bluuebunny/crossref_metadata_2025 huggingface dataset:
Decline of surface MHC I by adenoviral gene transfer of anti‐MHC I intrabodies in human endothelial cells—new perspectives for the generation of universal donor cells for tissue transplantation
Authors: Beyer Florian , Doebis Cornelia , Busch Annette , Ritter Thomas , Mhashilkar Abner , Marasco Wayne M. , Laube Horst , Volk Hans‐Dieter , Seifert Martina
Published: March 2004

Background

The seeding of small‐calibre vascular polytetrafluoroethylene (PTFE) grafts with endothelial cells provides an increase in biocompatibility of the graft surface. The harvest and ex vivo culture of autologous endothelial cells is highly delicate. Allogeneic human umbilical vein endothelial cells (HUVEC) could be a potential cell source—however, rejection might occur due to major histocompatibility complex (MHC) I mismatches. Lowering cell surface MHC I expression on endothelial cells by gene transfer of an anti‐MHC I intrabody might reduce graft failure. The intrabody consists of a single‐chain variable fragment (sFv) of an anti‐MHC I antibody, carrying a terminal KDEL sequence to retain the molecule together with the MHC I inside the endoplasmic reticulum.

Methods

Adenoviral gene transfer was used to express the intrabody in HUVEC. The MHC I surface expression was measured 48 h after transduction by flow cytometry. Functional effects of the intrabody expression were analyzed in a calcein release cytotoxicity assay.

Results

A transduction efficiency of more than 95% with EGFP‐adenovirus indicates a sufficient gene transfer into HUVEC. Intrabody‐adenovirus‐transduced HUVEC show a massive reduction in MHC I surface expression creating almost a complete ‘knockout’ phenotype. Stimulation with inflammatory cytokines could not overcome this effect. The cell lysis of anti‐MHC I intrabody‐expressing HUVEC in a cytotoxicity assay is reduced when compared with the level of the MHC mismatched control.

Conclusions

Our data indicate that HUVEC with reduced levels of MHC I might be used as universal donor cells for the seeding of vascular grafts. Copyright © 2004 John Wiley & Sons, Ltd.
Factors affecting retrovirus‐mediated gene transfer to human CD34+ cells
Authors: Hong Youngtae , Lee Karim , Yu Seung Shin , Kim Sujeong , Kim Joong‐Gon , Shin Hee Young , Kim Sunyoung
Published: March 2004

Background

Retrovirus‐mediated gene transfer is a useful technology in studying the biology of hematopoietic stem cells (HSCs) as well as in developing gene therapy products for a variety of human diseases. One of the most important factors determining the success of these studies is the number of HSCs receiving the gene of interest.

Methods

We tested various parameters for their influences on gene transfer efficiency to CD34+ cells derived from bone marrow. Based on a literature survey, three medium formulations of CD34+ cells have been compared for their effects on gene delivery efficiency and differentiation of them. We also tested whether FBS, used in the medium formulation, could be replaced with human serum or synthetic material.

Results

Formulation A, consisting of stem cell factor, Flt‐3 ligand, thrombopoietin, and IL‐3, provided optimum results in that it maintained the highest percentage of CD34+ cells during the culture as well as produced the highest gene delivery efficiency. It was found that the synthetic serum substitute containing bovine serum albumin, insulin and human transferrin could replace the fetal bovine serum present in the original formulation A without compromising gene transfer efficiency. When the transduction procedure was repeated three times, the gene could be delivered in up to 60% of the cell population. Gene delivery efficiency was comparable between CD34+ cells derived from bone marrow and mobilized peripheral blood.

Conclusions

Our data could be useful in designing a procedure for stem cell gene therapy and providing a basis for further improving the conditions for gene transfer to various HSCs. Copyright © 2004 John Wiley & Sons, Ltd.
Improving the survival of human CNS precursor‐derived neurons after transplantation
Authors: Le Belle J.E. , Caldwell M.A. , Svendsen C.N.
Published: March 2004

We have examined the effects of predifferentiation and energy substrate deprivation on long‐term expanded human neural precursor cells (HNPCs). The pre‐differentiation of HNPC cultures produced large numbers of neurons (>60%) and mature glial cells capable of generating glycogen stores that protected the neuronal population from experimental metabolic stress. When predifferentiated HNPCs were transplanted into intact adult rat hippocampus, fewer cells survived compared to undifferentiated HNPC transplants. This cell death was completely attenuated, however, when predifferentiated HNPC cultures were pretreated to boost glial energy stores and resulted in greatly increased neuronal survival in vivo. The transplanted cells primarily engrafted within the granular layer of the dentate gyrus, where a large proportion of the predifferentiated HNPCs co‐expressed neuronal markers whereas most HNPCs outside of the neuronal layer did not, indicating that the predifferentiated cells remained capable of responding to local cues in the adult brain. Undifferentiated HNPCs migrated more widely in the brain after grafting than did the predifferentiated cells, which generally remained within the hippocampus. © 2004 Wiley‐Liss, Inc.
Differentiation of embryonic stem cells to a neural fate: A route to re‐building the nervous system?
Authors: Lang Kenneth J.D. , Rathjen Joy , Vassilieva Svetlana , Rathjen Peter D.
Published: March 2004

