bluuebunny/crossref_metadata_2025 huggingface dataset:
Implications of cytokeratin 8/18 filament formation in stratified epithelial cells: Induction of transformed phenotype
Authors: Raul Ujwala , Sawant Sharada , Dange Prerana , Kalraiya Rajiv , Ingle Arvind , Vaidya Milind
Published: May 2004

The cytokeratin (CK) pair 8 and 18 is normally expressed in all simple epithelia. This pair is not normally seen in stratified epithelial cells. Squamous cell carcinomas derived from stratified epithelia show anomalous expression of this CK pair. It is not known whether CKs 8 and 18 in any way contribute to the malignant phenotype of these cells. We used an immortalised, nontransformed human foetal buccal mucosa (FBM) cell line that expresses significantly higher amounts of CK18 compared to CK8. FBM cells were transfected with the full‐length CK8 gene to study the role of CKs 8 and 18 in malignant transformation. Clones with higher expression of CK8 compared to untransfected FBM cells were studied for changes in their phenotypic characteristics. Immunofluorescence studies using antibodies to CKs 8 and 18 revealed well‐decorated filaments throughout the cytoplasm in CK8 gene–transfected cells vs. untransfected FBM cells. Transmission images showed that FBM cells were isolated while transfected cells were in groups of well‐spread cells with cellular projections. Transfected cells were independent of growth supplement requirements and showed anchorage‐independent growth in soft agar assay and significantly reduced doubling time compared to nontransfected FBM cells. DNA flow‐cytometric studies revealed increased DNA content and prolonged S phase in transfected clones vs. FBM cells. Injection of cells s.c. obtained from soft agar colonies developed from 2 of the clones resulted in tumour formation at the site of injection. In both cases, lung metastasis was also seen. Thus, in conclusion, it appears that increased expression of CK8 in some way changes the phenotypic characteristics of stratified epithelial cells, resulting in malignant transformation. © 2004 Wiley‐Liss, Inc.
Suppression of thymic lymphomas and increased nonthymic lymphomagenesis in Trp53‐deficient mice lacking inducible nitric oxide synthase gene
Authors: Tatemichi Masayuki , Tazawa Hiroshi , Masuda Mitsuharu , Saleem Mohammad , Wada Sayori , Donehower Lawrence A. , Ohgaki Hiroko , Ohshima Hiroshi
Published: May 2004

Trp53‐deficient mice spontaneously develop lymphomas, mainly of thymic origin, although the molecular mechanism remains largely unknown. As several interaction effects between p53 and iNOS have been reported, we hypothesized that iNOS activity in the thymus is causally linked to lymphomagenesis in Trp53‐deficient mice. We therefore created mouse strains with different combinations of the Trp53 and iNOS genes. Western blot and histologic analyses showed that the iNOS protein was constitutively expressed in the thymus independently of Trp53 status and its expression was enhanced in Trp53+/– and Trp53–/– mice compared to Trp53+/+ mice. Homozygous disruption of iNOS decreased the incidence of thymic lymphomas by almost 40% (p = 0.087) and 90% (p < 0.05) in Trp53–/– and Trp53+/– mice, respectively, compared to the respective iNOS wild‐type mice but significantly (p < 0.05) increased the development of nonthymic lymphomas in Trp53–/– and Trp53+/– mice. Although iNOS gene disruption did not affect the phenotype of thymic lymphomas, absence of the iNOS gene shifted the spectrum of nonthymic lymphoma from the B‐cell to the T‐cell lineage. RT‐PCR analysis revealed enhanced expression of IL‐10, which could have a promoting effect on lymphomagenesis, even without any stimulation, in the spleen of aging mice with the gene combinations Trp53–/–iNOS–/– and Trp53+/–iNOS–/– but not Trp53–/–iNOS+/+ or Trp53+/–iNOS+/+. These results suggest that iNOS could increase the development of thymic lymphomas in Trp53‐deficient mice. While iNOS may have protective effects against nonthymic lymphomagenesis, the regulation of cytokine production by iNOS may be involved in the underlying mechanism of antilymphomagenesis effects in the peripheral lymphoid organ. © 2004 Wiley‐Liss, Inc.
Test sensitivity of prostate‐specific antigen in the Finnish randomised prostate cancer screening trial
Authors: Auvinen Anssi , Määttänen Liisa , Finne Patrik , Stenman Ulf‐Håkan , Aro Jussi , Juusela Harri , Rannikko Sakari , Tammela Teuvo L.J. , Hakama Matti
Published: May 2004

We estimated the sensitivity of serum prostate‐specific antigen (PSA) as a screening test for prostate cancer in the Finnish randomised, population‐based prostate cancer screening trial. The study population consisted of 80,458 men aged 55–67 years identified from the national population registry and randomised to the screening or control arm of the trial. The screening algorithm was based on determination of serum PSA concentration. Test sensitivity was estimated based on interval cancer incidence during the first 4 years of follow‐up among screening participants with a negative screening test. Interval cancers were defined as those occurring among men with a negative screening test. Altogether, 19 interval cancers were detected among 17,897 men with serum PSA < 3 ng/ml during the first screening interval. A further 5 cases were diagnosed among 811 men with PSA 3.0–3.9 ng/ml with a benign digital rectal examination or free total PSA ratio ≥ 0.16. Test sensitivity based on serum PSA of 3 ng/ml was estimated to be 0.89 (95% confidence interval 0.84–0.93) and that based on PSA of 4 ng/ml combined with an ancillary test (digital rectal examination or free total PSA ratio in the PSA range 3.0–3.9) was 0.87 (0.82–0.92). Test sensitivity achieved with serum PSA in prostate cancer screening appears excellent in the context of a population‐based effectiveness trial. © 2004 Wiley‐Liss, Inc.
In vitro effects of native human acute myelogenous leukemia blasts on fibroblasts and osteoblasts
Authors: Glenjen Nils , Ersvær Elisabeth , Ryningen Anita , Bruserud Øystein
Published: May 2004

