bluuebunny/crossref_metadata_2025 huggingface dataset:
Stem cell biology of the central nervous system
Authors: Okano Hideyuki
Published: October 2002

Neural stem cells (NSCs) are multipotential progenitor cells that have self‐renewal activities. A single NSC is capable of generating various kinds of cells within the central nervous system (CNS), including neurons, astrocytes, and oligodendrocytes. Because of these characteristics, there is increasing interest in NSCs and neural progenitor cells from the aspects of both basic developmental biology and therapeutic applications to the damaged brain. This special issue, dedicated to understanding the nature of the NSCs present in the CNS, presents an introduction to several avenues of research that may lead to feasible strategies for manipulating cells in situ to treat the damaged brain. The topics covered by these studies include the extracellular factors and signal transduction cascades involved in the differentiation and maintenance of NSCs, the population dynamics and locations of NSCs in embryonic and adult brains, prospective identification and isolation of NSCs, the induction of NSCs to adopt particular neuronal phenotypes, and their transplantation into the damaged CNS. © 2002 Wiley‐Liss, Inc.
Msx2 and p21CIP1/WAF1 mediate the proapoptotic effects of bone morphogenetic protein‐4 on ventricular zone progenitor cells
Authors: Israsena Nipan , Kessler John A.
Published: October 2002

Treatment of cultured ventricular zone (VZ) progenitor cells with bone morphogenetic protein‐4 (BMP4) promoted cell death in a dose‐dependent manner. VZ progenitor cells became progressively more resistant to the proapoptotic effects of BMP4 between E10 and E16, and, by E18 and thereafter, BMP4 treatment no longer led to progenitor cell death. BMP4 treatment of E13 progenitor cells promoted expression of msx2 and p21CIP1/WAF1 (p21) and inhibition of expression of either gene prevented BMP4‐mediated apoptosis. Treatment of E18 cells with BMP4 failed to induce apoptosis but still induced expression of low levels of msx2 and p21. Knockout of bax significantly reduced but did not prevent BMP4‐mediated death of E13 murine progenitor cells. These observations indicate that msx2 and p21 mediate the proapoptotic effects of BMP4 on VZ progenitor cells and that each gene is necessary but insufficient to promote apoptosis. © 2002 Wiley‐Liss, Inc.
Hippocampal adult neurogenesis occurs in a microenvironment provided by PSA‐NCAM‐expressing immature neurons
Authors: Seki Tatsunori
Published: October 2002

Neurons continue to be generated in the adult hippocampus. In the present study, the early developmental processes of newly generated neurons in the adult rat hippocampus were examined by confocal laser scanning microscopy using a combination of bromodeoxyuridine (BrdU) labeling and immunohistochemistry for highly polysialylated neural cell adhesion molecule (PSA‐NCAM) and NeuroD, which are markers for immature neurons, and glial fibrillary acidic protein (GFAP). Rats were injected with BrdU and 2 hours, 1, 3, and 7 days after the injection, the hippocampus was processed for immunohistochemistry. One day after the injection, BrdU‐labeled cells were found frequently in clusters consisting of dividing cells, putative undifferentiated cells, NeuroD‐positive differentiated neurons, and GFAP‐positive astrocytes. Three days later, BrdU‐labeled cells were loosely aggregated and BrdU‐positive fragmented nuclei were sometimes observed, suggesting that apoptosis occurred in the clusters. These BrdU‐labeled nuclei were frequently associated in various ways with the processes of immature PSA‐positive granule cells. They are positioned along PSA‐positive apical and basal dendrites or surrounded by these processes. By 7 days after the injection, the number of the clusters was reduced and the BrdU‐labeled cells had developed dendrites. These cell‐to‐cell associations support the hypothesis that the clustering and a microenvironment provided by the PSA‐expressing immature neurons contribute to the early developmental events of adult neurogenesis, such as proliferation, differentiation, apoptosis, and neurophilic migration in the adult hippocampus. © 2002 Wiley‐Liss, Inc.
Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity
Authors: Kanemura Yonehiro , Mori Hideki , Kobayashi Satoshi , Islam Omedul , Kodama Eri , Yamamoto Atsuyo , Nakanishi Youko , Arita Norio , Yamasaki Mami , Okano Hideyuki , Hara Masayuki , Miyake Jun
Published: October 2002

To scale up human neural stem/progenitor cell (NSPC) cultures for clinical use, we need to know how long these cells can live ex vivo without losing their ability to proliferate and differentiate; thus, a convenient method is needed to estimate the proliferative activity of human NSPCs grown in neurosphere cultures, as direct cell counting is laborious and potentially inaccurate. Here, we isolated NSPCs from human fetal forebrain and prepared neurosphere cultures. We determined the number of viable cells and estimated their proliferative activity in long‐term culture using two methods that measure viable cell numbers indirectly, based on their metabolic activity: the WST‐8 assay, in which a formazan dye is produced upon reduction of the water‐soluble tetrazolium salt WST‐8 by dehydrogenase activity, and the ATP assay, which measures the ATP content of the total cell plasma. We compared the results of these assays with the proliferative activity estimated by DNA synthesis using the 5‐bromo‐2′‐deoxyuridine incorporation assay. We found the numbers of viable human NSPCs to be directly proportional to the metabolic reaction products obtained in the WST‐8 and ATP assays. Both methods yielded identical cell growth curves, showing an exponentially proliferative phase and a change in the population doubling time in long‐term culture. They also showed that human NSPCs could be expanded for up to 200 days ex vivo without losing their ability to proliferate and differentiate. Our findings indicated that indirect measurements of viable cells based on metabolic activity, especially the ATP assay, are very effective and reproducible ways to determine the numbers of viable human NSPCs in intact neurospheres. © 2002 Wiley‐Liss, Inc.
An immunochemical technique for the detection of ovalbumin in surimi‐derived products
Authors: Romero‐Rodríguez Ma Angeles , Barcia‐Vieitez Ramiro , Vázquez‐Odériz Ma Lourdes
Published: October 2002

Egg white is frequently used in surimi‐derived products. However, electrophoretic detection of egg white in such products is not reliable, since the egg white protein ovalbumin and the fish muscle protein actin have very similar molecular weights. We report the development of an immunoblotting (Western blotting) technique for the detection of ovalbumin in products of this type. We initially used a monoclonal antibody, but this did not bind effectively to ovalbumin in surimi‐derived products. We therefore switched to the use of a polyclonal antibody, which gave excellent results and showed very high sensitivity (detection limit 0.5 ng µl−1).