The many and varied proposed applications of cell replacement therapies in the treatment of human disease states, particularly those arising from cell loss or dysfunction, have been discussed widely in both the scientific and popular press. Although an attractive concept, cell therapies require the development of a readily available source of donor cells suitable for transplantation. Embryonic stem (ES) cells, with proven ability to differentiate to all cell populations of the embryo and adult in vitro, provide a potential source of therapeutic cells. The differentiation capability of mouse ES cells in vitro has been studied extensively over the last 20 years and the formation of neural precursors and neural cell lineages from mouse ES cells is well established. Cell populations highly enriched/homogenous in neural precursors have been achieved using a variety of chemical or biological inducing agents coupled with selective growth conditions. Preliminary reports suggest that similar neural enrichment is seen when these methodologies are applied to primate and human ES cells. ES cell‐derived neural precursors have been analyzed in vitro and in vivo and found to be functionally normal and, after introduction into rodent models of human neurodegenerative diseases, capable of effecting measurable disease recovery. We review progress in the formation of neural precursors from mouse ES cells, particularly the recent reports of directed differentiation of ES in response to biological inductive factors, and assess the transfer of these approaches to human ES cells. © 2004 Wiley‐Liss, Inc.
Multifaceted dialogue between graft and host in neurotransplantation
Authors: Ourednik Vaclav , Ourednik Jitka
Published: March 2004

Current restorative neurotransplantation research focuses mainly on the potential of the neural graft to replace damaged or missing cell populations and to deliver needed gene products in the form of transgenes. Because of this graft‐oriented bias of the procedure, possible dormant regenerative capabilities within the host have been largely underestimated and dismissed as insignificant. This review discusses existing evidence that neural grafts can have stimulating effects on host‐intrinsic plasticity that can help regeneration of the mammalian central nervous system. If confirmed, the synergistic interaction between graft and host might substantially enhance our therapeutic possibilities. © 2004 Wiley‐Liss, Inc.
Magnetic resonance tracking of transplanted bone marrow and embryonic stem cells labeled by iron oxide nanoparticles in rat brain and spinal cord
Authors: Jendelová Pavla , Herynek Vít , Urdzíková Lucia , Glogarová Kateřina , Kroupová Jana , Andersson Benita , Bryja Vítězslav , Burian Martin , Hájek Milan , Syková Eva
Published: March 2004

Nuclear magnetic resonance (MR) imaging provides a noninvasive method for studying the fate of transplanted cells in vivo. We studied, in animals with a cortical photochemical lesion or with a balloon‐induced spinal cord compression lesion, the fate of implanted rat bone marrow stromal cells (MSCs) and mouse embryonic stem cells (ESCs) labeled with superparamagnetic iron oxide nanoparticles (Endorem). MSCs were colabeled with bromodeoxyuridine (BrdU), and ESCs were transfected with pEGFP‐C1 (eGFP ESCs). Cells were either grafted intracerebrally into the contralateral hemisphere of the adult rat brain or injected intravenously. In vivo MR imaging was used to track their fate; Prussian blue staining and electron microscopy confirmed the presence of iron oxide nanoparticles inside the cells. During the first week postimplantation, grafted cells migrated to the lesion site and populated the border zone of the lesion. Less than 3% of MSCs differentiated into neurons and none into astrocytes; 5% of eGFP ESCs differentiated into neurons, whereas 70% of eGFP ESCs became astrocytes. The implanted cells were visible on MR images as a hypointense area at the injection site, in the corpus callosum and in the lesion. The hypointense signal persisted for more than 50 days. The presence of GFP‐positive or BrdU‐positive and nanoparticle‐labeled cells was confirmed by histological staining. Our study demonstrates that both grafted MSCs and eGFP ESCs labeled with a contrast agent based on iron oxide nanoparticles migrate into the injured CNS. Iron oxide nanoparticles can therefore be used as a marker for the long‐term noninvasive MR tracking of implanted stem cells. © 2004 Wiley‐Liss, Inc.
Base cation, aluminum, and phosphorus release potential in Danish forest soils
Authors: Callesen Ingeborg , Raulund‐Rasmussen Karsten
Published: March 2004

Long‐term nutrient supply in forest ecosystems is due to the dissolution of primary and secondary minerals in soils. The potential of nutrient release in 19 forest soils in a cool humid climate was examined. The soil profiles are classified as Alfisols (10), Spodosols (2), Entisols (4), Ultisols (1), and Mollisols (2), thus covering a gradient in soil fertility. Short‐term and long‐term release of calcium, magnesium, potassium, phosphorus, and aluminum was evaluated by a batch extraction using dilute nitric acid (0.1 M) for 2 hours, followed by 2 days (48 h), and 7 days (168 h). The solution was renewed after 2 and 50 hours extraction time. Nutrient pools expressed as g m–2 to soil depth 100 cm, and a base index (Ca2++ Mg2++ K+ (molc m–2) : Ca2+ + Mg2+ + K+ + Al3+ (molc m–2)) were interpreted in relation to soil texture classes. Subsoil texture classes: Coarse: < 5 % clay; medium 5–10 % clay or (> 5 % silt or > 50 % fine sand), or fine > 10 % clay were evaluated as an indicator of forest soil quality. Base cation and phosphorus release decreased in the order fine > medium > coarse. Texture classes explained base cation release by about 80 % of total variation, and phosphorus release by 40–50 %. The base index generally increased by extraction time for sandy soils and decreased for loamy soils. This indicated that sandy soils released accumulated reactive aluminum in the 0–2 hour extraction. Subsoil texture class is suggested as a pedotransfer function for long‐term nutrient release potential in Danish forest soils.
Modeling of the fine‐scale temperature response of arylsulfatase activity in soil
Authors: Elsgaard Lars , Vinther Finn P.
Published: March 2004

