bluuebunny/crossref_metadata_2025 huggingface dataset:
Students' conceptual structure, search process, and outcome while preparing a research proposal: A longitudinal case study
Authors: Pennanen Mikko , Vakkari Pertti
Published: May 2003

This article focuses on analysing students' information needs in terms of conceptual understanding of the topic they propose to study and its consequences for the search process and outcome. The research subjects were 22 undergraduates of psychology attending a seminar for preparing a research proposal for a small empirical study. They were asked to make searches in the PsycINFO database for their task in the beginning and end of the seminar. A pre‐ and postsearch interview was conducted in both sessions. The students were asked to think aloud in the sessions. This was recorded, as were the transaction logs. The results show that during the preparation of research proposals different features of the students' conceptual structure were connected to the search success. Students' ability to cover their conceptual construct by query terms was the major feature affecting search success during the whole process. In the beginning also the number of concepts and the proportion of subconcepts in the construct contributed indirectly via search tactics to retrieving partly useful references. Students' ability to extract new query terms from retrieved items improved search results.
Dermatomes and the central organization of dermatomes and body surface regions in the spinal cord dorsal horn in rats
Authors: Takahashi Yuzuru , Chiba Tanemichi , Kurokawa Masahiro , Aoki Yasuchika
Published: May 2003

Dermatomes and the associated central projection fields were studied with the application of fluorescent neurotracer, 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI), to 21 reference points on rat trunk and hindlimb skin. Segmental distribution and rostrocaudal central level of dorsal root ganglion (DRG) neurons innervating reference points were examined and DiI‐induced fluorescent areas were mapped in the horizontal plane through lamina II of the dorsal horn. Segmental levels of DRG neurons innervating reference points were generally identical to the level determined using dye‐extravasation methods. However, innervation of the first digit was situated in the L4 dermatome, not the L3 reported previously using those methods. Generally, afferents from a reference point projected to a single field in the ipsilateral dorsal horn. Reference points on ventral and dorsal median lines of the trunk were represented bilaterally. Afferents from reference points located on the ventral median line of the hindlimb projected to two separate fields: one on the medial margin of spinal cord segments L2–L5 and the other on the medial half of spinal cord segment L5. From the distribution of central projection fields of reference points, central projection fields of dermatomes were revealed as even in shape and located within corresponding spinal cord segments. The arrangement of peripheral and central fields of dermatomes and body surface regions suggests that peripheral and central projection fields of cutaneous afferent fibers are reshaped from the common prototypical pattern that exhibits an orderly and evenly sequenced arrangement. J. Comp. Neurol. 462:29–41, 2003. © 2003 Wiley‐Liss, Inc.
Expression and function of FGF10 in mammalian inner ear development
Authors: Pauley Sarah , Wright Tracy J. , Pirvola Ulla , Ornitz David , Beisel Kirk , Fritzsch Bernd
Published: May 2003

We have investigated the expression of FGF10 during ear development and the effect of an FGF10 null mutation on ear development. Our in situ hybridization data reveal expression of FGF10 in all three canal crista sensory epithelia and the cochlea anlage as well as all sensory neurons at embryonic day 11.5 (E11.5). Older embryos (E18.5) displayed strong graded expression in all sensory epithelia. FGF10 null mutants show complete agenesis of the posterior canal crista and the posterior canal. The posterior canal sensory neurons form initially and project rather normally by E11.5, but they disappear within 2 days. FGF10 null mutants have no posterior canal system at E18.5. In addition, these mutants have deformations of the anterior and horizontal cristae, reduced formation of the anterior and horizontal canals, as well as altered position of the remaining sensory epithelia with respect to the utricle. Hair cells form but some have defects in their cilia formation. No defects were detected in the organ of Corti at the cellular level. Together these data suggest that FGF10 plays a major role in ear morphogenesis. Most of these data are consistent with earlier findings on a null mutation in FGFR2b, one of FGF10's main receptors. Developmental Dynamics 227:203–215, 2003. © 2003 Wiley‐Liss, Inc.
Folic acid and homocysteine affect neural crest and neuroepithelial cell outgrowth and differentiation in vitro
Authors: Boot Marit J. , Steegers‐Theunissen Régine P.M. , Poelmann Robert E. , Van Iperen Liesbeth , Lindemans Jan , Gittenberger‐De Groot Adriana C.
Published: May 2003

The beneficial effect of additional folic acid in the periconceptional period to prevent neural tube defects, orofacial clefts, and conotruncal heart defects in the offspring has been shown. Folate shortage results in homocysteine accumulation. Elevated levels of homocysteine have been related to neural tube defects. We studied the behavior of neuroepithelial cells and cranial and cardiac neural crest cells in vitro. Neural tube explants were cultured for 24 and 48 hr in medium after addition of folic acid and/or homocysteine. Folic acid addition increased neuroepithelial cell outgrowth and increased neural crest cell differentiation into nerve and smooth muscle cells. Addition of homocysteine increased neural crest cell outgrowth and migration from the neural tube and inhibited neural crest cell differentiation. Our findings suggest that neural tube defects caused by folate deficiency and hyperhomocysteinemia develop due to increased neuroepithelial to neural crest cell transformation. This increased transformation leads to a shortage of neuroepithelial cells in the neural tube. Defects in orofacial and conotruncal development are explained by abnormal differentiation of neural crest cells in the presence of high homocysteine concentrations. Our findings supports a critical role for folic acid and homocysteine in the development of neural tube defects and neural crest related heart malformations. Developmental Dynamics 227:301–308, 2003. © 2003 Wiley‐Liss, Inc.
Molecular structure and developmental expression of three muscle‐type troponin T genes in zebrafish
Authors: Hsiao Chung‐Der , Tsai Wei‐Yuan , Horng Long‐Shyan , Tsai Huai‐Jen
Published: May 2003

