bluuebunny/crossref_metadata_2025 huggingface dataset:
Vibrational circular dichroism and IR absorption of DNA complexes with Cu2+ ions
Authors: Andrushchenko V. , Van De Sande J. H. , Wieser H.
Published: August 2003

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0–0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0–0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine‐cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine‐thymine (AT) base pairs, which favors a “sandwich” complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B‐form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A‐ or Z‐form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3‐dimensional structure of the DNA–metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone. © 2003 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 72: 374–390, 2003
Raman structural markers of tryptophan and histidine side chains in proteins
Authors: Takeuchi Hideo
Published: August 2003

The Raman spectrum of a protein contains a wealth of information on the structure and interaction of the protein. To extract the structural information from the Raman spectrum, it is necessary to identify and interpret the marker bands that reflect the structure and interaction in the protein. Recently, new Raman structural markers have been proposed for the tryptophan and histidine side chains by examining the spectra–structure correlations of model compounds. Raman structural markers are now available for the conformation, hydrogen bonding, hydrophobic interaction, and cation–π interaction of the indole ring of Trp. For His, protonation, tautomerism, and metal coordination of the imidazole ring can be studied by using Raman markers. The high‐resolution X‐ray crystal structures of proteins provide the basis for testing and modifying the Raman structural markers of Trp and His. The structures derived from Raman spectra are generally consistent with the X‐ray crystal structures, giving support for the applicability of most Raman structural makers. Possible modifications and limitations to some marker bands are also discussed. © 2003 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 72: 305–317, 2003
Die Winogradsky‐Säule: Modellversuch zur Mikrobiologie
Authors: Telgmann Gisela
Published: August 2003

In einer Winogradsky‐Säule entwickeln sich unterschiedliche Schwefel— und Sauerstoffgradienten. Die im Schlamm und Wasser vorhandenen Mikroorganismen siedeln sich entsprechend ihrer Ansprüche an ihre ökologische Nische in „Etagen”︁ an und vermehren sich im Verlauf der folgenden Wochen derart stark, dass sie als farbige Banden sichtbar werden. Mit Hilfe einfacher Identifizierungsmöglichkeiten können diese Banden bekannten Mikroorganismen zugeordnet werden. Die Kenntnis ihrer Stoffwechselleistungen und Lebensansprüche ermöglicht die Deutung der zeitlichen Abfolge bei der Bandenbildung und die Zuordnung wichtiger Vorgänge zu wesentlichen Teilprozessen des Kohlenstoff‐, Schwefel‐ und Stickstoffkreislaufes. Die Beobachtung über mehrere Wochen ermöglicht die Unterscheidung von Sukzession und Klimaxstadium und lässt so das Prozesshafte ökologischer Vorgänge erkennen.
Auf der Suche nach alten Bekannten: Sequenzvergleich durch Alignments
Authors: Haubold Bernhard
Published: August 2003

Die laufenden Genomprojekte bescheren den Molekularbiologen einen phantastischen, allgemein zugänglichen Datenreichtum. Sowohl die Erhebung dieser Daten als auch ihre Interpretation stützt sich auf ein in der Biologie althergebrachtes Verfahren, dem Vergleich. Relativ neu ist jedoch der starke Einsatz bioinformatischer Methoden zum genomweiten Sequenzvergleich. Dieser Aufsatz gibt eine Einführung in die Genomik und die sie unterstützende Bioinformatik.
Physikalische Uhren zur Frühzeit des Menschen: Archäochronometrie
Authors: Wagner Günther A. , Glasmacher Ulrich A.
Published: August 2003

Physikalische Datierungsverfahren basieren auf dem natürlichen Zerfall radioaktiver Mutternuklide in Tochternuklide. Für den Altersbereich seit dem Auftreten des Menschen (zwei bis drei Millionen Jahre) werden vor allem die K‐Ar, die Lumineszenz, die Kernspaltspuren, die Uranreihen und die Radiokohlenstoffmethode angewandt. Ständige Weiterentwicklungen dieser verschiedenen Methoden führen zu einer präziseren Altersbestimmung und ermöglichen die zeitliche Zuordnung von Funden und Prozessen, die bisher nicht datierbar waren. So konnte mit der 14C‐Datierung von Knochen und der TL‐Datierung von gebrannten Feuersteinen im Geißenklösterle bei Blaubeuren das erste Auftreten des Homo sapiens in Europa auf die Zeit um 40.000 Jahre vor heute festgelegt werden.
Schlüsselenzym der Nitratassimilation: Nitratreduktase
Authors: Appenroth Klaus‐J. , Lillo Cathrine
Published: August 2003

Das Schlüsselenzym der Nitratassimilation ist Nitratreduktase (NR). Über drei Redox‐Domänen werden Elektronen von NADH auf Nitrat übertragen, das zu Nitrit reduziert wird. Nitratassimilation ist sehr energieaufwendig, das Reaktionsprodukt Nitrit ist ein Zellgift. Nitratreduktase ist deshalb hochgradig reguliert: Die Expression des NR‐Gens ist lichtabhängig. In dunkelgewachsenen Pflanzen wird dieser Prozess durch Phytochrome kontrolliert, in lichtgewachsenen Pflanzen durch Photosynthese. Nach erfolgter Biosynthese wird die Aktivität der NR im Dunkeln verringert, indem das Enzym phosphoryliert wird und danach an ein inaktivierendes Protein (14‐3‐3) bindet. Auch im Tagesverlauf wird NR reguliert: Durch zirkadiane Steuerung kann die Erhöhung der Enzymaktivität schon vor Tagesbeginn vorbereitet werden. Man spricht von einer „Stickstoff‐Uhr”︁, weil alle notwendigen Komponenten einer biologischen Uhr bei der Nitratassimilation vorhanden sind. Biotechnologische Forschung zur Erzeugung von Pflanzen mit verbesserter Stickstoff‐Assimilation hat bisher noch nicht zu praktischen Ergebnissen geführt.
Strategien gegen die Schwerkraft: Marines Zooplankton
Authors: Schneider Gerald
Published: August 2003

