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PubMed2006

Extracellular matrix modulates expression of cell-surface proteoglycan genes in fibroblasts.

Cell-surface proteoglycans are involved in many functions, including interactions with components of the extracellular microenvironment. They also act as coreceptors that bind and modify the actions of various growth factors, cytokines, and the extracellular matrix (ECM). This study investigated the regulation by the ECM of the expression of cell-surface proteoglycans (CD44, syndecan-1-4, betaglycan, glypican-1). We examined the changes in the expression levels of cell-surface proteoglycan genes in intact tendon, monolayer culture, and under various culture conditions. There was a significant increase in the expression of CD44 and syndecan-4 mRNAs during cell isolation from the tendon. With the switch to a 3D culture environment, there was a significant increase in the expression of CD44 at each passage point relative to its expression in 2D at those passage points. Syndecan-4 mRNA also increased steadily at each passage point in 3D culture environment. This influence on cell surface proteoglycans gene expression may indicate that collagen gel culture mimics in vivo tendon environment. This study provides further insight into the regulation of cell-surface proteoglycans in ligament and tendon fibroblasts by the ECM and 3D culture conditions.

Animals↗

PubMed1994

Contribution of a proline residue and a salt bridge to the stability of a type I reverse turn in chymotrypsin inhibitor-2.

The contributions of the components of a type I reverse turn to the stability of chymotrypsin inhibitor-2 (Lys43-Pro44-Gly45) have been determined by protein engineering methods. A double-mutant cycle was used to determine the interaction between Lys43 and Glu45 by replacing them with alanine. We also mutated Pro44, which gives the geometry of the turn, to alanine and analysed the stability of the resulting mutants compared with wild-type chymotrypsin inhibitor-2, using equilibrium denaturation induced by guanidinium chloride. There are decreases in stability (in kcal/mol) of 0.64 +/- 0.06 for Lys43-->Ala, 0.57 +/- 0.15 for Glu45-->Ala, 0.95 +/- 0.06 for Lys43-->Ala/Glu45-->Ala and 1.93 +/- 0.09 for Pro44-->Ala. The free energy of interaction between Lys43 and Glu45 is calculated to be only 0.25 +/- 0.09 kcal/mol. From the changes in denaturation midpoint, Tm measured by circular dichroism, we estimate the energy of interaction between Lys43 and Glu45 to be 0.36 +/- 0.07 kcal/mol whereas the contribution of Pro44 is approximately 2.0 kcal/mol. The contribution of the salt bridge to the stability of the protein is very small and the residue Pro44 plays the key role in stabilizing the turn.

Amino Acid Sequence↗

PubMed2004

Expression of the C-terminus of HIV-1 reverse transcriptase p66 and p51 subunits as a single polypeptide with RNase H activity.

The C-terminus of the HIV-1 reverse transcriptase heterodimer was reconstructed into a single polypeptide. The construct encodes the p51 thumb (T) and connection (C) subdomains joined through a linker region to the p66 connection (C) and RNase H (R) domain. The TCCR protein was purified from insoluble fractions of Escherichia coli lysates. The TCCR construct maintains Mn(2+)-dependent RNase H activity and specifically cleaves the substrate mimicking the tRNA removal required for second-strand transfer reactions.

Blotting, Western↗

PubMed2004

Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting.

The observation of the regulation of fast protein dynamics in a cellular context requires the development of reliable technologies. Here, a signal regulation cascade reliant on the stimulus-dependent acceleration of the bidirectional flow of mitogen-activated protein kinase (extracellular signal-regulated kinase) across the nuclear envelope was visualized by reversible protein highlighting. Light-induced conversion between the bright and dark states of a monomeric fluorescent protein engineered from a novel coral protein was employed. Because of its photochromic properties, the protein could be highlighted, erased, and highlighted again in a nondestructive manner, allowing direct observation of regulated fast nucleocytoplasmic shuttling of key signaling molecules.

Active Transport, Cell Nucleus↗

PubMed2001

Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development.

In ventricular cells of the mouse telencephalon, differential expression of cadherin cell adhesion molecules defines neighbouring regions; R-cadherin delineates the future cerebral cortex, while cadherin-6 delineates the lateral ganglionic eminence. By using cell labelling analyses in the whole embryo culture system, we demonstrated that the interface between R-cadherin and cadherin-6 expression is a boundary for cell lineage restriction at embryonic day 10.5. Interestingly, when a group of cells with exogenous cadherin-6 were generated to straddle the cortico-straital boundary by electroporation at embryonic day 11.0, ectopic cadherin-6-expressing cortical cells were sorted into the striatal compartment, and the reverse was the trend for ectopic R-cadherin-expressing striatal cells. Although cadherin-6 gene knockout mice engineered in this study showed no obvious phenotype in telencephalic compartmentalisation, the preferential sorting of ectopic cadherin-6-expressing cells was abolished in this mutant background. Thus, the differential expression pattern of cadherins in the embryonic telencephalon is responsible for maintaining the cortico-striatal compartment boundary.