Troponin T (Tnnt), a troponin component, interacts with tropomyosin and is crucial to the regulation of striated muscle contraction. To gain insight into the molecular evolution and developmental regulation of Tnnt gene (Tnnt) in lower vertebrates, zebrafish Tnnt1 (slow Tnnt), Tnnt2 (cardiac Tnnt), and Tnnt3b (fast Tnnt isoform b) were characterized. The polypeptides of zebrafish Tnnt1, Tnnt2, and Tnnt3b were conserved in the central tropomyosin‐ and C‐terminal troponin I‐binding domains. However, the N‐terminal hypervariable regions were highly extended and rich in glutamic acid in polypeptides of Tnnt1 and Tnnt2, but not Tnnt3b. The Tnnt2 and Tnnt3b contain introns, whereas Tnnt1 is intron‐free. During development, large to small, alternatively spliced variants were detected in Tnnt2, but not in Tnnt1 or Tnnt3. Whole‐mount in situ hybridization showed zebrafish Tnnt1 and Tnnt2 are activated during early somitogenesis (10 hr postfertilization, hpf) and cardiogenesis (14 hpf), respectively, but Tnnt3b is not activated until middle somitogenesis (18 hpf). Tnnt2 and Tnnt3b expression was cardiac‐ and fast‐muscle specific, but Tnnt1 was expressed in both slow and fast muscles. We propose that three, distinct, muscle‐type Tnnt evolved after the divergence of fish and deuterostome invertebrates. In zebrafish, the developmental regulation of Tnnt during somitogenesis and cardiogenesis is more restricted and simpler than in tetrapods. These new findings may provide insight into the developmental regulation and molecular evolution of vertebrate Tnnt. Developmental Dynamics 227:266–279, 2003. © 2003 Wiley‐Liss, Inc.
The Substituent Effect of the Phosphaalkenyl Group
Authors: Termaten Arjan , van der Sluis Marcel , Bickelhaupt Friedrich
Published: May 2003

Several novel phosphaalkenes (E)‐Mes*P=CHC6H4X (6; X = Me3Si, Me3Sn, CHO, COOH, CN) were prepared and, together with other known members of this series, subjected to an analysis of linear free‐energy relationships based on the values of pKa and values of δ(13Cpara) of substituted benzoic acids and on the values of δ(13Cpara) of substituted bromobenzenes. These analyses indicate that the (E)‐Mes*P=CH group is a weak electron donor with a predominantly inductive effect (σp = −0.1) on a neighboring benzene ring; resonance interactions are of minor importance. This result shows that the P=C bond is surprisingly analogous to the C=C bond, and thus confirms the well‐known diagonal relationship in the Periodic System between carbon and phosphorus atoms. The relationship between X and the values of δ(31P) of 6 is quite pronounced. It confirms the dominance of inductive interactions on the value of δ(31P); only with strongly electron‐donating substituents may a certain degree of resonance interaction be involved. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Blocking Fluorine Substitution in Biotransformation of Nortricyclanyl N‐Phenylcarbamates with Beauveria bassiana
Authors: Haufe Günter , Wölker Dörthe
Published: May 2003

The biotransformation of tricyclo[2.2.1.02,6]hept‐3‐yl N‐phenylcarbamate (8) by a standard procedure using Beauveria bassiana gave a 7:1 mixture of optically active exo,exo‐ and exo,endo‐5‐hydroxytricyclo[2.2.1.02,6]hept‐3‐yl N‐phenylcarbamates 15 and 16 in 19% isolated yield. No ring opening of the three‐membered ring was observed. Substitution with a fluorine atom at the 5‐endo‐ or 5‐exo‐position prevented hydroxylation of any alicyclic position of the molecules, p‐hydroxylation of the aromatic ring occurring to a small extent instead. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Ten‐Membered Substituted Cyclic 2‐Oxecanone (Decalactone) Derivatives from Latrunculia corticata, a Red Sea Sponge
Authors: Řezanka Tomáš , Dembitsky Valery M.
Published: May 2003

Two novel glycosides, latrunculinosides A and B, containing substituted 2‐oxecanones (decalactones) were isolated from Latrunculia corticata collected in the Gulf of Aqaba, Israel. They were characterized by spectroscopic methods, predominantly 1H NMR, 13C NMR, MS, IR and UV, and by chemical degradation. Both glycosides contain unusual saccharides such as β‐D‐olivose, β‐L‐digitoxose, α‐L‐amicetose, and β‐D‐oliose. The compounds gave positive results in antifeeding activity assays. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Potential role of radial glia in adult neurogenesis of teleost fish
Authors: Zupanc Günther K.H. , Clint Sorcha C.
Published: May 2003

Persistence of radial glia within the adult central nervous system is a widespread phenomenon among fish. Based on a series of studies in the teleost species Apteronotus leptorhynchus, we propose that one function of this persistence is the involvement of radial glia in adult neurogenesis, i.e., the generation and further development of new neurons in the adult central nervous system. In particular, evidence has been obtained for the involvement of radial glia in the guidance of migrating young neurons in both the intact and the regenerating brain; for a possible role as precursor cells from which new neurons arise; and for its role as a source of trophic substances promoting the generation, differentiation, and/or survival of new neurons. These functions contribute not only to the potential of the intact brain to generate new neurons continuously, and of the injured brain to replace damaged cells by newly generated ones, but they also provide an essential part of the cellular substrate of behavioral plasticity. GLIA 43:77–86, 2003. © 2003 Wiley‐Liss, Inc.
Long‐term implantation of zinc‐releasing calcium phosphate ceramics in rabbit femora
Authors: Kawamura H. , Ito A. , Muramatsu T. , Miyakawa S. , Ochiai N. , Tateishi T.
Published: May 2003