Bone marrow stromal cells constitute a heterogeneous population, and in the present study we investigated intercellular crosstalk via release of soluble mediators between native human AML blasts and fibroblasts/osteoblasts. Coculture of nonleukemic stromal cells and AML blasts separated by a semipermeable membrane decreased proliferation of the fibroblast line HFL1, and the inhibition was maintained when HFL1 and AML cells were cultured in direct contact. A similar inhibitory effect was observed for osteoblastic sarcoma cell lines (Cal72, SJSA‐1) and normal osteoblasts. GM‐CSF was released by both nonleukemic cells and a subset of AML blast populations, and increased levels of GM‐CSF were detected in AML cocultures with fibroblasts and osteoblastic sarcoma cells when testing AML cell populations with constitutive GM‐CSF release. Furthermore, constitutive IL‐1β secretion by AML blasts was detected only for a subset of patients, whereas relatively high levels of IL‐1RA were observed for all patients; coculture of AML blasts with HFL1 fibroblasts and osteoblastic sarcoma cells increased IL‐1β levels for patients with constitutive IL‐1β secretion, whereas IL‐1RA levels were slightly decreased but still generally higher than IL‐1β levels (tested only for HFL1 fibroblasts). The bidirectional crosstalk between AML blasts and stromal cells with increased release of AML growth factors may be important in leukemogenesis, whereas the decreased stromal cell proliferation combined with the persistent release of IL‐1RA may in addition inhibit remaining normal hematopoiesis and thereby contribute to bone marrow failure in AML. © 2004 Wiley‐Liss, Inc.
Dietary agent indole‐3‐carbinol protects female rats against the hepatotoxicity of the antitumor drug ET‐743 (trabectidin) without compromising efficacy in a rat mammary carcinoma
Authors: Donald Sarah , Verschoyle Richard D. , Greaves Peter , Colombo Tina , Zucchetti Massimo , Falcioni Cristiano , Zaffaroni Marco , D'Incalci Maurizio , Manson Margaret M. , Jimeno Jose , Steward William P. , Gescher Andreas J.
Published: May 2004

ET‐743, an experimental antitumor drug with promising activity in sarcoma, breast and ovarian carcinoma, is currently under phase 2 clinical evaluation. It is hepatotoxic in animals and patients. We tested the hypothesis that indole‐3‐carbinol (I3C), the hydrolysis product of glucosinolates occurring in cruciferous vegetables, may protect against ET‐743‐induced hepatotoxicity in the female Wistar rat, the animal species with the highest sensitivity toward the adverse hepatic effect of this drug. Hepatotoxicity was adjudged by measurement of plasma levels of bilirubin, alkaline phosphatase (ALP) and aspartate aminotransferase (AST) and by liver histopathology. The effect of I3C on the kinetics of ET‐743 in rat plasma and liver was investigated by high‐pressure liquid chromatography. The effect of I3C on the antitumor efficacy of ET‐743 was explored in rats bearing the 13762 mammary carcinoma. ET‐743 (40 μg/kg i.v.) alone caused an elevation of plasma bilirubin, ALP and AST levels and degeneration and patchy focal necrosis of bile duct epithelial cells. Addition of I3C to the diet (0.5%) for 6 days prior to ET‐743 administration almost completely abolished manifestations of hepatotoxicity. In contrast, a dietary concentration of 0.1% I3C did not protect, nor did dietary diindolylmethane (0.2%), an acid‐catalyzed condensation product of I3C. Ingestion by rats of I3C for 6 days prior to ET‐743 (40 μg/kg i.v.) decreased plasma but not hepatic concentrations of ET‐743 compared to animals that received ET‐743 alone. I3C did not interfere with the antitumor efficacy of ET‐743. The results suggest that ingestion of I3C may counteract the unwanted effect of ET‐743 in the liver. I3C should be investigated as a hepatoprotectant in patients who receive ET‐743 therapy. © 2004 Wiley‐Liss, Inc.
Detection of circulating cancer cells expressing uroplakins and epidermal growth factor receptor in bladder cancer patients
Authors: Osman Iman , Kang Melissa , Lee Andy , Deng Fang‐Ming , Polsky David , Mikhail Maryann , Chang Caroline , David Dexter A. , Mitra Nandita , Wu Xue‐Ru , Sun Tung‐Tien , Bajorin Dean F.
Published: May 2004