© 2002 Society of Chemical Industry
Dietary fibre level and enzyme inclusion affect nutrient digestibility and excreta characteristics in grower pigs
Authors: Moeser Adam J , van Kempen Theo A T G
Published: October 2002

An experiment was conducted with 12 grower pigs to determine the effects of (1) manipulating dietary neutral detergent fibre (NDF) level and (2) dietary inclusion of fibre‐degrading enzymes on nutrient digestibility and excreta characteristics in pigs. Four diets were formulated to contain three levels of NDF: 1, 66 g kg−1 NDF (low fibre); 2, 121 g kg−1 NDF (moderate fibre); 3, 222 g kg−1 NDF (high fibre); 4, diet 3 plus 2.0 g kg−1 enzyme. Increasing levels of dietary NDF linearly decreased dry matter and energy digestibilities (r2 = 0.99, P < 0.05). Lowering dietary NDF from 222 to 121 g kg−1 improved nitrogen digestibility by 13% (P < 0.01). Faecal production was decreased by 9% for each 1% decrease in dietary NDF content (r2 = 0.99, P < 0.05). Inclusion of fibre‐degrading enzymes in the high‐fibre diet improved dry matter and energy digestibilities by 2 and 3% respectively, and reduced faecal production by 10% (P < 0.01). Faecal and manure (faeces plus urine) pH values from pigs fed the high‐fibre diet and the high‐fibre plus enzyme diet were lower than those from pigs fed the other experimental diets (P < 0.05). In conclusion, lowering the dietary NDF level or inclusion of fibre‐degrading enzymes in high‐fibre diets may offer relatively practical and easy methods for reducing waste production in pigs.

© 2002 Society of Chemical Industry
Identification and origin of the character‐impact compounds of raw oyster Crassostrea gigas
Authors: Pennarun Anne‐Laure , Prost Carole , Demaimay Michel
Published: October 2002

The French consume large amounts of raw oysters. The study of the aroma of oyster Crassostrea gigas is of economic interest because it is a good method of checking the sensory quality. Aromas were extracted by vacuum steam distillation at 20 °C using whole oyster flesh. This extract presented similar sensory characteristics to raw oyster. The odour‐active compounds were characterised by gas chromatography coupled with olfactometry using a panel of 10 judges trained in seafood aroma recognition. Fifty‐nine volatile compounds were identified in oyster aroma extract. Among these, 25 were responsible for the overall odour of raw oyster. Four compounds identified in oysters were characterised by fresh and marine odour: 3‐(E)‐hexen‐1‐ol, decanal, 2‐undecanone and 3,6‐(E,Z)‐nonadien‐1‐ol. Some compounds were identified for the first time in oysters: 4‐(Z)‐heptenal (white boiled fish odour), which comes from n‐3 polyunsaturated fatty acid oxidation, and 3‐octanol (moss and sulphury odour), 2‐nonanol (cucumber odour) and octanoic acid, which arise from n‐6 polyunsaturated fatty acid oxidation.

© 2002 Society of Chemical Industry
Chemical composition and nutritional attributes of selected newly developed lines of soybean (Glycine max (L) Merr)
Authors: Giami Sunday Y
Published: October 2002

The chemical composition and nutritional properties of raw, autoclaved and boiled samples of three promising advanced breeding lines of soybean (TGx 923 − 2EN, TGx 1019 − 2EN and TGx 1497 − 1D), part of a larger collection evaluated in agronomic field trials in Nigeria, were investigated. Protein quality was evaluated using weanling albino rats fed diets which were formulated to supply 10% protein using soybean samples, with casein as a control. Raw seeds contained 35.6–42.4% crude protein, 8.9–9.8 mg iron per 100 g, 8.62–18.21 mg trypsin inhibitor g−1 and 2.25–6.15 mg phytic acid g−1 seed flour. TGx 923 − 2EN possessed higher crude protein contents and lower amounts of trypsin inhibitor, polyphenol and phytic acid compared with TGx 1019 − 2EN or TGx 1497 − 1D. Boiling proved more effective than autoclaving for reducing the levels of antinutrients and improving the protein quality of the beans, as shown by the higher values for weight gain, protein efficiency ratio (PER), net protein ratio (NPR) and true digestibility (TD) of boiled samples. There were no significant (p > 0.05) differences between the values obtained for PER, NPR and TD of diets containing boiled samples of TGx 923 − 2EN and casein, indicating the nutritional superiority of this soybean line compared with TGx 1019 − 2EN and TGx 1497 − 1D.

