed both, electroosmotic flow and analyte adsorption on the capillary surface resulting in an excellent migration time reproducibility and a very good analyte peak symmetry. Additionally, the HEC addition significantly reduced the noise and long‐term fluctuations of the CCD baseline. The optimized electrophoretic separation method together with the CCD was proved to be a powerful technique for determination of amino acid profiles in various natural samples, like beer, yeast, urine, saliva, and herb extracts.
Differences in capillary electrophoresis profiles of urinary and recombinant erythropoietin
Authors: de Frutos Mercedes , Cifuentes Alejandro , Diez‐Masa Jose C.
Published: February 2003

Different profiles were obtained by capillary zone electrophoresis (CZE) of human erythropoietin (EPO) of recombinant and urinary origin. To unambiguously detect doping by EPO, direct methods able to determine the presence of the drug itself in a physiological fluid are required. Since the host cell line used for EPO production influences its glycosylation, the carbohydrate distribution of natural human EPO may be different from that of recombinant EPO. The different content in sialic acid groups between recombinant and endogenous EPO provide a basis for their distinction by CZE.
Separation of homologues and isomers of linear alkylbenzenesulfonates by capillary electrophoresis with sodium dodecyl sulfate, carboxylic acids and bile salts
Authors: Herrero‐Martínez José Manuel , Simó‐Alfonso Ernesto F. , Ramis‐Ramos Guillermo
Published: February 2003

The ability of several anionic compounds, including carboxylic and dicarboxylic acids, sodium dodecyl sulfate (SDS), and sodium deoxycholate (SDC) and other bile salts, to separate the C10–C13 homologues and the corresponding 20 positional isomers of linear alkylbenzenesulfonates (LAS) by capillary electrophoresis was studied. Up to 19 peaks and a shoulder were observed with a background electrolyte (BGE) containing 10 mM phosphate (pH 6.8), 30% acetonitrile and 40 mM SDS, and 18 peaks were obtained with a BGE containing 10 mM borate (pH 9), 40% ethanol and 40 mM palmitic acid (PA). Resolution increased with the alkyl chain length of the carboxylic acid. Dicarboxylic acids with a short alkyl chain, as azelaic acid, were useful to separate the homologues without distinguishing between the isomers. Up to 16 peaks and a shoulder were distinguished with SDC. Resolution decreased with the other bile salts. The 6‐C11/5‐C11 isomer pair was better resolved with SDC than with SDS, and the 2‐C12 isomer was isolated using both PA and SDC, but not with SDS. Only the 7‐C13/6‐C13 pair could not be resolved with any of the discriminating agents used.
Micellar electrokinetic chromatography with on‐line Fourier transform infrared detection
Authors: Kölhed Malin , Hinsmann Peter , Lendl Bernhard , Karlberg Bo
Published: February 2003

Micellar electrokinetic chromatography (MEKC) was successfully coupled to Fourier transform infrared (FTIR) detection, using a micromachined IR‐transparent flow cell with an optical path length of 15 μm for the on‐line detection of five neutral analytes. Tight connections between the flow cell and the capillaries were achieved by creating a small O‐ring of UV‐curing epoxy adhesive on the sharply cut capillary ends. The background electrolyte consisted of 15 mM phosphate buffer at pH 7 and 40 mM sodium dodecyl sulfate (SDS). Five analytes (paracetamol, caffeine, p‐nitro benzyl alcohol, m‐nitrophenol and p‐nitrophenol) were successfully separated, yielding detailed IR stack plots that could be used for quantification and identification. Linear calibration graphs were obtained for each individual analyte present in mixtures at concentrations up to 10 mM. The limit of detection (3 S/N) ranged between 1.1 and 1.5 mM (1.2–1.8 ng). Analytes were identified by comparing spectra obtained during the MEKC separation with those resulting from completely filling the capillary with each individual analyte dissolved in the micelle‐containing electrolyte. Information on the specific functional groups of all analytes could be elucidated from the spectra. Since FTIR is a nondestructive detection technique, a conventional on‐line UV detector was introduced directly after the developed IR flow cell to test the system's performance and to demonstrate that tandem FTIR and UV detection is feasible.
Chromatographic behavior of packing materials for capillary electrochromatography with a coating of different immobilized polysiloxanes
Authors: Zhang Guodong , li Yongshan , Fang Yi'ou , Han Nanyin , Xu Bingjiu
Published: February 2003

Octadecyl silyl silica (ODS) phase coated with immobilized polysiloxanes (OV1701, SE‐54, SE‐30) were synthesized, their characteristics as capillary electrochromatography (CEC) column packing materials were studied. It was found that, although the polysiloxane coatings were different in polarity, the resulting packing materials showed the highest efficiencies when the respective coating ratios (polysiloxane:ODS, w/w) were all 20–30%. As expected, packing materials coated with different polysiloxanes resulted in different selectivity on solute pairs. Separations on these stationary phases were studied with different factors such as pH values and acetonitrile contents of the mobile phases. It was found that all these kind of stationary phases could resist basic mobile phase with a pH value as high as 11.6. Tests were made to analyze polar, basic drugs with CEC using the stationary phases.
Determination of low‐aliphatic aldehydes indoors by micellar electrokinetic chromatography using sample dissolution manipulation for signal enhancement
Authors: Pereira Elisabete A. , Cardoso Arnaldo A. , Tavares Marina F. M.
Published: February 2003