Die Lebensgemeinschaften des Planktons unterliegen der Gefahr, in für die jeweilige Gruppe lebensfeindliche Wassertiefen abzusinken. Dagegen haben die Organismen verschiedene Strategien entwickelt. Bei marinem Zooplankton sind dies vor allem Dichte reduzierende Mechanismen wie ein möglichst niedriges Protein‐/Lipidverhältnis, ein hoher Wassergehalt und die teilweise Ersetzung von schweren Sulfationen durch leichtere Chloridionen in den Geweben. Neben solchen biochemischen Strategien kennen wir auch aktive und passive Auftriebskörper wie beispielsweise Gasblasen oder extrakorporale Gallertstrukturen, aber auch die Ausnutzung von widerstandsreichen Körpergeometrien und bestimmte Verhaltensweisen, die ein Absinken verhindern. Obwohl seit mehr als hundert Jahren über diese Zusammenhänge nachgedacht wird, erweisen sich neuere Forschungen über dieses Thema als produktiv.
Monarchfalter: Fliegen mit der Sonnenuhr
Authors: Irsch Wilhelm
Published: August 2003

Jedes Jahr ziehen im Herbst die nordamerikanischen Monarchfalter (Danaus plexippus), Tagschmetterlinge mit einer Flügelspannweite von bis zu zwölf Zentimetern, zu Millionen in ihr Winterquartier im Süden und treten im Frühjahr wieder die Rückreise an. Sie fliegen allein, in Gruppen oder großen Schwärmen und legen bis zu 3500 Kilometer zurück. Die Flugrichtung und der Zeitpunkt dieser Massenwanderung sind so genau vorhersagbar, dass an den Zugstraßen viele Städte das Eintreffen der Falter mit Volksfesten begehen.
Hyänen — sanfte Männchen sind erfolgreicher
Authors: Irsch Wilhelm
Published: August 2003

Tüpfelhyänen (Crocuta crocuta) leben in hochkomplexen Clans zusammen, bei denen in der Regel die Weibchen das Sagen haben. Verhaltensforscher des Berliner Instituts für Zoo‐ und Wildtierforschung haben nun nachweisen können, dass sanfte Männchen weit mehr Paarungserfolge vorweisen können als ihre aggressiveren Geschlechtsgenossen.
Genome schützen sich vor fremder DNA
Authors: Brennicke Axel
Published: August 2003

Unsere kleinsten biologischen Feinde sind DNA‐Stücke, die sich in unserem Erbgut festsetzen und sich von dort aus vermehren. Das Pantoffeltierchen und seine Verwandten liefern jetzt neue Erkenntnisse über Verteidigungsmaßnahmen gegen solche Invasoren. Über den RNA‐Umweg erkennt unser Genom die eingewanderte Fremd‐DNA und schneidet sie heraus.
Giftmörderin mit Appetit
Authors: Tillmann Urban
Published: August 2003

Fressen und Gefressen werden, dieser tägliche Kampf prägt bekanntermaßen das Leben. Während normalerweise die Rollen von Fressern und Futter klar verteilt sind, gibt es im Plankton offensichtlich bemerkenswerte Ausnahmen — die Alge Prymnesium parvum gehört dazu.
In fremde Haut gehüllt
Authors: Wendler Silke
Published: August 2003

Parasiten aus der Ordnung der Fächerflügler (Strepsiptera) hüllen sich aktiv in einen Sack aus Hautgewebe des Wirtes und entgehen so dessen Immunabwehr. Die Wirte werden durch diesen Befall mit Tarnkappe sterilisiert, weswegen man über einen Einsatz in der biologischen Schädlingsbekämpfung nachdenkt.
Georg Klebs
Authors: Penzlin Heinz
Published: August 2003

Vor 100 Jahren begründete Georg Klebs mit seinem Buch „Willkürliche Entwickelungsänderungen bei Pflanzen”︁ die botanische Entwicklungs‐ und Fortpflanzungsphysiologie. Heute ist der Forscher, der schon früh „das Beharrliche im Flusse der Erscheinungen”︁ erkannte, fast in Vergessenheit geraten.
Protein – DNA interactions: the story so far and a new method for prediction
Authors: Jones Susan , Thornton Janet M.
Published: August 2003

This review describes methods for the prediction of DNA binding function, and specifically summarizes a new method using 3D structural templates. The new method features the HTH motif that is found in approximately one‐third of DNA‐binding protein families. A library of 3D structural templates of HTH motifs was derived from proteins in the PDB. Templates were scanned against complete protein structures and the optimal superposition of a template on a structure calculated. Significance thresholds in terms of a minimum root mean squared deviation (rmsd) of an optimal superposition, and a minimum motif accessible surface area (ASA), have been calculated. In this way, it is possible to scan the template library against proteins of unknown function to make predictions about DNA‐binding functionality. Copyright © 2003 John Wiley & Sons, Ltd.
The prediction of membrane protein structure and genome structural annotation
Authors: Martelli Pier Luigi , Fariselli Piero , Tasco Gianluca , Casadio Rita
Published: August 2003

