Cell Culture Research Papers - Academia.edu (original) (raw)

We describe the finding of a novel viral haemorrhagic septicaemia virus (VHSV) Genotype III strain that caused disease of both a neurological and septicaemic nature in seawater-farmed rainbow trout Oncorhynchus mykiss in Storfjorden, Norway. In November 2007, an outbreak of VHS associated with slightly elevated mortality was confirmed at a seawater site rearing rainbow trout (90 to 440 g). Within 3 to 4 mo, the disease was recognised in 3 neighbouring sea sites with ongrowing rainbow trout. The clinical, gross pathological and histopathological findings were in accordance with VHS, and the diagnosis was confirmed by the detection of VHSV in brain and internal tissues by immunohistochemistry, cell culture and reverse transcriptase PCR (RT-PCR). Sequence analysis of the G-gene revealed that the isolated virus clustered with VHSV Genotype III and that the Norwegian isolate represents a unique strain of VHSV. The pathogenicity of the virus strain to rainbow trout and Atlantic salmon Salmo salar was examined using infection experiments. In immersion trials, the Norwegian isolate produced a cumulative mortality of 70% in rainbow trout, while nearly 100% mortality was obtained after intraperitoneal injection of the virus. For Atlantic salmon, no mortality was observed in immersion trials, whereas 52% mortality was observed after intraperitoneal injection. The Norwegian isolate thus represents the first VHSV of Genotype III pathogenic to rainbow trout.

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- Pathology, Epidemiology, Fisheries, Immunohistochemistry

The corrosion resistance of AISI 304 stainless steel (AISI 304 SS) and manganese stainless steel (low-nickel SS) brackets in artificial saliva was investigated. The cytotoxic effects of their corrosion products on L929 cell culture were compared by two assays, crystal violet, to evaluate cell viability, and MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide), for cell metabolism and proliferation. The atomic absorption spectroscopic analysis of the corrosion products demonstrated that nickel and manganese ion concentrations were higher for the AISI 304 SS-bracket immersion solution as compared with the low-nickel SS brackets. Scanning electron microscopy and energy-dispersive spectroscopy demonstrated less corrosion resistance for the AISI 304 SS brackets. Although none of the bracket extracts altered L929 cell viability or morphology, the AISI 304 SS-bracket extracts decreased cellular metabolism slightly. The results indicated that the low-nickel SS presents better i...

- by Marcos Lenza
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- Materials Science, Dentistry, Cytotoxicity, Medicine

In this study, a stable, differentiated culture format (primary hepatocytes cultured between two layers of collagen gel) was used to study the effect of the inducer 3,3',4,4'-tetrachlorobiphenyl (TCB) on the activity of cytochrome P4501Al. P450IAI (ethoxyresorufin O-deethylase) enzymatic activity was measured using the rate of conversion of ethoxyresorufin (ER) to resorufin (R). After I4 days of induction with 10mh M TCB, hepatocytes in the double collagen gel configuration exhibited a maximum activity of 180.3 + 46.8 pmol R/fig DNA/hr (3.6 + 0.9 nmol R/IO6 cells/hr) compared with 34.9 + 3.8 pmol R/pg DNA/hr for cells cultured on a single layer of gel. At a TCB level of IOms M, the P450IAI activity in the double-gel configuration peaked at 220.8 + 37.0 pmol R/pg DNA/hr. Cessation of 10e6 M TCB induction produced a decrease in activity to 25.8 + 4.1 pmol R/pg DNA/hr within 4 hr. Subsequent re-application of the inducer caused an increase in activity to 76.5 k-1 I. I pmol R/pg DNA/hr within 6 hr. reaching a maximal value of 131.0 + 38.6 nmol R/u~ DNAihr within I2 hr. Since TCB is ranidlv metabolized bv hepatocytes, a continuous perfusion cul&re system was developed to examine the effect of exposure to a constant level of TCB. Continuous perfusion of the cells with 10-s or IO-'M TCB, resulted in activities significantly higher than those of cultures induced by daily application of induction medium. A mechanistic model of TCB-dependent induction of P4501Al was developed using kinetic parameters estimated from static culture data. The model accurately predicted cyclic variations in P450IAl activity in static culture, and the steady-state activity level of perfusion cultures. This work describes procedures for exposing stable hepatocyte cultures to either continuous or declining levels of consumable inducers and for measuring the activity of cytochrome P450IAl in cultured hepatocytes by a non-invasive method.

- by Howard W.T. Matthew
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- Cell Culture, Steady state, In Vitro Toxicology, Enzymatic Activity
- by Laurent Corcos
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- Cell Culture, Dexamethasone, In Vitro, Gene

The entry of dengue virus-1 (DENV-1) strain Hawaii into mosquito C6/36 cells was analyzed using a variety of biochemical inhibitors together with electron microscopy. The treatment with ammonium chloride, chlorpromazine, dansylcadaverine and dynasore inhibited virus yields, determined by infectivity titrations, whereas nystatin and methyl-␤-cyclodextrin did not have any effect. The effect of the clathrin and dynamin inhibitors on DENV-1 entry was corroborated by detection of internalized virions using immunofluorescence staining. Furthermore, electron micrographs showed the incoming virions attached to electron-dense invaginations of the plasma membrane and within coated vesicles that resembled clathrin-coated pits and vesicles, respectively. The susceptibility to clathrin and dynamin inhibitors of clinical isolates from recent outbreaks was comparable to that shown by the cell culture-adapted reference strain. Similarly, DENV-3 strain H87 and DENV-4 strain 8124 were also inhibited in the presence of ammonium chloride, chlorpromazine and dynasore, allowing conclude that the infectious entry of DENV serotypes to mosquito cells occurs by low pH-dependent clathrin-mediated endocytosis.

