bluuebunny/crossref_metadata_2025 huggingface dataset:
Ligation of exogenous linear DNA after gene transfer in vitro and in vivo
Authors: Rittner Karola , Benavente Annie , Jacobs Eric
Published: August 2003

Background

We have analyzed the physical/topographical state of linear exogenous DNA after gene transfer in vitro and in vivo.

Methods and results

Linear DNA carrying a luciferase expression cassette, either intact or corrupted within the coding region, was tested in gene transfer experiments in vitro and in vivo. To this, a plasmid with a CMV‐IE1 promoter‐driven luciferase gene was rendered non‐functional by the insertion of a 1.2 kb EcoRV–EcoRV fragment. After removal of the insert by digestion with EcoRV, the resulting linear DNA fragments were used to transfect HeLa cells. The recovery of luciferase activity from these cells indicated functional reconstitution of the expression cassette. Recovery of low molecular weight DNA from HeLa cells allowed amplification of an intact luciferase gene, confirming accurate ligation of free DNA ends. In the mouse, rapid intravenous injection of plasmid DNA, linearized within the luciferase gene, resulted in significant luciferase activities in liver and lung. Ligation products could be detected by PCR.

Conclusions

These data suggest that linear DNA is efficiently circularized after gene transfer in vitro and in vivo. Secondly, equally high luciferase activities were observed in the mouse after rapid intravenous injection of luciferase expression cassettes, either consisting of linear DNA produced by PCR, or carried by linearized plasmid DNA. These findings encourage the use of linear DNA elements for gene transfer applications in vivo. Copyright © 2003 John Wiley & Sons, Ltd.
Osteogenesis in rats induced by a novel recombinant helper‐dependent bone morphogenetic protein‐9 (BMP‐9) adenovirus
Authors: Li Jin Zhong , Hankins Gerald R. , Kao Chinghai , Li Hongwei , Kammauff John , Helm Gregory A.
Published: August 2003

Background

Although recombinant first‐generation BMP adenoviruses can induce ectopic bone formation in immune deficient animals, the osteoinductive activity of these BMP vectors is reduced in immune competent animals. Helper‐dependent adenoviral vectors have been developed to decrease the immune response and, therefore, increase gene expression in immune competent animals compared with first‐generation vectors. In the present study, the osteoinductive activity of a helper‐dependent GFP and BMP‐9 adenoviral vector (ADGBMP9) was evaluated in vitro and in vivo.

Methods

Initially, purified ADGBMP9 was used to transduce human mesenchymal stem cells (hMSCs) and the alkaline phosphatase activity was determined as a measure of osteogenic activity. The vector was then injected into the thigh muscle of athymic nude and Sprague‐Dawley rats, and CT scans and histology were subsequently used to assess bone formation.

Results

In vitro, ADGBMP9 was capable of inducing alkaline phosphatase expression in hMSCs. In vivo in athymic nude and Sprague Dawley rats, ADGBMP9 initiated the process of bone formation 3 days after percutaneous injection into the thigh musculature. The rats demonstrated intramuscular ectopic ossification in CT scans as early as day 9 post viral injection and ultimately formed significant amounts of ectopic bone. Histologically, the induced bone was formed via normal endochondral mechanisms.

Conclusions

A helper‐dependent adenoviral vector containing the BMP‐9 and GFP genes has significant osteoinductive activity in both athymic nude and immune competent rats. Additional direct and ex vivo BMP gene therapy studies are required to assess the vector's activity in more animal models. Copyright © 2003 John Wiley & Sons, Ltd.
IONP‐PLL: a novel non‐viral vector for efficient gene delivery
Authors: Xiang Juan‐Juan , Tang Jing‐Qun , Zhu Shi‐Guo , Nie Xin‐Min , Lu Hong‐Bin , Shen Shou‐Rong , Li Xiao‐Ling , Tang Ke , Zhou Ming , Li Gui‐Yuan
Published: August 2003

Background

Non‐viral methods of gene delivery have been an attractive alternative to virus‐based gene therapy. However, the vectors that are currently available have drawbacks limiting their therapeutic application.

Methods

We have developed a self‐assembled non‐viral gene carrier, poly‐L‐lysine modified iron oxide nanoparticles (IONP‐PLL), which is formed by modifying poly‐L‐lysine to the surface of iron oxide nanoparticles. The ability of IONP‐PLL to bind DNA was determined by ratio‐dependent retardation of DNA in the agarose gel and co‐sedimentation assay. In vitro cytotoxic effects were quantified by MTT assay. The transfection efficiency in vitro was evaluated by delivering exogenous DNA to different cell lines using IONP‐PLL. Intravenous injection of IONP‐PLL/DNA complexes into mice was evaluated as a gene delivery system for gene therapy. The PGL2‐control gene encoding firefly luciferase and the EGFP‐C2 gene encoding green fluorescent protein were used as marker genes.

Results

IONP‐PLL could bind and protect DNA. In contrast to PLL and cationic liposomes, IONP‐PLL described here was less cytotoxic in a broad range of concentrations. In the current study, we have demonstrated that IONP‐PLL can deliver exogenous gene to cells in vitro and in vivo. After intravenous injection, IONP‐PLL transferred reporter gene EGFP‐C2 to lung, brain, spleen and kidney. Furthermore, we have demonstrated that IONP‐PLL transferred exogenous DNA across the blood‐brain barrier to the glial cells and neuron of brain.