Animals↗

PubMed2004

Bacterial sterilization and intracellular protein release by a pulsed electric field.

Several biotechnological applications of high-voltage pulsed electric field (PEF) are introduced. Electrical breakdown or disruption of a biological membrane by PEF is understood to occur by electromechanical compression, which results in the formation of transmembrane pores. If the total area of induced pores is small in relation to the total surface area of the membrane, the pores are able to close again mainly through the diffusion of the lipid molecules and rearrangement of the proteins (reversible disruption). If the total area of the pores becomes unfavorably large, the membrane is no longer able to repair these perturbations (irreversible disruption), and that results in sterilization. We have investigated effective sterilization by using PEF-induced irreversible disruption of biological membranes. The treatment temperature or growth temperature was found to have a great effect on PEF sterilization. The shape of the treatment chamber also proved important for effective PEF sterilization. Therefore, a number of reactors having novel structures were developed. We have also verified that this PEF-induced reversible disruption could be utilized for the selective release of intracellular proteins from yeast and certain gene-engineered Escherichia coli. The secretion of periplasmic protein from E. coli was achieved during cultivation.

Bacteria↗

PubMed2005

Genetic manipulation of stromal cell-derived factor-1 attests the pivotal role of the autocrine SDF-1-CXCR4 pathway in the aggressiveness of breast cancer cells.

Stromal cell-derived factor-1 (SDF-1), via its receptor CXCR4, has been implicated in metastasis of cancer, including breast cancer. Exogenous SDF-1 is known to regulate locomotion, chemotaxis and adhesion. The knowledge regarding the effect of autocrine SDF-1 on breast cancer cells is not available. The current study evaluated the effects of SDF-1 on the biological behaviour of breast cancer cells by genetically modifying the expression of SDF-1 in breast cancer cells. Two human breast cancer cell lines (MDA-MB-231 and MDA-MB-435s) and a human fetal lung fibroblast cell line (MRC5) were used. The expression of SDF-1 and the SDF-1 receptor, CXCR4 in the cell lines were studied. Expression cassettes of human SDF-1 and hammerhead ribozyme transgenes specifically targeting human SDF-1 were constructed and used to over-express SDF-1 or to knockout the expression of SDF-1 in cancer cells, respectively. Invasiveness, migration and growth of the genetically modified cells were assessed. SDF-1 was expressed in wild-type human breast cancer cell line MDA-MB-435s and fibroblast cell line MRC5, but not in MDA-MB-231 cell line. In contrast, CXCR4 expression was observed in all three cell lines tested. The ability of invasion and migration was significantly reduced in SDF-1 knockout MDA-MB-435s cells, compared with wild-type and vector control cells (p<0.01). On the other hand, SDF-1 transfected MDA-MB-231epsilonSDF1+/+ cells that stably expressed SDF-1 showed a different behaviour from MDA-MB-231SDF1+/- (plasmid control) and wild-type MDA-MB-231 cells, both being SDF-1 negative. MDA-MB-231epsilonSDF1+/+ cells displayed a higher degree of invasiveness and migration, compared with wild-type and MDA-MB-231SDF+/- cells (p<0.01). Furthermore, SDF1-knockout MDA-MB-435s cells showed a slower growth rate over a 7-day period compared with the respective control and wild-type MDA-MB-435s cells. In contrast, the growth of the SDF-1 transfected MDA-MB-231SDF1+/+ cells was markedly enhanced when compared with wild-type and vector control cells. Breast cancer cell lines, when equipped with the autocrine SDF-1-CXCR4 signal pathway, display aggressive behaviour, including an increase in invasiveness, migration together with faster growth. SDF-1, together with its receptor CXCR4 may provide important information for predicting the aggressive nature and constitute important therapeutic targets in human breast cancer.

Breast Neoplasms↗

PubMed1994

Introduction of foreign sequences into the genome of influenza A virus.