The prominent nitric oxide (NO) donor [Ru(terpy)(bdqi)NO](PF 6 ) 3 has been synthesized and evaluated with respect to noteworthy biological effects due to its NO photorelease, including vascular relaxation and melanoma cell culture toxicity. The potential for delivering NO in therapeutic quantities is tenable since the nitrosyl ruthenium complex (NRC) must first reach the "target tissue" and then release the NO upon stimulus. In this context, NRC-loaded lipid carriers were developed and characterized to further explore its topical administration for applications such as skin cancer treatment. NRC-loaded solid lipid nanoparticles (SLN) and nanostructured lipid carriers were prepared via the microemulsification method, with average diameters of 275 ± 15 nm and 211 ± 31 nm and zeta potentials of −40.7 ± 10.4 mV and −50.0 ± 7.5 mV, respectively. In vitro kinetic studies of NRC release from nanoparticles showed sustained release of NRC from the lipid carriers and illustrated the influence of the release medium and the lyophilization process. Stability studies showed that NO is released from NRC as a function of temperature and time and due to skin contact. The encapsulation of NRC in SLN followed by its lyophilization, significantly improved the complex stability. Furthermore, of particular interest was the fact that in the NO photorelease study, the NO release from the NRC-loaded SLN was approximately twice that of just NRC in solution. NRC-loaded SLN performs well enough at releasing and protecting NO degradation in vitro that it is a promising carrier for topical delivery of NO.

- by Renata Fonseca Vianna Lopez and +1
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- Analytical Chemistry, Kinetics, Photochemistry, Scanning Electron Microscopy

Teflon is utilized in neurosurgery as well as in plastic, vascular and heart surgery. Although the effect of Teflon on different types of cells and tissues has been previously studied, we are not aware of any study in which the effect of Teflon was tested on cells of the central nervous system. We have therefore examined the tissue compatibility of spongy and fibrous Teflon by directly exposing the Teflon to dissociated cerebellar cells containing both glia and neurons in tissue culture. Daily examination of the growth of the cells adjacent to Teflon fibers using an inverted phase contrast microscope revealed that Teflon has little or no effect on the growth of these cells. When the cells are fixed after 7 days in culture and stained by the Jenner-Giemsa method, adhesion of both gila and neurons to the surface of the Teflon was seen. Attachment of neural cells to the Teflon was not extensive, as was shown by indirect immunofluorescence technique in connection with double-label staining with anti-GFAP as glia marker and anti-M6 as mouse neuron marker. Thus, these experiments show that Teflon is relatively inert when used as an implant in the central nervous system.

- by Ahmed Amar
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- Surgery, Cell Adhesion, Cell Culture, Cerebellum

Methodology was developed for quantifying the photocytotoxicity of fluoranthene to a gill cell line from rainbow trout for future use in screening polycyclic aromatic hydrocarbons for their relative photocytotoxicity to fish. Solubilization in a modified culture medium was achieved with and without foetal bovine serum (FBS) and with and without dimethyl sulfoxide (DMSO). FBS caused most of the fluoranthene to remain in solution and blocked photocytotoxicity if present during UV irradiation. DMSO had little effect on fluoranthene distribution in cell cultures but caused cells to be slightly more sensitive to the phototoxicity of fluoranthene. The indicator dyes alamar Blue() and 5-carboxyfluorescein diacetate acetoxymethyl ester were used to quantify cytotoxicity in two different ways-singly in two separate assays, and mixed together in a novel single assay, which saved time and material. With UV irradiation for 2 hr at a photon fluence rate of either 1.4 mumol UV-B/m(2)/sec (UV-A:UV...

- by Niels Bols
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- Cell Culture, Cell line, Polycyclic Aromatic Hydrocarbon, Rainbow trout

To test this hypothesis, we used a retroviral vector to express IGFBP-rP1 cDNA in the IGFBP-rP1-deficient MCF-7 breast cancer cell line. Compared with control vector-transduced cells, cumulative cell numbers for IGFBP-rP1-transduced polyclonal or clonal cell cultures were reduced by 39 and 74%, respectively, after 1 week. Medium conditioned by IGFBP-rP1-producing cultures reduced cumulative cell numbers in parental MCF-7 cultures by 20% compared with medium from cultures of a control vector-transduced cell line. Nuclear fragmentation analysis and cell proliferation assays completed in the presence of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethylketone excluded apoptosis as the responsible mechanism. The percentage of cells containing senescence-associated ␤-galactosidase activity was doubled compared with control cell cultures. Flow cytometry analysis indicated that twice as many noncycling cells were present in the IGFBP-rP1-transduced MCF-7 cell cultures compared with controls. These findings indicate that IGFBP-rP1 is an inhibitor of MCF-7 breast cancer cell proliferation and may act via a cellular senescence-like mechanism. . The abbreviations used are: IGFBP, insulin-like growth factor binding protein; IGFBP-rP1, IGFBP-related protein 1; HMEC, human mammary epithelial cell; ER, estrogen receptor; TNF, tumor necrosis factor; zVADfmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone; SA-␤galactosidase, senescence-associated ␤-galactosidase; BrdUrd, 5bromodeoxyuridine; ␣MEM, ␣-minimal essential medium; FBS, fetal bovine serum.