Our purpose was to determine the clinical relevance of the detection of circulating tumor cells (CTCs) expressing urothelial and epithelial markers in bladder cancer patients. Sixty‐two patients who presented to Memorial Sloan‐Kettering Cancer Center between July 2000 and September 2001 were studied. Peripheral blood was tested by nested RT‐PCR assay for uroplakins (UPs) Ia, Ib, II and III as well as for epidermal growth factor receptor (EGFR). We determined the sensitivity and specificity of each individual marker and the combinations of UPIa/UPII and UPIb/UPIII. The latter strategy was based on our data, which showed that UPIa and UPIb form heterodimers with UPII and UPIII, respectively. Forty patients had clinically advanced bladder cancer and 22 had no evidence of disease at the time of assay. Eight of the 22 patients recurred during the follow‐up period. All 8 patients were positive at presentation for UPIa/UPII. The combination of UPIa/UPII provided the best sensitivity (75%) of detecting CTCs, with a specificity of 50%. The combination of UPIb/UPIII was the most specific (79%) but had modest sensitivity (31%). Detection of EGFR‐positive cells alone and in combination with UPs was inferior to that for UPIa/UPII. Combinations of urothelial markers are superior to single urothelial or epithelial markers in detecting CTCs in bladder cancer patients. Further efforts are under way to confirm the potential predictive value of these markers in a prospectively designed study of a larger cohort of patients. © 2004 Wiley‐Liss, Inc.
Viscoelastic characterizations of acellular dermal matrix (ADM) preparations for use as injectable implants
Authors: Ho Hsiu‐O , Tsai Yu‐Ting , Chen Ray‐Neng , Sheu Ming‐Thau
Published: May 2004

Viscoelastic characteristics of acellular dermal matrix (ADM) preparations with various additives were analyzed with creep curves, stress–strain relationships, and the storage modulus with reference to those of ADM preparations crosslinked with glutaraldehyde. Creep curves for all ADM preparations were determined to comply with the Kelvin–Voigt model. The stress–strain plots of all ADM preparations compared were described as linear. The storage modulus of all ADM preparations was maintained at a nearly constant level throughout the range of oscillating frequencies applied. ADM preparations crosslinked with glutaraldehyde showed that both Young's modulus (E) for the spring part and retardation time (τ) in the Kelvin–Voigt model, and hence viscosity (η) for the liquid part, increased with an increasing concentration of glutaraldehyde. Higher Young's modulus and viscosity and a greater extent of the “solid” response of ADM preparations crosslinked with glutaraldehyde might have been responsible for the longer persistence that was demonstrated after implantation. The increase in ADM concentration and the addition of various additives to ADM preparations, including α‐hydroxy acid (citric acid, lactic acid, and glycolic acid) and hyaluronic acid, resulted in similar effects on the viscoelastic characteristics of the ADM preparations, but they were less efficacious than those crosslinked with glutaraldehyde. Among them, increasing ADM concentration to >200 mg/mL and addition of glycolic acid at a concentration of >2% improved the viscoelastic characteristics of the resulting ADM preparations so that their level of persistence was closer to that of material crosslinked with glutaraldehyde. On the contrary, the influence on viscoelastic characteristics of adding PVP greatly differed from that of hyaluronic acid and was only apparent when adding concentrations of PVP of >10%. Similarly, viscoelastic characteristics of the ADM preparations examined were also so sensitive to temperature that the persistence of ADM preparations after implantation at body temperature would deteriorate. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 70A: 83–96, 2004
Prolonged ectopic calcification induced by BMP‐2–derived synthetic peptide
Authors: Saito Atsuhiro , Suzuki Yoshihisa , Ogata Shin‐Ichi , Ohtsuki Chikara , Tanihara Masao
Published: May 2004

Bone morphogenetic protein‐2 (BMP‐2) promotes the formation and regeneration of bone and cartilage, and therefore constitutes the most promising candidate for a bone repair material. However, it also has a wide range of functions, such as in organogenesis and apoptosis. Therefore, we investigated a novel synthetic peptide corresponding to residues 73–92 of BMP‐2. This peptide bound to a BMP‐2‐specific receptor and elevated both alkaline phosphatase activity and osteocalcin mRNA in the murine cell line, C3H10T1/2. The 73–92 peptide also induced ectopic calcification when conjugated to a covalently crosslinked alginate gel. Here we report that the 73–92 peptide‐conjugated alginate gel showed prolonged ectopic calcification for up to 7 weeks in rat calf muscle. In contrast, rhBMP‐2‐impregnated collagen gel showed maximum ectopic calcification at 3 weeks, and the calcified products that had formed disappeared after 5 weeks. Histological examination showed that the 73–92 peptide‐conjugated alginate gel induced many osteoblast‐like cells and few osteoclasts. In contrast, rhBMP‐2‐impregnated collagen gel induced many osteoclasts. These results suggest that the 73–92 peptide on alginate gel remains active at the implanted site, continuously induces differentiation of osteoblast precursor cells into osteoblasts, and activates osteoblasts to promote ectopic calcification. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 70A: 115–121, 2004
Influence of additive hyaluronic acid on the lubricating ability in the temporomandibular joint
Authors: Kawai Nobuhiko , Tanaka Eiji , Takata Takashi , Miyauchi Mutsumi , Tanaka Masao , Todoh Masahiro , van Eijden Theo , Tanne Kazuo
Published: May 2004

In synovial fluid, hyaluronic acid (HA) is an essential component for the lubrication of joints, thus preventing friction. The relationship between HA and joint friction is not unambiguously established yet. In the present study, the effect of the application of HA on the frictional coefficient in the temporomandibular joint was evaluated. After measuring the frictional coefficient in intact porcine joints (n = 10), the subsequent effect of phosphate‐buffered saline (PBS) washing and gauze scouring and the application of HA was examined. Compared with the intact joint, the frictional coefficient was significantly larger after PBS washing and gauze scouring. Subsequent application of HA resulted in a significant decrease (50–75%) of the frictional coefficient. However, it did not recover to the same value as in the intact joints. Observations by scanning electron microscopy showed that after PBS washing, the amorphous layer of the articular cartilage was still intact, whereas it was partially collapsed after gauze scouring. In conclusion, the addition of HA did reduce the coefficient of friction under the experimental conditions in this study; the relevance to the clinical condition and the duration of the treatment effect in vivo require further investigation. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 70A: 149–153, 2004
Opinions and trends in biomaterials education: Report of a 2003 Society for Biomaterials survey
Authors: Karp Jeffrey M. , Friis Elizabeth A. , Dee Kay C. , Winet Howard
Published: May 2004