Zinc is an essential trace element that has stimulatory effects on bone formation. Recently, we developed zinc‐releasing calcium phosphate ceramics in order to add the pharmacologic effect of zinc to calcium phosphate ceramics. In our previous study, we showed that the optimum zinc content for promoting bone formation was 0.316 wt %. Therefore a zinc composite ceramic of zinc‐containing β‐tricalcium phosphate and hydroxyapatite, with a zinc content of 0.316 wt %, was chosen for long‐term implantation. Cylindrical rods of the zinc composite ceramic were implanted in rabbit femora for 2 to 60 weeks. Using computer‐aided image analysis, a histomorphometric study was carried out to investigate bone formation and resorption around the implants. The control was a composite ceramic of β‐tricalcium phosphate and hydroxyapatite without zinc. The addition of zinc to the implant demonstrated both favorable and unfavorable effects on bone remodeling. The favorable effect was enhanced bone apposition to the implant surface, demonstrated by a significant increase in intramedullary bone apposition rate at 6 weeks and in cortical bone apposition rate at 24 and 60 weeks (p < 0.05). The unfavorable effect was increased bone resorption, demonstrated by a significant increase in medullary cavity area at 60 weeks (p < 0.05). In order to utilize the favorable effect and avoid the unfavorable effect of zinc, either a reduction in zinc content in the zinc composite ceramic or the selection of implantation sites that do not have excessive exposure to bone marrow are required. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 468–474, 2003
Relative influence of polymer fiber diameter and surface charge on fibrous capsule thickness and vessel density for single‐fiber implants
Authors: Sanders J. E. , Cassisi D. V. , Neumann T. , Golledge S. L. , Zachariah S. G. , Ratner B. D. , Bale S. D.
Published: May 2003

Single polypropylene microfibers plasma‐coated with polymers of different surface charge [N,N‐dimethylaminoethyl methacrylate (NN) (positive charge), methacrylic acid (MA) (negative charge), and hexafluoropropylene (HF) (neutral)] were implanted in the subcutaneous dorsum of Sprague‐Dawley rats for 5‐week intervals. Thee groups of fiber diameters were used: (I) 1.0 to 5.9 μm; (II) 6.0 to 10.9 μm; and (III) 11.0 to 15.9 μm. Fibrous capsule thickness and blood‐vessel density (number of vessels within 100 μm of the fiber) were assessed in tissue sections in the planes of microfiber cross‐sections. Results from a multifactorial analysis of variance demonstrated statistically significant main effects (p < 0.05) for microfiber diameter but not for surface‐charge coating. The mean differences in capsule thickness among the microfiber diameter groups were: between groups II and I: 5.4 μm; between groups III and I: 10.2 μm; and between groups III and II: 4.7 μm. The mean differences in capsule thickness among surface‐charge coatings were: between MA and NN: 0.7 μm; between MA and HF: 1.4 μm; and between NN and HF: 0.7 μm. Many of the 1.0 to 5.9 μm‐in‐diameter fibers had no capsule and no sign of a foreign‐body reaction. For the vessel density analysis, neither microfiber diameter nor surface‐charge coating had a statistically significant effect. Thus the geometric feature of microfiber diameter was more important than was surface charge relative to fibrous capsule formation but not relative to local vessel density. This ranking of the relative influence of design features in relation to tissue response provides useful information for prioritization in biomaterial design. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 462–467, 2003
Enhancing the vascularization of three‐dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres
Authors: Perets Anat , Baruch Yaacov , Weisbuch Felix , Shoshany Gideon , Neufeld Gera , Cohen Smadar
Published: May 2003

Site‐specific delivery of angiogenic growth factors from tissue‐engineered devices should provide an efficient means of stimulating localized vessel recruitment to the cell transplants and would ensure cell survival and function. In the present article, we describe the construction of a novel porous alginate scaffold that incorporates tiny poly (lactic‐co‐glycolic acid) microspheres capable of controlling the release of angiogenic factors, such as basic fibroblast growth factor (bFGF). The microspheres are an integral part of the solid alginate matrix, and their incorporation does not affect the scaffold porosity or pore size. In vitro, bFGF was released from the porous composite scaffolds in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of cardiac fibroblasts. The controlled delivery of bFGF from the three‐dimensional scaffolds accelerated the matrix vascularization after implantation on the mesenteric membrane in rat peritoneum. The number of penetrating capillaries into the bFGF‐releasing scaffolds was nearly fourfold higher than into the control scaffolds (those incorporating microspheric BSA and heparin but not bFGF). At day 10 posttransplantation, capillary density in the composite scaffolds was 45 ± 3/mm2 and it increased to 70 ± 7/mm2 by day 21. The released bFGF induced the formation of large and matured blood vessels, as judged by the massive layer of mural cells surrounding the endothelial cells. The control over bFGF delivery and localizing its effects to areas of need, may aid in the wider application of bFGF in therapeutic angiogenesis as well as in tissue engineering. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 489–497, 2003
Effect of biphasic calcium phosphate on human macrophage functions in vitro
Authors: Silva S. N. , Pereira M. M. , Goes A. M. , Leite M. F.
Published: May 2003

