bluuebunny/crossref_metadata_2025_split huggingface dataset:
A bicistronic SIN‐lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines
Authors: Richard Emmanuel , Géronimi Fabien , Lalanne Magalie , Ged Cécile , Redonnet‐Vernhet Isabelle , Lamrissi‐Garcia Isabelle , Gerson Stanton L , de Verneuil Hubert , Moreau‐Gaudry François 
Published: August 2003

AbstractBackgroundErythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells.MethodsWe have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O6‐methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency.ResultsEPP lymphoblastoid (LB) cell lines, K562 and cord‐blood‐derived CD34+ cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O6‐benzylguanine (BG)/N,N′‐bis(2‐chloroethyl)‐N‐nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus‐containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation.ConclusionsThis study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells. Copyright © 2003 John Wiley & Sons, Ltd.
Electrophysiologically “complex” glial cells freshly isolated from the hippocampus are immunopositive for the chondroitin sulfate proteoglycan NG2
Authors: Schools Gary P. , Zhou Min , Kimelberg Harold K. 
Published: August 2003

AbstractWe have recently described a subgroup of isolated glial fibrillary acidic protein‐positive (GFAP+) hippocampal astrocytes that predominantly express outwardly rectifying currents (which we term “ORAs” for outwardly rectifying astrocytes), which are similar to the currents already described for hippocampal GFAP− “complex glia.” We now report that post‐recording staining of cells that were first selected as “complex” by morphology and then confirmed by their electrophysiological characteristics were NG2+ ∼90% of the time. Also, the morphology of freshly isolated NG2+ cells differs from that of isolated GFAP+ ORAs in having a smaller and round cell body with thinner processes, which usually are collapsed back onto the soma. Upon detailed examination, NG2+ cells were found to differ quantitatively in some electrophysiological characteristics from GFAP+ ORAs. The outward, transient K+ currents (IKa) in the NG2+ cells showed a slower decay than the IKa in ORAs, and their density decreased in NG2+ cells from older animals. The other two major cation currents, the voltage‐activated Na+ and outwardly delayed rectifier K+ currents, were similar in NG2+ cells and ORAs. To further distinguish isolated complex cells from outwardly rectifying GFAP+ astrocytes, we performed immunocytochemistry for glial markers in fixed, freshly isolated rat hippocampal glia. NG2+ cells were negative for GFAP and also for the astrocytic glutamate transporters GLT‐1 and GLAST. Thus the isolated hippocampal NG2+ glial cells, though having an electrophysiological phenotype similar to that of ORAs, are an immunologically and morphologically distinct glial cell population and most likely represent NG2+ cells in situ. © 2003 Wiley‐Liss, Inc.
Bcl‐2 deficiency deprives peripheral nerves of neurotrophic activity against injured optic nerve
Authors: Kotulska Katarzyna , Lewin‐Kowalik Joanna , Larysz‐Brysz Magdalena , Marcol Wiesław , Fus Zbigniew 
Published: August 2003

AbstractOptic nerve injury leads to retinal ganglion cell apoptosis, thus preventing fiber regeneration. Peripheral nerve grafts are known to promote survival and regeneration in injured adult mammalian central nervous system, including optic nerve, but the mechanisms of their activity remain unclear. It is likely that they attenuate the apoptotic cascade triggered by axotomy in retinal ganglion cells. The aim of this work was to examine the role of the antiapoptotic gene bcl‐2 in the optic nerve regeneration induced by such grafts. Experiments were carried out on bcl‐2‐deficient and wild‐type mice. We have reported previously that predegeneration markedly enhances neurotrophic activity of peripheral nerve grafts, so we applied both predegenerated and non‐predegenerated implants to the transected optic nerves. We studied the neurotrophic effects of bcl‐2‐deficient grafts on wild‐type and bcl‐2 knock‐out optic nerves, as well as wild‐type grafts on both strains of mouse optic nerves. After application of fluorescent dye to the end of the graft, we counted the stained retinal ganglion cells. Predegenerated wild‐type grafts promoted survival and outgrowth of retinal ganglion cells axons in both types of mice. By contrast, non‐predegenerated and predegenerated bcl‐2‐deficient grafts induced little or no regeneration in the optic nerves. These results indicate that the lack of bcl‐2 gene does not deprive retinal ganglion cells of their regenerative potential. At the same time, we found that bcl‐2 knock‐out dispossesses peripheral nerves of their neurotrophic activity. © 2003 Wiley‐Liss, Inc.
Increased turnover of Na‐K ATPase molecules in rat brain after rapid eye movement sleep deprivation
Authors: Majumdar Sudipta , Faisal Mohd , Madan Vibha , Mallick Birendra N. 
Published: August 2003