Conclusions

IONP‐PLL, a low‐toxicity vector, appears to have potential for fundamental research and genetic therapy in vitro and in vivo, especially for gene therapy of CNS disease. Copyright © 2003 John Wiley & Sons, Ltd.
Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat
Authors: Chen Jieli , Li Yi , Katakowski Mark , Chen Xiaoguang , Wang Lei , Lu Dunyue , Lu Mei , Gautam Subhash C. , Chopp Michael
Published: August 2003

The present study investigates the induction of neurogenesis, reduction of apoptosis, and promotion of basic fibroblast growth factor (bFGF) expression as possible mechanisms by which treatment of stroke with bone marrow stromal cells (MSCs) improves neurological functional recovery. Additionally, for the first time, we treated cerebral ischemia in female rats with intraveneous administration of MSCs. Female rats were subjected to 2 hr of middle cerebral artery occlusion (MCAo), followed by an injection of 3 × 106 male (for Y chromosome labeling) rat MSCs or phosphate‐buffered saline (PBS) into the tail vein 24 hr after MCAo. All animals received daily injection of bromodeoxyuridine (BrdU; 50 mg/kg, i.p.) for 13 days after treatment for identification of newly synthesized DNA. Animals were sacrificed at 14 days after MCAo. Behavioral tests (rotarod and adhesive‐removal tests) were performed. In situ hybridization, immunohistochemistry, and terminal deoxynucleotidyltransferase (TdT)‐mediated dUTP‐biotin nick‐end labeling (TUNEL) were performed to identify transplanted MSCs (Y chromosome), BrdU, bFGF, and apoptotic cells in the brain. Significant recovery of behavior was found in MSC‐treated rats at 7 days in the somatosensory test and at 14 days in the motor test after MCAo compared with control, PBS‐treated animals (P < .05). MSCs were found to survive and preferentially localize to the ipsilateral ischemic hemisphere. Significantly more BrdU‐positive cells were located in the subventricular zone (P < .05), and significantly fewer apoptotic cells and more bFGF immunoreactive cell were found in the ischemic boundary area (P < .05) of MSC‐treated rats than in PBS‐treated animals. Here we demonstrate that intravenously administered male MSCs increase bFGF expression, reduce apoptosis, promote endogenous cellular proliferation, and improve functional recovery after stroke in female rats. © 2003 Wiley‐Liss, Inc.
Dysfunctional oligodendrocyte progenitor cell (OPC) populations may inhibit repopulation of OPC depleted tissue
Authors: Chari D.M. , Huang W.L. , Blakemore W.F.
Published: August 2003

We have attempted to extend a previously described rat model of focal oligodendrocyte progenitor cell (OPC) depletion, using 40 Gy X‐irradiation (Chari and Blakemore [2002] Glia 37:307–313), to the adult mouse spinal cord, to examine the ability of OPCs present in adjacent normal areas to colonise areas of progenitor depletion. In contrast to rat, OPCs in the mouse spinal cord appeared to be a comparatively radiation‐resistant population, as 30–35% of OPCs survived in X‐irradiated tissue (whereas <1% of OPCs survive in X‐irradiated rat spinal cord). The numbers of surviving OPCs remained constant with time indicating that this population was incapable of regenerating itself in response to OPC loss. Additionally, these OPCs did not contribute to remyelination of axons when demyelinating lesions were placed in X‐irradiated tissue, suggesting that the surviving cells are functionally impaired. Importantly, the length of the OPC‐depleted area did not diminish with time, as would be expected if progressive repopulation of OPC‐depleted areas by OPCs from normal areas was occurring. Our findings therefore raise the possibility that the presence of a residual dysfunctional OPC population may inhibit colonisation of such areas by normal OPCs. © 2003 Wiley‐Liss, Inc.
Glia‐conditioned medium induces de novo synthesis of tyrosine hydroxylase and increases dopamine cell survival by differential signaling pathways
Authors: de Bernardo S. , Canals S. , Casarejos M. J. , Rodriguez‐Martin E. , Mena M. A.
Published: August 2003

The mesencephalic astroglia‐conditioned medium (GCM) greatly increases dopamine (DA) phenotype expression, and it also protects from spontaneous and toxin‐induced cell death in midbrain cultures. In this study, we have investigated the signaling pathways implicated in those effects. Genistein at 5 μM, an inhibitor of tyrosine kinase receptors, and KT‐5720, a protein kinase A inhibitor, blocked the GCM‐induced effects on DA phenotype expression and DA cell survival but did not abolish the increased astrocytic (glial fibrillary acidic protein‐positive; GFAP+) processes. We analyzed the role of phosphatidylinositol‐3 kinase (PI‐3K) on TH induction and cell survival, with the PI‐3K inhibitors LY‐294002 and wortmannin, and the role of the phosphorylation of mitogen‐activated protein kinase (MAPK) with PD‐98059, a p‐ERK1/2 MAPK inhibitor. LY‐294002 at 20–30 μM blocked the GCM‐induced effects on TH expression and DA cell survival but did not abolish the increased astrocytic processes. PD‐98059 at 20 and 40 μM blocked the GCM‐induced effects on DA phenotype, cell survival, and GFAP expression. However, staurosporine at 10 nM, a protein kinase C inhibitor, only blocked the protective effects induced by GCM on midbrain cell apoptosis. The data presented herein show that tyrosine kinase receptors, cAMP‐dependent protein kinase, PI‐3K, and MAPK signaling pathways are implicated in de novo synthesis of TH+cells induced by GCM as well as in DA cell apoptosis and that these effects are unrelated to increased GFAP expression. PKC inhibitors only abolished the GCM‐induced effects on midbrain neuronal survival, suggesting that signaling pathways for DA phenotype expression and survival may be independent. © 2003 Wiley‐Liss, Inc.
Dietary lithium induces regional increases of mRNA encoding cysteine string protein in rat brain
Authors: Cordeiro Mara L. , Gundersen Cameron B. , Umbach Joy A.
Published: August 2003