Bioceramics may initiate several and complex biological reactions in host tissues. The cell–biomaterial interaction can determine macrophage activation that may elicit and sustain inflammatory response at the implant site. The current study describes some of the in vitro phenomena regarding the effect of surface reactivity of biphasic calcium phosphate (BCP) granules on human macrophages locomotion and secretion. X‐ray diffraction analysis indicated that the synthesized ceramic presented 80% hydroxyapatite and 20% tricalcium phosphate. When BCP was put in contact with human macrophage cells, we observed that cells and BCP granules attached to each other. Cells attached to BCP presented a higher intracellular free Ca2+ concentration compared with nonattached neighbors and secreted calcium phosphate particles into the medium. Energy dispersive X‐ray analysis showed that the secreted particles presented a calcium/phosphorus ratio of 1.64 ± 0.05, similar to hydroxyapatite. We propose that the secreted particles create a transition zone that allows further macrophage adhesion. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 475–481, 2003
Increased gap junctional intercellular communication is directly related to the anti‐tumor effect of all‐trans‐retinoic acid plus tamoxifen in a human mammary cancer cell line
Authors: Sáez Claudia G. , Velásquez L. , Montoya M. , Eugenín E. , Alvarez M.G.
Published: May 2003

Additive effects against tumor cells might be achieved by combining anti‐neoplastic agents directed against one or more altered mechanisms in cancer. We investigated the participation of gap junctional intercellular communication (GJIC), which is commonly dysfunctional in tumor cells as a possible mediating mechanism of the effect of all‐trans‐retinoic acid (RA) and tamoxifen (Tx) in MCF‐7 human breast cancer cell lines. The combination of RA + Tx stimulated GJIC in approximately 53 ± 3% of MCF‐7 cells as early as after 6 h of treatment remaining communicated through 144 h of culture. The GJIC enhancement occurred along with immunolocalization of Cx26 and 43 at the membrane of contacting cells and correlated with higher protein levels. Cx40 immunoreactive plaques were detected at cell‐to‐cell contacts during 48 h of RA + Tx treatment that did not involve higher protein expression, to the contrary, a downregulation occurred after 72 h of treatment. Cell proliferation inhibition upon RA + Tx exposure was observed with optimal effects at 96–120 h of culture with an accumulation of cells primarily in G2/M and G0/G1 cell cycle boundaries. An enhancement of the pre‐existing E‐cadherin levels was observed after drug exposure along with a downregulation of Bcl‐2 and C‐myc protein levels and a reduction of telomerase activity, suggesting partial tumor phenotype reversion. Blockage of the RA + Tx‐induced GJIC with 18‐β‐glycyrrhetinic acid (β‐Gly) prevented in 34% the inhibition of MCF‐7 proliferation and the E‐cadherin increment in 30% at 96 h of culture. GJIC blockage did not alter the downregulation of Bcl‐2, c‐Myc, or telomerase activity induced by RA + Tx. Our results showed the participation of GJIC as a mediator mechanism of the combined action of RA and Tx in MCF‐7 cells. The chemopreventive modulation of GJIC might represent an approachable alternative for the improvement of cancer therapy. J. Cell. Biochem. 89: 450–461, 2003. © 2003 Wiley‐Liss, Inc.
Regulation of p27 in the process of neuroblastoma N2A differentiation
Authors: Muñoz Juan P. , Sánchez Jaime R. , Maccioni Ricardo B.
Published: May 2003

Neuronal differentiation implies morphological and biochemical changes to generate a specialized neuron. N2A neuroblastoma cells can be promoted to undergo differentiation associated to neurites outgrowth, a process linked to the arrest of cell division. Using N2A cells as a model, we investigated the detailed molecular aspects on the involvement of p27 in dibutyryl cAMP‐induced neuronal differentiation. In the undifferentiated N2A phenotype, an unusually high level of accumulated p27 protein mass was evidenced. Data suggest that in proliferating cells, p27 could be sequestered by direct interaction with cyclin D1, thus preventing its inhibitory action on cell cycle Cdks. Studies also indicate that p27 is functionally active and that its loss of action on Cdks in proliferating cells is due to its strong association with cyclin D1. Therefore, when cell differentiation is triggered, the action of p27 on Cdks seems to depend on both p27 and cyclin D1 degradation during the early steps of differentiation followed by late events of re‐synthesis of active p27. In this context, an overexpression of p27 after N2A transfection with a mouse p27 clone induces the outgrowth of neurites associated with a decrease in cyclin D1 expression. On the other hand, treatment of N2A undifferentiated cells with c‐myc antisense oligonucleotides led to a decrease in p27 and cyclin D1 levels, similar events as those in early stages of cell differentiation. Studies suggest that blockage in c‐myc expression triggers early events in neuronal differentiation. These studies are of the utmost importance to elucidate regulatory mechanisms of molecules that play a critical role in the transition from a proliferating phenotype to differentiated cells. © 2003 Wiley‐Liss, Inc.
Nitric oxide synthase I mediates osteoclast activity in vitro and in vivo
Authors: Jung Jae Y. , Lin Aaron C. , Ramos Lisette M. , Faddis Brian T. , Chole Richard A.
Published: May 2003

Bone resorption is responsible for the morbidity associated with a number of inflammatory diseases such as rheumatoid arthritis, orthopedic implant osteolysis, periodontitis and aural cholesteatoma. Previous studies have established nitric oxide (NO) as a potentially important mediator of bone resorption. NO is a unique intercellular and intracellular signaling molecule involved in many physiologic and pathologic pathways. NO is generated from L‐arginine by the enzyme nitric oxide synthase (NOS). There are three known isoforms of NOS with distinct cellular distributions. In this study, we have used mice with targeted deletions in each of these isoforms to establish a role for these enzymes in the regulation of bone resorption in vivo and in vitro. In a murine model of particle induced osteolysis, NOS I‐/‐ mice demonstrated a significantly reduced osteoclast response. In vitro, osteoclasts derived from NOS I‐/‐ mice were larger than wild type controls but demonstrated decreased resorption. Although NOS I has been demonstrated in osteoblasts and osteocytes as a mediator of adaptive bone remodeling, it has not previously been identified in osteoclasts. These results demonstrate a critical role for NOS I in inflammatory bone resorption and osteoclast function in vitro. © 2003 Wiley‐Liss, Inc.
Expression of genes for certain enzymes of pyrimidine and purine salvage pathway in peripheral blood leukocytes collected from patients with Graves' or Hashimoto's disease
Authors: Karbownik Małgorzata , Brzeziańska Ewa , Zasada Krzysztof , Lewiński Andrzej
Published: May 2003