Fine‐scale (1.0–2.2 °C) temperature dependence of soil arylsulfatase activity (arylsulfate sulfohydrolase, EC 3.1.6.1) was measured at 0 to 75 °C in a Danish sandy, arable soil. Assays were done with field‐moist soil samples in the absence of toluene as plasmolytic agent – a procedure that primarily measures the extracellular enzymes. The aim was to evaluate the use of temperature models to describe the temperature response of soil arylsulfatase activity. In addition, we searched for increases in activity at high temperatures (e.g., 50–60 °C), which might be associated with unmasking (exposure) of intracellular enzymes. Arylsulfatase activities ranged from 1.1 to 60.3 μg p‐nitrophenol (g dry weight soil)–1 h–1, with an optimum temperature at 58.1 °C. The temperature response below 58.1 °C could be described by the Arrhenius equation (r2 = 0.978, n = 83) and the simple Ratkowsky equation (r2 = 0.977, n = 83). The expanded Ratkowsky equation, which covered the entire temperature range (0–75 °C), was less satisfactory (r2 = 0.958, n = 90) because the model underestimated the reaction rates near the optimum temperature. The activation energy (Ea) calculated from the Arrhenius equation was 42.2 kJ mol–1. This was higher than previously found for other soils (16.5–34.7 kJ mol–1), possibly due to the use of toluene in these studies. Further analysis of the temperature response showed that no increase in activity occurred due to potential unmasking of intracellular enzymes by disintegration of bacterial cell membranes at high temperatures. Thus, the use of high incubation temperatures did not facilitate the differentiation between intra‐ and extracellular enzyme activity.
Preconcentration of the herbicide glyphosate and its metabolite AMPA by Immobilised Metal Ion Affinity Chromatography (IMAC)
Authors: Rios Carolina , Salvadó Victòria , Hidalgo Manuela
Published: March 2004

A method based on Immobilised Metal Ion Affinity Chromatography (IMAC) using a chelating resin (Spheron Oxine 1000) loaded with Pd(II) is evaluated for the extraction and preconcentration of glyphosate and AMPA from natural water samples. The efficiency of the metal‐loaded resin in retaining glyphosate and AMPA and the ability of different reagents to recover them is investigated. The most effective elution solution for the recovery of both analytes from the resin is found to be a mixture of 0.1 M HCl and 1 M NaCl. The effect of flow rate, analyte concentration, and sample volume is evaluated. The optimised experimental conditions are then used in the extraction of the analytes from spiked natural waters. The use of the Pd loaded resin led to recoveries ranging from 80–90% for glyphosate and 60–80% for AMPA.
Karst rocky desertification in southwestern China: geomorphology, landuse, impact and rehabilitation
Authors: Wang S.‐J. , Liu Q.‐M. , Zhang D.‐F.
Published: March 2004

Karst rocky desertification is a process of land degradation involving serious soil erosion, extensive exposure of basement rocks, drastic decrease in soil productivity, and the appearance of a desert‐like landscape. It is caused by irrational, intensive land use on a fragile karst geo‐ecological environment. The process is expanding rapidly, and it is daily reducing the living space of residents and is the root of disasters and poverty in the karst areas of southwestern China. The tectonic, geomorphic and environmental background to karst rocky desertification is analysed. Population pressure and the intensive land use that have led to this serious land degradation are described. Although the problem concerns the Chinese Government and some profitable experience in the partial restoration or reconstruction of the ecological environment has been gained, effective remedial action has not been achieved on a large scale. Copyright © 2004 John Wiley & Sons, Ltd.
Growing three aromatic grasses in different alkali soils in semi‐arid regions of northern India
Authors: Dagar J. C. , Tomar O. S. , Kumar Y. , Yadav R. K.
Published: March 2004

Alkali lands in India occupy about 3 million ha. Due to poor physical properties, excessive exchangeable sodium and high pH, most of these lands support a very poor vegetation cover. Many of the medicinal and aromatic plants are in great demand for both internal requirements and export. But since these crops are non‐conventional in nature, it is not always possible for them to be produced on fertile lands, which can be used for arable crops. The marginal lands, specifically the lands affected by salinity or sodicity problems where profitable returns are not possible from agricultural crops, could be successfully utilized for the cultivation of these high‐value crops with marginal inputs. Results reported in this paper clearly indicated that out of three aromatic grasses, palmarosa (Cymbopogon martinii (Roxb.) Wats.) and lemon grass (C. flexuosus (Steud.) Wats.) could successfully be grown on moderatly alkali soils of pH up to 9·2 while vetiver (Vetiveria zizanioides (L.) Nash), which withstands both high pH and stagnation of water, could successfully be grown without significant yield reduction on highly alkali soils. These grasses not only produce essential oils used for industrial purposes but also ameliorate the soil. Copyright © 2004 John Wiley & Sons, Ltd.
Rehabilitation of a lignite mine‐disturbed area in the Indian desert
Authors: Sharma K. D. , Kumar Praveen , Gough L. P. , Sanfilipo J. R.
Published: March 2004