The ability to apply reverse genetics technologies to influenza virus now allows us to construct novel viruses containing heterologous sequences. We have engineered two neuraminidase (NA) genes of influenza A/WSN/33 virus containing additional sequences which were inserted downstream of the open reading frame of the NA. These NA genes, NA/EMC and NA/EMC-NS1, possess a 546 and a 917 nucleotide (nt) insertion respectively. Transfectant viruses were rescued following ribonucleoprotein (RNP) transfection of the engineered NA genes into influenza helper-virus-infected cells. The transfectant viruses maintained their artificially introduced sequences stably during three passages. The rescued virus containing the NA/EMC-NS1 gene produced one log fewer infectious virus particles in MDBK cells than did wild type A/WSN/33 virus. The growth characteristics in tissue culture of the virus containing the NA/EMC gene was indistinguishable from that of wild-type influenza virus. Based on these results we conclude that influenza A viruses can tolerate heterologous insertions of at least a kilobase in the NA gene.

Animals↗

PubMed2006

Epidermal morphogenesis: the transcriptional program of human keratinocytes during stratification.

The epidermis serves to protect the body against environmental assaults and at the same time is able to survive and replenish itself under harsh conditions. The epidermis accomplishes this feat via a well-orchestrated program of stratification and terminal differentiation that provides barrier against infection, radiation, and water loss. Despite significant progress in skin biology, many molecules and pathways that are involved in stratification and barrier formation remain unknown. Here, we employed tissue-engineered models of complete versus impaired epidermal stratification to discover the genes that may be important in this process. Transcriptional profiling at different stages of development showed significant differences in transcription, signaling, and most important metabolism-associated genes between fully stratified and poorly stratified epithelia. These transcriptional changes correlated well with functional data on cell proliferation, expression of adhesion molecules, and utilization of metabolic pathways, ultimately leading to different phenotypes. Our data identified genes that were not previously known to play a role in epidermis and established a link between metabolism and morphogenesis in skin epithelium.

Carbohydrate Metabolism↗

PubMed2004

Mechanical stimulation promotes osteogenic differentiation of human bone marrow stromal cells on 3-D partially demineralized bone scaffolds in vitro.

Bone is a dynamic tissue that is able to sense and adapt to mechanical stimuli by modulating its mass, geometry, and structure. Bone marrow stromal cells (BMSCs) are known to play an integral part in bone formation by providing an osteoprogenitor cell source capable of differentiating into mature osteoblasts in response to mechanical stresses. Characteristics of the in vivo bone environment including the three dimensional (3-D) lacunocanalicular structure and extracellular matrix composition have previously been shown to play major roles in influencing mechanotransduction processes within bone cells. To more accurately model this phenomenon in vitro, we cultured human BMSCs on 3-D, partially demineralized bone scaffolds in the presence of four-point bending loads within a novel bioreactor. The effect of mechanical loading and dexamethasone concentration on BMSC osteogenic differentiation and mineralized matrix production was studied for 8 and 16 days of culture. Mechanical stimulation after 16 days with 10 nM dexamethasone promoted osteogenic differentiation of BMSCs by significantly elevating alkaline phosphatase activity as well as alkaline phosphatase and osteopontin transcript levels over static controls. Mineralized matrix production also increased under these culture conditions. Dexamethasone concentration had a dramatic effect on the ability of mechanical stimulation to modulate these phenotypic and genotypic responses. These results provide increased insight into the role of mechanical stimulation on osteogenic differentiation of human BMSCs in vitro and may lead to improved strategies in bone tissue engineering.

Adult↗

PubMed2003

A nanoporous molecular magnet with reversible solvent-induced mechanical and magnetic properties.

Interest in metal-organic open-framework structures has increased enormously in the past few years because of the potential benefits of using crystal engineering techniques to yield nanoporous materials with predictable structures and interesting properties. Here we report a new efficient methodology for the preparation of metal-organic open-framework magnetic structures based on the use of a persistent organic free radical (PTMTC), functionalized with three carboxylic groups. Using this approach, we create an open-framework structure Cu3(PTMTC)2(py)6(CH3CH2OH)2(H2O), which we call MOROF-1, combining very large pores (2.8-3.1 nm) with bulk magnetic ordering. MOROF-1 shows a reversible and highly selective solvent-induced 'shrinking-breathing' process involving large volume changes (25-35%) that strongly influence the magnetic properties of the material. This magnetic sponge-like behaviour could be the first stage of a new route towards magnetic solvent sensors.

Copper↗

PubMed2005

Gelatin/chondroitin-6-sulfate copolymer scaffold for culturing human nucleus pulposus cells in vitro with production of extracellular matrix.