AbstractIt has been shown that rapid eye movement (REM) sleep deprivation increases Na‐K ATPase activity. Based on kinetic study, it was proposed that increased activity was due to enhanced turnover of enzyme molecules. To test this, anti‐α1 Na‐K ATPase monoclonal antibody (mAb 9A7) was used to label Na‐K ATPase molecules. These labeled enzymes were quantified on neuronal membrane by two methods: histochemically on neurons in tissue sections from different brain areas, and by Western blot analysis in control and REM sleep‐deprived rat brains. The specific enzyme activity was also estimated and found to be increased, as in previous studies. The results confirmed our hypothesis that after REM sleep deprivation, increased Na‐K ATPase activity was at least partly due to increased turnover of Na‐K ATPase molecules in the rat brain. © 2003 Wiley‐Liss, Inc.
Zebrafish Cx35: Cloning and characterization of a gap junction gene highly expressed in the retina
Authors: McLachlan Elizabeth , White Thomas W. , Ugonabo Chioma , Olson Carl , Nagy James I. , Valdimarsson Gunnar 
Published: August 2003

AbstractThe vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304‐amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT‐PCR, at 2 days post‐fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos. © 2003 Wiley‐Liss, Inc.
Characterization of activated retinal microglia following optic axotomy
Authors: Zhang Cheng , Tso Mark O.M. 
Published: August 2003

AbstractMicroglia are prominently involved in neural degenerative diseases of the CNS and the retina. In this study, we determined the activation and phagocytotic function of different subtypes of retinal microglial cells at 1 week and 1 month following optic axotomy. Fluorescent DiI crystals were placed at the stumps of the cut optic nerves of Lewis rats to retrolabel retinal ganglion cells. Microglial cells were indirectly labeled as they phagocytosed the dye particles in the dying ganglion cells. OX‐42, 5D4, ED1, and OX‐6 antibodies were used for immunohistochemical study. The OX‐42‐ and 5D4‐positive microglial cells were increased in the inner retinal layers after optic axotomy. The increase of OX‐42‐positive cells was considerably greater than that of 5D4‐positive cells. The 5D4‐positive cells were ramified in shape, whereas OX‐42‐positive cells were ameboid and ovoid. Both 5D4‐ and OX‐42‐positive cells phagocytosed dying ganglion cells at 1 week and 1 month after axotomy. Scattered ameboid ED1‐positive cells were detected in the normal retina and showed phagocytotic activity at 1 month after optic axotomy. The number of ED1‐positive cells in the retina was unchanged after axotomy. In optic axotomy, three types of microglial cells were activated, namely, 5D4‐positive ramified cells and OX‐42‐ and ED1‐positive ameboid cells. All of them exhibited the phagocytosis of dying ganglion cells. Insofar as the blood–retinal barrier presumably remained intact in optic axotomy, the OX‐42‐ and 5D4‐positive cells might derive from resident microglial cells. The ED1‐positive cells, presumably recently blood‐borne macrophage in the CNS, remained the same number in the axotomized retina. © 2003 Wiley‐Liss, Inc.
Transcription factor activator protein‐1 expressed by kainate treatment can bind to the non‐coding region of mitochondrial genome in murine hippocampus
Authors: Ogita Kiyokazu , Fujinami Yoshiaki , Kitano Masahiro , Yoneda Yukio 
Published: August 2003