Lithium salts are used to treat manic‐depressive disorders; however, the mechanism by which lithium produces its therapeutic benefit remains obscure. The action of lithium may involve alterations of proteins important for regulating synaptic function. In this context, we observed recently that lithium at therapeutically relevant concentrations enhanced expression of cysteine string protein (csp) at the level of both mRNA and protein, in cell culture and in rat brain. Several lines of evidence have shown that csps are vital components of the regulated secretory pathway. We were interested whether lithium modulates expression of csp in specific brain regions. To study this issue, we analyzed the effects of chronic lithium administration (21 days) on csp mRNA levels in rat brain using in situ hybridization. Densitometric analysis revealed that lithium upregulated csp mRNA in several brain areas that are important for mood and behavior. This effect may be germane to understanding the beneficial action of lithium in mood disorders. © 2003 Wiley‐Liss, Inc.
Selective disappearance of an axonal protein, 440‐kDa ankyrinB, associated with neuronal degeneration induced by methylmercury
Authors: Sakaue Motoharu , Takanaga Hiromi , Adachi Tatsumi , Hara Shuntaro , Kunimoto Manabu
Published: August 2003

The 440‐kDa isoform of brain ankyrin, 440‐kDa ankyrinB, is a neuron‐specific protein and is confined to axons. Cerebellum is one of the areas characteristically altered by methylmercury intoxication both in the adult and during development. When rat cerebellar neurons matured for 7 days in vitro were exposed to methylmercury at 0.03 μM for 48 hr, viability of the cells was unaffected. However, the immunocytochemical staining of 440‐kDa ankyrinB diminished drastically, whereas that of microtubule‐associated protein‐2, which is localized in dendrites and cell bodies, and of glial fibrillary acidic protein (GFAP), a marker for astroglial cells coexisting in the culture, remained unchanged. To confirm these observations, a simplified dot blot assay was established to determine 440‐kDa ankyrinB and several other marker proteins in cultured cell samples. With this assay, we found that methylmercury at a submicromolar range induced a decrease of 440‐kDa ankyrinB and an increase of GFAP in a dose‐dependent manner in cerebellar cells in primary culture. Surprisingly, another axonal protein, tau, remained mostly in its intact molecular sizes even in the presence of 0.3–1.0 μM methylmercury, though its immunocytochemical localization was substantially altered. These results indicate that selective loss of the axonal protein 440‐kDa ankyrinB is associated with the early stage of degeneration of cerebellar neurons induced by methylmercury. Therefore, 440‐kDa ankyrinB should be useful as a specific and sensitive marker for the neurotoxicity of methylmercury. © 2003 Wiley‐Liss, Inc.
Targeted transgene expression in rat brain using lentiviral vectors
Authors: Jakobsson Johan , Ericson Cecilia , Jansson Maria , Björk Elin , Lundberg Cecilia
Published: August 2003

Direct gene transfer to the adult brain is dependent on vectors that transduce non‐dividing cells, such as lentiviral vectors. Another aspect of the development of gene therapy to the brain is the need for cell‐specific transgene expression. Expression from vesicular stomatitis virus G‐protein (VSV‐G) pseudotyped lentiviral vectors has been reported to be mainly neuron specific in the brain. We constructed cell‐specific lentiviral vectors using the neuron‐specific enolase (rNSE) or the glial fibrillary acidic protein (hGFAP) promoters and compared them to the ubiquitous human cytomegalovirus promoter (hCMV), a hybrid CMV/β‐actin promoter (CAG) and the promoter for human elongation factor 1α (EF1α). Our results showed that the hGFAP promoter was expressed only in glial cells, whereas rNSE was purely neuron specific, showing that VSV‐G is pantropic in the rat striatum. We conclude that the VSV‐G allows transduction of both glial and neuronal cells and the promoter dictates in what cell type the transgene will be expressed. The expression of transgenes exclusively in astrocytes would allow for local delivery of secreted transgene products, such as glial cell line‐derived neurotrophic factor (GDNF), circumventing the anterograde transport that may induce unwanted side effects. © 2003 Wiley‐Liss, Inc.
Caspase activity is essential for long‐term potentiation
Authors: Gulyaeva Natalia V. , Kudryashov Igor E. , Kudryashova Irina V.
Published: August 2003

Slices from rat hippocampus were incubated with the caspase‐3 inhibitor N‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp fluoromethylketone (Z‐DEVD‐FMK) or with the inactive peptide N‐benzyloxycarbonyl‐Phe‐Ala fluoromethylketone (Z‐Phe‐Ala‐FMK) for 30 min. The peptides changed neither input–output curves nor paired‐pulse effects at 70‐msec interpulse intervals, nor amplitudes of pop spikes in the CA1 region 1.0–6.9 hr after the incubation. Slices taken 1.0–1.4 hr after Z‐DEVD‐FMK or inactive peptide treatment demonstrated similar long‐term potentiation (LTP) curves; however, LTP was suppressed significantly (P < 0.001) 1.5–3.4 hr after Z‐DEVD‐FMK treatment when compared to the corresponding inactive peptide group. LTP magnitude correlated with time after Z‐DEVD‐FMK (r = −0.74; P < 0.02) but did not depend on time after the inactive peptide treatment. After 3.5 hr, LTP was blocked completely. Z‐DEVD‐FMK did not have a significant effect on presynaptic function. The results are the first evidence that inhibition of caspase‐3 significantly decreases or fully blocks LTP in the CA1 region and suggest that caspase‐3 is essential for LTP. Candidate caspase‐3 substrates that may be cleaved for LTP induction and maintenance are discussed. © 2003 Wiley‐Liss, Inc.
Relationship between NMDA receptor expression and MPP+ toxicity in cultured dopaminergic cells
Authors: Church William H. , Hewett Sandra J.
Published: August 2003