New methods, essentially based on hidden Markov models (HMM) and neural networks (NN), can predict the topography of both β‐barrel and all‐α membrane proteins with high accuracy and a low rate of false positives and false negatives. These methods have been integrated in a suite of programs to filter proteomes of Gram‐negative bacteria, searching for new membrane proteins. Copyright © 2003 John Wiley & Sons, Ltd.
Multiple sequence alignments as tools for protein structure and function prediction
Authors: Valencia Alfonso
Published: August 2003

Multiple sequence alignments have much to offer to the understanding of protein structure, evolution and function. We are developing approaches to use this information in predicting protein‐binding specificity, intra‐protein and protein‐protein interactions, and in reconstructing protein interaction networks. Copyright © 2003 John Wiley & Sons, Ltd.
The 4th Bologna Winter School: hot topics in structural genomics
Authors: Casadio Rita
Published: August 2003

The 4th Bologna Winter School on Biotechnologies was held on 9–15 February 2003 at the University of Bologna, Italy, with the specific aim of discussing recent developments in bioinformatics. The school provided an opportunity for students and scientists to debate current problems in computational biology and possible solutions. The course, co‐supported (as last year) by the European Science Foundation program on Functional Genomics, focused mainly on hot topics in structural genomics, including recent CASP and CAPRI results, recent and promising genome‐wide predictions, protein–protein and protein–DNA interaction predictions and genome functional annotation. The topics were organized into four main sections (http://www.biocomp.unibo.it). Published in 2003 by John Wiley & Sons, Ltd.
Hybrid methods for electromagnetic scattering simulations on overlapping grids
Authors: El Hachemi M. , Hassan O. , Morgan K. , Rowse D. P. , Weatherill N. P.
Published: August 2003

The numerical simulation of practical electromagnetic scattering problems often involves geometries that are complex in shape and that may contain small scale features. The result is a large scale simulation and this requires solution techniques that are efficient, in terms of both computer time and computer memory requirements. In addition, it is advantageous if the method adopted exhibits a high degree of flexibility from the viewpoint of mesh generation. Hybrid methods, based upon the coupling of the structured grid finite difference time domain approach with an unstructured finite element or finite volume time domain procedure, appear ideally suited to meet these requirements. The structured grid method is memory and time efficient, while the unstructured grid methods readily handle general geometries, allowing detailed definition of small scale features. This paper presents the results of an initial investigation into the possibility exploiting these advantages in electromagnetic scattering simulations. Copyright © 2003 John Wiley & Sons, Ltd.
Novel multiparameter flow cytometry assay using Syto16 for the simultaneous detection of early apoptosis and apoptosis‐corrected P‐glycoprotein function in clinical samples
Authors: van der Pol Marjolein A. , Broxterman Henk J. , Westra Guus , Ossenkoppele Gert J. , Schuurhuis Gerrit J.
Published: August 2003

Background

The fluorescent probe Syto16 has been used successfully to measure P‐glycoprotein (Pgp) function and, separately, early apoptosis and cell death. The present study was designed to evaluate whether the combined use of Syto16, the Pgp blocker PSC833, and 7‐AAD allows simultaneous detection of all parameters, with emphasis on applications in acute myeloid leukemia (AML).

Methods

Pgp negative/positive KB cell lines treated with tumor necrosis factor α/hyperthermia and frozen–thawed AML samples were used as apoptosis/Pgp models.

Results

For the accurate assessment of apoptosis in samples with unknown Pgp status, it was essential to include a sample with PSC833: in such samples, viable cells always show a Syto16high and apoptotic cells a Syto16low fluorescence. Apoptotic cells loose their Pgp activity early on; in Pgp‐positive cells, the Syto16low apoptotic cells then colocalize with the Syto16low viable cells in the situation minus PSC833. We have developed a gating strategy that, apart from quantifying apoptosis, allowed gating out these apoptotic cells for proper Pgp assessment. By using this strategy, no differences in Pgp activity were found in the treated versus the untreated samples (KB cells: P = 0.779, n = 10; AML cells: P = 0.525, n = 45).

Conclusions

The use of the combination Syto16/PSC833/7‐AAD provides a sensitive multiparameter flow cytometry method that enables accurate assessment of both apoptosis, cell death, and Pgp function in clinical samples. Cytometry Part B (Clin. Cytometry) 55B:14–21, 2003. © 2003 Wiley‐Liss, Inc.
Correlation of protein expression and gene expression in acute leukemia
Authors: Kern Wolfgang , Kohlmann Alexander , Wuchter Christian , Schnittger Susanne , Schoch Claudia , Mergenthaler Susanne , Ratei Richard , Ludwig Wolf‐Dieter , Hiddemann Wolfgang , Haferlach Torsten
Published: August 2003

Background

Flow cytometry (FC) is a standard method for diagnosing and subclassifying acute myeloid (AML) and acute lymphoblastic (ALL) leukemias and allows the analysis of cell surface and intracellular proteins. In the future, diagnostic procedures may include oligonucleotide microarray analysis (MA) to detect expression patterns of large numbers of specific genes.

Methods

For comparison between methods, we performed FC and MA by using the Affymetrix GeneChip HG‐U133A microarray in parallel and correlated protein expression levels and mRNA abundance of 39 relevant genes in 113 patients with newly diagnosed AML and ALL and four normal bone marrow samples.

Results

In 1,512 of 2,187 (69.1%) comparisons between methods, congruent results were obtained with regard to positivity or negativity of expression, respectively. Specifically, there was a significant correlation between protein expression and mRNA abundance for genes essential for diagnosing and subclassifying AML and ALL with regard to positivity and expression.