Tissue-engineering approaches for treating degenerative intervertebral discs aim to regenerate intervertebral disc tissues in order to retard or even reverse the degenerative process. This study was designed to investigate the feasibility of the glutaraldehyde crosslinked gelatin/chondroitin-6-sulfate copolymer scaffold to serve as a bioactive scaffold for culturing human nucleus pulposus (NP) cells in vitro with preservation of the cell viability, cell proliferation, and production of important extracellular matrix, including glycoaminoglycans (GAG) and Type II collagen. Each experimental sample was seeded with 1 x 10(6) human NP cells, and then the cell-scaffold hybrids were cultured in vitro for 6 or 12 weeks. SEM showed a highly porous structure with an average pore size of 100 microm in the copolymer scaffold. Immediately after cell seeding, SEM showed that the seeded cells penetrated deeply and distributed evenly in the copolymer scaffold. Water-soluble tetrazolium salt-1 (WST-1) assay showed good viability and active proliferation of cultured human NP cells in the copolymer scaffolds up to 12 weeks. The cell-scaffold hybrids contained significantly higher levels of sulfated GAG than the control samples (41.29 mug vs 6.04 mug per scaffold). Immunohistochemical study showed Type II collagen fibrils on the surface of scaffold substrate after 6 weeks of cultivation. More abundant deposition of Type II collagen could be detected after 12 weeks. The results achieved in this study indicate that the gelatin/chondroitin-6-sulfate copolymer scaffold is a promising bioactive scaffold for regeneration of nucleus pulposus tissue.

Biocompatible Materials↗

PubMed2006

Adipogenic differentiation of adipose tissue derived adult stem cells in nude mouse.

We evaluated the use of a combination of adipose tissue derived adult stem cells (ADSCs) obtained from liposuction and injectable poly(lactic-co-glycolic acid) (PLGA) spheres for adipose tissue engineering. Adipogenesis was examined in nude mice injected subcutaneously with ADSCs (group I), PLGA spheres (group II), or ADSCs attached PLGA spheres (group III) cultured in adipogenic medium for 7 days. After 4 and 8 weeks, newly formed adipose tissue was observed in groups II and III but not in group I. Oil red O staining of newly formed tissue showed that there was substantially more tissue regeneration and adipogenic differentiation in group III than in group II. RT-PCR confirmed that, after 8 weeks, the PLGA-attached ADSCs had fully differentiated into adipocytes. This study provides significant evidence that ADSCs and PLGA spheres can be used in a clinical setting to generate adipose tissue as a noninvasive soft tissue filler.

Adipose Tissue↗

PubMed2002

Comparison of mixed cell culture containing genetically engineered BGMK and CaCo-2 cells (Super E-Mix) with RT-PCR and conventional cell culture for the diagnosis of enterovirus meningitis.

BACKGROUND: Enteroviral meningitis has traditionally been diagnosed by cell culture. More recently, molecular techniques and shell vials with a mixture of human colon carcinoma and genetically engineered buffalo green monkey kidney cells (BGMK cells) (Super E-Mix) have been described. OBJECTIVE: We compared the results of this new cell culture technique with two reverse transcriptase polymerase chain reaction (RT-PCR) techniques and conventional cell culture to assess the accuracy of these various methods. STUDY DESIGN: 2 ml of cerebrospinal fluid (CSF) was obtained from 72 patients with suspected viral meningitis. 600 microl was used for conventional cell culture and 200 microl was inoculated to each of two Super E-Mix vials. One vial was incubated for 24 h, then stained with pan-enterovirus antibody, and the other was examined for CPE daily for 5 days and stained when positive or at the end of incubation. The final aliquot (500 microl) was centrifuged at 25,000g, and the pellet in 140 microl of supernatant was used for ribonucleic acid (RNA) extraction and tested by commercial single-step (Chemicon, Temecula, CA) and two-step (Argene, Varilhes, France) RT-PCR methods. RESULTS: Conventional culture was 51% sensitive and 100% specific. The Super E-Mix was slightly higher (sensitivity 76%, specificity 100%). The Chemicon and Argene PCR methods had a sensitivity of 93% and 88% and specificity of 90% and 97%, respectively. CONCLUSION: The Super E-Mix procedure had greater sensitivity than conventional cell culture, but RT-PCR was the most sensitive technique with this type of specimen. The RT-PCR methods performed equally.

Adolescent↗

PubMed2003

High-frequency phenotypic reversion and pathogenicity of an acyclovir-resistant herpes simplex virus mutant.

A double-guanine-insertion mutation within a run of guanines in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. This mutation was engineered into strain KOS, and stocks were generated from single plaques. Plaque autoradiography revealed that most plaques in such stocks exhibited low levels of TK activity, while approximately 3% of plaques exhibited high levels of TK activity, indicating a remarkably high frequency of phenotypic reversion. This virus was able to reactivate from latency in mouse ganglia; a fraction of the reactivating virus expressed a high level of TK activity due to an additional G insertion, suggesting that the observed genetic instability contributed to pathogenicity.