Extensive lignite mining in the Indian (Thar) Desert commenced within the past decade. Accompanying extraction of this valuable resource there have been visible, important environmental impacts. The resultant land degradation has prompted concern from both public and regulatory bodies. This research assesses the success of rehabilitation plans implemented to revegetate a lignite mine‐disturbed area, near the village of Giral in western Rajasthan State. Rehabilitation success was achieved within the environmental constraints of this northwest Indian hot‐desert ecosystem using a combination of: (1) backfilling (abandoned pits) with minespoil and of covering the backfilled‐surfaces with fresh topsoil to a thickness of about 0·30 m; (2) use of micro‐catchment rainwater harvesting (MCWH) technique; (3) soil profile modification approaches; (4) plant establishment methodologies; and (5) the selection of appropriate germplasm material (trees, shrubs and grasses). Preliminary results indicate that the resulting vegetative cover will be capable of self‐perpetuation under natural conditions while at the same time meeting the land‐use requirements of the local people. The minespoil is alkaline in nature and has high electrical conductance. The average content of organic carbon, N, P and K is lower than in the regional topsoil. However, the concentration of Ca, Mg, Na and total S in the minespoil is much higher than in the topsoil. Further, the spoil material has no biological activity. Enhanced plant growth was achieved in MCWH plots, compared to control plots, where minespoil moisture storage was improved by 18–43 per cent. The rehabilitation protocol used at the site appears to have been successful because, in addition to the planted species, desirable native invasive species have become established. This study developed methods for the rehabilitation of lignite mine‐disturbed areas and has also resulted in an understanding of rehabilitation processes in arid regions with an emphasis on the long‐term monitoring of rehabilitation success. Copyright © 2004 John Wiley & Sons, Ltd.
Social and economic factors affecting the adoption of soil and water conservation in West Usambara highlands, Tanzania
Authors: Tenge A. J. , De Graaff J. , Hella J. P.
Published: March 2004

Accelerated soil erosion is one of the major constraints to agricultural production in many parts of the Tanzanian highlands. Although several soil and water conservation technologies have been developed and promoted, the adoption of many recommended measures is minimal and soil erosion continues to be a problem. This research was conducted in order to determine the social and economic factors that influence adoption of soil and water conservation (SWC) measures in the West Usambara highlands, Tanzania. For this research a household survey, group discussions and transect walks were undertaken. A total of 104 households were interviewed and several fields were visited during the transect walks. Data was analysed with the use of cross‐tabulation, cluster analysis, factor analysis and chi‐squared methods. The results obtained indicate that involvement in off‐farm activities, insecure land tenure, location of fields and a lack of short‐term benefits from SWC are among the major factors that negatively influence adoption of SWC measures. Membership in farmer groups, level of education, contacts with extension agents and SWC programmes were found to be positively influencing the adoption of SWC measures. Recommendations to facilitate adoption of different SWC measures include: integration of social and economic factors into SWC plans; the creation of more awareness among farmers of soil‐erosion effects and long‐term benefits of SWC; the development of flexible SWC measures to cater for different farm patterns and a participatory approach to SWC at catchment level rather than at individual farmers' fields. Copyright © 2004 John Wiley & Sons, Ltd.
Influence of erosion on soil microbial biomass, abundance and community diversity
Authors: Mabuhay J. A. , Nakagoshi N. , Isagi Y.
Published: March 2004

This study aimed to determine microbial biomass carbon and microbial abundance immediately after, and two years after, forest soil erosion, so as to estimate the degree of damage, including the rate of recovery of microorganisms, in each area. It also aimed to determine the community diversity, and to establish relationships between microbial biomass, microbial abundance and the physico‐chemical properties of the soil.

Three different study areas in Hiroshima Prefecture, Japan, were used. One undisturbed area and two eroded areas (one immediately after and one two years after erosion). The analysis of variance showed a highly significant difference in microbial biomass carbon and abundance between the study areas. The undisturbed area showed the highest value, followed by the area eroded two years ago, then lastly the area studied immediately after the erosion. The biomass carbon was highly correlated with gram positive bacteria with r2 = 0·983, p < 0·01. The biomass carbon and microbial population were shown to be significantly correlated to the soil's physico‐chemical properties, such as pH, moisture content, water‐holding capacity and CN ratio. However, CN ratio proved to be closely correlated to biomass carbon with r2 = −0·978, p < 0·01, to Gram‐positive bacteria with r2 = −0·977, p < 0·01, to Gram‐negative bacteria with r2 = −0·989, p < 0·01 and to fungi with r2 = −0·977, p < 0·01. The undisturbed area showed a highly diverse community in both of the restriction enzymes used, followed by the area affected by erosion two years ago, then the area immediately after erosion. Copyright © 2004 John Wiley & Sons, Ltd.
Ste20‐like kinase SLK displays myofiber type specificity and is involved in C2C12 myoblast differentiation
Authors: Storbeck Christopher J. , Daniel Kate , Zhang Yi‐Hong , Lunde John , Scime Anthony , Asakura Atsushi , Jasmin Bernard , Korneluk Robert G. , Sabourin Luc A.
Published: March 2004