Conclusions

These data suggest that protein expression is highly correlated to mRNA abundance in AML and ALL. Further, expression patterns of specific genes provide important information at diagnosis for patients with AML and ALL that may be used for the discrimination from other leukemias. Cytometry Part B (Clin. Cytometry) 55B:29–36, 2003. © 2003 Wiley‐Liss, Inc.
Sensitivity of hemozoin detection by automated flow cytometry in non‐ and semi‐immune malaria patients
Authors: Grobusch Martin P. , Hänscheid Thomas , Krämer Benedikt , Neukammer Jörg , May Jürgen , Seybold Joachim , Kun Jürgen F. J. , Suttorp Norbert
Published: August 2003

Background

Cell‐Dyn automated blood cell analyzers use laser flow cytometry technology, allowing detection of malaria pigment (hemozoin) in monocytes. We evaluated the value of such an instrument to diagnose malaria in febrile travelers returning to Berlin, Germany, the relation between the instrument's performance and the patients' immune status, and the capacity to increase its sensitivity.

Methods

Malaria diagnosis was routinely established by thick‐film microscopy. The patients' immune status was determined by an indirect fluorescent antibody test. Hemozoin detection was performed with a Cell‐Dyn 3000. To assess the capacity for sensitivity increase, the relative frequencies of pigment‐containing monocytes were determined for a subgroup of patients with the Cell‐Dyn 3000 in comparison with a flow cytometer specifically adapted to rare‐event analysis.

Results

Of 403 patients screened microscopically, 107 had malaria. Overall sensitivity with the Cell‐Dyn 3000 reached 48.6% (73.7% in semi‐immune and 28.6% in nonimmune individuals; P < 0.0001). Specificity was 96.2%. The detection limit was at a relative concentration of 2 × 10−4 pigment‐containing monocytes (PCMs). By employing rare‐event flow cytometry, the detection limit decreased to 3.25 × 10−5, thus yielding a considerably increased sensitivity for the subgroup studied.

Conclusions

The correlation between immune status and relative concentration of PCMs explains the failure of the routine instrument for nonimmune patients and its significantly higher sensitivity for semi‐immune individuals. The technique can be significantly improved by rare‐event flow cytometry. Cytometry Part B (Clin. Cytometry) 55B:46–51, 2003. © 2003 Wiley‐Liss, Inc.
Flow cytometry in primary breast carcinomas: Prognostic impact of multiploidy and hypoploidy
Authors: Michels Jean‐Jacques , Duigou Françoise , Marnay Jacques , Denoux Yves , Delozier Thierry , Chasle Jacques
Published: August 2003

Background

The aims of the present work were to study the prognostic impact of multiploidy and/or hypoploidy in breast cancers and their relation to other classic clinicopathologic prognostic factors (T, grade, receptors, and lymph node status).

Methods

From 3 January 1990 to 7 January 1999, 1984 previously untreated, invasive breast carcinoma samples were snap frozen for flow‐cytometry.

Results

Multiploid tumors had the same prognosis as the aneuploid ones, and those with one hypoploid peak had a better prognosis than did the other aneuploid tumors. However, the presence of both multiploid and hypoploid peaks was correlated with a poor outcome, even after multivariate analysis. In this series after quality control, 93.4% of the histograms could be evaluated concerning ploidy; of these 81.6% could be assessed concerning S‐phase fraction (SPF) in the entire population and 77.1% in the multiploid population. In the entire population, we performed a multivariate analysis including all relevant prognostic factors remaining after monovariate analysis by using a compound factor (proliferative activity) regrouping SPF and mitotic activity. This analysis showed that lymph node status and proliferative activity correlates with every type of survival, whereas receptor status correlates with all types of survival except recurrence free survival size, correlated with non‐metastasis and overall survival. Grade and age correlated only with overall survival and vascular permeations only with disease‐free survival.

Conclusions

SPF is a valuable predictor of survival, can be confidently assessed in multiploid histograms, and thus improves the yield of flow cytometry. When combined with mitotic activity, the prognostic impact of SPF is the same as that of lymph node status. Tumors that are hypoploid and multiploid have a significantly worse prognosis. Cytometry Part B (Clin. Cytometry) 55B:37–45, 2003. © 2003 Wiley‐Liss, Inc.
Comparison of intracellular cytokine production with extracellular cytokine levels using two flow cytometric techniques
Authors: Schuerwegh A. J. , De Clerck L. S. , Bridts C. H. , Stevens W. J.
Published: August 2003

BACKGROUND

We investigated the relation between intracellular cytokine production and extracellular cytokine levels by using two flow cytometric techniques.

METHODS

A two‐color flow cytometric technique was used to measure interleukin (IL)–1β, IL‐6, tumor necrosis factor α (TNF‐α), IL‐10, and IL‐12 production blocked intracellularly with brefeldin A in lipopolysaccharide (LPS)–stimulated CD14+ monocytes and IL‐2, IL‐4, and IFN‐γ production in phorbol‐12‐mirystate‐13‐acetate (PMA)–stimulated CD3+ T lymphocytes in samples from patients with rheumatoid arthritis. A flow cytometric microsphere‐based immunoassay was performed to detect cytokine secretion in plasma of PMA‐ and LPS‐stimulated whole blood samples.

RESULTS

There was a strong linear correlation between extracellular quantitative (pg/ml) and intracellular semiquantitative detection of LPS‐stimulated IL‐1β, IL‐6, IL‐10, and IL‐12 production (r > 0.9). For lymphocytes, extracellularly detected IL‐2 and IFN‐γ correlated well with percentages of cytokine‐producing cells (r > 0.8). The percentages of IL‐4–positive T cells were moderately correlated with the secreted amounts of IL‐4 as detected with the microsphere‐based immunoassay (r = 0.7).