© 2002 Society of Chemical Industry
Loss of tocopherols and formation of degradation compounds at frying temperatures in oils differing in degree of unsaturation and natural antioxidant content
Authors: Barrera‐Arellano Daniel , Ruiz‐Méndez Victoria , Velasco Joaquín , Márquez‐Ruiz Gloria , Dobarganes Carmen
Published: October 2002

Samples of oils of different degrees of unsaturation, namely palm olein, olive oil, high‐linoleic sunflower oil, high‐oleic sunflower oil, rapeseed oil and soybean oil, were heated at 180 °C for 2, 4, 6, 8 and 10 h in the presence or absence of their natural antioxidants. Also, tocopherol‐stripped oils were supplemented with α‐tocopherol (500 mg kg−1), δ‐tocopherol (500 mg kg−1) or a mixture of α‐, β‐, γ‐ and δ‐tocopherols (250 mg kg−1 each) and heated under the same conditions. Losses of tocopherols and formation of polymeric triacylglycerols were followed. Total polar compounds were also evaluated after 10 h of heating. Results demonstrated that tocopherols were lost very rapidly, in the expected order, with α‐tocopherol being the least stable. Polymeric and polar compound formation during heating was inhibited to a variable extent, being more dependent on the natural content and type of tocopherols than on the degree of unsaturation of the oil. For example, polymeric and polar compound contents in soybean oil were significantly lower than those found in high‐linoleic sunflower oil. However, the expected influence of the degree of unsaturation was evident when oils were unprotected or possessed identical initial antioxidant contents. Finally, levels of degradation compounds after 10 h of heating were not dependent on the remaining content of antioxidants.

© 2002 Society of Chemical Industry
Aesthetic and functional advantages of the anterolateral thigh flap in reconstruction of tumor‐related scalp defects
Authors: Lutz Barbara S.
Published: October 2002

Eleven patients underwent free‐flap reconstruction of tumor‐related defects of the scalp, forehead, and temporal region. Flap selection aimed at achieving acceptable functional and aesthetic results combined with negligible donor‐site morbidity. Ten males and one female, aged 61.3 ± 14.3 years, were included in this study. Eight patients presented with tumor recurrences after previous surgery, irradiation, and/or chemotherapy. The average extension of defects was 169.5 (range, 30–600) qcm. Free flaps employed for reconstruction included antero‐lateral thigh flaps (8), suprafascial radial forearm flap (1), lateral arm flap (1), latissimus dorsi muscle flap (1), and myocutaneous vertical rectus abdominis flap (1). Other procedures included nerve grafts to the facial nerve (2), ectropion correction (2), and fascia lata slings for static procedure in facial palsy (2). There was no pedicle revision and no flap failure. Donor‐site morbidity was negligible. Hospitalization averaged 9.2 ± 1.7 days. The anterolateral thigh perforator flap offers excellent coverage of tumor‐related defects of the scalp, which require a thin flap for adequate contouring. The customized harvested myocutaneous anterolateral thigh flap is regarded as an elegant option for covering defects which consist of both deep and superficial areas. Fascia lata and nerve grafts are available at the same donor site. This easily allows additional procedures for cosmetic and functional improvement that are of high benefit for patients. © 2002 Wiley‐Liss, Inc.MICROSURGERY 22:258–264 2002
Numerical methods for the topology optimization of structures that exhibit snap‐through
Authors: Bruns T. E. , Sigmund O. , Tortorelli D. A.
Published: October 2002

The inclusion of non‐linear elastic analyses into the topology optimization problem is necessary to capture the finite deformation response, e.g. the geometric non‐linear response of compliant mechanisms. In previous work, the non‐linear response is computed by standard non‐linear elastic finite element analysis. Here, we incorporate a load–displacement constraint method to traverse non‐linear equilibrium paths with limit points to design structures that exhibit snap‐through behaviour. To accomplish this, we modify the basic arc length algorithm and embed this analysis into the topology optimization problem. Copyright © 2002 John Wiley & Sons, Ltd.
Learning to suck eggs, or is there a case for a postgraduate development studies benchmark?
Authors: Copestake James
Published: October 2002

Many academic disciplines in the UK have recently introduced subject benchmarks that specify core or minimum content for particular degrees. The article suggests that efficiency, quality, and transparency arguments in favour of such benchmarks may be undermined by problems in specification, identification, and implementation. By contrasting the programme specification for the Master's in development studies at Bath (overspecified) with the benchmark for Master's in management and business administration (underspecified), it suggests that an intermediate benchmark for development studies could usefully enhance transparency about what development studies is and what it has to offer. Copyright © 2002 John Wiley & Sons, Ltd.
Building e‐government in East and Southeast Asia: Regional rhetoric and national (in)action
Authors: Holliday Ian
Published: October 2002

Among many regional policy initiatives taken by states in East and Southeast Asia in the wake of the 1997 financial crisis, one central project launched by the Association of Southeast Asian Nations (ASEAN), and taken up by its dialogue partners in East Asia, was promotion of information and communication technology. While part of ASEAN's 1999–2004 action plan focused on services for business, another part sought to put public sectors online, and to promote electronic government, or e‐government. Taking the 16 states and quasi‐states of East and Southeast Asia, this article evaluates progress at the action plan's mid‐point in January 2002. It begins by defining e‐government and reviewing three academic literatures on the information age, developmental states, and Confucian societies. It then describes the major policy initiatives taken by ASEAN and its partner states, and surveys implementation progress through an analysis of government homepages and sites. Its main finding is that e‐government activity in East and Southeast Asia is highly diverse, reflecting national strengths and weaknesses rather than regional capacity for policy change. The article argues for increased attention to national implementation strategies. Copyright © 2002 John Wiley & Sons, Ltd.
Frequent expression of the Epstein–Barr virus (EBV)‐induced gene, EBI3, an IL‐12 p40‐related cytokine, in Hodgkin and Reed–Sternberg cells
Authors: Niedobitek Gerald , Päzolt Doreen , Teichmann Martina , Devergne Odile
Published: October 2002