- by Lebris Quinn
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- Flow Cytometry, Cell Culture, Cell Division, Cell line

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- by Dan Lindholm
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- Chemistry, Flow Cytometry, Fluorescence Microscopy, Biology

This work was aimed to record the concentrations of eight polycyclic aromatic hydrocarbons (PAHs): (benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, indeno[1,2,3-c,d]pyrene, benzo[g,h,i]perylene and chrysene) in meats and fishes sold in Abobo market in Abidjan, Côte d'Ivoire. The amount of PAHs present in each sample was quantified using high-performance liquid chromatography (HPLC) equipped with ultraviolet (UV) detector. PAHs were present in all samples in variable quantity. More over benzo[a]pyrene (B[a]P) was present in majority of samples, in quantity above the limit fixed by European Union. With regard to cooking processes, smoking produce more PAHs compared to frying or grilled cooking. Concerning the nature of the matrices, no significant differences were found between meat and fish except benzo[g,h,i]perylene. The study declared that PAHs contamination in the tested foods exceeded the acceptable limit. Health risks linked with the consumption of these foods is a real danger that requires further study.

- by Ed Overton
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- Analytical Chemistry, Archives, Asphalt, Environmental Monitoring

Telomerase is a ribonucleoprotein enzyme that can elongate telomeric DNA, which is thought to be required for the development of cellular immortality and oncogenesis in mammals. We examined telomerase activity in tissues and primary cultured lymphoid cells of adult penaeid shrimps. Using the telomeric repeat amplification protocol (TRAP), we studied the characteristics of a putative novel telomerase in Penaeus japonicus. This telomerase could be inactivated by heating or treatment with RNase A or proteinase K. At elongation, this telomerase required dATP, dGTP, and dTTP, but not dCTP, as substrates. Sequence analysis of the TRAP product revealed that this telomerase synthesized (TTAGG) n repeated sequences. The activity of this telomerase was decreased but still readily detectable in 100 ng of protein extract from lymphoid tissue. The telomerase activity was detected in all examined tissues including testis, ovary, lymphoid, heart, hepatopancreas, and muscle. The highest telomerase activity was in the extract of ovarian tissues. In primary cultured lymphoid cells, the telomerase activity was retained. Thus, primary cultured lymphoid cells of Penaeus japonicus possess one of the factors necessary for cell line establishment.

- by Nakao Nomura
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- Technology, Sequence Analysis, Cell Culture, Telomerase

A tissue-culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the epididymal duct in the cat. A widely used culture protocol for rat epididymal epithelium was used as a starting point and subsequently modified. The cellular population of the cat's epididymal epithelium was isolated by successive collagenase and trypsin digestion. A high yield of isolated cells obtained with good viability, were cultured in DMEM/F12 medium supplemented with foetal bovine serum, in absence or in presence of additional dihydrotestosterone (1 nM). The plated primary cultures reached confluence within 5-8 days, producing a monolayer of cohesive cells. Samples taken after 6 days in culture were processed for transmission and scanning electron microscopies. Immunocytochemical staining was used to estimate the purity of the epithelial cell population in the monolayers. The cell cultures displayed several functional traits of in vivo epithelia, including [ 35 S] hypotaurine and [ 35 S] taurine production. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually mature cats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell functions, metabolic activities and their regulation in cats. #

We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-1, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. Biotechnol. Bioeng. 2010; 106: 106–118. © 2010 Wiley Periodicals, Inc.

- by Tiago Fernandes
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- Chemical Engineering, Chemistry, Reference, Stem Cells

Voltage sensitive fluorescent dyes have long been used to measure physiological voltages in live cell cultures. However dyes suffer from poor contrast and limited recording duration due to photobleaching. A photostable voltage sensitive cellular label, such as a noble metal nanoparticle, would potentially allow for indefinite recording from neural and other live cell cultures. Noble metals possess an inherent voltage sensitivity: their optical properties depend on their density of free electrons, which can be modulated in an aqueous environment by charging or discharging the double layer capacitance with an applied voltage. This manuscript contains a simple analysis of the expected voltage sensitivity using gold nanospheres and nanoshells in both darkfield and photothermal detection modalities and concludes that high bandwidth voltage measurement is fundamentally achievable.

- by Mark Pitter
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- Cell Culture, Double Layer, Lorentz
- by Willibald Schliemann and +1
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- Phytochemistry, Stem Cell, Cell Culture, Biological Sciences

There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several hu-manized monoclonal... more

- by H. Butler
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- Production, Biotechnology, Process Control, Apoptosis
- by Johan Vehof
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- Materials Science, Biomaterials, Titanium, Medicine

Loss of genomic material from chromosomal band 13q14.3 is the most common genetic imbalance in B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, pointing to the involvement of this region in a tumor suppressor mechanism. From the minimally deleted region, 3 candidate genes have been isolated, RFP2, BCMS, and BCMSUN. DNA sequence analyses have failed to detect small mutations in any of these genes, suggesting a different pathomechanism, most likely haploinsufficiency. We, therefore , tested B-CLL patients for epigenetic aberrations by measuring expression of genes from 13q14.3 and methylation of their promotor region. RB1, CLLD7, KPNA3, CLLD6, and RFP2 were downregulated in B-CLL patients as compared with B cells of healthy donors, with RFP2 showing the most pronounced loss of expression. To test whether this loss of gene expression is associated with methylation of CpG islands in the respective promotor regions, we performed methylation-sensitive quantitative polymerase chain reaction analyses and bisulfite sequencing on DNA from B-CLL patients. No difference in the methylation patterns could be detected in any CpG island of the minimally deleted region. Downregulation of genes within chromosomal band 13q14.3 in B-CLL is in line with the concept of haploinsufficiency, but this tumor-specific phenomenon is not associated with DNA methylation. (Blood. 2002; 99:4116-4121)