The Society for Biomaterials (SFB) aims to serve its members through acting as a forum for the exchange of information and ideas. To aid in the practical development of the SFB and more specifically biomaterials education, all active, associate, and student members were surveyed. In general, the survey asked questions regarding respondent demographics, experiences and activities with the SFB, and opinions about biomaterials education. Perceptions and needs of biomaterials‐related education and career‐related training practices were a specific focus of the survey. A total of 140 individuals responded to the survey for a response rate of 18%. Members from industry felt that new hires, in general, should be better trained in product development, regulatory issues for new materials and devices, and in the relevant testing required. When asked what was missing from their professional education, many respondents commented that business training in areas such as negotiations, management, and understanding the needs outside of academia was lacking. Also, many respondents seemed to have trouble identifying with what they were supposed to know and felt a “lack of set professional knowledge.” This study has raised many ideas and questions that require further discussion. The results should ultimately be useful for helping the SFB decide how best to focus future efforts in biomaterials education. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 70A: 1–9, 2004
Transitions in replication timing in a 340 kb region of human chromosomal R‐Band 1p36.1
Authors: Brylawski Bruna P. , Cohen Stephanie M. , Horne Hisani , Irani Neville , Cordeiro‐Stone Marila , Kaufman David G.
Published: May 2004

DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase (Cohen et al. [1998]: Exp Cell Res. 245:321–329). In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1–2 h later. Using a quantitative PCR‐based assay to measure sequence abundance in size‐fractionated (900–1,700 nt) nascent DNA (Giacca et al. [1994]: Proc Natl Acad Sci USA. 91:7119), we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed. © 2004 Wiley‐Liss, Inc.
Backup pathways of NHEJ are suppressed by DNA‐PK
Authors: Perrault Ronel , Wang Huichen , Wang Minli , Rosidi Bustanur , Iliakis George
Published: May 2004

In cells of higher eukaryotes double strand breaks (DSBs) induced in the DNA after exposure to ionizing radiation (IR) are rapidly rejoined by a pathway of non‐homologous end joining (NHEJ) that requires DNA dependent protein kinase (DNA‐PK) and is therefore termed here D‐NHEJ. When this pathway is chemically or genetically inactivated, cells still remove the majority of DSBs using an alternative, backup pathway operating independently of the RAD52 epistasis group of genes and with an order of magnitude slower kinetics (B‐NHEJ). Here, we investigate the role of DNA‐PK in the functional coordination of D‐NHEJ and B‐NHEJ using as a model end joining by cell extracts of restriction endonuclease linearized plasmid DNA. Although DNA end joining is inhibited by wortmannin, an inhibitor of DNA‐PK, the degree of inhibition depends on the ratio between DNA ends and DNA‐PK, suggesting that binding of inactive DNA‐PK to DNA ends not only blocks processing by D‐NHEJ, but also prevents the function of B‐NHEJ. Residual end joining under conditions of incomplete inhibition, or in cells lacking DNA‐PK, is attributed to the function of B‐NHEJ operating on DNA ends free of DNA‐PK. Thus, DNA‐PK suppresses alternative pathways of end joining by efficiently binding DNA ends and shunting them to D‐NHEJ. © 2004 Wiley‐Liss, Inc.
TGF‐β1 enhances βig‐h3‐mediated keratinocyte cell migration through the α3β1 integrin and PI3K
Authors: Jeong Ha‐Won , Kim In‐San
Published: May 2004

βig‐h3 is an extracellular matrix (ECM) protein whose expression is highly induced by transforming growth factor beta1 (TGF‐β1). We previously demonstrated that βig‐h3 has two α3β1 integrin‐interacting motifs, which promote adhesion, migration, and proliferation of human keratinocytes. Both βig‐h3 and TGF‐β1 have been suggested to play important roles in the healing of skin wounds. In this study, we demonstrate that TGF‐β1 enhances keratinocyte adhesion and migration toward βig‐h3 through the α3β1 integrin. TGF‐β1 did not increase the amount of the α3β1 integrin on the cell surface, but rather increased its affinity for βig‐h3. LY294002, an inhibitor of PI3K, blocked the basal and TGF‐β1‐enhanced cell migration but not adhesion to βig‐h3. A constitutively active mutant of PI3K stimulated cell migration but not adhesion to βig‐h3. The PI3K pathway is also not associated with the affinity of the α3β1 integrin to βig‐h3. TGF‐β1 induced phosphorylation of AKT and FAK. Taken together, these data suggest that TGF‐β1 increases affinity of the α3β1 integrin to βig‐h3, resulting in enhanced adhesion and migration of keratinocytes toward βig‐h3. TGF‐β1 also enhances migration through PI3K, but PI3K is not associated with either the binding affinity of the α3β1 integrin or its adhesion to βig‐h3. © 2004 Wiley‐Liss, Inc.
Developmental expression of OAP‐1/Tspan‐3, a member of the tetraspanin superfamily
Authors: Tiwari‐Woodruff Seema K. , Kaplan Roman , Kornblum Harley I. , Bronstein Jeff M.
Published: May 2004