Epstein–Barr virus (EBV)‐associated Hodgkin lymphoma (HL) and nasopharyngeal carcinoma (NPC) usually occur in patients without clinically manifest deficiencies in anti‐viral immunity. In spite of expressing viral proteins, both tumours are apparently able to escape EBV‐specific immunity in vivo. EBI3 is an EBV‐induced cytokine homologous to the interleukin (IL)‐12 p40 subunit and can heterodimerize with IL‐12 p35. It has been suggested that EBI3 may function to antagonize IL‐12 and to inhibit the development of a Th1 immune response. EBI3 expression has been studied in tumour entities frequently associated with EBV infection to examine if EBI3 might contribute to local modulation of the immune response. It is shown that EBI3 is strongly expressed in Hodgkin and Reed–Sternberg cells in 32 of 33 HL cases, independently of the EBV status of the tumour cells. Furthermore, EBI3 expression was detected in the epithelial tumour cells of six of 40 NPC biopsies but not in Burkitt lymphomas. The results suggest that EBI3 may be an additional component of the repertoire employed by Hodgkin and Reed–Sternberg cells to inhibit an effective anti‐tumour or anti‐viral immune response. Copyright © 2002 John Wiley & Sons, Ltd.
Inter‐laboratory and inter‐observer reproducibility of immunohistochemical assessment of the Ki‐67 labelling index in a large multi‐centre trial
Authors: Mengel Michael , von Wasielewski Reinhard , Wiese Birgitt , Rüdiger Thomas , Müller‐Hermelink Hans Konrad , Kreipe Hans
Published: October 2002

Proliferative activity of tumour cells, as assessed by the Ki‐67 labelling index, has been suggested as a potential prognostic indicator in many neoplastic diseases. Meaningful application of the immunohistochemically determined tumour cell growth fraction in clinical decision‐making requires information about its inter‐laboratory reproducibility. To assess the reproducibility of Ki‐67 determined growth fraction, a multi‐centre immunohistochemical trial was performed with 172 participating laboratories, each testing 30 different tissue samples. Evaluating 5160 Ki‐67 labelling indices with a newly developed tissue microarray, good inter‐observer reproducibility but high inter‐laboratory variability was found. Reassessment of all stainings revealed considerable inter‐laboratory differences in the intensity and frequency of labelled nuclei, suggesting that antigen retrieval or staining techniques are predominantly responsible for the inter‐laboratory variability found in this trial. Consequently, cut‐off levels for Ki‐67, suggested to distinguish prognostic subgroups in tumours, appear to have limited reproducibility in a multi‐centre approach. It is concluded that there is a need to standardize the immunohistochemical determination of the Ki‐67 labelling index when it is used as a prognostic indicator in surgical pathology. Copyright © 2002 John Wiley & Sons, Ltd.
Cancer‐specific survival of patients with multiple cancers: an application to patients with multiple breast cancers
Authors: Heinävaara S. , Teppo L. , Hakulinen T.
Published: October 2002

In the analysis of cause‐specific survival, the causes of death must be known. For single‐cancer patients with a known cause of death, the estimation of the cause‐specific survival rate is straightforward. For multiple‐cancer patients with two primary cancers, however, the analysis of cause‐specific survival rates is more complex, particularly if the cancers are of the same primary site. In these situations, a concept of cancer‐specific survival may also be distinguished from cause‐specific survival. Cancer‐specific survival rates are studied here by introducing two models, the primary one where the death from cancer is attributed to one of the cancers, and an alternative where such an attribution is not necessary. The models are illustrated using data on patients with multiple breast cancers. The model‐based survival rates are compared with each other and with the corresponding relative survival rates based on analogous modelling of relative survival. The results show that for the subsequent breast cancer, the cancer‐specific survival rates based on the alternative, where the distinction between the cancers as a cause of death was not necessary, tended to be higher than those based on that distinction. It is thus possible that the subsequent cancer was too often coded as a cause of death, particularly when being localized at diagnosis. Copyright © 2002 John Wiley & Sons, Ltd.
On the potential of measurement error to induce differential bias on odds ratio estimates: an example from radon epidemiology
Authors: Heid I. M. , Küchenhoff H. , Wellmann J. , Gerken M. , Kreienbrock L. , Wichmann H. E.
Published: October 2002

It is well established that odds ratios estimated by logistic regression are subject to bias if exposure is measured with error. The dependence of this bias on exposure parameter values, particularly for multiplicative measurement error, and its implications in epidemiology are not, however, as fully acknowledged. We have been motivated by a German West case‐control study on lung cancer and residential radon, where restriction to a subgroup exhibiting larger mean and variance of exposure than the entire group has shown higher odds ratio estimates as compared to the full analysis. By means of correction formulae and simulations, we show that bias from additive classical type error depends on the exposure variance, not on the exposure mean, and that bias from multiplicative classical type error depends on the geometric standard deviation (in other words on the coefficient of variation of exposure), but not on the geometric mean of exposure. Bias from additive or multiplicative Berkson type error is independent of exposure distribution parameters. This indicates that there is a potential of differential bias between groups where these parameters vary. Such groups are commonly compared in epidemiology: for example when the results of subgroup analyses are contrasted or meta‐analyses are performed. For the German West radon study, we show that the difference of measurement error bias between the subgroup and the entire group exhibits the same direction but not the same dimension as the observed results. Regarding meta‐analysis of five European radon studies, we find that a study such as this German study will necessarily result in smaller odds ratio estimates than other studies due to the smaller exposure variance and coefficient of variation of exposure. Therefore, disregard of measurement error can not only lead to biased estimates, but also to inconsistent results and wrongly concluded effect differences between groups. Copyright © 2002 John Wiley & Sons, Ltd.
GD2 ganglioside biosynthesis is a distinct biochemical event in human melanoma tumor progression
Authors: Thurin Jan , Thurin Magdalena , Herlyn Meenhard , Elder David E. , Steplewski Zenon , Clark Wallace H. , Koprowski Hilary
Published: October 2002