- by Infinity Valencia
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- Oncology, Molecular Medicine, Cancer, Biology

A cross-linking reagent is required to improve mechanical strength and degradation properties of biopolymers for tissue engineering. To find the optimal preparative method, we prepared diverse genipin-cross-linked chitosan/collagen scaffolds using different genipin concentrations and various cross-linking temperatures and cross-linking times. The compressive strength increased with the increasing of genipin concentration from 0.1 to 1.0%, but when concentration exceeded 1.0%, the compressive strength decreased. Similarly, the compressive strength increased with the increasing of temperature from 4 to 20°C, but when temperature reached 37°C, the compressive strength decreased. Showing a different trend from the above two factors, the effect of cross-linking time on the compressive strength had a single increasing tendency. The other results also demonstrated that the pore size, degradation rate and swelling ratio changed significantly with different cross-linking conditions. Based on our study, 1.0% genipin concentration, 20°C cross-linking temperature and longer cross-linking time are recommended. Long Bi and Zheng Cao contributed equally to this work.

- by Monica Sandri
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- Materials Engineering, Materials Science, Biomedical Engineering, Chitosan

Approximately 2 million people in the United States suffer from Alzheimer's disease (AD), which is the most common cause of chronic dementia among the aging population. During the last 7 yr, excellent opportunities to screen drugs against AD have been provided by animal models of the disease. Because even in the fastest model, AD pathology does not start before the end of the second month, it has been necessary to wait at least until that age to inject drugs into the animal to assess whether they prevent, reduce, or revert synaptic impairment, plaque formation, and increase of β-amyloid (Aβ) levels, the main features of the disease. A solution to the problems mentioned above is achieved by the present fast, efficient, and reproducible cultured cell system from animal models of AD or Aβ-associated diseases, for the screening and testing of compounds for the treatment and therapy of AD or Aβ-associated diseases.

- by Joel Jacob
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- Cognitive Science, Molecular Neuroscience, Membrane Proteins, Synaptic Plasticity

Vimentin is a type III Intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and poor prognosis. We have analysed migration and adhesion to collagenous matrix of a panel of carcinoma cell lines. In vitro invasiveness was highest in vimentin-positive SW480 colon cancer and MDA-MB-231 breast cancer cells and the role of vimentin in these cell lines was investigated by RNA interference. Down-regulation of vimentin expression resulted in impaired migration in both scratchwound experiments and in invasion assays through cell culture inserts coated with collagen gel. Compromised migration was observed in both cell lines, whereas cell attachment assays revealed impaired adhesion to fibrillar collagen in MDA-MB-231 cells while the adhesion of vimentin-ablated SW480 cells, that express both vimentin and keratin intermediate filaments was not affected. In conclusion, ablation of vimentin expression inhibits migration and invasion of colon and breast cancer cell lines.

- by Arto Määttä
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- Cell Migration, Cell Adhesion, Cell Culture, Signal Transduction

Trifluoroacetylthiophene carboxamides have recently been reported to be class II HDAC inhibitors, with moderate selectivity. Exploration of replacements for the carboxamide with bioisosteric pentatomic heteroaromatic like 1,3,4-oxadiazoles, 1,2,4-oxadiazoles and 1,3-thiazoles, led to the discovery that 2-trifluoroacetylthiophene 1,3,4-oxadiazole derivatives are very potent low nanomolar HDAC4 inhibitors, highly selective over class I HDACs , and moderately stable in HCT116 cell culture.

- by Maria Orsale
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- Organic Chemistry, Cell Culture, Cell line, In Vitro

The Arabidopsis Nucleolar Protein Database (http://bioinf.scri.sari.ac.uk/cgi-bin/atnopdb/home) provides information on 217 proteins identified in a proteomic analysis of nucleoli isolated from Arabidopsis cell culture. The database is organized on the basis of the Arabidopsis gene identifier number. The information provided includes protein description, protein class, whether or not the plant protein has a homologue in the most recent human nucleolar proteome and the results of reciprocal BLAST analysis of the human proteome. In addition, for one-third of the 217 Arabidopsis nucleolar proteins, localization images are available from analysis of full-length cDNA–green fluorescent protein (GFP) fusions and the strength of signal in different parts of the cell—nucleolus, nucleolus-associated structures, nucleoplasm, nuclear bodies and extra-nuclear—is provided. For each protein, the most likely human and yeast orthologues, where identifiable through BLASTX analysis, are given with links to relevant information sources.

- by Paul Shaw
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- Proteomics, Cell Culture, Biological Sciences, Environmental Sciences

A marriage between two first cousins who have the same 2/7 balanced translocation is reported. The chromosome rearrangement was primarily detected in amniotic fluid cells cultured for prenatal chromosome analysis because of advanced maternal age. The translocation was also found in the couple's two normal children and in three other members of the family. The possible zygotic chromosome constitutions following 2:2 meiotic segregation in consanguineous parents with the same transloeation are discussed.

- by Maria Tibiletti
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- Genetics, Human Genetics, Complementary and Alternative Medicine, Cell Culture

A microchip-based cell culture system was developed and a primary culture of rat hepatocytes was realized in the system. The microchip was made of glass plates and had a microchannel and a microculture flask inside. The flask inner surface was coated using collagen solution; then FBS and DMEM were added successively. Rat hepatocytes suspended in a medium was introduced into the microchip and incubated at 37 • C in a humidified atmosphere with 5% CO 2 . Because of the shortage of dissolved oxygen, the cultured cells in the microchip resulted in a significant decrease in viability. To overcome this, a continuous medium flow oxygen and nutrition supplying system was designed and constructed. The system realized good cell growth for at least 4 days. Liver-specific functions, such as the synthesis of albumin and urea from hepatocytes were confirmed.