It has been suggested that excitotoxicity could be contributing to dopamine cell loss after methylphenylpyridinium ion (MPP+) exposure, although the literature regarding this is contradictory. Given that in cell culture excitotoxicity has been reported to be dependent on culture age, we postulated that these discrepant results might be explained by a difference in developmental expression of N‐methyl‐D‐aspartate (NMDA) receptors. To test this, mesencephalic cells were cultured and the number of dopaminergic neurons (tyrosine hydroxylase‐immunoreactive cells [TH‐IR] cells) expressing the NMDA R1 subunit (NR1) was determined using double‐label immunofluorescence microscopy. An increase in the percentage of TH‐IR cells expressing NR1 occurred over time in culture and this correlated with the toxicity of NMDA. At 7 days in vitro (DIV 7), only 17% (n = 167 cells/4 experiments) of TH‐IR cells expressed NR1 and these cells were insensitive to NMDA toxicity. This increased to 80% (n = 254 cells/6 experiments) by DIV 11 and cultures were now susceptible to NMDA‐induced injury. Cultures grown for either 7 or 11 days were treated for 48 hr with increasing concentrations of MPP+ (0.5–20 μM) and the loss of dopaminergic neurons was determined by cell counting. Cultures at DIV 7 were more sensitive to MPP+ than 11‐day‐old cultures (LD50 = ∼0.75 μM vs. 15 μM, respectively). Co‐exposure to MK‐801 (5 μM) did not protect against MPP+ toxicity in young cultures, but attenuated MPP+ toxicity in the older cultures, becoming statistically significant at 20 μM MPP+. These data indicate that the activation of NMDA receptors is not required for, but can contribute to, MPP+‐induced neurodegeneration of dopaminergic cells in culture. © 2003 Wiley‐Liss, Inc.
Palaeoecological evidence of changes in vegetation and climate during the Holocene in the pre‐Polar Urals, northeast European Russia
Authors: Kultti Seija , Väliranta Minna , Sarmaja‐Korjonen Kaarina , Solovieva Nadia , Virtanen Tarmo , Kauppila Tommi , Eronen Matti
Published: August 2003

This study investigated Holocene tree‐line history and climatic change in the pre‐Polar Urals, northeast European Russia. A sediment core from Mezhgornoe Lake situated at the present‐day alpine tree‐line was studied for pollen, plant macrofossils, Cladocera and diatoms. A peat section from Vangyr Mire in the nearby mixed mountain taiga zone was analysed for pollen. The results suggest that the study area experienced a climatic optimum in the early Holocene and that summer temperatures were at least 2°C warmer than today. Tree birch immigrated to the Mezhgornoe Lake area at the onset of the Holocene. Mixed spruce forests followed at ca. 9500–9000 14C yr BP. Climate was moist and the water level of Mezhgornoe Lake rose rapidly. The hypsithermal phase lasted until ca. 5500–4500 14C yr BP, after which the mixed forest withdrew from the Mezhgornoe catchment as a result of the climate cooling. The gradual altitudinal downward shift of vegetation zones resulted in the present situation, with larch forming the tree‐line. Copyright © 2003 John Wiley & Sons, Ltd.
Radiocarbon constraints on the Holocene flood deposits of the Ning‐Zhen Mountains, lower Yangtze River area of China
Authors: Yu Shi‐Yong , Zhu Cheng , Wang Fubao
Published: August 2003

Lying athwart both the temperate and subtropical zones, the Ning‐Zhen Mountains are particularly prone to extreme floods in the summer months when cold fronts collide with the subtropics‐derived warm airmasses. The Holocene flood deposits in the region may provide a long‐term perspective on hydrographical change and its palaeoclimatic implications. Radiocarbon dates on carbonised wood preserved in flooding sediments reveal that the region has experienced a number of catastrophic floods throughout the middle Holocene. These extreme flooding events cluster into three periods: (i) 9200–8200 cal. yr BP, (ii) 7600–5800 cal. yr BP and (iii) 5200–4000 cal. yr BP, corresponding to the times when the East Asian monsoon has been intensified under warm conditions. The significant falls in flooding frequency around 8200 cal. yr BP, 5800 cal. yr BP and 4000 cal. yr BP may have resulted from substantial reduction in regional precipitation, probably associated with meridional displacement of the planetary frontal system. Copyright © 2003 John Wiley & Sons, Ltd.
Glaciotectonised Quaternary sediments at Cape Shpindler, Yugorski Peninsula, Arctic Russia: implications for glacial history, ice movements and Kara Sea Ice Sheet configuration
Authors: Lokrantz Hanna , Ingólfsson Ólafur , Forman Steven L.
Published: August 2003

The coastal cliffs of Cape Shpindler, Yugorski Peninsula, Arctic Russia, occupy a key position for recording overriding ice sheets during past glaciations in the Kara Sea area, either from the Kara Sea shelf or the uplands of Yugorski Peninsula/Polar Urals. This study on Late Quaternary glacial stratigraphy and glaciotectonic structures of the Cape Shpindler coastal cliffs records two glacier advances and two ice‐free periods older than the Holocene. During interglacial conditions, a sequence of marine to fluvial sediments was deposited. This was followed by a glacial event when ice moved southwards from an ice‐divide over Novaya Zemlya and overrode and disturbed the interglacial sediments. After a second period of fluvial deposition, under interstadial or interglacial conditions, the area was again subject to glacial overriding, with the ice moving northwards from an inland ice divide. The age‐control suggests that the older glacial event could possibly belong to marine oxygen isotope stage (MOIS) 8, Drenthe (300–250 ka), and that the underlying interglacial sediments might be Holsteinian (>300 ka). One implication of this is that relict glacier ice, buried in sediments and incorporated into the permafrost, may survive several interglacial and interstadial events. The younger glacial event recognised in the Cape Shpindler sequence is interpreted to be of Early‐to‐Middle Weichselian age. It is suggested to correlate to a regional glaciation around 90 or 60 ka. The Cape Shpindler record suggests more complex glacial dynamics during that glaciation than can be explained by a concentric ice sheet located in the Kara Sea, as suggested by recent geological and model studies. Copyright © 2003 John Wiley & Sons, Ltd.
Early and Middle Pleistocene vegetation history of the Médoc region, southwest France
Authors: O'Brien C. E. , Jones R. L.
Published: August 2003