Gangliosides from cell cultures established from melanocytic lesions, representing different stages of melanoma tumor progression, were analyzed by chemical and immunological means on thin‐layer chromatograms. The GD2 ganglioside and N‐acetylgalactosaminyl transferase, which catalyzes the biosynthesis of GD2 from its precursor GD3, were detected in cultures established from advanced primary and metastatic melanomas, but not in cultures of normal melanocytes. Immunohistochemical studies on tissue sections from all progression stages confirmed GD2 expression only in these advanced lesions. A distinct biochemical event thus coincides with the onset of faster growth and acquisition of metastatic competence in human melanoma tumor progression.
Intracellular ADP activates K+ channels that are inhibited by ATP in an insulin‐secreting cell line
Authors: Dunne Mark J. , Petersen Ole H.
Published: October 2002

the effect of ADP on ATP‐sensitive K+ channels in the insulin‐secreting RINm5F cell line has been investigated with the help of single‐channel current recording from saponin‐permeabilized cells. ADP (100–500 μM) markedly activates K+ channels when added to the bath solution in contact with the membrane inside. ADP‐β‐S cannot mimick this effect. During sustained ATP (500 μM)‐evoked inhibition of K+ channel opening, 500 μM ADP markedly and reversibly activates the channels. Conversely ATP markedly reduces the opening probability of ADP‐activated channels. It is suggested that the physiological control of K+ channel opening in the insulin‐secreting cells is mediated by changes in ATP/ADP ratio rather than being solely determined by the ATP concentration.
Creatine kinase‐catalyzed ATP‐phosphocreatine exchange:
Authors: Kupriyanov V.V. , Lyulina N.V. , Steinschneider A.Ya. , Zueva M.Yu. , Saks V.A.
Published: October 2002

Unidirectional fluxes from ATP to phosphocreatine, catalyzed by the MM isoenzyme of creatine kinase, were measured by both the 31P‐NMR saturation transfer technique and radioisotope tracer ([γ‐32P]ATP) method. It was found that at 30–37°C and pH 7.4, over a wide range of[phosphocreatine]/[creatine] (from 0.2 to 5.0) ratios, both methods gave the same results, showing that magnetization transfer allows determination of real fluxes under ‘physiological’ conditions. However, at[PCr]/[Cr] ratios higher than 5 ([ADP]free < 30 μM) or at lower temperatures (t < 15°C,[PCr]/[Cr] ∼ 1), the fluxes assessed by saturation transfer were somewhat faster than those detected by the radioisotope tracer method. These data imply that under physiological conditions phosphoryl group transfer is actually the rate‐determining step of the creatine kinase reaction. In contrast, at high[PCr]/[Cr] ratios or at lower temperatures, control may be shifted from phosphoryl group transfer or distributed among other steps of the reaction.
Guanine nucleotides are potent secretagogues in permeabilized parathyroid cells
Authors: Oetting Marguerite , LeBoff Meryl , Swiston Linda , Preston Joseph , Brown Edward
Published: October 2002

We studied the effects of GTP and its' analogues on PTH release in permeabilized parathyroid cells to assess their role in mediating the unusual inverse relationship between Ca2+ and PTH release in intact parathyroid cells. Both 10‐5 M GppNHp and GTPγS, nonhydrolysable analogues of GTP, produce up to an 8‐fold enhancement of PTH release, which is dose‐dependent. This effect is specific for GTP analogues as we could not mimic it with other nucleotides. 10−3 M GDPβS, a nonhydrolysable GDP analogue, completely abolishes GppNHp‐stimulated hormone release, providing further support for mediation of this effect by a guanine‐nucleotide regulatory protein. In GppNHp‐stimulated cells, PTH release is maximal at free [Ca2+] less than 200 nM and progressively decreases as the free [Ca2+] increases from 300 nM to 100 μM. These results suggest the presence of a guanine‐nucleotide binding protein in the parathyroid cell that may play an important role in the regulation of PTH secretion by Ca2+ and perhaps other secretagogues.
Adenine nucleotide compartmentation in foetal rat hepatocytes
Authors: Roncero César , Estrela José M. , Benito Manuel
Published: October 2002

In the presence of lactate plus pyruvate, or glucose or alanine as substrates, ATP/ADP ratios in the cytosol were higher than in mitochondria in isolated rat foetal hepatocytes. The cytosolic ATP/ADP ratios were dependent on substrate (lactate + pyruvate > glucose > alanine). Oleate increased the cytosolic ATP/ADP ratios in the presence of the other substrates studied. Atractyloside decreased the cytosolic ATP/ADP ratios, oligomycin decreasing these values in both compartments. Isoproterenol, phenylephrine and Ca2+ ionophore decreased the cytosolic ATP/ADP ratio, without altering this value in mitochondria.
Protease inhibitor controls prophenoloxidase activation in Manduca sexta
Authors: Saul Steven J. , Sugumaran Manickam
Published: October 2002

Prophenoloxidase from the hemolymph of tobacco hornworm Manduca sexta can be activated by a specific activating enzyme found in the cuticle. Inhibition studies with benzamidine, diisopropyl phosphofluoridate and p‐nitrophenyl‐p′‐guanidinobenzoate indicate that the activating enzyme is a trypsin‐like serine protease. An endogenous protease inhibitor, isolated from the hemolymph of Manduca larvae, inhibits the prophenoloxidase activation mediated by this enzyme. These results indicate that the probable physiological role of endogenous protease inhibitor is to control the undesired activation of prophenoloxidase in the hemolymph.
Differential reactivity of 9‐NH2‐ellipticine on apurinic and apyrimidinic sites in circular DNA
Authors: Malvy Claude , Bertrand Jean Remi
Published: October 2002