This work describes a novel approach for the analysis of selected aldehydes (formaldehyde, acetaldehyde, propionaldehyde, and acrolein) and acetone in environmental samples using micellar electrokinetic chromatography (MEKC). The method is based on the reaction of carbonyl compounds with 3‐methyl‐2‐benzothiazoline hydrazone (MBTH) that gives an azine intermediate with maximum absorbance at 216 nm. A systematic evaluation of sample dissolution medium was conducted as a means to enhancing sensitivity. In the best condition, samples were dissolved in 0.030 mol·L−1 tetraborate solution. This condition presented enhancement factors in the range of 35–54 for the aldehydes under investigation, computed as the improvement of the concentration limits of detection (LODs) with reference to the sample dissolved in pure water. The running buffer was 0.020 mol·L−1 tetraborate, pH 9.3, containing 0.050 mol·L−1 sodium dodecyly sulfate (SDS). The overall methodology presented several advantages over established methods for aldehydes. Worthy mentioning that MBTH is available in high purity degree, dispensing laborious reagent purification procedures. A few method validation parameters were determined revealing good migration time repeatability (< 2.5% coefficient of variation, CV) and area repeatability (< 4% CV), excellent linearity (20–120 μg/L, r > 0.995) and adequate sensitivity for environmental applications. The LODs with respect to each single aldehyde were in the range of 0.54–4.0 μg·L−1 and 11 μg·L−1 for acetone. The methodology was applied to the determination of aldehydes indoors. Samples were collected in an impinger flask containing 0.05% MBTH solution, at a flow rate of 0.80 L·min−1, during 2.5 h, at different times during the day. The most abundant carbonyls in the samples were acetone, followed by formaldehyde and acetaldehyde, with estimate peak concentrations of 452, 5.2 and 2.2 ppbv, respectively.
Mixed micellar electrokinetic capillary chromatography separation of depolymerized grape procyanidins
Authors: Herrero‐Martínez José Manuel , Ràfols Clara , Rosés Martí , Torres Josep Lluís , Bosch Elisabeth
Published: February 2003

Oligomeric procyanidins are potent antioxidant polyphenols of potential interest as disease‐preventing agents. Their efficiency depends on the size and composition of their oligomeric structures. The mean degree of polymerization of these compounds is usually estimated by thiolysis with thiol‐α‐toluene followed by analysis using high‐performance liquid chromatography (HPLC). We show the development of a mixed micellar electrokinetic chromatography (MEKC) method for the separation of the major components obtained after thiolysis with cysteamine (catechins and their cysteamine conjugates). MEKC studies using sodium dodecyl sulfate (SDS as pseudostationary phase led to long migration times, e.g., with 100 mM SDS, at pH 7, the solutes were separated in about 40 min), while the use of sodium cholate (SC) produced an elution window relatively short. Using a mixed micellar SC‐SDS system (50 mM phosphate at pH 7 containing 40 mM SC and 10 mM SDS), it is possible to separate these compounds in less than 15 min. The proposed method is useful to separate the major components of the thiolysate in effluents from food processing (e.g., skins and seeds from grape and apple) considered as potential procyanidin sources.
Ultrafast analysis of oligosaccharides on microchip with light‐emitting diode confocal fluorescence detection
Authors: Dang Fuquan , Zhang Lihua , Hagiwara Hisashi , Mishina Yoshinori , Baba Yoshinobu
Published: February 2003

We have developed a new method for the high‐speed separation and high‐sensitivity detection of complex oligosaccharides based on microchip electrophoresis (ν‐CE) with light‐emitting diode (LED) confocal fluorescence detection. Oligosaccharides labeled with 8‐aminopyrene‐1,3,6‐trisulfonate (APTS) were found to strongly adsorb to the surface of polymethylmethacrylate (PMMA) microchips. Accordingly, three classes of major dynamic coating additives were systematically investigated, and cellulose derivatives were found to specifically suppress such adsorption and allow high‐performance separation on PMMA chips. Additive concentration, buffer pH and applied field strength were found to be key factors in the high‐performance separation& of APTS‐labeled oligosaccharides on PMMA chips. Under optimal conditions, 15 oligosaccharides in dextrin hydrolysate can be separated within 45 s with an electrophoretic separation efficiency of over 400 000 theoretical plates per meter. The relative standard deviation (RSD) values of migration times of fourteen oligosaccharides were less than 0.50% between six different channels, and the detection limit for APTS‐labeled glucose was about 1.98×10−8 mol/L or 8.61 amol with a signal‐to‐noise ratio (S/N) of 3. The high speed, high efficiency and high sensitivity of this μ‐CE‐based method indicate that it can be widely applied to analysis of complex oligosaccharides.