Pleistocene deposits, together with their pollen, plant macrofossil, foraminiferal, dinoflagellate and coleopteran remains, from five sites along the Atlantic coast of the Médoc Peninsula are described and discussed. Sediments making up the Négade Formation are shown to have been laid down under either estuarine or lagoonal conditions when closed Quercus–Pinus‐Tsuga canadensis regional woodland existed. Comparison with plant records from The Netherlands indicates that these deposits are most likely attributable to either the Early Pleistocene Bavel Interglacial (marine oxygen isotope stage (MIS) 31), or an interglacial of the Waalian (MIS 37–49) or Tiglian (MIS 63–79). In addition, clays assigned to the Argiles du Gurp sensu stricto, were similarly deposited in either an estuary or lagoon, which subsequently was cut off from the sea. A freshwater lake with vegetation dominated by Azolla filiculoides then developed. This was succeeded by reedswamp and an organic mud (termed Lignite in the corresponding French stratigraphical records) formed. Regional Quercus–Abies woodland was replaced by one with Pinus dominant and Pterocarya a minor component. Comparison with plant records from France and other parts of Europe suggest that the clays and organic mud might be correlated with the Holsteinian (Praclaux) Interglacial (MIS 11c). Copyright © 2003 John Wiley & Sons, Ltd.
Deformation styles as a key for interpreting glacial depositional environments
Authors: McCarroll Danny , Rijsdijk Kenneth F.
Published: August 2003

Lithostratigraphical and lithofacies approaches used to interpret glacial sediments often ignore deformation structures that can provide the key to environment of formation. We propose a classification of deformation styles based on the geometry of structures rather than inferred environment of formation. Five styles are recognised: pure shear (P), simple shear (S), compressional (C), vertical (V) and undeformed (U). These dictate the first letter of the codes; the remaining letters conveying the evidence. This information can be used to reconstruct palaeostress fields and to infer physical properties of sediments when they deformed. Individual structures are not diagnostic of particular environments but the suite of structures, their relative scale, stratigraphical relationships, and orientation relative to palaeoslopes and to palaeoice‐flow directions can be used to infer the environment in which they formed. This scheme is applied at five sites in west Wales. The typical succession is interpreted as subglacial sediments overlain by meltout tills, flow tills and sediment flows. Paraglacial redistribution of glacial sediments is widespread. Large‐scale compressional deformation is restricted to sites where glaciers readvanced. Large‐scale vertical deformation occurs where water was locally ponded near the ice margin. There is no evidence for glaciomarine conditions. Copyright © 2003 John Wiley & Sons, Ltd.
Effect of processing and storage time on in vitro digestibility and resistant starch content of two bean (Phaseolus vulgaris L) varieties
Authors: Osorio‐Díaz Perla , Bello‐Pérez Luis A , Sáyago‐Ayerdi Sonia G , Benítez‐Reyes María del Pilar , Tovar Juscelino , Paredes‐López Octavio
Published: August 2003

Seeds from two commercial bean varieties were cooked and stored for different times and analysed for chemical composition and in vitro starch digestibility. Parallel portions of cooked seeds were dried at 55 °C, milled and stored as flours. In general, protein and ash contents in both samples did not change with storage time, but statistical differences were shown between the two varieties (p < 0.05). Available starch (AS) contents in flours from the ‘negro’ variety did not change (p < 0.05) with storage time and, in general, were higher than in ‘flor de mayo’ samples, whose AS levels decreased during storage. The lower AS in ‘flor de mayo’ flour could be the consequence of formation of resistant starch due to retrogradation. Samples of whole ‘negro’ seeds did not show differences in AS content at 0, 24 and 48 h of storage compared with the corresponding flours, but at 72 and 96 h the AS increased in the whole samples. ‘Flor de mayo’ showed a similar pattern in flour and whole samples, with slightly higher values in the whole seeds. In general, total resistant starch (RS) content in the two varieties was higher in the flours than in ‘whole’ seeds, a fact that is not easy to explain at present. ‘Negro’ flour presented an RS content around 65.0 g kg−1, and approximately 55.0 g kg−1 was recorded in ‘flor de mayo’, with slight changes when storage time increased. Whole ‘flor de mayo’ showed significant levels of the retrograded portion of resistant starch (RRS), which did not change with storage time (p < 0.05). However, values were lower than in the flours. A pattern similar to that of the ‘negro’ variety was obtained for ‘flor de mayo’, since the flour exhibited higher amounts of RRS; however, in this variety, the RRS content in ‘whole’ samples decreased after prolonged storage. Flours presented higher amylolysis rates than whole samples, and the ease of digestion increased with storage time. Copyright © 2003 Society of Chemical Industry
Formation of paramagnetic products in leaves of wheat (Triticum aestivum) as a result of ozone‐induced stress
Authors: Goodman Bernard A , Reichenauer Thomas G
Published: August 2003

Electron paramagnetic resonance (EPR) measurements on light‐harvested samples of wheat leaves that had been exposed to toxic ozone levels showed the production of a single‐peak free radical signal and a large increase in intensity of the Fe(III) (g = 4.3) signal compared with control samples. These changes were accompanied by a decrease in the intensity of the more complex free radical signal that is associated with a tyrosine‐based radical centre in photosystem II (PSII) protein. No qualitative changes in the Mn(II) signal were seen as a result of ozone exposure. Identification of the stress‐induced free radical is discussed in the context of previous speculations in the literature, and it is concluded that there is still insufficient evidence for a positive identification. A similar situation exists with the Fe(III) component, where the ligand(s) remain(s) unidentified, although the complex is confirmed as a mononuclear species. Copyright © 2003 Society of Chemical Industry
Relationship between physicochemical and cooking properties, and effects of cooking on antinutrients, of yellow field peas (Pisum sativum)
Authors: Wang Ning , Daun James K , Malcolmson Linda J
Published: August 2003