CONCLUSION

Overall, there was a good correlation between semiquantitative intracellular detection of cytokines and the secreted amounts of cytokines detected with the microsphere based immunoassay. Cytometry Part B (Clin. Cytometry) 55B:52–58, 2003. © 2003 Wiley‐Liss, Inc.
Comparative immunophenotypic features of EBV‐positive and EBV‐negative atypical lymphocytosis
Authors: Hudnall S. David , Patel Jyoti , Schwab Hanna , Martinez José
Published: August 2003

Background

Atypical lymphocytosis is a common peripheral blood abnormality seen not only in Epstein‐Barr virus (EBV)‐associated acute infectious mononucleosis but also in other conditions, including other viral infections, cancer, immune reactions, etc. Despite numerous reports of individual immunophenotypic alterations in EBV‐positive infectious mononucleosis, a detailed comparative analysis of the immunophenotypic changes of peripheral blood lymphocyte subsets in infectious mononucleosis and other forms of atypical lymphocytosis is lacking.

Methods

Multiparametric flow immunocytometry with 26 monoclonal antibodies was performed on peripheral blood lymphocytes from 97 cases of atypical lymphocytosis and 37 normal controls. Atypical lymphocytosis was defined as absolute lymphocytosis with >10% atypical lymphocytes. Absolute or relative mean values of various lymphocyte subsets from EBV‐positive cases, EBV‐negative cases, and normal controls were compared with the Student's t‐test.

Results

Highly significant abnormalities detected in atypical lymphocytosis include increases in CD3+/CD8+, CD3−/CD16/56+, CD3+/γδ+, CD8+/CD48−, CD8+/CD57−, CD8+/CD95+, CD4+/CCR5+ CD4+/CD7−, CD4+/CD43−, CD4+/CD48−, and CD4+/CD62L− subsets. In contrast, no change in absolute CD4+ T cell and CD19+ B cell counts is seen. When compared with EBV‐negative cases, EBV‐positive cases are characterized by younger age, and increased numbers of absolute lymphocytes, atypical lymphocytes, CD8+ cells, NK cells, γδ T cells, CD8+/CD45RO+ cells, CD8+/CD57− cells, and CD8+/CD28+ cells.

Conclusions

All forms of atypical lymphocytosis are characterized by a marked increase in activated CD8‐positive T cells, a moderate increase in NK cells, and no increase in CD4‐positive T cells and B cells. Although morphologically indistinguishable, EBV‐associated mononucleosis is characterized by several significant differences in peripheral blood lymphocyte subsets when compared with EBV‐negative atypical lymphocytosis, most notably increased numbers of CD57‐negative CD8 T cells and γδ T cells. Cytometry Part B (Clin. Cytometry) 55B:22–28, 2003. © 2003 Wiley‐Liss, Inc.
Erratum
Authors: Marti Gerald E.
Published: August 2003

The original article to which this Erratum refers was published in Cytometry Part B: Clinical Cytometry(2003) 52B(1) 1–12.
Flow rate calibration II: A clinical evaluation study using PanLeucoGating as a single‐platform protocol
Authors: Storie Ian , Sawle Alex , Whitby Liam , Goodfellow Karen , Granger Vivian , Reilly John T. , Barnett David
Published: August 2003

Background

CD4+ T‐lymphocyte enumeration is vital for monitoring disease progression in individuals positive for the human immunodeficiency virus (HIV), and as a result, there is a need to develop cost‐effective protocols that provide accuracy, precision, and affordability. Recently, PanLeucoGating has been shown to fulfill these requirements; however, although comparable to state‐of‐the‐art single‐platform protocols (SP), there is still a requirement for an accurate total white cell count. To overcome this limitation, we recently developed a flow‐rate based calibration method that enables the PanLeucoGating protocol to be used as a SP approach, and in this study show that this approach can be used for CD4+ T‐lymphocyte enumeration.

Methods

A total of 113 HIV samples were analyzed using three protocols: (a) state‐of‐the art SP bead‐based method (MultiSet; predicate protocol), (b) PanLeucoGating protocol used as a dual‐platform (DP) approach, and (c) the newly developed flow rate–based SP approach. We demonstrate that flow rate calibration can be achieved easily and that the method is highly comparable to the state‐of‐the‐art SP method.

Results

A high correlation was observed between the predicate protocol and the SP PanLeucoGating approach over the whole range of CD4 counts tested (r2 = 0.9928; bias 8 cells/μl), including the clinically relevant range (e.g., 0–200 CD4 cells/μl; bias 0 cells/μl). For batched samples, the cost of providing a CD4+ T‐lymphocyte count was reduced to approximately US $1.

Conclusions

The SP PanLeucoGating is a cost‐effective approach to CD4+ T‐lymphocyte enumeration that maintains accuracy and precision. Cytometry Part B (Clin. Cytometry) 55B:8–13, 2003. © 2003 Wiley‐Liss, Inc.
Flow rate calibration I: A novel approach for performing absolute cell counts
Authors: Storie Ian , Sawle Alex , Goodfellow Karen , Whitby Liam , Granger Vivian , Reilly John T. , Barnett David
Published: August 2003

Background

Reports suggest that flow rate (FR) is constant on bench top flow cytometers. Therefore, if FR is constant, the volume acquired in a fixed time period will also be constant, enabling absolute leucocyte counting using flow rate calibration (FRC).

Methods

FR stability was ascertained on a standard FACSCalibur by counting TruCount beads suspended in phosphate buffered saline over 120 s. Studies using two lysing solutions (FACS lysing solution and PharM Lyse) and corresponding sample lysates established a lysing solution calibration factor (CF). Absolute CD4+T‐lymphocyte counts on 10 peripheral blood samples determined using FRC were compared with the predicate method TruCount/MultiTEST, incorporating MultiSET software. Linearity studies were also performed at three different flow rates.