Increased activities of some enzymes, which participate in pyrimidine and purine salvage pathway, were found in blood fractions of patients suffering from different autoimmunological diseases, thyroid diseases included. The aim of the study was to estimate the expression of genes, specific for deoxycytidine kinase (dCK, EC 3.7.1.74), thymidine kinase 1 (TK1; EC 2.7.1.21), and adenosine deaminase (ADA, EC 3.5.4.4) in blood leukocytes, collected from patients with autoimmunological thyroid diseases (AITD), i.e., Graves' or Hashimoto's disease. The total mRNA was isolated from peripheral blood leukocytes and, afterwards, submitted to reverse transcription (RT), with the following amplification of genes encoding for particular examined enzymes and β‐actin, as a supervisory gene [RT‐polymerase chain reaction (RT‐PCR)]; ADA gene was amplified with the use of three different primer pairs (ADA3, ADA4, and ADA5). PCR products were electrophoresed in 8% polyacrylamide gel and then, submitted to densitometric analysis. The levels of expression of all the examined genes in leukocytes from patients with either Graves' or Hashimoto's disease were significantly increased when compared to those in controls; above a twofold elevation of expression of TK1, ADA4, and ADA5 genes was observed. In conclusion, the changes of activities of salvage enzymes in patients with AITD occur likely at transcription level; the measurement of gene expression for purine and pyrimidyne salvage enzymes may likely help explain the mechanism of autoimmune diseases, being also significant in the diagnostics and/or monitoring of AITD. J. Cell. Biochem. 89: 550–555, 2003. © 2003 Wiley‐Liss, Inc.
Fatal toxicity following radio‐ and chemotherapy of medulloblastoma in a child with unrecognized Nijmegen Breakage Syndrome
Authors: Distel Luitpold , Neubauer Susann , Varon Raymonda , Holter Wolfgang , Grabenbauer Gerhard
Published: May 2003

Background

In large‐scale pediatric chemo‐ and radiotherapy trials a proportion of patients as high as 10–15% is usually reported as having severe treatment related toxicity occasionally resulting in toxic death. Little is known on the underlying predisposition of the individual child. Several hereditary disorders including immunodeficiency (ID) syndromes or repair disorders, Ataxia Telangiectasia (AT), and Nijmegen breakage syndrome (NBS) were associated with an elevated risk for severe treatment related toxicity.

Procedure

This report involves the case of a 7‐year‐old boy with medulloblastoma who suffered from remarkably severe side effects during and after postoperative radio‐ and chemotherapy. Several months following craniospinal radiation with a total dose of 36 Gy, late normal tissue side effects were observed within the treated volume. Eighteen months after initiation of treatment the patient died due to protracted cardiopulmonary failure.

Results

To quantify the intrinsic radiation sensitivity, lymphoblastoid cells were used to examine chromosomal aberrations by fluorescence in situ hybridization detecting between two to ninefold higher chromosomal breakage rates in comparison to cells of average cancer patients. Skin fibroblasts showed in the clonogenic survival assays a twofold increased sensitivity. Western blotting demonstrated a typical lack of Nbs1. PCR‐SSCP analysis followed by direct sequencing of positive samples revealed a homozygous truncating mutation of the NBS1 gene (657del5).

Conclusions

This case highlights that severe treatment related complications in pediatric cancer patients may be the result of increased intrinsic radio‐ and chemosensitivity due to NBS, AT, and other ID syndromes. It is suggested to exclude such conditions in all patients with anthropometric parameters below the 3rd centile and other signs suggestive for repair disorders or ID syndromes. Med Pediatr Oncol 2003;41:44–48. © 2003 Wiley‐Liss, Inc.
Expression of the mouse and rat mas proto‐oncogene in the brain and peripheral tissues
Authors: Metzger Rainer , Bader Michael , Ludwig Thomas , Berberich Christof , Bunnemann Bernd , Ganten Detlev
Published: May 2003

We isolated the mas proto‐oncogene from a mouse genomic library. Sequence analysis showed that it contains an open reading frame without intervening sequences. The amino acid sequence deduced confirms the seven‐transmembrane‐domain structure and exhibits 97% and 91% amino acid homology with the rat and the human Mas, respectively. In mice and rats, mas mRNA was detected in the testis, kidney, heart, and in the brain regions: hippocampus, forebrain, piriform cortex, and olfactory bulb. Testicular mas mRNA from rats increases markedly during development, while cerebellar mRNA is high postnatally but completely disappears at later stages. We conclude that the product of the mouse mas gene may be involved in the development of the brain and testis.
Characterisation of the single copy trefoil peptides intestinal trefoil factor and pS2 and their ability to form covalent dimers
Authors: Chinery Rebecca , Bates Paul A. , De Amitabha , Freemont Paul S.
Published: May 2003

A bacterial recombinant expression system was established to produce biologically active rat Intestinal Trefoil factor (rITF). Characterisation of purified rITF shows that both monomers and dimers can be observed under reducing and non‐reducing conditions, respectively. Site‐directed mutagenesis studies show that Cys57 is necessary for rITF dimer formation. Samples of human gastrointestinal tissue following biopsy also demonstrated the presence of reducible human pS2 and ITF covalent dimers. Three‐dimensional models for pS2 and ITF support the hypothesis that both pS2 and ITF can exist as disulphide‐linked dimers in vivo and that any proposed function for these peptides must take dimer formation into account.
Tissue inhibitor of metalloproteinases‐2 (TIMP‐2) in rat liver cells is increased by lipopolysaccharide and prostaglandin E2
Authors: Roeb Elke , Rose-John Stefan , Erren Andrea , Edwards Dylan R. , Matern Siegfried , Graeve Lutz , Heinrich Peter C.
Published: May 2003