- by Masayuki Yamato
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- Engineering, Technology, Cell Culture, Urea

Mitochondrial dysfunction is a hallmark of cancer cells. Consistent with this phenotype mutations in mitochondrial genome have been reported in all cancers examined to date. However, it is not clear whether mitochondrial genomic status in human cells affects nuclear genome stability and whether proteins involved in inter-genomic cross talk are involved in tumorigenesis. Using cell culture model and cybrid cell technology, we provide evidence that mitochondrial genetic status impacts nuclear genome stability in human cells. In particular our studies demonstrate 1) that depletion of mitochondrial genome (rho 0 ) leads to chromosomal instability (CIN) reported to be present in variety of human tumors and 2) rho 0 cells show transformed phenotype. Our study also demonstrates that mitochondrial genetic status plays a key role in regulation of a multifunctional protein APE1 (also known as Ref1 or HAP1) involved in transcription and DNA repair in the nucleus and the mitochondria. Interestingly we found that altered expression of APE1 in rho 0 cells and tumorigenic phenotype can be reversed by exogenous transfer of wild type mitochondria in rho 0 cells. Furthermore, we demonstrate that APE1 expression is altered in variety of primary tumors. Taken together, these studies suggest that inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis and that APE1 mediates this process. D

- by Mariola Kulawiec
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- Genetics, Immunohistochemistry, Mitochondria, DNA repair

A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, Denmark and N. Ireland. The antigenic profiles of these viruses were determined by indirect immunofluorescence assays using polyclonal antisera and monoclonal antibodies (mAbs) prepared against previously isolated PCVs. A rapid and convenient PCR-based test was developed and used for the genotyping of these PCV isolates. These PCV isolates were found to be antigenically and genomically similar to previously reported isolates of PCV from pigs with wasting disease (PCV2), but distinct from the isolate of PCV from continuous PK/15 cell cultures (PCV1). # 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 -1 1 3 5 ( 9 9 ) 0 0 0 0 4 -8

- by Brian Meehan and +1
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- Microbiology, Veterinary Microbiology, Veterinary, Cell Culture

Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disorder triggered by autoreactive T cells directed to pancreas beta-cell antigens. In this disorder, more than 90% of beta cells are destroyed. Cell death may be mediated via soluble or membrane-bound cell death ligands. One of these ligands may be tumour necrosis factor (TNF)-related apoptosisinducing ligand (TRAIL), a member of the TNF-α superfamily. In the present study, we examined whether TRAIL had cytotoxic effects on adult rat pancreas beta cell cultures and INS1-E rat insulinoma cell line cultures or not. In this study, cell destruction models were built with TRAIL concentrations of 10, 100 and 1000 ng. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for evaluating cell viability. It was detected that cell cultures with TRAIL added showed no differences statistically when compared with control cultures containing no toxic additions. These results showed that TRAIL did not have significant cytotoxic effects on pancreas beta cell culture and INS-1E rat insulinoma cell line cultures. Detection of the expression of TRAIL receptors and natural apoptosis inhibitor proteins will be favourable to investigate the resistance mechanisms to TRAIL-induced cell death in this cell culture system.

- by Cetin Pekcetin
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- Apoptosis, Cell Culture, Cell line, Cell Death

An in vitro quanta1 assay (TCID,,) for a non-occluded baculo-like virus isolate from naturally infected Penaeus juponicws obtained from China and experimentally infected P. stylirostris was developed using primary shrimp lymphoid cell cultures in Primaria TM 24-well tissue culture plates. The virus caused cytopathogenic effect (CPE) in the cell cultures as early as 2 day post-infection (p.i.). Initially, the cells rounded up and finally detached from the culture vessel as the infection progressed. At the present time, there is no established quantitative in vitro cell culture protocol for the assay of this baculo-like virus which has been reported by our laboratory to be highly pathogenic for P. stylirostris and P. vannamei, the two species of penaeid shrimp commercially cultured in Hawaii and the Western hemisphere. This quanta1 assay thus provides a simple and convenient method for the detection and assay of infectious virus in cultured penaeid shrimp. Copyright 0 1997 Elsevier Science B.V.

- by Cesar Nadala and +1
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- Microbiology, Medical Microbiology, Crustacea Decapoda, Cell Culture

Background: Selective neutralization of the IL21/IL21R signaling pathway is a promising approach for the treatment of a variety of autoimmune diseases. Ab-01 is a human neutralizing anti-IL21R antibody. In order to ensure that the activities of Ab-01 are restricted to neutralization even under in vitro cross-linking and in vivo conditions, a comprehensive assessment of agonistic potential of Ab-01 was undertaken.