Results

A high degree of linearity over a wide range of counts (50 to >1,600 CD4+T lymphocytes/μl) at all three pressures was observed. Importantly, there was no significant difference from the predicate method when appropriate lysing solution CF was used.

Conclusions

Using a simple calibration procedure and incorporation of an appropriate lysing solution CF, we show that FRC can easily be performed. The technical details that underpin this novel approach for absolute leucocyte enumeration are provided. Cytometry Part B (Clin. Cytometry) 55B:1–7, 2003. © 2003 Wiley‐Liss, Inc.
Patterning of embryonic blood vessels
Authors: LaRue Amanda C. , Mironov Vladimir A. , Argraves W. Scott , Czirók András , Fleming Paul A. , Drake Christopher J.
Published: August 2003

Morphometric methods were developed to characterize the geometry of vascular patterns in avian and murine embryos. By using these methods, we found that networks of blood vessels formed during vasculogenesis share similar geometric properties (i.e., mean blood vessel diameters and avascular space diameters) regardless of developmental stage, location, or species in which they form. We also found that endothelial cell density within a unit area of an embryonic vasculature could be used to accurately distinguish between a small diameter, capillary‐like vascular network (low endothelial cell density) and a large diameter, presinusoidal network (high endothelial cell density). Furthermore, we show that endothelial cell size remains constant in small and large diameter vessels, indicating that increased endothelial cell size is not the basis for diversity in vessel diameter. These observations serve as a foundation for future studies seeking to evaluate the effects of agents or genetic mutations on aspects of vasculogenesis. Developmental Dynamics 228:21–29, 2003. © 2003 Wiley‐Liss, Inc.
Timeless in lung morphogenesis
Authors: Xiao Jing , Li Changgong , Zhu Nian‐Ling , Borok Zea , Minoo Parviz
Published: August 2003

The Clock gene, timeless, regulates circadian rhythm in Drosophila, but its vertebrate homolog is critical to embryonic development. Timeless was shown to be involved in murine urethral bud branching morphogenesis. We generated a polyclonal antibody to mouse TIMELESS (mTIM) and studied its distribution and its potential role during lung development, which also requires branching morphogenesis. In the early mouse embryo, TIM was localized to all organs, especially the neural epithelium. In embryonic day (E) 9.5 embryos, TIM was present in both epithelial and mesenchymal cells at the onset of lung morphogenesis. In E15 embryos, TIM decreased in the mesenchyme but remained pronounced in the epithelium of both large and small airways. Later, TIM was localized to a specific subset of epithelial cells with alveolar type 2 phenotype. This finding was verified by immunostaining of isolated alveolar type 2 cells. In the proximal airways, TIM was colocalized with CCSP to nonciliated columnar epithelial cells. Antisense oligonucleotides to mTim specifically inhibited branching morphogenesis of embryonic lungs in explant culture without affecting SpC expression an alveolar type 2 cell marker. In cultured lung cells, expression of TIM is independent of cell cycle and proliferation. These studies indicate that the function of Timeless is highly conserved in organs whose formation requires branching morphogenesis. Developmental Dynamics 228:82–94, 2003. © 2003 Wiley‐Liss, Inc.
Analysis of transcription regulatory regions of embryonic chicken pepsinogen (ECPg) gene
Authors: Watanuki Kumiko , Yasugi Sadao
Published: August 2003

Genes encoding pepsinogens, zymogens of digestive enzyme pepsins, are expressed specifically in the gland epithelial cells of the vertebrate stomach, and their expression is also developmentally regulated, therefore providing a good model for the analysis of transcriptional regulation of genes. In the development of chicken embryonic stomach, the epithelium invaginates into the mesenchyme and forms glands and gland epithelial cells then begin to express embryonic chicken pepsinogen (ECPg) gene. It has been shown that cGATA5 binds directly GATA binding sites located within 1.1‐kbp upstream of ECPg gene and activates its transcription. To find more precisely the sequences necessary for ECPg gene transcription, we carried out deletion and mutation analysis with 1.1‐kbp upstream region. The results suggest that binding of GATA factor to three GATA binding sites within the upstream region −656 to −419 synergistically regulates ECPg expression in the gland epithelial cells. Developmental Dynamics 228:51–58, 2003. © 2003 Wiley‐Liss, Inc.
Chromatin reorganization accompanying cellular dedifferentiation is associated with modifications of histone H3, redistribution of HP1, and activation of E2F‐target genes
Authors: Williams Leor , Zhao Jing , Morozova Nadya , Li Yan , Avivi Yigal , Grafi Gideon
Published: August 2003

The remarkable regeneration capacity of plant cells is based on their capability to dedifferentiate. We recently reported that cellular dedifferentiation proceeds through two distinct phases, each accompanied by chromatin decondensation: acquisition of competence for fate switch followed by a signal‐dependent reentry into S phase (Zhao et al. [2001] J. Biol. Chem. 276:22772‐22778). The purpose of this study was to (1) characterize changes in chromatin factors associated with chromatin decondensation, and (2) study the relationship between chromatin decondensation and transcriptional activation of pRb/E2F‐regulated genes. We show that plant cells competent for fate switch display a disruption of nucleolar domain appearance associated with condensation of 18S ribosomal DNA, as well as modifications of histone H3 and redistribution of heterochromatin protein 1 (HP1). We further show that the pRb/E2F‐target genes RNR2 and PCNA are condensed and silent in differentiated leaf cells but become decondensed, although not yet activated, as cells acquire competence for fate switch; transcriptional activation becomes evident during progression into S phase, concomitantly with pRb phosphorylation. We propose that chromatin reorganization is central for reversion of the differentiation process leading to resetting of the gene expression program and activation of silent genes. Developmental Dynamics 228:113–120, 2003. © 2003 Wiley‐Liss, Inc.
Vesicle‐associated protein‐A is differentially expressed during intestinal smooth muscle cell differentiation
Authors: Gabetta Vijayalakshmi , Trzyna Wendy , Phiel Christopher , McHugh Kirk M.
Published: August 2003