AbstractWe have demonstrated previously that the transcription factor activator protein‐1 (AP‐1) complex is translocated into mitochondria into the nucleus in murine hippocampus after systemic kainate injection (Ogita et al. [2002] J. Neurosci. 22:2561–2570). The present study investigates whether the mitochondrial AP‐1 complex translocated in response to kainate treatment binds to AP‐1‐like sites located at the non‐coding region of the mitochondrial genome in mouse hippocampus. There are 10 sites with sequences similar to the nuclear AP‐1 site in the non‐coding region. Of 10 pieces (MT‐1–MT‐10) of synthesized double‐stranded oligonucleotides, each containing a mitochondrial AP‐1‐like site, MT‐3, MT‐4, and MT‐9 were effective in inhibiting mitochondrial AP‐1 DNA binding enhanced by kainate. Electrophoresis mobility shift analysis using radiolabeled MT‐3 and MT‐9 probes demonstrated marked enhancement with binding of these 2 probes in hippocampal mitochondrial extracts prepared 2–6 hr after kainate treatment. Unlabeled AP‐1 probe was more potent than unlabeled MT‐9 probe in inhibiting the mitochondrial MT‐9 binding. Supershift analysis revealed participation of particular Fos/Jun family proteins, such as c‐Fos, Fos‐B, c‐Jun, Jun‐B, and Jun‐D, in MT‐9 binding in hippocampal mitochondrial extracts prepared 4 hr after kainate treatment. Immunoprecipitation analysis using anti‐c‐Fos antibody demonstrated that c‐Fos associated with the mitochondrial genome in hippocampal mitochondria prepared from kainate‐treated animals. These results suggest that the AP‐1 complex expressed by in vivo kainate treatment would bind to AP‐1‐like sites in the non‐coding region of the mitochondrial genome after translocation into mitochondria from murine hippocampus. © 2003 Wiley‐Liss, Inc.
Increases in matrix metalloproteinase‐9 and tissue inhibitor of matrix metalloproteinase‐1 mRNA after cerebral contusion and depolarisation
Authors: von Gertten Christina , Holmin Staffan , Mathiesen Tiit , Sandberg Nordqvist Ann‐Christin 
Published: August 2003

AbstractMatrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play major roles in physiological extracellular matrix turnover during normal development and in pathological processes. In brain, increases in MMP activity occur, for example, in multiple sclerosis, Alzheimer's disease, and after head trauma. We examined MMP‐9 and TIMP‐1, ‐2, and ‐3 in events after head trauma. A time‐course study was carried out using two different rat injury models, cerebral contusion and depolarisation. Brains were analysed by RT‐PCR and in situ hybridisation. We observed a distinct and time‐dependent upregulation of MMP‐9 and TIMP‐1 mRNA in ipsilateral cortical areas. MMP‐9 mRNA levels were upregulated 1 day after cerebral contusion with a peak at Day 4. Depolarisation per se, which also occurs after traumatic brain injury, lead to delayed increase of MMP‐9 mRNA, 4 days post application. At Day 14, MMP‐9 mRNA levels were indistinguishable from controls in both models. TIMP‐1 mRNA increases were observed in both models 4 hr after injury, and increased further at Days 1 and 4. At Day 14, mRNA levels declined and were no higher than control levels. No alterations in mRNA levels were noted for TIMP‐2 or ‐3. Our results support earlier reports on MMP‐9 involvement in brain injury. It also shows a role for TIMP‐1 in the mechanisms of trauma, where depolarisation could be the mechanism responsible for this upregulation. © 2003 Wiley‐Liss, Inc.
Optimization of hot water extraction of roselle juice using response surface methodology: a comparative study with other extraction methods
Authors: Wong Peng‐Kong , Yusof Salmah , Ghazali Hasanah Mohd , Che Man Yaakob bin 
Published: August 2003