Cell growth and terminal differentiation are controlled by complex signaling cascades that regulate the expression of specific subsets of genes implicated in cell fate and morphogenic processes. We have recently cloned and characterized a novel Ste20‐like kinase termed SLK that is associated with adhesion structures during cell adhesion and spreading. However, the specific function of SLK is poorly understood. To gain further insight into the role of SLK, we have characterized its activity, expression, and distribution in skeletal muscle and during the in vitro differentiation of C2C12 myoblasts. Although SLK is expressed ubiquitously in adult tissues, our results show that it is predominantly expressed in muscle masses during development. Furthermore, SLK activity is upregulated during the differentiation of C2C12 myoblasts. In addition, we have found that SLK localizes presynaptically at neuromuscular junctions and that it is preferentially expressed in types I and IIA myofibers at major myofibrillar striations. Supporting a role in myoblast function and differentiation, SLK expression is induced in Myf5‐ and Pax7‐positive activated satellite cells during regeneration and expression of dominant negative SLK in C2C12 cultures impairs myoblast fusion, suggesting a role for SLK in muscle cell differentiation. Muscle Nerve 29: 553–564, 2004
Trigeminal nerve repetitive stimulation in myasthenia gravis
Authors: Rubin Devon I. , Harper C. Michel , Auger Raymond G.
Published: March 2004

The aim of this study was to evaluate the utility of repetitive nerve stimulation (RNS) of the trigeminal nerve in assessing patients with myasthenia gravis (MG). In 26 normal controls and 21 patients with myasthenia gravis (MG), 2‐Hz repetitive stimulation of the trigeminal nerve was performed using a monopolar needle for percutaneous nerve stimulation and recording over the surface of the masseter. In the MG patients, repetitive stimulation of the ulnar, spinal accessory, and facial nerves was also performed. The mean percent decrement in the compound muscle action potential (CMAP) amplitude among the different nerves at rest were: ulnar, 4.3%; spinal accessory, 10.1%; facial, 14%; and trigeminal, 17.3%. The facial nerve demonstrated abnormal decrement in 57% of all patients, compared with the spinal accessory (48%), trigeminal (43%), and ulnar (20%) nerves. All patients tolerated trigeminal RNS better than or as well as facial RNS. The study demonstrates that trigeminal RNS is a safe, reliable, efficient, and well‐tolerated technique that provides another cranial nerve–muscle combination that can be used to supplement repetitive stimulation of other limb or cranial nerves in the evaluation of patients with bulbar or generalized MG. Muscle Nerve 29: 591–596, 2004
New technologies in radiation therapy for pediatric brain tumors: The rationale for proton radiation therapy
Authors: Kirsch David G. , Tarbell Nancy J.
Published: March 2004

Background

Pediatric brain tumors are frequently treated with radiation therapy and often cured. The long‐term side effects of treatment with high‐energy X‐rays (photons) can be substantial. Proton radiation therapy may limit these late effects.

Procedure

The physical difference between photon and proton irradiation is compared. The clinical benefits of the superior physical properties of proton beam radiation therapy are explained for children with brain tumors.

Results

At biologically equivalent doses, proton radiation therapy offers tumor control similar to photon radiation therapy. The superior physical properties of proton beams make this mode of radiation therapy less likely to cause late effects.

Conclusions

For many children with brain tumors, proton beam radiation therapy may limit the late effects of radiation therapy and therefore offer an advantage over techniques using photons. © 2004 Wiley‐Liss, Inc.
Staged informed consent for a randomized clinical trial in childhood leukemia: Impact on the consent process
Authors: Angiolillo A.L. , Simon C. , Kodish E. , Lange B. , Noll R.B. , Ruccione K. , Matloub Y.
Published: March 2004

Background

Children Cancer Group (CCG) 1991 is the first childhood acute lymphoblastic leukemia trial within CCG that allowed the utilization of a staged approach to the consent process.

Methods

One hundred and forty subjects participated in the Project on Informed Consent which compared the primary outcome measures in the consent process of patients enrolled in CCG‐1991 with those enrolled in other CCG leukemia studies.

Results

The parents' trust scores were higher for the CCG‐1991 compared with other protocols. Eighty percent of parents enrolled in CCG‐1991 understood the distinction between the randomized clinical trial and the standard treatment arm, compared with 62.5% in the other studies, P = 0.05. Multiple other outcome measures suggested a positive impact from staged informed consent.

Conclusions

Our results suggest that a consent process with a staged approach can help investigators obtain a more truly informed consent. Future research is needed to confirm the benefits of the staged approach to the informed consent process. © 2004 Wiley‐Liss, Inc.
Immunologic monitoring of maintenance therapy for acute lymphoblastic leukaemia in children—preliminary report
Authors: Luczynski Wlodzimierz , Stasiak‐Barmuta Anna , Krawczuk‐Rybak Maryna
Published: March 2004

Background

In the study we evaluated immune reconstitution during maintenance therapy for acute lymphoblastic leukaemia (ALL) in relation to different treatment protocols and response of the immune system to the accompanying infections.

Procedure

The study group consisted of 40 children. The BFM protocol'90 was used in the standard risk group, while the New York protocol—in the high risk group. Assessment of the immune system was based on the analysis of peripheral blood mono‐nuclear cells by flow cytometry and concentrations of immunoglobulins: G, M, A and IgE. Each patient was examined at 1–3 months' intervals.

Results

Following cessation of intensive therapy, the successive months of maintenance treatment showed: (1) a considerable depletion of B lymphocytes, a durable decrease in IgM, IgA and gradually increasing IgG; (2) a correlation between the time passing from the cessation of intensive therapy and increased numbers and percentage of B cells, and the helper/suppressor cell ratio. In the group of children treated according to the high risk protocol, compared to the low‐risk group patients, we found lower levels of the following parameters: IgG, % lymphocytes: B and T lymphocytes (including CD4/CD8 ratio and “naive”/“memory” ratio) and NK cells (% and count). During infection: (1) a significant increase was noted in the percentage of T cells with HLA co‐expression and monocytes with ICAM‐1 co‐expression, (2) the percentage of CD3+CD45RO+ “memory” T cells was found to increase.