OSP/claudin‐11‐associated protein (OAP‐1/Tspan‐3), originally isolated by yeast two‐hybrid screening using OSP/claudin‐11 (oligodendrocyte‐specific protein) as bait, is a member of the tetraspanin superfamily and OAP‐1/Tspan‐3, OSP/claudin‐11, and β1 integrin form a protein complex that seems to be involved in oligodendrocyte proliferation and migration. This study investigated the temporal and regional expression, glycosylation status, and tissue distribution of OAP‐1/Tspan‐3. OAP‐1/Tspan‐3 mRNA was expressed as a single transcript throughout brain development, with high levels of expression in the germinal zones. OAP‐1/Tspan‐3 protein contains N‐terminal glycosylation sites in extracellular loop 2 and deglycosylation studies indicated a decrease in apparent molecular weight of OAP‐1/Tspan‐3, consistent with removal of N‐glycans. Similar to OSP/claudin‐11, OAP‐1/Tspan‐3 is expressed in all stages of oligodendrocyte development and in the myelin sheath. Unlike OSP/claudin‐11, however, it is expressed in all cell types tested in the central nervous system (CNS), including neurons and astrocytes. The association of OAP‐1/Tspan‐3 with OSP/claudin‐11 and β1 integrin, its subcellular distribution as a cell surface, membrane‐spanning glycoprotein, and its widespread distribution supports its potential role in cell migration, proliferation, and interactions between cells and extracellular matrix. © 2004 Wiley‐Liss, Inc.
Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: Disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype
Authors: Neuhuber Birgit , Gallo Gianluca , Howard Linda , Kostura Lisa , Mackay Alastair , Fischer Itzhak
Published: May 2004

Bone marrow stromal cells (MSC), which represent a population of multipotential mesenchymal stem cells, have been reported to undergo rapid and robust transformation into neuron‐like phenotypes in vitro following treatment with chemical induction medium including dimethyl sulfoxide (DMSO; Woodbury et al. [2002] J. Neurosci. Res. 96:908). In this study, we confirmed the ability of cultured rat MSC to undergo in vitro osteogenesis, chondrogenesis, and adipogenesis, demonstrating differentiation of these cells to three mesenchymal cell fates. We then evaluated the potential for in vitro neuronal differentiation of these MSC, finding that changes in morphology upon addition of the chemical induction medium were caused by rapid disruption of the actin cytoskeleton. Retraction of the cytoplasm left behind long processes, which, although strikingly resembling neurites, showed essentially no motility and no further elaboration during time‐lapse studies. Similar neurite‐like processes were induced by treating MSC with DMSO only or with actin filament‐depolymerizing agents. Although process formation was accompanied by rapid expression of some neuronal and glial markers, the absence of other essential neuronal proteins pointed toward aberrantly induced gene expression rather than toward a sequence of gene expression as is required for neurogenesis. Moreover, rat dermal fibroblasts responded to neuronal induction by forming similar processes and expressing similar markers. These studies do not rule out the possibility that MSC can differentiate into neurons; however, we do want to caution that in vitro differentiation protocols may have unexpected, misleading effects. A dissection of molecular signaling and commitment events may be necessary to verify the ability of MSC transdifferentiation to neuronal lineages. © 2004 Wiley‐Liss, Inc.
Induction of bone marrow stromal cells to neurons: Differentiation, transdifferentiation, or artifact?
Authors: Lu Paul , Blesch Armin , Tuszynski Mark H.
Published: May 2004

Differentiation of stem cells toward a neuronal lineage normally involves a gradually progressive restriction in developmental potential and is regulated by a diverse set of specific and temporally precise genetic events. However, recent studies have indicated that both rodent and human bone marrow stromal cells (MSCs) can be rapidly (within minutes to hours) induced to differentiate into neurons in vitro by relatively simple chemical means (using β‐mercaptoethanol [BME] or dimethylsulfoxide [DMSO] and butylated hydroxyanisol [BHA]; Woodbury et al. [ 2000] J. Neurosci. Res. 61:364–370). The ability to transdifferentiate an easily accessible cell source into neurons could have substantial potential for promoting neural repair. We therefore explored the potential of simple chemical methods to transdifferentiate other cell types, including primary rat fibroblasts, primary human keratinocytes, HEK293 cells, rat PC‐12 cells, and as positive control rat bone marrow stromal (BMS) cells. Surprisingly, all cells except for keratinocytes adopted at least partial “neuron‐like” pyramidal cell morphology with fine‐cellular extensions resembling neurites upon stimulation with BME or DMSO/BHA. However, time‐lapse microscopy indicated that the chemical exposure of MSCs did not result in new neurite growth but rather cellular shrinkage, with retraction of the majority of existing cell extensions, leaving only few, fine neurite‐like processes. To determine whether the chemically induced transdifferentiation resulted from simple cellular toxicity, MSCs were exposed to various stressors, including detergents, high‐molarity sodium chloride, and extremes of pH. In all cases, cellular shrinkage and adoption of pseudoneuronal morphology were observed. Concomitantly with cellular shrinkage, apparent increases in immunolabeling for the neuronal markers NSE and NeuN were detected in the cell soma that could not be confirmed by RT‐PCR. Furthermore, blockade of protein synthesis with cycloheximide did not prevent cells from adopting “neuron‐like” morphology after chemical induction. Thus, morphological changes and increases in immunolabeling for certain cellular markers upon “chemical induction” of MSCs are likely the result of cellular toxicity, cell shrinkage, and changes in the cytoskeleton and do not represent regulated steps in a complicated cellular differentiation process. © 2004 Wiley‐Liss, Inc.
Effects of NF‐κB oligonucleotide “Decoys” on gene expression in P7 rat hippocampus after hypoxia/ischemia
Authors: Qiu Jingxin , Hu Xiaoming , Nesic Olivera , Grafe Marjorie R. , Rassin David K. , Wood Thomas G. , Perez‐Polo J. Regino
Published: May 2004