- by Edward Lavallie
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- Gene expression, Translational Medicine, Cell Culture, Signal Transduction
- by Orlaith Brennan
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- Global Health, Minerals, Calcium, Micronutrients

Bone marrow derived hematopoietic stem cells can function as endothelial progenitor cells. They are recruited to malignant tumors and differentiate into endothelial cells. This mechanism of neovascularization termed vasculogenesis is distinct from proliferation of preexisting vessels. To better understand vasculogenesis we developed a cell culture model with expansion and subsequent endothelial differentiation of human CD133 + progenitor cells in vitro. a v b 3-integrins are expressed by endothelial cells and play a role in the attachment of endothelial cells to the extracellular matrix. We investigated the effect of Cilengitide, a peptide-like, high affinity inhibitor of a v b 3-and a v b 5-integrins in our in vitro system. We could show expression of a v b 3-integrin on 60 ± 9% of non-adherent endothelial progenitors and on 91 ± 7% of differentiated endothelial cells. a v b 3-integrin was absent on CD133 + hematopoietic stem cells. Cilengitide inhibited proliferation of CD133 + cells in a dose-dependent manner. The development of adherent endothelial cells from expanded CD133 + cells was reduced even stronger by Cilengitide underlining its effect on integrin mediated cell adhesion. Expression of endothelial antigens CD144 and von Willebrand factor on differentiating endothelial precursors was decreased by Cilengitide. In summary, Cilengitide inhibits proliferation and differentiation of human endothelial precursor cells underlining its anti-angiogenic effects.

- by Walter Fiedler
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- Cell Adhesion, Extracellular Matrix, Snake venoms, Cell Culture

We obtained callus, cell suspension and hairy root cultures of Tropaeolum majus and we demonstrated their ability to produce glucotropaeolin and myrosinase. In hairy roots glucotropaeolin content and myrosinase activity were higher in comparison with callus, cell suspension and leaves of intact plants. In hairy root cultures the highest glucotropaeolin contents were detected on the 9th day of culture. In

- by Marzena Wielanek
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- Production, Plant Biology, Cell Culture, In vitro culture

toxicity has not been well elucidated. Understanding the Cellular Uptake of Trivalent Arsenite and Pentavalent Arsenate differential toxic mechanisms of As(III) and As(V) is critiin KB Cells Cultured in Phosphate-Free Medium. HUANG, R. N., cally important in assessing the risk from arsenic exposure. AND LEE, T. C. (1996). Toxicol. Appl. Pharmacol. 136, 243-249.

- by Rong Nan Huang
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- Metabolism, Arsenic, Cell Culture, Cell line

F Magnetic resonance imaging High performance liquid chromatography coupled with Ultra Violet a b s t r a c t The paper describes ex vivo applications of colchicine derivatives for the treatment of human T-Lymphoblastoid (CEM) cells. Moreover, the role of the substitutions of ring A at C-1 and C-7 side chain of colchicine analogues was probed by the synthesis and examination of their effects on the three-dimensional (3-D) CEM cells' growth. The CEM cells were cultured in the hollow fiber bioreactor (HFB) device. We used 1 H and 19 F magnetic resonance imaging (MRI) to monitor changes in 3-D CEM cell culture. 19 F MRI was used for visualization of the cellular uptake of new fluorine derivatives. Before and after treatment CEM cells profile was investigated with high performance liquid chromatography (HPLC-UV). Crown j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o o r g pounds used to the synthesis of colchicines derivatives were purchased from Sigma-Aldrich (Oakville, ON).

- by B Gino Fallone
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- Organic Chemistry, Magnetic Resonance Imaging, MRI, Cell Culture
- by Janusz Zwiazek
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- Plant Biology, Cell Culture, Growth, Plant Physiology

N-Acetylaspartylglutamate (NAAG) is the most abundant neuropeptide in the mammalian nervous system. Considerable data support the hypothesis that N A A G is synaptically released in a manner consistent with neurotransmission. Primary murine brain cultures containing neurons and glia expressed 1.2-3.5 nmol of NAAG/mg of protein. In contrast to conclusions drawn from immunohistochemistry, pure glial cultures also expressed high levels of N A A G (0.6-2.1 I nmol/mg of protein). These data suggest that although a subpopulation of neurons contains very high N A A G levels, micromolar concentrations of the pep tide also are present in glia. Both culture types demonstrated robust extracellular peptidase activity when incubated with NAAG, as well as peptide transport. Uptake of [3H]NAAG was both temperature and sodium dependent, yet relatively insensitive to the presence of extracellular glutamate. These results indicate that synaptically released NAAG, as well as that which may be released from glia, is removed from the extracellular space by direct uptake as well as the robust enzymatic degradation of the peptide. A kinetic analysis of this N A A G transport (estimated K , = 1.8 p M ) suggests a high-affinity N A A G transport system. The balance of the two processes of direct peptide uptake and peptide hydrolysis would markedly influence the sequence of receptor-mediated events that follow N A A G release. Key Words: Neuropeptide-Uptake-Glutamate-Neurotransmitter-N-Acetylaspartylglutamate-Cell culture. Cassidy M. and Neale J. H. Localization and transport of N-acetylaspartylglutamate in cells of whole murine brain in primary culture.

- by Joseph Neale
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- Neurochemistry, Immunohistochemistry, Cell Culture, Glutamate

The present work evaluates a newly developed silated hydroxypropylmethylcellulose (Si-HPMC)-based hydrogel as a scaffold for 3D culture of osteogenic cells. The pH variation at room temperature catalyzes the reticulation and self-hardening of the viscous polymer solution into a gelatine state. We designed reticulation time, final consistency and pH in order to obtain an easy handling matrice, suitable for in vitro culture and in vivo injection. Three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D inside this Si-HPMC hydrogel. We show here that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines, indicate that all the cells cultured in 3D into this hydrogel, exhibited a more mature differentiation status than cells cultured in monolayer on plastic. This study demonstrates that this Si-HPMC hydrogel is well suited to support osteoblastic survival, proliferation and differentiation when used as a new scaffold for 3D culture and represents also a potential basis for an innovative bone repair material. r

- by Patrick Gaudray
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- Biomaterials, Tissue Engineering, Hydrogels, Gene expression