To explore the functional role of TIMP‐2 in liver, we determined TIMP‐2 mRNA levels in primary rat hepatocytes and in total rat liver. Rat hepatocytes constitutively express TIMP‐2 mRNA at a low level. Incubation with dexamethasone, prostaglandin E2 and a combination of inflammatory cytokines leads to an up‐regulation of TIMP‐2 mRNA. In rats in vivo we found a dramatic increase of TIMP‐2 expression after intraperitoneal injection of lipopolysaccharide. Compared to our previous findings on TIMP‐1 we conclude that TIMP‐2 mRNA expression is regulated in a distinct and partially opposite manner. Over‐production of TIMP‐2 could inhibit the activity of metalloproteinases and thus lead to matrix accumulation. Dysregulation of TIMP‐2 synthesis might be involved in the development of liver fibrosis.
Crystallization and preliminary crystallographic studies of recombinant dimerization cofactor of transcription factor HNF1/pterin‐4α‐carbinolamine dehydratase from liver
Authors: Ficner Ralf , Sauer Uwe H. , Ceska Thomas A. , Stier Gunter , Suck Dietrich
Published: May 2003

The bi‐functional protein dimerization cofactor of HNF1 (DCoH)/pterin‐4α‐carbinolamine dehydratase (PCD) is found in liver cell nuclei bound to the transcription factor hepatocyte nuclear factor 1 (HNF1) as well as in the cytoplasm acting as an enzyme involved in the phenylalanine hydroxylation system. Deficiency of DCoH/PCD activity in liver causes an atypical hyperphenylalaninemia and deficiency in human epidermis is related to the depigmentation disorder vitiligo. DCoH/PCD from rat liver, which is identical to the human protein, was expressed in E. coli, purified to homogeneity and crystallized. The crystals belong to the trigonal space group P3121 (or P3221) with unit cell dimensions of . Native crystals diffract to a resolution of 2.5 Å.
The high potential iron‐sulfur protein (HiPIP) from Rhodoferax fermentans is competent in photosynthetic electron transfer
Authors: Hochkoeppler Alejandro , Ciurli Stefano , Venturoli Giovanni , Zannoni Davide
Published: May 2003

The functional role of the High Potential Iron‐sulfur Protein (HiPIP) from the photosynthetic bacterium Rhodoferax fermentans was investigated. We demonstrated that the HiPIP increased the rate of light‐induced oxygen reduction mediated by the photosynthetic reaction center (RC); this stimulation reached half‐saturation at [HiPIP]/[RC] ca. 15. The capability of the HiPIP in delivering electrons to the reaction center of Rhodoferax fermentans was demonstrated through kinetic spectrophotometry of cytochrome c‐556 oxidation in the presence or in the absence of HiPIP. It is concluded that the HiPIP is competent in the photosynthetic electron transfer chain of Rhodoferax fermentans.
Purification and characterization of an iron superoxide dismutase from the nitrogen‐fixing Azotobacter vinelandii
Authors: Pagani Silvia , Colnaghi Rita , Palagi Arianna , Negri Armando
Published: May 2003

Two electrophoretically distinct forms of superoxide dismutase (SOD; EC 1.15.1.1) which show different inhibition patterns to hydrogen peroxide have been identified in Azotobacter vinelandii. The SOD inhibited by hydrogen peroxide was purified to homogeneity, and turned out to be an iron superoxide dismutase. The enzyme is present in only one molecular form with an isoelectric point of 4.1, and it is composed of two identical subunits with an apparent molecular weight of 21,000 Da. Spectroscopic analyses indicated that this enzyme contains ferric iron (1.4–1.6 mol/mol protein) in the typical high‐spin form present in other prokaryotic Fe‐SODs. N‐Terminal sequence alignments (up to the 49th residue) showed that A. vinelandii Fe‐SOD has high similarity with other prokaryotic Fe‐SODS.
DAMGO, a μ‐opioid receptor selective agonist, distinguishes between μ‐ and δ‐opioid receptors around their first extracellular loops
Authors: Onogi Tatsuhiro , Minami Masabumi , Katao Yoshikazu , Nakagawa Takayuki , Aoki Yasuhide , Toya Takashi , Katsumata Seishi , Satoh Masamichi
Published: May 2003

The structural basis of μ‐opioid receptor (OPR) for the specificity in its ligand binding was investigated using chimeric μ/δ‐OPRs. Replacement of the region around the first extracellular loop of δ‐OPR with the corresponding region of μ‐OPR gave the resultant chimeric receptor the similar affinity to DAMGO compared with the native μ‐OPR. The reciprocal replacement deprived the high affinity to DAMGO from μ‐OPR. These results indicate that the difference(s) in the structure around the first extracellular loop is critical for DAMGO to distinguish between μ‐ and δ‐OPRs. Furthermore, displacement studies revealed that this region is partly involved in the discrimination between μ‐ and δ‐OPRs by other peptidic μ‐selective ligands, such as dermorphin, morphiceptin and CTOP, but not by non‐peptidic ligands, such as morphine and naloxone.
Changes in subcellular and zonal distribution of glucokinase in rat liver during postnatal development
Authors: Toyoda Yukiyasu , Miwa Ichitomo , Kamiya Masahiro , Ogiso Saiko , Okuda Jun , Nonogaki Tsunemasa
Published: May 2003