Endonucleases for apurinic sites as well as chemical compounds reacting with aldehydes do not generally differentiate between apurinic and apyrimidinic sites. We have studied the effect of the apurinic site reagent, 9‐NH2‐ellipticine, on apyrimidinic sites enzymatically generated on PBR322 DNA and compared it to its' action on apurinic PM2 and PBR322 DNAs. In conditions where this compound induces breakage of apurinic sites, it does not display any action on apyrimidinic sites.
MAP2: a sensitive cross‐linker and adjustable spacer in dendritic architecture
Authors: Friedrich P. , Aszódi A.
Published: October 2002

Microtubule‐associated protein 2 (MAP2), a long, filamentous molecule thought to cross‐link dendritic cytoskeleton, is rich in PEST sequences, putative signals for rapid proteolytic degradation. It is suggested that MAP2 is indeed highly susceptible to protease, e.g. calpain, attack, which is needed for a plastic change, but actual breakdown depends on the regulation of protease(s). Phosphorylation is expected to make the molecule longer and rigid, similarly to what was observed with the related tau protein. Such a structural transition may provide a mechanism for the putative role of MAP2 in dendritic branching.
Epidermal growth factor (EGF) decreased endothelin‐2 (ET‐2) production in human renal adenocarcinoma cells
Authors: Tokito Fumino , Suzuki Nobuhiro , Hosoya Masaki , Matsumoto Hirokazu , Ohkubo Shoichi , Fujino Masahiko
Published: October 2002

Production of immunoreactive (ir‐) endothelin‐2 (ET‐2) in renal adenocarcinoma cells, ACHN, was reduced by transforming growth factor‐β, basic fibroblast growth factor, transforming growth factor‐α and, most strikingly, by epidermal growth factor (EGF). These growth factors did not show such inhibitory effects on the secretion of ir‐ET‐1 in ET‐1‐producing cells, indicating that the production of ET‐2 and ET‐1 is regulated differently by the growth factors. EGF specifically reduced the secretion of not only ir‐ET‐2 but also ir‐big ET‐2 with only a small decrease in total protein synthesis. Northern blot analysis indicated that EGF controls the ET‐2‐production at the transcription levels of ET‐2 gene.
Direct observation of the biphasic conformational change of DNA induced by cationic polymers
Authors: Minagawa Keiji , Matsuzawa Yukiko , Yoshikawa Kenichi , Matsumoto Mitsuhiro , Doi Masao
Published: October 2002

The interaction between T4 DNA and basic polypeptides was observed using fluorescence microscopy. Free DNA molecules exhibited random Brownian motion accompanying the conformational change. With the addition of polycation, such as histone and polyarginine. DNA molecules tended to shrink to become spherical shapes. The persistent lengths and the distributions of long axis lengths of DNA‐polyarginine complexes were determined from the video images at various polyarginine concentrations. It is demonstrated that the conformation of DNA changes in a biphasic manner in the presence of polyarginine.
Gene expression of type I phospholipase A2 in pancreatic beta cells
Authors: Metz S. , Holmes D. , Robertson R.P. , Leitner W. , Draznin B.
Published: October 2002

Messenger RNA from intact rat pancreatic islets, or from transformed hamster beta (HIT) cells, hybridized with the cDNA probe for type I (but not type II) phospholipase A2. The levels of phospholipase A2 mRNA increased in islets from fasted rats: they decreased in islets cultured in a high glucose concentration (control values at 5.5 mM glucose = 150±6% of those at 22 mM) which impaired subsequent insulin secretion (reduction in second‐phase release = 70±11%). These studies uniquely demonstrate that type I phospholipase A2 is expressed specifically in beta cells and that nutrient availability modulates transcript levels, an effect which could contribute to the detrimental influence of prolonged hyperglycemia on islet function.
Two isoforms of the muscle acetylcholine receptor α‐subunit are translated in the human cell line TE671
Authors: Morris A. , Beeson D. , Jacobson L. , Baggi F. , Vincent A. , Newsom-Davis J.
Published: October 2002

We have previously reported the existence of 2 forms of mRNA for the human muscle acetylcholine receptor (AChR) α‐subunit, thought to be generated by alternate splicing of a primary transcript and to encode 2 α‐subunit protein isoforms [1]. The 2 predicted α‐subunit isoforms, differing by the insertion of 25 amino acids at position , have been synthesized from cRNA transcripts using rabbit reticulocyte lysates: these protein isoforms could be differentiated by immunoprecipitation using antibodies raised against synthetic peptides. The antibodies were used to demonstrate translation of both AChR α‐subunit isoforms in the rhabdomyosarcoma (muscle) cell line TE671, in an approximate 1:1 ratio.
A novel alternatively spliced viral mRNA transcribed in cells infected with human T cell leukemia virus type 1 is mainly responsible for expressing p21X protein
Authors: Orita Satoshi , Saiga Akihiko , Takagi Shin , Tanaka Tatsuro , Okumura Koichi , Aono Yuko , Hinuma Yorio , Igarashi Hisanaga
Published: October 2002

The pX sequence of human T cell leukemia virus type 1 (HTLV‐1) has been thought to be expressed as a doubly spliced mRNA that codes for p40tax, p27rex and p21X. However, we identified a novel alternatively spliced mRNA in the HTLV‐1 infected cells by using reverse transcription followed by the polymerase chain reaction. This mRNA contains only the first and third exons of the doubly spliced mRNA and encodes only p21X. Our data that this mRNA is responsible for expressing p21X exists in most of HTLV‐1 infected cells strongly suggests that p21X may play a crucial role for HTLV‐1 replication.
Augmentation of macrophage recognition of oxidatively damaged erythrocytes by substratum‐bound fibronectin and macrophage surface fibronectin
Authors: Beppu Masatoshi , Masa Hideo , Hora Masachiyo , Kikugawa Kiyomi
Published: October 2002