Conclusions

Our findings indicate quantitative and qualitative changes of the immunity in children with ALL during maintenance therapy. © 2004 Wiley‐Liss, Inc.
Alendronate regulates cell invasion and MMP‐2 secretion in human osteosarcoma cell lines
Authors: Cheng Y.Y. , Huang L. , Lee K.M. , Li K. , Kumta S.M.
Published: March 2004

Background

Osteosarcoma is the most common malignant bone tumor of childhood. Significant proportions of these patients eventually develop pulmonary metastases and succumb to their disease even after conventional multi‐agent chemotherapy and surgical excision. Matrix metalloproteinase (MMP)‐2 induced degradation of blood vessel basement membranes is an important pre‐requisite for tumor invasion and metastasis. Bisphosphonates (BPs) have been known to inhibit tumor growth and metastasis in some tumors such as breast cancer, renal cell carcinoma, and prostate cancer, and may do so through inhibition of MMP secretion. We, therefore, tested the effect of BPs on tumor cell invasion, MMP‐2 secretion, and apoptosis of osteosarcoma cell lines.

Procedure

Two osteosarcoma cell lines (SaOS‐2, U2OS) were treated with alendronate (50, 100, and 150 μM) for 24 and 48 hr. Matrigel invasion assay was used to investigate the invasive potential of osteosarcoma cell lines before and after alendronate treatment. Real‐time quantitative RT‐PCR was used to determine the mRNA level of MMP‐2 with and without alendronate treatment. Enzyme‐linked immunosorbent assay (ELISA) was used to quantify the cytokine level of MMP‐2 secreted in the condition medium. BP‐induced cell apoptosis was evaluated by fluorescent flow cytometric analysis.

Results and Conclusions

The results showed that alendronate inhibited cell invasion of both osteosarcoma cell lines in a dose‐dependent manner. Alendronate reduced the mRNA level and cellular level of MMP‐2 in both cell lines in a time and dose‐dependent manner. Alendronate also induced significant apoptosis in both cell lines. Our finding suggests that alendronate downregulates MMP‐2 secretion and induces apoptosis in osteosarcoma cells, which may both contribute to the reduction of invasive potential of the tumor cells. © 2004 Wiley‐Liss, Inc.
Fetal open spina bifida: a natural history of disease progression in utero
Authors: Biggio Joseph R. , Wenstrom Katharine D. , Owen John
Published: March 2004

Objective

To determine the natural history of the prenatal development of ventriculomegaly and talipes in fetuses with open spina bifida.

Study design

All fetuses with isolated open spina bifida and managed at our center between January 1996 and March 2000 were retrospectively evaluated. Ultrasonographic images and reports were reviewed from examinations performed every 3 to 4 weeks from the time of diagnosis to delivery for lesion level and type, ventriculomegaly (defined as an atrial width of ≥10 mm), and lower extremity appearance.

Results

Of the 53 pregnancies identified, 20 (38%) were electively terminated. In the 33 ongoing gestations, the lesions ranged from lower thoracic to sacral; 79% were characterized as meningomyeloceles and 21% as myeloschises. Fifty‐five percent (n = 18) had ventriculomegaly at diagnosis (early onset, mean gestational age at diagnosis 22 ± 5 weeks), 33% (n = 11) subsequently developed ventriculomegaly (late onset, mean 29 ± 6 weeks), and 12% (n = 4) had normal ventricle size at the last sonogram before birth (mean 38 ± 1 weeks). The ventricular size prior to delivery was significantly smaller with late‐onset ventriculomegaly than with early‐onset: 15 ± 4 mm versus 28 ± 10 mm, (p = 0.001). Only 6% (n = 2) had talipes at the initial sonogram, and 18% (n = 6) were subsequently determined to have talipes (mean 30 ± 6 weeks).

Conclusion

Most fetuses with open spina bifida develop ventriculomegaly, and the majority do so by 21 weeks' gestation. Fetuses that develop ventriculomegaly later in gestation have less severe ventricular dilation at birth. In contrast, a minority of fetuses have congenital talipes, and because most cases develop after 20 weeks, they are not predicted by early midtrimester sonographic evaluation. Copyright © 2004 John Wiley & Sons, Ltd.
Structural organization and drawability in polyamide blends
Authors: Persyn O. , Miri V. , Lefebvre J. M. , Depecker C. , Gors C. , Stroeks A.
Published: March 2004

The thermal behavior of polyamide 6/amorphous polyamide blends has been investigated as a function of blend composition and temperature. Crystal phase nature and stability are probed by a combination of thermal analysis, X‐ray diffraction and Fourier transform infrared spectroscopy. Ductility appears to be strongly affected by the addition of the amorphous polyamide, but the corresponding decrease of the yield stress, either taken at the same draw temperature or under a comparable state of mobility of the amorphous phase, may not be simply accounted for by the reduction of overall ordered phase content.
Bubble removal in rotational molding
Authors: Gogos George
Published: March 2004