AbstractRoselle has regained the attention of many fruit juice manufacturers in Malaysia as a product that contains high ascorbic acid and anthocyanins. However, ascorbic acid and anthocyanin pigments can be easily destroyed during processing of fruit juice. Therefore, in the present study, the effects of different processing methods, namely hot water extraction (HWE), hot water blending, cold water blending and screw press, on the changes in anthocyanins and ascorbic acid contents of roselle juice were evaluated. The anthocyanins and ascorbic acid contents of roselle were determined using the pH differential method and high‐performance liquid chromatography respectively. The physico‐chemical characteristics and sensory properties of roselle juice were also evaluated. The results of the study indicate that the HWE method is the most effective extraction method, resulting in high anthocyanins and ascorbic acid contents of 43 g l−1 (as delphinidin‐3‐glucoside) and 2.34 g kg−1 respectively. The optimum juice extraction conditions were 3.5 h at 60 °C. Copyright © 2003 Society of Chemical Industry
Soil moisture stress‐induced alterations in bioconstituents determining tea quality
Authors: Jeyaramraja PR , Pius PK , Raj Kumar R , Jayakumar D 
Published: August 2003

AbstractThe impact of water stress on the biochemical constituents that determine black tea quality was investigated. Phenylalanine ammonia lyase (PAL) activity was highest in the drought tolerant ‘Assam’ cultivar UPASI‐2, followed by UPASI‐8 and UPASI‐9, under non‐stress conditions. Under soil moisture stress a reduction in PAL activity was found in all three clones investigated. A strong positive correlation was observed between an increase in soil moisture deficit and a decrease in PAL activity. Lower PAL activity correlated well with lower synthesis of flavanols such as epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), which are important precursors of theaflavin‐3,3′‐digallate that determines final tea quality. Altered synthesis of EGCG and ECG could be due to their molecular rearrangement at elevated leaf temperature during drought. Synthesis of quality constituents such as gallic acid and caffeine declined significantly owing to both drought and waterlogging stress. The reduction in gallic acid due to water stress could lead to lower synthesis of theaflavin fractions such as epitheaflavic acid, epitheaflavic acid‐3′‐gallate and theaflavic acid and, thereby, quality deterioration. Similarly to drought, flooding stress was also found to alter the biochemical constituents necessary for tea quality. Copyright © 2003 Society of Chemical Industry
Modified method for the determination of pyruvic acid with dinitrophenylhydrazine in the assessment of onion pungency
Authors: Anthon Gordon E , Barrett Diane M 
Published: August 2003

AbstractModifications to the commonly used Schwimmer and Weston procedure for determining pyruvic acid in onions are proposed. These modifications involve changes in the concentrations and volumes of the reagents and the use of 515 rather than 420 nm as the wavelength for measurements. These changes improve both the linearity and the sensitivity of the assay and eliminate interference from coloured material found in yellow onions. With these changes, more reliable determinations of pyruvic acid and thus of onion pungency should be possible. Copyright © 2003 Society of Chemical Industry
Removing τ‐fluvalinate residues from press‐extracted honey
Authors: Waliszewski Stefan M , Infanzón Rosa M , Carvajal Octavio , Maxwell Hart Mary 
Published: August 2003

AbstractHoney is obtained from the hives of European honeybees. Honeybees can be infested by Varroa parasitism, which affects honey production. The acaricide τ‐fluvalinate used in beehives to control Varroa mites, can leave its residues in honey, pollen and wax. A monitoring study was undertaken to determine the τ‐fluvalinate residues in honey samples taken from treated hives. The τ‐fluvalinate residues were determined by gas chromatography using a short, non‐polar packed column with electron capture detection. Analysis of 50 samples showed an average residue level of 15 µg kg−1. The same samples were filtered through Whatman filter paper to separate the honey from residual suspended particles. The filtered honeys showed no acaricide residues above the detection limit of 3 µg kg −1, while the average residue level determined in the sediments was 392 µg kg −1. Thus the residues of τ‐fluvalinate in honey can be eliminated by separating the honey from residual suspended particles that contain acaricide residues. Copyright © 2003 Society of Chemical Industry
Antioxidant capacity and phenolic content in blueberries as affected by genotype and growing season
Authors: Howard Luke R , Clark John R , Brownmiller Cindi 
Published: August 2003