Thioglycollate‐induced mouse peritoneal macrophages plated on a coverglass bind oxidized mouse erythrocytes in the absence of serum. Macrophages plated on a coverglass pre‐coated with fibronectin (FN) were more active in binding of the oxidized erythrocytes. This effect of FN‐coated coverglass was due to specific binding of an RGD‐containing sequence of FN to FN‐receptors on the macrophage, since GRGDSP hexapeptide in solution inhibited this effect, and GRGDSP‐coated coverglass exhibited the same effect as FN‐coated coverglass. Removal of FN originally present on the macrophage surface by trypsinization, prior to attachment to the coverglass, resulted in diminution of their ability of recognition of the oxidized erythrocytes, but the diminished ability was restored when the trypsinized macrophages were plated on a FN‐coated coverglass, indicating that the cell surface FN is required for the macrophage recognition. Attachment to the coverglass was necessary for the cell surface FN to be effective. These results suggest that solid‐phase FN, produced either by deposition of soluble FN to substratum or attachment of macrophage surface FN to substratum, activates the macrophages and augments their ability to recognize the oxidized erythrocytes.
Protein phosphatase 2A from Xenopus oocytes
Authors: Cormier Patrick , Osborne H.Beverley , Bassez Thérèse , Poulhe Robert , Bellé Robert , Mulner-Lorillon Odile
Published: October 2002

A polyclonal antibody was raised against bacterially produced catalytic α subunit of protein phosphatase 2A (PP2AC) cloned from Xenopus ovarian library. The amount of PP2AC in Xenopus oocytes determined by Western blot analysis was 1 ng/μg of cytosolic protein. The antibody depleted PP2AC from oocyte extracts in association with 6 components (40, 62, 65, 80, 85 and 90 kDa). Prophase‐ and metaphase‐arrested oocytes contained identical amounts of PP2AC. Metaphase oocytes showed one specific change in the 62 kDa protein associated with PP2AC.
A single activity carboxyl methylates both farnesyl and geranylgeranyl cysteine residues
Authors: Volker Craig , Lane Pamela , Kwee Cynthia , Johnson Mark , Stock Jeff
Published: October 2002

Members of the Ras superfamily of small GTP‐binding proteins, γ‐subunits of heterotrimeric G proteins and nuclear lamin B are subject to a series of post‐translational modifications that produce prenylcysteine methylester groups at their carboxyl termini. The thioether‐linked polyisoprenoid substituent can be either farnesyl (C15) or geranylgeranyl (C20). Small molecule prenylcysteine derivatives with either the C15 or C20 modification, such as N‐acetyl‐S‐trans,trans‐farnesyl‐L‐cysteine (AFC), S‐trans,trans‐farnesylthiopropionate (FTP), as well as the corresponding geranylgeranyl derivatives (AGGC and GGTP) are substrates for the carboxyl methyltransferase. Saccharomyces cerevisiae ste 14 mutants that lack RAS and a‐factor carboxyl methyltransferase activity are also unable to methylate farnesyl and geranylgeranylcysteine derivatives. Moreover, C20‐substituted cysteine analogs directly compete for carboxyl methylation with the C15‐substituted cysteine analogs and vice versa. Finally, AGGC is even more effective than AFC as an inhibitor of Ras carboxyl methylation, despite the fact that Ras is methylated at a farnesylcysteine rather than a geranylgeranylcysteine residue.
Conformational changes in melittin upon complexation with an anionic melittin analog
Authors: Ramalingam Kalaiyarasi , Bello Jake , Aimoto Saburo
Published: October 2002

Melittin and its Glu‐(7,21,22,23,24) analog upon mixing in equimolar concentrations form a hybrid oligomer with significant helical structure, in conditions in which each peptide separately adopts a largely disordered structure. The hybrid exhibits both cold‐ and heat‐induced denaturations similar to the phenomena exhibited by proteins. The hybrid also retains significant residual structure at higher temperature, similar to the ‘molten globular state’ that has been suggested for proteins. Melittin, at concentrations in which it forms helical tetramers, also exhibits these phenomena and may be used as a model for protein‐denaturation studies.
Differential methylation of a CpG‐island concatemer in hemi‐ and homozygous transgenic mice
Authors: Gundersen Glenn , Kolstø Anne-Brit , Prydz Hans
Published: October 2002

Methylation‐free islands (MFIs), clusters of non‐methylated CpG‐dinucleotides in mammalian genomes, are associated with a majority of studied genes. By which precise mechanism they maintain their unmethylated status is unknown. The behaviour of transgenic MFIs may contribute to unveil this enigma. We have generated a high‐copy number transgenic line with the MFI from the murine Thy‐1.1 allele. A stable, minor fraction of this otherwise non‐methylated DNA became completely methylated in all adult tissues tested. Furthermore, individuals homozygous for the transgene showed a significantly higher proportion of methylated copies compared to the hemizygous state. These findings support the hypothesis that a limited pool of trans‐acting factors are involved in maintaining the hypomethylated state.
Hyperphosphorylation and accumulation of neurofilament proteins in Alzheimer disease brain and in okadaic acid‐treated SY5Y cells
Authors: Wang Jian-zhi , Tung Yunn Chyn , Wang Yipeng , Li Xian Tau , Iqbal Khalid , Grundke-Iqbal Inge
Published: October 2002