Closed form solutions have been obtained for bubble dissolution in typical polymer melts encountered in rotational molding. The solutions are in excellent agreement with experimental data available in the literature. Using these solutions, it is shown that under typical rotational molding conditions the polymer melts may be almost saturated. As a result, bubble shrinkage occurs over long periods. Depending on the degree of saturation, surface tension may contribute substantially to the concentration gradient that drives bubble shrinkage. It is also shown that a pressure increase imposed on a nearly saturated polymer melt leads to a steep concentration gradient at the bubble/melt interface that can cause extremely fast bubble shrinkage. Applied to the rotational molding process, such a pressure increase can result in substantial cycle‐time shortening through elimination (or reduction) of the currently used excessive heating. A further benefit may be that additional resins, which at present cannot be used because of oxidation at sustained high‐temperatures, can become available to the rotational molding industry. Under the under‐saturated conditions created by a pressure increase, the effect of surface tension on the rate of bubble shrinkage is negligible.
Effect of high concentration of Co (II) on Enterobacter liquefaciens strain C‐1: A bacterium highly resistant to heavy metals with an unknown genome.
Authors: Marrero Jeannette , González Luis Javier , Sánchez Aniel , Ayala Marta , Paz‐Lago Dalila , González Wendy , Fallarero Adyary , Castellanos‐Serra Lila , Coto Orquídea
Published: March 2004

Heavy metals are required as nutrients for essential functions in microorganisms. However, higher concentrations of these cations are generally toxic and may produce contrasting effects on living organisms. Enterobacter liquefaciens strain C‐1, a bacterium isolated from the Moa mine in Cuba, is able to survive in the presence of high concentrations of heavy metals. The proteomes of Enterobacter liquefaciens strain C‐1, grown under aerobic conditions in the presence and absence of Co (II) were compared using two‐dimensional gel electrophoresis analysis in the isoelectric point range of 4–7 and the mass range of 15–120 kDa. Significant changes in the expression level (> two‐fold) were detected for 13 spots: seven and six were up‐ and down‐regulated, respectively. Because the genome of this bacterium is unknown, identification by peptide mass fingerprinting only succeeded in four cases and most of the cross‐species identifications were supported by de novo sequencing of tryptic peptides followed by sequence alignment using the MS BLAST program. Twelve different proteins were identified, ten are involved in cellular antioxidant defence probably induced by the presence of Co (II). This is the first step towards understanding the role of proteins participating in the mechanism of resistance to heavy metals in this bacterium.
Proteomics analysis of human endothelial cell line EA.hy926 after exposure to GSM 900 radiation
Authors: Nylund Reetta , Leszczynski Dariusz
Published: March 2004

The human endothelial cell line EA.hy926 was exposed to mobile phone radiation and the effect on protein expression was examined using two‐dimensional electrophoresis (2‐DE). Up to 38 various proteins have statistically significantly altered their expression levels following the irradiation. Four proteins were identified with matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS). Two of the affected proteins were determined to be isoforms of cytoskeletal vimentin. This finding supports our earlier presented working hypothesis which indicated that the mobile phone radiation might affect the cytoskeleton and might have an effect on the physiological functions that are regulated by the cytoskeleton.
Selective growth of epithelial basal cells from human prostate in a three‐dimensional organ culture
Authors: Papini Sandra , Rosellini Alfredo , Campani Daniela , DeMatteis Anna , Selli Cesare , Revoltella Roberto Paolo
Published: March 2004

BACKGROUND

A three‐dimensional organotypic culture method has been developed for selectively growing epithelial basal cells from human benign prostate.

METHODS

Tissue fragments were cultured on sponges for several weeks and the viability of luminal and basal epithelium and cellular responses to 4,5α‐dihydrotesterone (DHT) stimulation were studied.

RESULTS

The gland tissue could be successfully maintained showing preservation of ducts and lobules as in vivo. Without DHT, a progressive hyperplasia of basal cells was observed: these cells proliferated with retention of the lumen or forming nests with squamous differentiation, lining the surface of the fragment and migrating to the underlying sponge. In contrast, secretory cells disappeared. Epithelial cells isolated from long‐term cultures showed a typical basal cell‐immunophenotype. DHT‐treated tissues maintained a much higher percentage of luminal cells than untreated tissues.

CONCLUSIONS

These systems allow the study of proliferation and differentiation of basal cells within their natural microenvironment as well as prostate pathobiology. © 2004 Wiley‐Liss, Inc.
The extended left‐handed helix: A simple nucleic acid‐binding motif
Authors: Hicks Joshua M. , Hsu Victor L.
Published: March 2004

The poly‐proline type II extended left‐handed helical structure is well represented in proteins. In an effort to determine the helix's role in nucleic acid recognition and binding, a survey of 258 nucleic acid‐binding protein structures from the Protein Data Bank was conducted. Results indicate that left‐handed helices are commonly found at the nucleic acid interfacial regions. Three examples are used to illustrate the utility of this structural element as a recognition motif. The third K homology domain of NOVA‐2, the Epstein‐Barr nuclear antigen‐1, and the Drosophila paired protein homeodomain all contain left‐handed helices involved in nucleic acid interactions. In each structure, these helices were previously unidentified as left‐handed helices by secondary structure algorithms but, rather, were identified as either having small amounts of hydrogen bond patterns to the rest of the protein or as being “unstructured.” Proposed mechanisms for nucleic acid interactions by the extended left‐handed helix include both nonspecific and specific recognition. The observed interactions indicate that this secondary structure utilizes an increase in protein backbone exposure for nucleic acid recognition. Both main‐chain and side‐chain atoms are involved in specific and nonspecific hydrogen bonding to nucleobases or sugar‐phosphates, respectively. Our results emphasize the need to classify the left‐handed helix as a viable nucleic acid recognition and binding motif, similar to previously identified motifs such as the helix‐turn‐helix, zinc fingers, leucine zippers, and others. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Identification of structural proteins from shrimp white spot syndrome virus (WSSV) by 2DE‐MS
Authors: Zhang Xiaobo , Huang Canhua , Tang Xuhua , Zhuang Ying , Hew Choy Leong
Published: March 2004