Subcellular and zonal distribution of glucokinase in rat liver during postnatal development was examined immunohistochemically. Before day 11 after birth, only some hepatocytes were immunostained, and a positive immunostaining was found in the cytoplasm but not in the nucleus. No zonal distribution of glucokinase was observed in livers of such pups. From day 15, at which time a dietary change from milk to laboratory chow begins to take place, glucokinase immunoreactivity increased; this increase was associated with increases in glucokinase activity and in glucokinase protein, and also the immunostaining was observed mainly in the nuclei. At day 21, the glucokinase immunoreactivity was found almost exclusively in the perivenous zone. At day 30, an intense immunostaining was seen both in the perivenous zone and in the periportal zone, being slightly predominant in the former. The present results indicate that dramatic changes in the distribution of glucokinase in developing rat liver may be related to dietary change.
Control of apoptosis by the cellular ATP level
Authors: Richter Christoph , Schweizer Matthias , Cossarizza Andrea , Franceschi Claudio
Published: May 2003

Apoptosis is a physiological form of cell death. Its causes and execution mechanisms are not clearly understood. Oxidative stress, nitric oxide and its congeners, Ca2+, proteases, nucleases, and mitochondria are considered mediators of apoptosis. At present their importance and exact role are elusive but it is clear that mitochondria are both the target and the source of oxidative stress, nitric oxide, and Ca2+. The mitochondrial membrane potential (ΔΨ), which is the driving force for mitochondrial ATP synthesis, declines during apoptosis, and maintenance of ΔΨ prevents apoptosis. Since apoptosis is highly regulated and involves the activity of hydrolytic enzymes, chromatin condensation and vesicle formation apoptosis is likely to have a high energy demand. We propose that the cellular ATP level is an important determinant for cell death. This hypothesis is supported by circumstantial evidence, is consistent with the available data, has a corrolary in aging, and is amenable to direct experimental testing particularly with flow cytometry as a promising tool.
Enzymatic activity of the ribosome‐bound nascent polypeptide
Authors: Makeyev Eugeny V. , Kolb Vyacheslav A. , Spirin Alexander S.
Published: May 2003

Firefly luciferase was shown to be completely folded and thus enzymatically active immediately upon release from the ribosome [Kolb et al. (1994) EMBO J. 13, 3631–3637]. However, no luciferase activity was observed while full‐length luciferase was attached to the ribosome as a peptidyl‐tRNA, probably because the C‐terminal portion of the enzyme is masked by the ribosome and/or ribosome‐associated proteins. Here we have demonstrated that the ribosome‐bound enzyme acquires the enzymatic activity when its C‐terminus is extended by at least 26 additional amino acid residues. The results demonstrate that the acquisition of the final native conformation by a nascent protein does not need the release of the protein from the ribosome.
Inhibition of the catalytic activity of human transaldolase by antibodies and site‐directed mutagenesis
Authors: Banki Katalin , Perl Andras
Published: May 2003

Transaldolase is a key enzyme of the pentose phosphate pathway. While antibody (Ab) 169, directed against the N‐terminal 139 residues of human transaldolase (TAL‐H), had no effect on enzyme activity, Ab 12484 raised against full length and functional recombinant TAL‐H inhibited catalytic activity. This tentatively mapped the catalytic site to the C‐terminal 140–336 amino acid portion of TAL‐H. Dihydroxyacetone transfer reactions catalyzed by transaldolase depend on Schiff base formation by a lysine residue. Replacement of lysine‐142 by glutamine using site‐directed mutagenesis resulted in a complete loss of enzyme activity, suggesting that lysine‐142 is essential for the catalytic activity of TAL‐H.
Schizosaccharomyces pombe possesses an unusual and a conventional hexokinase: biochemical and molecular characterization of both hexokinases
Authors: Petit Thomas , Blázquez Miguel A. , Gancedo Carlos
Published: May 2003

Two hexokinases were characterized in Schizosaccharomyces pombe: hexokinase 1, with a low phosphorylation coefficient on glucose (Km 8.5 mM) and hexokinase 2, a kinetically conventional hexokinase. Genes hxk1
+ and hxk2
+ encoding these enzymes were cloned and sequenced. Disruption of hxk1
+ had no effect on growth but disruption of hxk2
+ doubled the generation time in glucose. Spores carrying the double disruption hxk1
+ hxk2
+ did not grow on glucose or fructose after one week. Expression of hxk1
+ increased strongly during growth in fructose or glycerol. Expression of hxk2
+ was highest during growth in glycerol. A NADP‐dependent glucose dehydrogenase was detected, but not a glucokinase.
Dansyl cadaverine regulates ligand induced endocytosis of interleukin‐8 receptor in human polymorphonuclear neutrophils
Authors: Ray Ena , Samanta Ajoy Kumar
Published: May 2003

Interleukin‐8 (IL‐8), a neutrophil chemotactic agent, acts as a key mediator in a large number of acute and chronic inflammatory diseases. At 37°C, the receptor for IL‐8 is rapidly internalized with its ligand. But no specific inhibitor of this ligand induced internalization of the receptor has been reported so far. We have found that monodansyl cadaverine (MDC) inhibited about 70% of IL‐8 induced endocytosis and caused 70% and 66% inhibition of IL‐8 mediated chemotaxis and respiratory burst response, respectively, in neutrophils. The uninternalized receptor was detected by anti IL‐8R antibody in MDC treated cells. The endocytosis of IL‐8R was strongly inhibited under Ca2+ depleted conditions which was restored on addition of 1 mM CaCl2 indicating the critical involvement of a Ca2+ ion in the process. Absence of receptor internalisation makes the MDC treated neutrophils suitable for studying the interaction of IL‐8R with potential therapeutic agents e.g. for in vitro screening of anti‐inflammatory agents.
Identification and analysis of catalytic TIM barrel domains in seven further glycoside hydrolase families
Authors: Rigden Daniel J , Jedrzejas Mark J , de Mello Luciane V
Published: May 2003