Beauveria bassiana colonizes insect hosts initially through a yeast phase, which is common in some artificial liquid cultures, but not reported on artificial solid media. We describe a yeast-like phase for B. bassiana isolate 447 (ATCC 20872) on MacConkey agar and its virulence toward Diatraea saccharalis and Tetranychus urticae. The yeast-like cells of B. bassiana developed by budding from germinating conidia after 24-h incubation. Cells were typically and fungal colonies were initially circular and mucoid, but later were covered with mycelia and conidia. Ability to produce yeast-like cells on MacConkey medium was relatively common among different B. bassiana isolates, but growth rate and timing of yeast-like cell production also varied. Metarhizium anisopliae and Paecilomyces spp. isolates did not grow as yeast-like cells on MacConkey medium. Yeast-like cells of B. bassiana 447 were more virulent against D. saccharalis than conidia when 107 cells/ml were used. At 108 cells/ml, the estimated mean survival time was 5.4 days for the yeast suspension and 7.7 days for the conidial suspension, perhaps due to faster germination. The LC50 was also lower for yeast than conidial suspensions. Yeast-like cells and conidia had similar virulence against T. urticae; the average mortalities with yeast-like cells and conidia were, respectively, 42.8 and 45.0%, with 107 cells/ml, and 77.8 and 74.4%, with 108 cells/ml. The estimated mean survival times were 3.6 and 3.9 for yeast and conidial suspensions, respectively. The bioassay results demonstrate the yeast-like structures produced on MacConkey agar are effective as inoculum for B. bassiana applications against arthropod pests, and possibly superior to conidia against some species. Obtaining well-defined yeast phase cultures of entomopathogenic hyphomycetes may be an important step in studies of the biology and nutrition, pathogenesis, and the genetic manipulation of these fungi.

- by Rogerio Lopes
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- Genetics, Zoology, Cell Cycle, Scanning Electron Microscopy

The anti-in ammatory and antioxidant activities of the aerial part of Helichrysum italicum extracts have been established in various in-vivo and in-vitro experimental models. The results obtained on the acute oedemas induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) and ethyl phenylpropiolate in the mouse ear, by serotonin and phospholipase A 2 (PLA 2 ) in the mouse paw, on chronic in ammation induced by repeated application of TPA in the mouse ear and on the delayed-type hypersensitivity induced by sheep red blood cells suggest that said anti-in ammatory activity is due to the effects of compounds expressed via a corticoid-like mechanism. In addition, the antioxidant activity of the extracts seems to be implicated in this anti-in ammatory activity, as the former inhibits enzymatic and non-enzymatic lipid peroxidation and has free-radical scavenger properties. We conclude that the antiin ammatory activity of Helichrysum italicum can be explained by multiple effects, including in ammatory enzyme inhibition, free-radical scavenging activity and corticoid-like effects.

- by Fabricia Petronilho and +1
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- Pharmacy, Immunology, Water, Complementary and Alternative Medicine

Three-dimensional porous scaffolds prepared from regenerated silk fibroin using either an all-aqueous process or a process involving an organic solvent, hexafluoroisopropanol (HFIP), have shown promise in cell culture and tissue engineering applications. However, their biocompatibility and in vivo degradation have not been fully established. The present study was conducted to systematically investigate how processing method (aqueous vs. organic solvent) and processing variables (silk fibroin concentration and pore size) affect the short-term (up to 2 months) and long-term (up to 1 year) in vivo behavior of the protein scaffolds in both nude and Lewis rats. The samples were analyzed by histology for scaffold morphological changes and tissue ingrowth, and by real-time RT-PCR and immunohistochemistry for immune responses. Throughout the period of implantation, all scaffolds were well tolerated by the host animals and immune responses to the implants were mild. Most scaffolds prepared from the all-aqueous process degraded to completion between 2 and 6 months, while those prepared from organic solvent (hexafluoroisopropanol (HFIP)) process persisted beyond 1 year. Due to widespread cellular invasion throughout the scaffold, the degradation of aqueous-derived scaffolds appears to be more homogeneous than that of HFIP-derived scaffolds. In general and especially for the HFIP-derived scaffolds, a higher original silk fibroin concentration (e.g. 17%) and smaller pore size (e.g. 100-200 mm) resulted in lower levels of tissue ingrowth and slower degradation. These results demonstrate that the in vivo behavior of the three-dimensional silk fibroin scaffolds is related to the morphological and structural features that resulted from different scaffold preparation processes. The insights gained in this study can serve as a guide for processing scenarios to match desired morphological and structural features and degradation time with tissue-specific applications.

- by Carl Kirker-head
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- Biomaterials, Tissue Engineering, Immune response, Immunohistochemistry

Articular cartilage lacks self-repair capacity. Currently, two methods employing autologous cells are used to stimulate repair of articular cartilage. Micro-fracture induced repair induces autologous mesenchymal cell migration from bone marrow. Autologous chondrocytes' transplantation involves in vitro expansion of chondrocytes, and later implantation. In 15 patients de-differentiated chondrocytes obtained by cartilage biopsy were compared to cells derived from repair tissue induced by micro-fracture. These patients all underwent micro-fracture during the cartilage biopsy procedure. Autologous chondrocytes' transplantation was performed at least two months later then the biopsy. Tissue bits from articular cartilage and micro-fracture repair tissue were incubated in-vitro and explant cell cultures established. The cell cultures were assessed by immunohistochemistry and induced to differentiate. Differentiation into bone tissue was stimulated by addition of basic fibroblast gr...