AbstractBlueberry fruits from five commercial cultivars and 13 breeding selections grown at the same locations over two growing seasons were analysed for total phenolics (TPH), total anthocyanins (ACY), total hydroxycinnamic acids (HCA), total flavonols (FLA), fruit weight and oxygen radical‐absorbing capacity (ORAC). Variation in ORAC, TPH, ACY, HCA, FLA and fruit weight among genotypes was much greater than that observed between growing seasons, indicating that genetics plays a more important role than growing season in influencing ORAC and phenolic content in blueberries. Significant main effects for growing season and genotype × growing season observed for ORAC, ACY, HCA and fruit weight demonstrate that environmental growing conditions can impact levels of phenolics and ORAC in blueberries and that certain genotypes vary in their capacity to synthesise phenolics under different growing conditions. In general, genotypes with smaller berries had higher ORAC values and levels of TPH, ACY, HCA and FLA than large‐berried genotypes. Over both growing seasons, ORAC correlated highly with TPH, ACY, HCA and FLA, while fruit weight correlated inversely with all phenolics measured. Our results indicate that blueberry genotypes should be screened over multiple growing seasons in order to identify antioxidant‐ and phenolic‐rich germplasm. Copyright © 2003 Society of Chemical Industry
Decreased expression of human papillomavirus E2 protein and transforming growth factor‐β1 in human cervical neoplasia as an early marker in carcinogenesis
Authors: Torng Pao‐Ling , Chan Wing‐Yee , Lin Chin‐Tang , Huang Su‐Cheng 
Published: August 2003

AbstractBackground and ObjectivesHuman papillomavirus (HPV) is thought to be one of the possible causative factors in cervical carcinogenesis, and cervical carcinoma cells are refractory to tumor transforming growth factor (TGF)‐β1. The purpose of this study is to investigate the possible cause–effect association between HPV and TGF‐β1 during cervical tumorigenesis.MethodsWe assessed the expression of HPV capsid proteins, HPV‐16 E7, HPV‐16 E2 (C and N terminals), TGF‐β1, and their receptors TGF‐β RI and RII by immunohistochemistry in 48 paraffin‐embedded blocks of tumor tissue derived from patients of cervical neoplasia.ResultsExpression of TGF‐β1 decreased as tumor cells progressed from cervical intraepithelial neoplasia (CIN)1, CIN2, CIN3, to microinvasive carcinoma (P < 0.05). Levels of TGF‐βRI and TGFβ‐RII stayed the same in all cases. HPV was found in 89.6% of the studied sections, and cervical lesions without HPV infection expressed significantly less TGF‐β1 (P < 0.05). By comparing the expression pattern of TGF‐β1 and HPV in the neoplastic cells with that of normal cervical epithelium in each section, we found loss of HPV‐16 E2 higher in CIN3 (15/24) than in CIN1 or CIN2 (3/7), and there is a significant trend that loss of HPV‐16 E2 expression correlated with a >50% loss of TGF‐β1 at the lesion site (P < 0.05).ConclusionsOur result showed co‐suppression of HPV and TGF‐β1 expression during progression of cervical squamous cell cancer. Using antibody against HPV‐16 E2 may be an auxiliary tool for the investigation of cervical tumor progression. J. Surg. Oncol. 2003;84:17–23. © 2003 Wiley‐Liss, Inc.
Primary malignant melanoma of the lip
Authors: Froix Anthony J. , Salti George I. 
Published: August 2003

AbstractMelanoma of the lip is a very rare clinical entity. As such, no large studies are available to help in diagnosis and treatment. We report our results on 11 patients with lip melanoma. Patients with lip melanoma should be approached in a similar fashion to patients with other cutaneous melanomas. J. Surg. Oncol. 2003;84:7–9. © 2003 Wiley‐Liss, Inc.