Gastrointestinal (GI) smooth muscle diseases represent a major health concern affecting in excess of 2 million people each year. Little is currently known regarding the molecular mechanisms controlling either normal or pathogenic GI smooth muscle development. In an effort to identify the specific gene products responsible for modulating GI smooth muscle cell (SMC) differentiation, we performed differential display on distinct intestinal SMC (ISMC) phenotypes. This analysis identified over 40 unique transcripts that appeared to be differentially expressed in distinct SMC phenotypes. One such transcript that appeared to be preferentially expressed in immature smooth muscle myocytes was identified as vesicle‐associated membrane protein, associated protein A (VAP‐A). Northern blot analysis confirmed that VAP‐A was expressed threefold higher in immature smooth muscle myocytes when compared with both smooth muscle myoblasts and mature smooth muscle myocytes. VAP‐A mRNA was differentially expressed during normal rat development and showed peak levels of expression in the intestine during late embryogenesis and early neonatal development. These observations provide the first evidence that VAP‐A–mediated membrane trafficking may play an important role in modulating ISMC differentiation. Developmental Dynamics 228:11–20, 2003. © 2003 Wiley‐Liss, Inc.
HOXB5 expression is spatially and temporarily regulated in human embryonic gut during neural crest cell colonization and differentiation of enteric neuroblasts
Authors: Fu Ming , Chi Hang Lui Vincent , Har Sham Mai , Nga Yin Cheung Annie , Kwong Hang Tam Paul
Published: August 2003

HOX genes from paralogous groups 4 and 5 are particularly relevant to the gut neuromusculature development because these genes are expressed at the splanchnic mesoderm surrounding the gut diverticulum, and at the level of the neural tube from where the vagal neural crest cells (NCCs) originate. In this study, we examined the migration and differentiation of NCCs, and investigated the expression patterns of HOXB5 in human embryonic guts. Human embryos of gestational week‐4 to ‐8.5 were studied. Vagal NCCs enter the esophagus, migrate, and colonize the entire gut in a rostrocaudal manner between week‐4 and week‐7. The migrating NCCs in gut express HOXB5. Two separate and discontinuous mesenchymal expression domains of HOXB5 were detected in the gut: the distal domain preceding the migratory NCCs; and the proximal domain overlapping with the NCCs. The two expression domains shift caudally in parallel with the rostrocaudal migration of NCCs between week‐4 and week‐5. Neuron and glia differentiation of NCCs are concomitant with HOXB5 down‐regulation in NCCs and the mesenchyme. By week‐7, myenteric plexuses have formed; HOXB5 expression is switched on in the plexuses. We found that (1) the migratory route of NCCs in human embryonic gut was similar to that in mice and chicks; and (2) the expression pattern of HOXB5 correlated with the migration and differentiation of NCCs, suggesting a regulatory role of HOXB5 in the development of NCCs. Developmental Dynamics 228:1–10, 2003. © 2003 Wiley‐Liss, Inc.
Pak1 and its T212 phosphorylated form accumulate in neurones and epithelial cells of the developing rodent
Authors: Zhong Julia L. , Banerjee Monisha D. , Nikolic Margareta
Published: August 2003

The serine/threonine kinase Pak1 is a target of the RhoGTPases Rac and Cdc42 and an important regulator of cell morphology and migration. Recent work from several laboratories has indicated that Pak1 controls microtubule dynamics as well as the organisation of F‐actin microfilaments. Pak1 is phosphorylated on T212 by the p35/Cdk5 or cyclin B1/Cdc2 kinase in postmitotic neurones and mitotic cells, respectively. To understand its function during development, we have carried out a detailed temporal and spatial analysis of Pak1 expression and phosphorylation on T212. In the embryonic forebrain, Pak1 and Pak1T212(PO4) were seen to accumulate in the corpus callosum, intermediate zone, lateral olfactory tracts, and anterior commissures. Epithelial cells of the mouse embryo lung, kidney, intestine, and skin also exhibited high levels of Pak1 and Pak1T212(PO4), suggesting a previously unsuspected role in epithelial differentiation. Pak1T212(PO4) was undetectable in all adult tissues. Together, these data indicate a specific, developmentally regulated role of the Pak1 kinase. Developmental Dynamics 228:121–127, 2003. © 2003 Wiley‐Liss, Inc.
Temporospatial gene expression and protein localization of matrix metalloproteinases and their inhibitors during mouse molar tooth development
Authors: Yoshiba Nagako , Yoshiba Kunihiko , Stoetzel Corinne , Perrin‐Schmitt Fabienne , Cam Yves , Ruch Jean Victor , Lesot Hervé
Published: August 2003