White spot syndrome virus (WSSV) is a major shrimp pathogen that also infects many other species of crustaceans. Its 305‐kb double‐stranded DNA genome has the capacity to encode 181 presumptive proteins. In an attempt to identify the viral proteins from the 181 theoretical proteins, proteins of the purified WSSV were separated by two‐dimensional electrophoresis (2‐DE). More than 60 protein spots were revealed, as detected by silver staining, from which 12 viral proteins were identified by mass spectrometry. In total, 25 WSSV proteins, including those reported in one of our earlier studies (Huang et al., Mol Cell Proteomics 2002;1:223–231), were revealed by this proteomic approach, and their corresponding genes were further confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR). Two of them were characterized to be WSSV envelope proteins using immuno‐electron microscopy. Our study showed that the proteomic approach is a powerful method for discovering the viral structural proteins and their corresponding genes. Proteins 2004;9999:000–000. © 2004 Wiley‐Liss, Inc.
High‐resolution prediction of protein helix positions and orientations
Authors: Li Xin , Jacobson Matthew P. , Friesner Richard A.
Published: March 2004

We have developed a new method for predicting helix positions in globular proteins that is intended primarily for comparative modeling and other applications where high precision is required. Unlike helix packing algorithms designed for ab initio folding, we assume that knowledge is available about the qualitative placement of all helices. However, even among homologous proteins, the corresponding helices can demonstrate substantial differences in positions and orientations, and for this reason, improperly positioned helices can contribute significantly to the overall backbone root‐mean‐square deviation (RMSD) of comparative models. A helix packing algorithm for use in comparative modeling must obtain high precision to be useful, and for this reason we utilize an all‐atom protein force field (OPLS) and a Generalized Born continuum solvent model. To reduce the computational expense associated with using a detailed, physics‐based energy function, we have developed new hierarchical and multiscale algorithms for sampling the helices and flanking loops. We validate the method using a test suite of 33 cases, which are drawn from a diverse set of high‐resolution crystal structures. The helix positions are reproduced with an average backbone RMSD of 0.6 Å, while the average backbone RMSD of the complete loop–helix–loop region (i.e., the helix with the surrounding loops, which are also repredicted) is 1.3 Å. Proteins 2004;55:000–000. © 2004 Wiley‐Liss, Inc.
A novel method of resistance for influenza against a channel‐blocking antiviral drug
Authors: Astrahan Peleg , Kass Itamar , Cooper Matthew A. , Arkin Isaiah T.
Published: March 2004

Effective antivirals are few and far between, and as such, the appearance of resistance toward such treatments is an obvious medical concern. In this article, we analyze the mechanism by which influenza attains resistance toward amantadine, a blocker of the viral M2 H+ channel. Binding analyses of amantadine to M2 peptides from different viral strains showed that the virus has developed two alternate routes to avoid blockage of its channel: (1) a conventional route, in which the channel no longer binds the blocker and, hence, the blocker cannot exert its inhibitory function; and (2) a novel mechanism, in which binding of the blocker is retained, yet the function of the protein is unaffected. Pore diameter profiles revealed the molecular mechanism by which the virus may attain this novel type of resistance: an increase in the size of the channel. Thus, despite the drug binding the channel, it may not be able to block the pore, since the channel diameter has increased. Our findings may have broad ramifications in the design of new antivirals, and of novel blockers against malfunctioning human channels implicated in disease. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Analysis of CDK2 active‐site hydration: A method to design new inhibitors
Authors: Kříž Zdeněk , Otyepka Michal , Bártová Iveta , Koča Jaroslav
Published: March 2004

The interactions between the protein and the solvent were analyzed, and protein regions with a high density of water molecules, as well as structural water molecules, were determined by using molecular dynamics (MD) simulations. A number of water molecules that were in contact with the protein for the whole trajectory were determined. Their interaction energies and hydrogen bonds with protein residues were analyzed. Altogether, 39, 27, 49, and 32 water molecules bound to the protein were found for trajectories of the free CDK2, CDK2/ATP, CDK2/roscovitine, and CDK2/isopentenyladenine complexes, respectively. Positions of observed water molecules were compared with X‐ray crystallography data. Special attention was paid to water molecules in the active site of the enzyme, and especially to the deep pocket, where the N9 roscovitine side‐chain is buried. Exchange of active‐site water molecules with bulk water through the tunnel from the pocket was observed. In the CDK2/isopentenyladenine complex simulation, two water molecules that arrange interaction between the inhibitor and the enzyme via an H‐bond were observed. Two stable water molecules in the trajectory of the free CDK2 were found that occupy the same position as the nitrogens N3 and N9 of the isopentenyladenine or N1 and N6 nitrogens of the adenosine triphosphate (ATP). The positions of structural water molecules were compared with the positions of substrate polar groups and crystallographic water molecules found in the Brookhaven Protein Data Bank for various CDK2 complexes. It was concluded that tracing tightly bound water molecules may substantially help in designing new inhibitors. Proteins 2004. © 2004 Wiley‐Liss, Inc.