“Decoy” oligonucleotides can be used as gene‐specific nuclear factor (NF‐κB) inhibitors to regulate gene expression. We applied two different decoy oligonucleotides that contained the NF‐κB binding consensus sequences present in the immunoglobulin G (IgG)‐κB and Bcl‐x promoter into 7‐day‐old (P7) rat lateral ventricles before hypoxia/ischemia (HI) and compared their effects on gene expression in hippocampi to saline‐treated, scrambled decoy‐treated, or untreated hippocampi exposed to HI. Left hippocampi were collected at 12 hr after HI. Electrophoretic mobility shift assays (EMSAs) showed that the two decoy treatments had different effects on NF‐κB binding to the IgG‐κB and Bcl‐x promoter‐specific consensus sequences, respectively. We assessed the decoys' effects on gene expression 12 hr after HI using ribonuclease protection assays (RPAs) and Affymetrix DNA microarrays. RPAs showed that both decoys significantly decreased interleukin (IL)‐1α mRNA levels but had no impact on IL‐1β, IL‐6, and IL‐10 mRNA levels. IgG‐κB decoys significantly decreased tumor necrosis factor (TNF)‐α and TNF‐β mRNA levels compared to minimal changes after treatment with Bcl‐x decoys. DNA microarray analyses showed that Bcl‐x decoy treatment significantly decreased Bcl‐xL mRNA levels. The decreased Bcl‐xL mRNA levels after Bcl‐x decoy treatment was confirmed by RPA analysis. DNA microarray data also indicated that several other genes were affected by both decoys. Our results suggest that different NF‐κB decoy treatments could differentially regulate transcriptional responses to central nervous system trauma. Careful design of decoy sequences, however, is essential to acquire selective effects on cell death outcome. © 2004 Wiley‐Liss, Inc.
Matrix metalloproteinase‐9 promotes nerve growth factor‐induced neurite elongation but not new sprout formation in vitro
Authors: Shubayev Veronica I. , Myers Robert R.
Published: May 2004

Matrix metalloproteinase‐9 (MMP‐9) is a basal‐lamina‐degrading protease that we have recently shown to be localized in regenerating sciatic nerve. We now demonstrate that MMP‐9 colocalizes with growth‐associated protein GAP‐43 in regenerating nerves in vivo and is involved in vitro in axonal sprouting. By using a PC12 cell model for neuronal sprouting, we analyzed the effects of recombinant MMP‐9, MMP‐9‐neutralizing antibody, and a broad‐spectrum MMP inhibitor (Ro 31‐9790) on sprout formation, elongation, and branching. Quantitative phase‐contrast microscopy showed that MMP‐9 elongated neuronal sprouts by 67% and increased their branching by 14% but did not change the number of sprouts relative to nerve growth factor (NGF) treatment. Double immunofluorescence for GAP‐43, a marker for growth cones, and α‐tubulin, a marker for axonal microtubules, showed that MMP‐9‐treated cells had increased distribution of α‐tubulin but no effect on GAP‐43. Western blot analyses of cell lysates demonstrated that the NGF‐induced increase in GAP‐43 was unchanged with MMP‐9 treatment or inhibition, confirming that MMP‐9 had no effect on new sprout formation. However, Ro 31‐9790 reduced GAP‐43 levels to those seen in untreated cells, suggesting that an MMP other than MMP‐9 is important for sprout formation. Finally, phosphorylated neurofilament M (NFM‐p), a marker for regenerative elongation, was induced with MMP‐9 treatment and was inhibited by the anti‐MMP‐9 antibody treatment, confirming the role of MMP‐9 in axonal elongation. NFM‐p colocalized with MMP‐9 in regenerating sciatic nerve fibers. These findings suggest that MMP‐9 regulates neurite extension in regenerating peripheral nerve fibers and, therefore, might be of therapeutic value in promoting regeneration in vivo. © 2004 Wiley‐Liss, Inc.
Exercise‐induced behavioral recovery and neuroplasticity in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐lesioned mouse basal ganglia
Authors: Fisher Beth E. , Petzinger Giselle M. , Nixon Kerry , Hogg Elizabeth , Bremmer Samuel , Meshul Charles K. , Jakowec Michael W.
Published: May 2004