Acyclovir↗

PubMedDate not supplied

Human cell lines engineered for tetracycline-regulated expression of tumor suppressor candidate genes from a frequently affected chromosomal region, 3p21.

BACKGROUND: We modified a tetracycline-regulated system that can control the activity of individual genes quantitatively and reversibly in transgenic mammals. Despite these advances, there remained one problem in the intensive use of the tet-system: the limited range of acceptor cell lines, expressing a tetracycline-controlled transcriptional activator (tTA). This study describes in detail new vectors and a unifying strategy to generate tTA-expressing cell lines. METHOD: Two retroviral vectors pLNCtTA-hCMV and pLNCtTA-EF1alpha coding for the tTA were used to engineer cell lines to constitutively express tTA. New expression vectors pETE-Hyg and pETE-Bsd were also created that replicate in episomal form in human cells and facilitate tetracycline-regulated expression of targeted genes. RESULTS: The primate-tropic retroviruses efficiently delivered the regulatory tTA gene into 12 selected human cancer cell lines. Two candidate tumor suppressor genes from the human 3p21-p22 region MAPKAPK3 (3pK) and MLH1 were cloned into the episomal vector and transfected into engineered A9 and KRC/Y cells. The transfectants were subcutaneously grown in SCID mice, and the expression of the transgene was successfully controlled in vivo by tetracycline administered ad libitum in drinking water. The experiments demonstrated that both transgenes did not antagonize the tumorous growth of these cells. CONCLUSIONS: New retroviral and episomal vectors appear particularly suited for tight regulation of genes that cause suppression of cell growth. The generated cell lines can be used in various applications to study the effect of an inducible transgene in human cancer cells.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Infectious transcripts of tick-borne encephalitis virus, generated in days by RT-PCR.

Construction of infectious clones of flaviviruses can be problematic owing to instability, toxicity, and recombination events occurring while cloning cDNA in the bacterial vectors. To overcome these difficulties we have devised a rapid and simple method for producing an infectious genetically engineered tick-borne encephalitis virus in less than 10 days using viral RNA from an unpurified virus suspension. The experimental protocol utilized the high fidelity reverse transcription-polymerase chain reaction to produce two long (5.7 and 5.2 kb) overlapping cDNA segments. To produce full-length cDNA the two overlapping segments were either ligated or fused by polymerase chain reaction. The cDNA was then transcribed and the derived full-length RNA was injected intracerebrally into young mice which reproduced the infectious virus within 8-20 days. To differentiate the engineered virus from parent virus, a Sunl restriction site was introduced by substituting nucleotides at positions 5688 and 5691 of the viral genome. This restriction site was present in the engineered virus recovered from infected mice. Antigenic and electrophoretic analysis of the proteins recovered from the engineered virus confirmed that it was indistinguishable from parent virus. In addition to its applicability as a rapid method of producing infectious engineered virus, this protocol offers the opportunity to introduce changes by site-directed mutagenesis without needing to clone the viral DNA. The method should be applicable to most viruses possessing an infectious RNA molecule and reduces the time required to produce a genetically engineered virus from years to days. When appropriate, the choice of mice for transfection of RNA has the advantage of being extremely simple, very sensitive, and producing high titers of stable virus.

Animals↗

PubMedDate not supplied

Propagation of human nasal chondrocytes in microcarrier spinner culture.

OBJECTIVE: The aim of this study was to test the effectiveness of nasal septal chondrocytes, propagated in microcarrier spinner culture, as an alternative tissue source of chondrocytic cells for cartilage grafts for head and neck surgery and for articular cartilage repair. METHODS: We harvested chondrocytes from 159 patients, ranging in age from 15 to 80 years and undergoing repair of a deviated nasal septum, and propagated the cells in a microcarrier spinner culture system. The nasal chondrocytes proliferated and produced extracellular matrix components similar to that produced by articular chondrocytes. RESULTS: In microcarrier spinner culture on collagen beads, chondrocyte numbers increased up to 14-fold in 2 weeks. After a month, the microcarriers seeded with nasal chondrocytes began to aggregate, producing a dense cartilage-like material. The newly synthesized extracellular matrix was rich in high molecular weight proteoglycans, and the chondrocytes expressed type II collagen and aggrecan but not type I collagen. CONCLUSION: These studies support the feasibility of engineering cartilage tissue using chondrocytes harvested from the nasal septum. Injectable and solid formulations based on this technology are being evaluated for applications in craniomaxillofacial reconstructive surgery and for plastic and orthopedic surgery practices.

Adolescent↗