The relationship between the physicochemical and cooking properties of yellow peas was examined in this study. A positive correlation was found between seed weight and water hydration capacity. The Peleg model, which was modified, could be used to describe the water absorption characteristics of peas and could be used to predict the rate of water absorption in the initial water absorption period. Cooking time could be measured objectively using the Mattson cooker. Cooking time was found to decrease with an increase in water hydration capacity. Hardness of cooked peas, measured using a texture analyser, was negatively correlated with both seed weight and water hydration capacity. Seed coats had a significant effect on water hydration and cooking quality of peas. Peas with relatively thin seed coats exhibited higher water hydration capacity, shorter cooking times and softer texture after cooking. The effects of soaking and cooking on trypsin inhibitor activity (TIA) and oligosaccharide levels in peas were also studied. Cooking was more effective than soaking in reducing TIA levels and oligosaccharides (raffinose, stachyose and verbascose) in peas. Copyright © 2003 Society of Chemical Industry
Carotenoids in yellow‐ and red‐fleshed papaya (Carica papaya L)
Authors: Chandrika U Gamage , Jansz Errol R , Wickramasinghe SMD Nalinie , Warnasuriya Narada D
Published: August 2003

Vitamin A deficiency is a disorder of public health importance in Sri Lanka. A recent national survey revealed that 36% of preschool children in Sri Lanka have vitamin A deficiency (serum retinol <0.2 µg ml−1). In view of its well‐established association with child morbidity and mortality, this is a reason for concern. One of the main fruits which has been recommended for prevention of vitamin A deficiency in Sri Lanka is papaya (Carica papaya L). In this study the carotenoid profiles of yellow‐ and red‐fleshed papaya were analysed by medium‐pressure liquid chromatography (MPLC) and UV‐vis spectrophotometry. A section of yellow‐fleshed papaya showed small carotenoid globules dispersed all over the cell, whereas in red‐fleshed papaya the carotenoids were accumulated in one large globule. The major carotenoids of yellow‐fleshed papaya were the provitamin A carotenoids β‐carotene (1.4 ± 0.4 µg g−1 dry weight (DW)) and β‐cryptoxanthin (15.4 ± 3.3 µg g−1 DW) and the non‐provitamin A carotenoid ζ‐carotene (15.1 ± 3.4 µg g−1 DW), corresponding theoretically to 1516 ± 342 µg kg−1 DW mean retinol equivalent (RE). Red‐fleshed papaya contained the provitamin A carotenoids β‐carotene (7.0 ± 0.7 µg g−1 DW), β‐cryptoxanthin (16.9 ± 2.9 µg g−1 DW) and β‐carotene‐5,6‐epoxide (2.9 ± 0.6 µg g−1 DW), and the non‐provitamin A carotenoids lycopene (11.5 ± 1.8 µg g−1 DW) and ζ‐carotene (9.9 ± 1.1 µg g−1 DW), corresponding theoretically to 2815 ± 305 µg kg−1 DW mean RE. Thus the carotenoid profile and organisation of carotenoids in the cell differ in the two varieties of papaya. This study demonstrates that carotenoids can be successfully separated, identified and quantified using the novel technique of MPLC. Copyright © 2003 Society of Chemical Industry
In vitro degradability of mature and immature leaves of tropical forage legumes differing in condensed tannin and non‐starch polysaccharide content and composition
Authors: Barahona Rolando , Lascano Carlos E , Narvaez Nelmy , Owen Emir , Morris Phillip , Theodorou Mike K
Published: August 2003

A study was designed to examine the relationships between protein, condensed tannin and cell wall carbohydrate content and composition and the nutritional quality of seven tropical legumes (Desmodium ovalifolium, Flemingia macrophylla, Leucaena leucocephala, L pallida, L macrophylla, Calliandra calothyrsus and Clitoria fairchildiana). Among the legume species studied, D ovalifolium showed the lowest concentration of nitrogen, while L leucocephala showed the highest. Fibre (NDF) content was lowest in C calothyrsus, L Leucocephala and L pallida and highest in L macrophylla, which had no measurable condensed tannins. The highest tannin concentration was found in C calothyrsus. Total non‐structural polysaccharides (NSP) varied among legumes species (lowest in C calothyrsus and highest in D ovalifolium), and glucose and uronic acids were the most abundant carbohydrate constituents in all legumes. Total NSP losses were lowest in F macrophylla and highest in L leucocephala and L pallida. Gas accumulation and acetate and propionate levels were 50% less with F macrophylla and D ovalifolium as compared with L leucocephala. The highest levels of branched‐chain fatty acids were observed with non‐tanniniferous legumes, and negative concentrations were observed with some of the legumes with high tannin content (D ovalifolium and F macrophylla). Linear regression analysis showed that the presence of condensed tannins was more related to a reduction of the initial rate of gas production (0–48 h) than to the final amount of gas produced or the extent (144 h) of dry matter degradation, which could be due to differences in tannin chemistry. Consequently, more attention should be given in the future to elucidating the impact of tannin structure on the nutritional quality of tropical forage legumes. Copyright © 2003 Society of Chemical Industry
Indigestible fraction of edible marine seaweeds
Authors: Rupérez Pilar , Toledano Gema
Published: August 2003