Physical activity has been shown to be neuroprotective in lesions affecting the basal ganglia. Using a treadmill exercise paradigm, we investigated the effect of exercise on neurorestoration. The 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐lesioned mouse model provides a means to investigate the effect of exercise on neurorestoration because 30–40% of nigrostriatal dopaminergic neurons survive MPTP lesioning and may provide a template for neurorestoration to occur. MPTP‐lesioned C57 BL/6J mice were administered MPTP (four injections of 20 mg/kg free‐base, 2 hr apart) or saline and divided into the following groups: (1) saline; (2) saline + exercise; (3) MPTP; and (4) MPTP + exercise. Mice in exercise groups were run on a motorized treadmill for 30 days starting 4 days after MPTP lesioning (a period after which MPTP‐induced cell death is complete). Initially, MPTP‐lesioned + exercise mice ran at slower speeds for a shorter amount of time compared to saline + exercise mice. Both velocity and endurance improved in the MPTP + exercise group to near normal levels over the 30‐day exercise period. The expression of proteins and genes involved in basal ganglia function including the dopamine transporter (DAT), tyrosine hydroxylase (TH), and the dopamine D1 and D2 receptors, as well as alterations on glutamate immunolabeling were determined. Exercise resulted in a significant downregulation of striatal DAT in the MPTP + exercise compared to MPTP nonexercised mice and to a lesser extent in the saline + exercised mice compared to their no‐exercise counterparts. There was no significant difference in TH protein levels between MPTP and MPTP + exercise groups at the end of the study. The expression of striatal dopamine D1 and D2 receptor mRNA transcript was suppressed in the saline + exercise group; however, dopamine D2 transcript expression was increased in the MPTP + exercise mice. Immunoelectron microscopy indicated that treadmill exercise reversed the lesioned‐induced increase in nerve terminal glutamate immunolabeling seen after MPTP administration. Our data demonstrates that exercise promotes behavioral recovery in the injured brain by modulating genes and proteins important to basal ganglia function. © 2004 Wiley‐Liss, Inc.
Cytotoxic mechanisms by M239V presenilin 2, a little‐analyzed Alzheimer's disease‐causative mutant
Authors: Abe Yoichiro , Hashimoto Yuichi , Tomita Yusuke , Terashita Kenzo , Aiso Sadakazu , Tajima Hirohisa , Niikura Takako , Matsuoka Masaaki , Nishimoto Ikuo
Published: May 2004

Although neurotoxic functions are well characterized in familial Alzheimer's disease (FAD)‐linked N141I mutant of presenilin (PS)2, little has been known about M239V‐PS2, another established FAD‐causative mutant. We found that expression of M239V‐PS2 caused neuronal cytotoxicity. M239V‐PS2 exerted three forms of cytotoxicity: one was sensitive to both an antioxidant glutathione‐ethyl‐ester (GEE) and a caspase inhibitor Ac‐DEVD‐CHO (DEVD); the second was sensitive to GEE but resistant to DEVD; and the third was resistant to both. The GEE/DEVD‐sensitive cytotoxicity by M239V‐PS2 was likely through NADPH oxidase and the GEE‐sensitive/DEVD‐resistant cytotoxicity through xanthine oxidase (XO). Both mechanisms by M239V‐PS2 were suppressed by pertussis toxin (PTX) and were mediated by Gαo, but not by Gαi. Although Aβ1–43 itself induced no cytotoxicity, Aβ1–43 potentiated all three components of M239V‐PS2 cytotoxicity. As these cytotoxic mechanisms by M239V‐PS2 are fully shared with N141I‐PS2, they are most likely implicated in the pathomechanism of FAD by PS2 mutations. Notably, cytotoxicity by M239V‐PS2 could be inhibited by the combination of two clinically usable inhibitors of superoxide‐generating enzymes, apocynin and oxypurinol. © 2004 Wiley‐Liss, Inc.
Comparative study of cell culture and purification methods to obtain highly enriched cultures of proliferating adult rat Schwann cells
Authors: Mauritz Christina , Grothe Claudia , Haastert Kirsten
Published: May 2004

We present here a fast protocol that could be used to obtain highly purified cultures of maximal proliferating adult rat Schwann cells. These adult rat Schwann cells can be transfected in a nonbiological way using the physical transfection method of electroporation. Schwann cells are decisive in recovery of peripheral nerves after injury. In a clinical context, the use of enriched adult Schwann cells is necessary for autologous cell transplantation within nerve transplants for peripheral nerve repair. Different parameters such as tissue preparation, culture conditions, and protocols for enrichment, elevation of proliferation rates, and transfection were evaluated in cell cultures harvested from adult rat peripheral nerves. Cell preparation from in vivo predegenerated adult rat sciatic nerves combined with the use of melanocyte growth medium supplemented with forskolin, fibroblast growth factor (FGF)‐2, and pituitary extract as a selective, serum‐free culture medium, with a secondary cell‐enrichment step using specific detachment, resulted in highly enriched cultures of adult rat Schwann cells (>90%) with enhanced proliferation rates (≥40%). About 20% of these adult Schwann cells could be modified genetically using an optimized electroporation protocol. © 2004 Wiley‐Liss, Inc.
Optimising the determination of maize endosperm vitreousness by a rapid non‐destructive image analysis technique
Authors: Erasmus Corinda , Taylor John RN
Published: May 2004

A rapid non‐destructive image analysis technique was developed and optimised for the determination of maize endosperm vitreousness without the need for sample preparation. Maize kernel translucency measurements were optimised for a light system consisting of positioning whole kernels on top of round illuminated areas smaller than the projected areas of the kernels, allowing light to shine through the kernels. A correction factor to allow for constant illumination of kernels was developed to adjust for kernel size variation in relation to constant light area. The intensity of translucency in maize is linearly correlated to the percentage of kernel illumination (r = 0.99, p < 0.001). Significant correlations were found between corrected translucency values and endosperm yields determined by hand dissection. These were: translucency as a percentage of the whole kernel and vitreous endosperm (mass%), r = 0.77; translucency as a percentage of the whole kernel and opaque endosperm (mass%), (r = −0.72); translucency as a percentage of endosperm and vitreous endosperm (mass%), r = 0.81; and translucency as a percentage of endosperm and opaque endosperm (mass%), r = −0.77; with n = 245 in all instances. Translucency varied by 29.5% between the lowest and highest values, and vitreous endosperm (mass%) varied by 16.8% between the lowest and highest values. Correlations increased significantly after corrections for kernel thickness. A thickness increase of 1 mm in the maize will cause a decrease of 21.86% in translucency and vice versa. The method allows for large samples (at least 49 kernels min−1) to be analysed for individual vitreous and opaque endosperm contents with no interferences with kernel structure. With further development of suitable automated sampling techniques, it could become a suitable potential method for in‐line quantification of maize endosperm contents. Copyright © 2004 Society of Chemical Industry
Physicochemical, cooking and textural characteristics of some Indian black gram (Phaseolus mungo L) varieties
Authors: Singh Narpinder , Kaur Maninder , Sandhu Kawaljit Singh , Sodhi Navdeep Singh
Published: May 2004