The gene expression and protein distribution of matrix metalloproteinase (MMP) ‐2, ‐9, membrane type‐1 MMP (MT1‐MMP), as well as of TIMP‐1, ‐2, and ‐3 were analyzed during mouse molar development. Immunohistochemical data demonstrated that all the MMPs investigated were expressed in the dental epithelium and mesenchyme. In contrast, gene and protein expression analysis for TIMPs showed that they were differentially expressed. TIMP‐1 was expressed in the dental epithelium and mesenchyme between E13 and E16 and was transiently up‐regulated at E14, the cap stage. TIMP‐1 expression was also detected in differentiating odontoblasts. TIMP‐2 RNA transcripts were found in the peridental and dental mesenchyme, odontoblasts, and ameloblasts. Protein analysis revealed high expression on the lingual side of the dental epithelium and underlying mesenchyme together with transient expression in the enamel knot at E14 and expression in the gingival tissue and enamel matrix postnatally. TIMP‐3 RNA transcripts were found in discrete regions of the dental epithelium, including at high levels in the cervical loop at E16. Expression was also detected in preodontoblasts at E16 and transiently during ameloblast differentiation. Analysis of the protein distribution revealed a lower level of TIMP‐3 on the lingual side of the dental epithelium at E14. MT1‐MMP was expressed in the dental mesenchyme between E13 and E16, at relatively high levels in the cervical loop at E14, and in the odontoblasts and ameloblasts. The distinct temporospatial distribution patterns of the TIMPs suggest that these inhibitors play several intrinsic roles during tooth development. Developmental Dynamics 228:105–112, 2003. © 2003 Wiley‐Liss, Inc.
Mouse pigpen encodes a nuclear protein whose expression is developmentally regulated during craniofacial morphogenesis
Authors: Alappat Sylvia R. , Zhang Meifeng , Zhao Xiang , Alliegro Mary Anne , Alliegro Mark C. , Burdsal Carol A.
Published: August 2003

Pigpen, a nuclear protein with RNA‐binding motifs and a putative transcriptional activation domain (TAD), is expressed at high levels in proliferating endothelial cells and expression is down‐regulated when cells adopt a quiescent or differentiated phenotype. We cloned the mouse homolog of pigpen and investigated the regulation of its expression during embryogenesis. In situ hybridization demonstrated that a broad pattern of pigpen expression became restricted during tooth formation in the mandible. In the eye, pigpen showed a spatial restriction to the more proliferating and less differentiated regions of the lens and neural retina. Expression was also restricted in the developing vibrissae, lung, and kidney, all sites where epithelial‐mesenchymal interactions are vital for morphogenesis. In vitro assays, that focused on the mandible and tooth development, indicated that epithelial signals, mediated by fibroblast growth factor‐8, were required to maintain pigpen expression in the mandibular mesenchyme, whereas bone morphogenetic protein‐4 negatively regulated expression in that tissue during early odontogenesis. At the protein level, immunocytochemistry demonstrated that Pigpen was expressed diffusely in the cytoplasm and more concentratedly in focal granules within the nuclei of mouse embryonic cells. Lastly, CAT reporter assays showed that the N‐terminus of mouse pigpen encodes an active TAD. These data suggest that mouse Pigpen may activate transcription in vivo in response to specific growth factor signals and regulate proliferation and/or differentiation events during mouse organogenesis. Developmental Dynamics 228:59–71, 2003. © 2003 Wiley‐Liss, Inc.
Ephs and ephrins during early stages of chick embryogenesis
Authors: Baker Robert K. , Antin Parker B.
Published: August 2003

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are membrane‐bound proteins that mediate bidirectional signals between adjacent cells. By modulating cytoskeleton dynamics affecting cell motility and adhesion, Ephs and ephrins orchestrate cell movements during multiple morphogenetic processes, including gastrulation, segmentation, angiogenesis, axonal pathfinding, and neural crest cell migration. The full repertoire of developmental Eph/ephrin functions remains uncertain, however, because coexpression of multiple receptor and ligand family members, and promiscuous interactions between them, can result in functional redundancy. A complete understanding of expression patterns, therefore, is a necessary prerequisite to understanding function. Here, we present a comprehensive expression overview for 10 Eph and ephrin genes during the first 48 hr of chick embryo development. First, dynamic expression domains are described for each gene between Hamburger and Hamilton stages 4 and 12; second, comparative analyses are presented of Eph/ephrin expression patterns in the primitive streak, the somites, the vasculature, and the brain. Complex spatially and temporally dynamic expression patterns are revealed that suggest novel functions for Eph and ephrin family members in both known and previously unrecognized processes. This study will provide a valuable resource for further experimental investigations of Eph and ephrin functions during early embryonic development. Developmental Dynamics 228:128–142, 2003. © 2003 Wiley‐Liss, Inc.
Coordinated and conserved expression of alphoid repeat and alphoid repeat‐tagged coding sequences
Authors: Li Yin‐Xiong , Kirby Margaret L.
Published: August 2003

We have found an alpha‐like simple‐sequence DNA repeat that is differentially expressed during early embryogenesis in both chick and zebrafish. Before and during the primitive streak stage, transcripts of the alphoid repeat sequence were ubiquitously expressed throughout zebrafish and chick embryos. After headfold formation, expression was limited to the cardiac neural crest, the head, and the heart. Two types of alphoid repeat sequence transcripts were identified: alphoid repeat RNA and alphoid repeat‐tagged mRNA (ESαT). Several of the ESαTs were identified by (1) searching expressed sequence tag databases, (2) arbitrary rapid amplification of cDNA ends (RACE), and (3) screening embryonic cDNA libraries. The alphoid element was located in the 3′ untranslated region of one ESαT that was obtained by RACE. The ESαT sequences encoded a variety of different types of proteins, but all were expressed within tissues that were positive for the alphoid repeat RNA. The presence of two types of coordinately expressed alphoid‐like repeat transcripts in maternal RNA with subsequent restriction to the head and heart, and the conservation of these features in disparate vertebrate embryos, suggest that the alphoid repeat sequence may serve as a control element in the gene regulation network. Developmental Dynamics 228:72–81, 2003. © 2003 Wiley‐Liss, Inc.