As dietary fibre constitutes only part of the non‐digestible components of plant foods, there is a general trend to extend the fibre concept to include all the main food constituents that reach the colon undegraded. In this study the major non‐digestible components of five edible brown and red Spanish marine seaweeds—Fucus vesiculosus. Laminaria digitata (Kombu), Undaria pinnatifida (Wakame), Chondrus crispus (Irish moss) and Porphyra tenera (Nori)—were determined under simulated physiological conditions by an in vitro enzymatic method (Sauro‐Calixto F et al, J Agric Food Chem 48: 3342–3347 (2000)) developed as an alternative to dietary fibre analysis. Two indigestible fractions, soluble (SIF) and insoluble (IIF), were obtained after enzymatic treatments of seaweeds. SIF amounted to 40–170 g kg−1 algal dry weight. Gravimetric values of IIF were 246–599 g kg−1 and its major components were resistant protein (37–133 g kg−1), minerals (13–78 g kg−1) and non‐starch polysaccharides plus klason lignin (196–415 g kg−1). The total indigestible fraction determined by colorimetry (335–516 g kg−1) was similar to the total dietary fibre content of seaweeds determined previously by the AOAC method. However, the distribution of the soluble and insoluble fractions differed between the two procedures. Estimation of the indigestible fraction of edible seaweeds could be useful from both nutritional and commercial points of view. Copyright © 2003 Society of Chemical Industry
New considerations for an overall approach to treat hepatocellular carcinoma in cirrhotic patients
Authors: Donckier Vincent , Van Laethem Jean Luc , Van Gansbeke Daniel , Ickx Brigitte , Lingier Pierre , Closset Jean , El Nakadi Issam , Feron Pascale , Boon Nathalie , Bourgeois Nadine , Adler Michael , Gelin Michel
Published: August 2003

Background

Increasing numbers of cases and organ shortage justify reconsidering the global therapeutic approach for hepatocelluar carcinoma in cirrhotic patients.

Methods

Recent literature was reviewed, focused on new therapeutic technologies such as radiofrequency.

Results

For small tumors, liver transplantation offers theoretically the best chance for cure. However, organ shortage may eliminate this advantage, because of tumor progression while waiting for a graft. For small tumors, arising on compensated cirrhosis, resection or radiofrequency ablation may provide efficient local tumor control without precluding subsequent transplantation in case of tumor recurrence and/or cirrhosis decompensation.

Conclusions

For small tumors and compensated cirrhosis, resection or radiofrequency could represent acceptable first line treatments. In addition to permit safe and immediate tumor control, this strategy would allow a preferential redistribution of grafts to patients with decompensated cirrhosis in whom transplantation is the only possibility. J. Surg. Oncol. 2003;84:36–44. © 2003 Wiley‐Liss, Inc.
Micronutrients, antioxidants, and carcinoma of the gallbladder
Authors: Shukla Vijay Kumar , Adukia Tarun Kumar , Singh Sureshwer Prasad , Mishra Chandra Pati , Mishra Ravindra Nath
Published: August 2003

Objectives

Nutrient deficiency in developing countries can be considered a significant contributory factor modifying the multistage process of carcinogenesis. Studies from different parts of the world have shown the deficiency of various micronutrients to be significantly associated with cancer. This study was undertaken to test the above hypothesis in patients with carcinoma of the gallbladder.

Methods

Selenium (Se), zinc (Zn), copper (Cu), manganese (Mn), ascorbic acid (vitamin C), and α‐tocopherol (vitamin E) were estimated in the serum, bile, and gallbladder tissue of 30 patients each of carcinoma of the gallbladder (group‐I), cholelithiasis (group‐II), and only in the serum of 30 age‐ and sex‐matched healthy controls (group‐III). The minerals were analyzed by atomic absorption spectrophotometer and vitamins by spectrophotometry.

Results

The mean serum levels of Se, Zn, Mn, vitamin E, and vitamin C were significantly lower (P < 0.001) in group‐I when compared with groups II and III. The mean biliary levels of Se and Zn (0.29, 3.45 mg/L) were reduced significantly (P < 0.001) in group‐I when compared with group II (0.51, 5.2 mg/L). Mean tissue levels of Se and Zn were also significantly lower (P < 0.001) in group I (2.75, 43.09 μg/g) compared to group II (3.90, 61.37 μg/g). However, no significant difference was observed in tissue concentration of Mn, vitamin C, and vitamin E. Cu levels and Cu/Zn ratio showed a highly significant (P < 0.001) increase in serum, bile, and gallbladder tissue in carcinoma of the gallbladder compared to the other two groups.

Conclusions

The data supports an association between lower levels of Se, Zn, vitamin E, and risk of carcinoma of the gallbladder and suggest that Cu/Zn ratio could be a useful parameter in evaluating the patients of carcinoma of the gallbladder. J. Surg. Oncol. 2003;84:31–35. © 2003 Wiley‐Liss, Inc.
Acute ultrastructural changes of the trabecular meshwork after selective laser trabeculoplasty and low power argon laser trabeculoplasty
Authors: Cvenkel Barbara , Hvala Anastazija , Drnovšek‐Olup Brigita , Gale Nina
Published: August 2003

Background and Objectives

To compare the histopathological changes in the human trabecular meshwork after low power argon laser trabeculoplasty (ALT) and selective laser trabeculoplasty (SLT) with a Q‐switched, frequency‐doubled, neodymium:yttrium–aluminium–garnet (Nd:YAG) laser.

Study Design/Materials and Methods

In gonioscopically normal trabecular meshwork of three patients awaiting enucleation due to malignant melanoma of the choroid, SLT and ALT were performed 1–5 days prior to enucleation. In each eye, the lower half of trabecular meshwork received SLT, one quadrant low power (460 mW) ALT and one quadrant was left untreated. Specimens were evaluated with light and transmission electron microscopy.