Physicochemical, cooking and textural properties of black gram varieties were studied and related to each other using Pearson correlation. Different varieties showed significant variation in their physicochemical, cooking and textural properties. Varieties having higher seed weight and seed volume had higher values of cooking time, swelling capacity, hydration capacity and hardness. The relationships between textural parameters of cooked grains from different black gram varieties showed a significant positive correlation of hardness with cohesiveness (r = 0.472), gumminess (r = 0.938) and chewiness (r = 0.859). Swelling index, cohesiveness and gumminess of black gram varieties were observed to be related to their fibre content. Swelling capacity and swelling index correlated well with cooking time, hardness and gumminess. Cooking time had a positive correlation with hardness (p < 0.05) and gumminess (p < 0.05) and a negative correlation with springiness. Copyright © 2004 Society of Chemical Industry
Effectiveness of antioxidant‐impregnated film in retarding lipid oxidation
Authors: Lee Youn Suk , Shin Han‐Seung , Han Jong‐Koo , Lee Myunghoon , Giacin Jack R
Published: May 2004

The effectiveness of an antioxidant‐impregnated film to retard autoxidation of a packaged model product containing linoleic acid, via an evaporation/sorption mechanism, was evaluated as a function of storage time and temperature. The rate of loss of antioxidant from the package film structure was described by a first‐order expression. The first‐order rate constants were dependent on the initial concentration of antioxidant in the film. The rate of loss of 3,5‐di‐tert‐butyl‐4‐hydroxytoluene (BHT) from the package film structure was found to be much higher than the rate of loss of α‐tocopherol at both storage conditions (23 and 45 °C, 50% relative humidity) studied. A freeze‐dried model food product system was developed as the source for the autoxidation of linoleic acid in storage stability studies. The storage stability of this model food system packaged with antioxidant‐impregnated film pouches was evaluated. Hexanal as an index of oxidation from the model product system was collected by a dynamic purge and trap system and quantified by a gas chromatography/mass spectrometry procedure. The BHT‐impregnated laminate pouch showed a notable effectiveness in retarding lipid oxidation of the model product at 45 °C as a function of storage time. The control (non‐antioxidant) and α‐tocopherol‐impregnated laminate pouch structures showed no effect on retarding lipid oxidation of the model product during storage at 45 °C. Copyright © 2004 Society of Chemical Industry
The foaming of mixtures of albumin and hordein protein hydrolysates in model systems
Authors: Bamforth CW , Milani C
Published: May 2004

Although hydrolysed albumin and hydrolysed hordein derived from barley are independently capable of stabilising foams in model beer solutions, when both are present together, the net foam stability is less than anticipated. In particular, it seems that hordein, even in an unhydrolysed state, interferes with the ability of albumin‐derived polypeptides to stabilise foam. It appears, therefore, that the observed foam stability of a product such as beer is not only dependent on the absolute level of individual foam‐stabilising polypeptides but also on the relative proportions of polypeptides derived from the albumin and hordein protein subsets. Copyright © 2004 Society of Chemical Industry
Storage affects the phenolic profiles and antioxidant activities of cherries (Prunus avium L) on human low‐density lipoproteins
Authors: Gonçalves Berta , Landbo Anne‐Katrine , Let Mette , Silva Ana Paula , Rosa Eduardo , Meyer Anne S
Published: May 2004

Four sweet cherry cultivars (cvs), Burlat, Saco, Summit and Van, were analysed at harvest and after storage at 2 and 15 °C for 30 and 6 days respectively. Phenolic profiles in methanolic extracts of freeze‐dried samples of the fresh and differently stored cherries were quantified by high‐performance liquid chromatography. Hydroxycinnamates dominated in all samples and represented 60–74% by weight of the phenols in the fresh and stored samples of the cvs Saco, Summit and Van, and 45% by weight of the phenols in the cv Burlat samples, which were richer in anthocyanins. The relative and total levels of hydroxycinnamates, anthocyanins, flavonols and flavan‐3‐ols varied among cultivars and during storage. Storage at 15 °C increased the phenol levels, particularly the cyanidin‐3‐rutinoside concentration. Cold storage induced decreased total phenol levels in the cvs Summit and Van but increased total phenol levels in the cvs Burlat and Saco. Phenolic cherry extracts inhibited low‐density lipoprotein oxidation in vitro in a dose‐dependent manner. Extracts of freshly harvested cherries exhibited significantly higher antioxidant activities than extracts of stored samples. The cv Summit samples had the highest antioxidant activity. Differences in the antioxidant effects of the cherry samples were positively correlated with their levels of p‐coumaroylquinic acid (p < 0.1) but negatively correlated with their cyanidin‐3‐rutinoside levels (p < 0.05). Copyright © 2004 Society of Chemical Industry