- by Dror Robinson
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- Cell Migration, Immunohistochemistry, Cell Culture, Dexamethasone

The cellular branch of the Drosophila larval innate immune system consists of three immunosurveillance (haemocyte) cell types: plasmatocytes, crystal cells, and lamellocytes. In order to examine haemocyte cytoskeletal dynamics or migration, most researchers use embryos or in vitro cell culture systems, but very little is known about the behaviour of post-embryonic haemocytes. The current method employs an ex vivo system, in which post-embryonic haemocytes are isolated for short-term analysis, in order to investigate various aspects of their behaviour during events requiring cytoskeleton dynamics and Rho GTPase signalling.

- by Christopher Sampson
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- Drosophila melanogaster, Cell Culture, Time-Lapse, Primary Cell Culture

Cognitive deficits are a core feature of patients with methamphetamine (METH) abuse. It has been reported that repeated METH treatment impairs long-term recognition memory in the novel object recognition test (NORT) in mice. Recent studies indicate that silibinin, a flavonoid derived from the herb milk thistle, has potent neuroprotective effects in cell cultures and several animal models of neurological diseases. However, its effect on the cognitive deficit induced by METH remains unclear. In the present study, we attempt to clarify the effect of silibinin on impairments of recognition memory caused by METH in mice. Mice were co-administered silibinin with METH for 7 days and then cognitive function was assessed by NORT after 7-day withdrawal. Tissue levels of dopamine and serotonin as well as their metabolites in the prefrontal cortex and hippocampus were measured 1 day after NORT. Silibinin dose-dependently ameliorated the impairment of recognition memory caused by METH treatment in mice. Silibinin significantly attenuated the decreases in the dopamine content of the prefrontal cortex and serotonin content of the hippocampus caused by METH treatment. We also found a correlation between the recognition values and dopamine and serotonin contents of the prefrontal cortex and hippocampus. The effect of silibinin on cognitive impairment may be associated with an amelioration of decreases in dopamine and serotonin levels in the prefrontal cortex and hippocampus, respectively. These results suggest that silibinin may be useful as a pharmacological tool to investigate the mechanisms of METH-induced cognitive impairments.

- by Toshitaka Nabeshima
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- Cognition, Recognition memory, Prefrontal Cortex, Cell Culture

We have established primary cell culture of the marine demosponge Dysidea avara and Suberites domuncula . Microbial contamination was controlled by the use of a pool of antibiotics confirming the goodness of this procedure. Effect of pH, temperature and light was studied to establish the better growth conditions. The comparison of lipid composition of sponge and cells suggested a series of experiments to optimise the medium. A glucose dose-dependent experiment showed that the ideal glucose concentration is 1 g l (1 . Supplementing the medium with unsaturated fatty acid and retinol, no promotion of growth was observed, but the compounds were totally metabolised by cells. Increments from 70 to 160% in the number of cells were observed, supplementing the medium with different concentration of cholesterol. These results suggest that the analysis of the chemical composition of sponge and cells give indication on the composition of the nutrient media. #

- by Salvatore De Rosa
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- Engineering, Technology, Biotechnology, Porifera

Recombinant antibodies and their derivatives are increasingly being used as therapeutic agents. Clinical applications of antibodies often require large amounts of highly purified molecules, sometimes for multiple treatments. The development of very efficient expression systems is essential to the full exploitation of the antibody technology. Production of recombinant protein in the milk of transgenic dairy animals is currently being tested as an alternative to plasma fractionation for the Ž manufacture of a number of blood factors human antithrombin, human alpha-1-antitrypsin, human serum albumin, factor . IX . The ability to routinely yield mgrml levels of antibodies and the scale-up flexibility make transgenic production an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. The following review examines the potential of transgenic expression for the production of recombinant therapeutic antibodies. q 1999 Elsevier Science B.V. All rights reserved.

- by Eszter Birck-Wilson
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- Immunology, Gene expression, Cell Culture, Germ Cells

Cultured purified oligodendroglia and astroglia exposed to heat stress (45 "C, 10 or 20 min) synthesized a 68-kDa heat-stress protein, which migrates on twodimensional gel electrophoresis and reacts with a specific monoclonal antibody suggesting it is similar to a major 72-kDa heat-shock protein previously reported in other cell types. This protein was not detected in control glial cultures. Actinomycin D prevented synthesis of this protein demonstrating an absolute requirement €or newly synthesized mRNA. The response was prolonged by increasing the period of heat stress from 10 to 20 min. In addition to the 68-kDa HSP protein, the incorporation of radioactivity into 70-, 89-, and 97-kDa proteins was also increased after heating, but in contrast to the 68 kDa protein these proteins appeared to be made in control glial cultures.

- by Barney Dwyer
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- Neuroscience, Psychology, Cell Culture, Glial Cell

Laminate construction of microfluidic devices offers a means to incorporate both additive and subtractive manufacturing techniques for the development of multiplexed fluidic devices that have, for example, porous membranes formed-in-place (FIP). Important applications for these hybrid devices includes sample preparation, separations, and containment of samples fed by a single inlet and outlet, such as multiple samples of suspended cells in culture contained in their respective wells. We describe the development and performance of a fluidic card for the NASA Ames GeneSat-1 program for autonomous cell culture experiments in space. The fluidic card was constructed as a multi-layer laminate of acrylic and bio-compatible bonding adhesive, using laser ablation to form the channels with porous membranes placed at the inlet and outlet of each sample well to contain individual samples of E. coli and prevent cross contamination.

- by Leanna Levine
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- Microfluidics, Cell Culture, Laminates