We investigated the role of neurofilament (NF) proteins in Alzheimer disease (AD) neurofibrillary degeneration. The levels and degree of phosphorylation of NF proteins in AD neocortex were determined by Western blots developed with a panel of phosphorylation‐dependent NF antibodies. Levels of all three NF subunits and the degree of phosphorylation of NF‐H and NF‐M were significantly increased in AD as compared to Huntington disease brains used as control tissue. The increase in the levels of NF‐H and NF‐M was 1.7‐ and 1.5‐fold (P<0.01) as determined by monoclonal antibody SMI33, and was 1.6‐fold (P<0.01) in NF‐L using antibody NR4. The phosphorylation of NF‐H and NF‐M in AD was increased respectively at the SMI31 epitope by 1.6‐ and 1.9‐fold (P<0.05) and at the SMI33 epitope by 2.7‐ and 1.3‐fold (P<0.01 and P<0.05). Essentially similar effects were observed in SY5Y human neuroblastoma cells when treated with okadaic acid, an inhibitor of protein phosphatase (PP)‐2A and ‐1. This is the first biochemical evidence which unambiguously demonstrates the hyperphosphorylation and the accumulation of NF subunits in AD brain, and shows that the inhibition of PP‐2A/PP‐1 activities can lead to the hyperphosphorylation of NF‐H and NF‐M subunits.
Oxidation products of polyamines induce mitochondrial uncoupling and cytochrome c release
Authors: Maccarrone M , Bari M , Battista N , Di Rienzo M , Falciglia K , Finazzi Agrò A
Published: October 2002

Spermine is shown to uncouple isolated mitochondria and to trigger the selective release of cytochrome c. Pargyline, an inhibitor of amine oxidase (AO), fully prevented these effects of spermine, which instead were potentiated by exogenous AO. Hydrogen peroxide, an oxidation product of spermine, mimicked the effects of spermine on mitochondria, while the addition of catalase prevented them. Spermidine and putrescine also caused mitochondrial uncoupling and triggered cytochrome c release, with a potency which correlated with the substrate preference of mitochondrial AO. Pargyline protected human lymphoma U937 cells against UVB‐induced apoptosis, by reducing AO activity, mitochondrial uncoupling and cytochrome c release.
Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p
Authors: Barth Henning , Meiling-Wesse Khuyen , Epple Ulrike D , Thumm Michael
Published: October 2002

We here report the identification of AUT10 as a novel gene required for both the cytoplasm to vacuole targeting of proaminopeptidase I and starvation‐induced autophagy. aut10Δ cells are impaired in maturation of proaminopeptidase I under starvation and non‐starvation conditions. A lack of Aut10p causes a defect in autophagy prior to vacuolar uptake of autophagosomes. Homozygous aut10Δ diploids do not sporulate. Vacuolar acidification indicated by accumulation of quinacrine is normal in aut10Δ cells and mature vacuolar proteinases are present. A biologically active Ha‐tagged Aut10p, chromosomally expressed from its endogenous promoter, localizes in indirect immunofluorescence microscopy in the cytosol and on granulated structures, which appear clustered around the vacuolar membrane. This localization differs from known autophagy proteins.
Both proline‐rich sequences in the TH region of Bruton's tyrosine kinase stabilize intermolecular interactions with the SH3 domain
Authors: Hansson Henrik , Smith C.I.Edvard , Härd Torleif
Published: October 2002

The Tec homology (TH) region located N‐terminal to the Src homology 3 (SH3) domain of Bruton's tyrosine kinase (Btk) contains two proline‐rich SH3‐binding sequences (PRRs). We have previously demonstrated that the TH region acts to stabilize intermolecular interactions in N‐terminally extended SH3 (PRR–SH3) fragments. Here, we analyze six PRR–SH3 fragments with different proline‐to‐alanine substitutions in the two PRRs. Gel permeation chromatography and nuclear magnetic resonance spectroscopy show that both PRRs can stabilize self‐association. This observation provides an explanation to why the TH region of Btk makes intermolecular interactions, whereas the corresponding interaction in the related Itk kinase with only one PRR, is intramolecular.
Tyrosine phosphorylation of the catalytic subunit p110 of phosphatidylinositol‐3 kinase induced by HMG‐CoA reductase inhibitor inhibits its kinase activity in L6 myoblasts
Authors: Nakagawa Hiroto , Mutoh Tatsuro , Kumano Takanori , Kuriyama Masaru
Published: October 2002

Previous studies from this laboratory have shown that 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitor (HCRI) causes apoptotic cell death of a muscle cell‐derived cell line, L6 myoblasts, by involving the phosphatidylinositol‐3 (PI‐3) kinase pathway and tyrosine phosphorylation of several cellular proteins, although the relationship between PI‐3 kinase pathway and tyrosine phosphorylation responses remained to be elucidated. Here, we show that HCRI induces tyrosine phosphorylation of catalytic subunit p110 of PI‐3 kinase as early as 5 min after addition of HCRI into culture medium. We could not detect the tyrosine phosphorylation of the regulatory subunit p85 of PI‐3 kinase under the present experimental conditions. Concomitantly, the kinase activity toward PI in p110 immunoprecipitates was decreased with a similar time course. Furthermore, both herbimycin A and genistein, potent inhibitors of tyrosine kinase activity, inhibited HCRI‐induced inhibition of PI‐3 kinase activity as well as HCRI‐induced apoptotic cell death. Once the catalytic subunit p110 becomes tyrosine‐phosphorylated, the regulatory subunit p85 appears to be dissociated from the catalytic subunit, because we observed a decreasing amount of p85 regulatory subunits in p110 immunoprecipitates in response to HCRI treatment. These results strongly suggest the novel function of tyrosine phosphorylation of catalytic subunit p110 of PI‐3 kinase in the regulation of its kinase activity. The tyrosine phosphorylation of these catalytic subunits may play an important role in the intracellular signal transduction of apoptotic cell death. To our knowledge, this is the first report that tyrosine phosphorylation of p110 catalytic subunit acts as a negative regulator of its kinase activity.