Fold recognition results allocate catalytic triose phosphate isomerase (TIM) barrels to seven previously unassigned glycoside hydrolase (GH) families, numbers 29, 44, 50, 71, 84, 85 and 89, enabling prediction of catalytic residues. Modelling of GH family 50 suggests that it may be the common evolutionary ancestor of families 42 and 14. TIM barrels now comprise the catalytic domains of more than half of the assigned GH families, and catalyse a much larger variety of GH reactions than any other catalytic domain architecture. Only 327 GH sequences still have no structurally identified catalytic domain.
Microbial isoprenoid biosynthesis and human γδ T cell activation
Authors: Eberl Matthias , Hintz Martin , Reichenberg Armin , Kollas Ann-Kristin , Wiesner Jochen , Jomaa Hassan
Published: May 2003

Human Vγ9/Vδ2 T cells play a crucial role in the immune response to microbial pathogens, yet their unconventional reactivity towards non‐peptide antigens has been enigmatic until recently. The break‐through in identification of the specific activator was only possible due to recent success in a seemingly remote field: the elucidation of the reaction steps of the newly discovered 2‐C‐methyl‐D‐erythritol‐4‐phosphate (MEP) pathway of isoprenoid biosynthesis that is utilised by many pathogenic bacteria. Unexpectedly, the intermediate of the MEP pathway, (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl‐pyrophosphate) (HMB‐PP), turned out to be by far the most potent Vγ9/Vδ2 T cell activator known, with an EC50 of 0.1 nM.
Adrenomedullin provokes endothelial Akt activation and promotes vascular regeneration both in vitro and in vivo
Authors: Miyashita Kazutoshi , Itoh Hiroshi , Sawada Naoki , Fukunaga Yasutomo , Sone Masakatsu , Yamahara Kenichi , Yurugi-Kobayashi Takami , Park Kwijun , Nakao Kazuwa
Published: May 2003

We previously reported that adrenomedullin (AM), a vasodilating hormone secreted from blood vessels, promotes proliferation and migration of human umbilical vein endothelial cells (HUVECs). In this study, we examined the ability of AM to promote vascular regeneration. AM increased the phosphorylation of Akt in HUVECs and the effect was inhibited by the AM antagonists and the inhibitors for protein kinase A (PKA) or phosphatidylinositol 3‐kinase (PI3K). AM promoted re‐endothelialization in vitro of wounded monolayer of HUVECs and neo‐vascularization in vivo in murine gel plugs. These effects were also inhibited by the AM antagonists and the inhibitors for PKA or PI3K. The findings suggest that AM plays significant roles in vascular regeneration, associated with PKA‐ and PI3K‐dependent activation of Akt in endothelial cells, and possesses therapeutic potential for vascular injury and tissue ischemia.
An E2F site in the 5′‐promoter region contributes to serum‐dependent up‐regulation of the human proliferating cell nuclear antigen gene
Authors: Li Yuan-Yuan , Wang Lei , Lu Chang-De
Published: May 2003

The synthesis of proliferating cell nuclear antigen (PCNA) is strictly regulated during the cell cycle. To investigate the contribution of the promoter region to the up‐regulation of human PCNA expression at the onset of S phase, we have examined 17 putative elements with reporter assays in quiescent L‐O2 cells and following serum stimulation. The E2F‐like sequence 5′‐TTCCCCGCAA‐3′ located at −84 to −75 is required for the serum‐induced transactivation. In electrophoretic mobility shift assays, nuclear extracts from asynchronous L‐O2 cells exhibit two binding activities toward the −75 E2F oligonucleotide, and the minor band, whose formation could be interfered with by E2F‐1 antibody, represents an S phase‐specific complex. This is the first demonstration of the E2F site in the human PCNA 5′ promoter as a serum‐responsive element.
Kinetics of inhibition of sperm β‐acrosin activity by suramin
Authors: Hermans Josephine M. , Haines Dianne S. , James Peter S. , Jones Roy
Published: May 2003

Sperm β‐acrosin activity is inhibited by suramin, a polysulfonated naphthylurea compound with therapeutic potential as a combined antifertility agent and microbicide. A kinetic analysis of enzyme inhibition suggests that three and four molecules of suramin bind to one molecule of ram and boar β‐acrosins respectively. Surface charge distribution models of boar β‐acrosin based on its crystal structure indicate several positively charged exosites that represent potential ‘docking’ regions for suramin. It is hypothesised that the spatial arrangement and distance between these exosites determines the capacity of β‐acrosin to bind suramin.
Early changes in glucose and phospholipid metabolism following apoptosis induction by IFN‐γ/TNF‐α in HT‐29 cells
Authors: Lutz Norbert W , Tome Margaret E , Cozzone Patrick J
Published: May 2003

The effects of apoptosis induction on glucose and phospholipid metabolite levels in cancer were studied using human colon adenocarcinoma cells (HT‐29). Apoptosis was induced by co‐incubation with 200 U/ml tumor necrosis factor (TNF)‐α for 4, 8 or 15 h, after sensitization with 500 U/ml interferon (IFN)‐γ for 7 h. Perchloric acid extracts were analyzed by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Significantly increased lactate and NTP (all nucleoside 5’‐triphosphates) signals were detected 4 h after apoptosis‐inducing IFN‐γ/TNF‐α treatment, but not in cells which were TNF‐α‐treated without IFN‐γ preincubation. Simultaneous lactate and NTP changes, if confirmed in vivo, may serve as early, non‐invasive markers of treatment response in some tumors.