Results

A sharp demarcation line was visible between the laser treated and untreated intact trabecular meshwork after ALT and SLT. Both lasers caused disruption of trabecular beams, but the extent of the damage was smaller after SLT. The collagen component of trabecular beams was mostly amorphous, the long‐spacing collagen was scanty after ALT, but more abundant after SLT. In the intertrabecular spaces fragmented cells and tissue debris with only a few pigmented cells were observed. Some endothelial cells were desquamated, but appeared slightly better preserved after SLT than ALT.

Conclusions

Our ultrastructural comparison of the morphological changes after low power ALT and SLT in patients demonstrated that both lasers caused splitting and fragmentation of the trabecular beams of the trabecular meshwork, but the extent of the damage was smaller and the preservation of long‐spacing collagen better after SLT than after ALT. Lasers Surg. Med. 33:204–208, 2003. © 2003 Wiley‐Liss, Inc.
Role of p53 and ATM in photodynamic therapy‐induced apoptosis
Authors: Heinzelmann‐Schwarz Viola , Fedier André , Hornung René , Walt Heinrich , Haller Urs , Fink Daniel
Published: August 2003

Background and Objectives

Photodynamic therapy (PDT) induces cell death through a laser light‐activated photosensitizer and is a treatment option for tumors resistant to radio‐ and chemo‐therapy.

Study Design/Materials and Methods

We investigated whether m‐THPC‐PDT induces cell death by necrosis and/or apoptosis, and whether these responses are modulated by p53 and/or ATM, two cancer‐associated genes. Sensitivity of atm+/+p53+/+, atm+/+p53−/−, and atm−/−p53−/− mouse embryonic fibroblasts to m‐THPC‐PDT performed at a wavelength of 652 nm was determined by the MTT assay, trypan blue‐exclusion, and the TUNEL and caspase3‐cleavage apoptosis assays. c‐Abl protein level was determined by immunoblotting.

Results

m‐THPC‐PDT rapidly induced cell death in a substantial fraction of cells by p53‐ and Ataxia telangiectasia mutated (ATM)‐independent non‐apoptotic processes. However, in the subset of apoptotic cells, apoptosis was reduced by loss of p53 and was even more reduced by the additional loss of ATM. Apoptosis correlated inversely with c‐Abl level.

Conclusions

p53 and ATM are not required for necrosis, but may be required for PDT‐mediated apoptosis. Lasers Surg. Med. 33:182–189, 2003. © 2003 Wiley‐Liss, Inc.
Effects of low power Er:YAG laser on the tooth pulp‐evoked jaw‐opening reflex
Authors: Zeredo Jorge L. , Sasaki Katia M. , Fujiyama Rie , Okada Yukio , Toda Kazuo
Published: August 2003

Background and Objectives

Analgesic properties of laser irradiation have been of great interest in the field of dentistry. This study aimed at evaluating the analgesic effects of the Er:YAG laser system in rats during and after laser irradiation.

Study Design/Materials and Methods

A pulsed Er:YAG laser was applied to the oral mucosa of the mandibular incisor at an energy density of approximately 0.1 J/cm2/pulse for 10 minutes at 10 Hz, and the integrated digastric muscle electromyogram in tooth pulp‐evoked jaw‐opening reflex was used as an index of the nociceptive response.

Results

Significant reflex suppression was observed 10 minutes after laser irradiation. The reflex amplitude started to return to its original level about 45 minutes after cessation of laser irradiation.

Conclusions

The Er:YAG laser used at low output levels presented inhibitory effects on the tooth pulp‐evoked jaw‐opening reflex, suggesting that this laser system may be of use for pain control during various dental treatments. Lasers Surg. Med. 33:169–172, 2003. © 2003 Wiley‐Liss, Inc.
Electrospray mass spectrometry of phospholipids
Authors: Pulfer Melissa , Murphy Robert C.
Published: August 2003

I.
Introduction
332

II.
Techniques and Methods
334

A.  Sample Preparation
334

B.  ESI‐MS/MS
336

    1.  Glycerophosphocholine (GPCho) Lipids
336

    2.  Sphingomyelin
341

    3.  Glycerophosphoethanolamine (GPEtn) Lipids
342

    4.  Glycerophosphatidic Acid (GPA) Lipids
344

    5.  Glycerophosphoinositol (GPI) Lipids
346

    6.  Glycerophosphoglycerol (GPGro) Lipids
349

    7.  Glycerophosphoserine (GPSer) Lipids
349

    8.  Cardiolipin
351

C.  Matrix‐Assisted Laser Desorption/Ionization (MALDI) Analysis
353

D.  Precursor‐Ion and Neutral‐Loss Scanning
353

E.  Quantitation
355

III.
Research Applications
356

IV.
Conclusion
359

Acknowledgments
360

Abbreviations
360

References
360

Phospholipids play a central role in the biochemistry of all living cells. These molecules constitute the lipid bilayer defining the outer confines of a cell, but also serve as the structural entities which confine subcellular components. Mass spectrometry has emerged as a powerful tool useful for the qualitative and quantitative analysis of complex phospholipids, including glycerophospholipids and the sphingolipid, sphingomyelin. Collision induced decomposition of both positive and negative molecular ion species yield rich information as to the polar head group of the phospholipid and the fatty‐acyl substituents esterified to the glycerophospholipid backbone. This review presents the current level of understanding of the mechanisms involved in the formation of various product ions following collisional activation of molecular ion species generated by electrospray ionization of the common glycerophospholipids, including phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, cardiolipin, and sphingomyelin. Recent advances in the application of matrix assisted laser desorption ionization is also considered. Several applications of mass spectrometry applied to phospholipid analysis are presented as they apply to physiology as well as pathophysiology. © 2003 Wiley Periodicals, Inc., Mass Spec Rev 22:332–364, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mas.10061