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Results for “reverse engineering”

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At least 667 records · Page 37Linked to original sources
PubMed2002

The identification and subcellular localization of human MRK.

A novel human kinase gene, human MRK (hMRK), was cloned by using degenerate RT-PCR. The hMRK encoded a putative 632 amino acids protein and was highly homologous to rat MRK (rMRK) in the entire coding region. The hMRK was located at chromosome 6p12.3 by RH-PCR analysis. The hMRK was generally expressed a single approximately 6.3 kb transcript at a low level in a variety of tissues except at a high level in testis. The full-length hMRK protein was fused to C-terminal of GFP and expressed in Hela cells. The fluorescence microscopy results identified its nuclear localization.

Amino Acid Sequence↗
PubMedDate not supplied

Synthesis and characterization of biocompatible thermo-responsive gelators based on ABA triblock copolymers.

The synthesis of biocompatible, thermo-responsive ABA triblock copolymers in which the outer A blocks comprise poly(N-isopropylacrylamide) and the central B block is poly(2-methacryloyloxyethyl phosphorylcholine) is achieved using atom transfer radical polymerization with a commercially available bifunctional initiator. These novel triblock copolymers are water-soluble in dilute aqueous solution at 20 degrees C and pH 7.4 but form free-standing physical gels at 37 degrees C due to hydrophobic interactions between the poly(N-isopropylacrylamide) blocks. This gelation is reversible, and the gels are believed to contain nanosized micellar domains; this suggests possible applications in drug delivery and tissue engineering.

Acrylic Resins↗
PubMed2005

Controlling the thermal stability of thin films by interfacial engineering.

The quantized electronic structure in Pb films on Si(111) varies substantially as the film thickness increases. The changes in electronic energy cause the thermal stability of the films to oscillate with an approximate bilayer period. The phase of the oscillations can be controlled by interfacial engineering. Comparison of Pb films prepared on Si(111) terminated by In, Au, and Pb as interfactants reveals a phase reversal. For , films made of odd numbers of atomic layers (5, 7, and 9) are more stable than the even ones. This trend is reversed for the other two cases.

Journal Article↗
PubMedDate not supplied

In vitro induction and transplantation of eye during early Xenopus development.

A vertebrate eye was induced via a series of coordinated inductive interactions. Here, we describe a novel in vitro system to induce eye formation at high frequency using Xenopus early gastrulae. The eye formed in vitro is morphologically similar to the normal eye. When the in vitro eye was transplanted into a stage-33 tadpole, the optic nerve was seen extending from the grafted eye to the tectum of the host brain and the in vitro eye graft was retained after metamorphosis. In addition, we transplanted the eye formed in vitro into a tadpole with both eyes removed. The resultant juvenile frogs could perceive brightness using the grafted eye and thereby control their skin color, suggesting that the eye formed in vitro could function normally.

Animals↗
PubMed2002

TGF-beta1-stimulated osteoblasts require intracellular calcium signaling for enhanced alpha5 integrin expression.

The osteoactive factor, transforming growth factor beta1 (TGF-beta1), influences osteoblast activity and bone function. We recently characterized a Smad-independent TGF-beta1-induced Ca(2+) signal in human osteoblasts (HOB) and demonstrated its importance in cell adhesion. Here, we further elucidate the role of the TGF-beta1 Ca(2+) signal in the mechanics of HOB adhesion. Osteoblast interaction with fibronectin (FN) through alpha5beta1 integrin is principally responsible for osteoblast-substrate adhesion. Our results show that the TGF-beta1 intracellular Ca(2+) signal is responsible, in part, for stimulation of alpha5 integrin expression, but not beta1 integrin or FN expression. Increased alpha5 integrin protein and mRNA expression was seen as early as 12 h after TGF-beta1 treatment, but was inhibited by cotreatment with nifedipine, a Ca(2+) channel blocker. TGF-beta1 increased both FN and beta1 integrin protein production within 48 h, independent of nifedipine cotreatment. Immunofluorescence observations revealed that TGF-beta1 increased alpha5 integrin staining, clustering, and colocalization with the actin cytoskeleton, effects that were blocked by nifedipine. The TGF-beta1 Ca(2+) signal, a pathway crucial for HOB adhesion, enhances alpha5 integrin expression, focal contact formation, and cytoskeleton reorganization. These early events are necessary for osteoblast adhesion; thus they determine the fate of the cell and ultimately affect bone function.

Actins↗
PubMed2002

Bacillus probiotics: spore germination in the gastrointestinal tract.

Spores of Bacillus species are being used commercially as probiotics and competitive exclusion agents. Unlike the more commonly used Lactobacillus-type probiotics, spores are dormant life forms. To address how spore probiotics might function we have investigated whether spores can germinate in the gastrointestinal tract by using a murine model. Using a genetically engineered chimeric gene, ftsH-lacZ, which is strongly expressed only in vegetative cells, we have developed a sensitive competitive reverse transcription-PCR assay which has enabled detection of as few as 10(2) vegetative bacteria in the mouse gut. Using this method we have administered doses of ftsH-lacZ spores to groups of mice and shown that spores can germinate in significant numbers in the jejunum and ileum. The levels of detection we obtained suggest that spores may colonize the small intestine, albeit briefly.

Animals↗
PubMed2000

Multiple blocks to human immunodeficiency virus type 1 replication in rodent cells.

The recent identification of human gene products that are required for early steps in the human immunodeficiency virus type 1 (HIV-1) life cycle has raised the possibility that rodents might be engineered to support HIV-1 infection. Therefore, we have examined the ability of modified mouse, rat, and hamster cell lines to support productive HIV-1 replication. Rodent cells, engineered to support Tat function by stable expression of a permissive cyclin T1 protein, proved to be able to support reverse transcription, integration, and early gene expression at levels comparable to those observed in human cell lines. Surprisingly, however, levels of CD4- and coreceptor-dependent virus entry were reduced to a variable but significant extent in both mouse and rat fibroblast cell lines. Additional posttranscriptional defects were observed, including a reduced level of unspliced HIV-1 genomic RNA and reduced structural gene expression. Furthermore, the HIV-1 Gag precursor is generally inefficiently processed and is poorly secreted from mouse and rat cells in a largely noninfectious form. These posttranscriptional defects, together, resulted in a dramatically reduced yield of infectious virus (up to 10,000-fold) over a single cycle of HIV-1 replication, as compared to human cells. Interestingly, these defects were less pronounced in one hamster cell line, CHO, which not only was able to produce infectious HIV-1 particles at a level close to that observed in human cells, but also could support transient, low-level HIV-1 replication. Importantly, the blocks to infectious virus production in mouse and rat cells are recessive, since they can be substantially suppressed by fusion with uninfected human cells. These studies imply the existence of one or more human gene products, either lacking or nonfunctional in most rodent cells that are critical for infectious HIV-1 virion morphogenesis.

Animals↗
PubMed2004

Selection in vitro of allosteric ribozymes.

The capacities of RNA for both catalysis and molecular recognition have been appreciated for some time. Recently, a number of studies have shown that these distinct functional classes may be combined to generate multipartite ribozymes in which the catalytic and ligand-binding elements act coordinately. In this chapter, we describe a procedure known as allosteric selection, in which an RNA catalyst in conjunction with a random sequence domain is subjected to iterative in vitro selection in order selectively to recover ligand-dependent ribozymes.

Allosteric Regulation↗
PubMed2005

Increased virulence and competitive advantage of a/alpha over a/a or alpha/alpha offspring conserves the mating system of Candida albicans.

The majority of Candida albicans strains in nature are a/alpha and must undergo homozygosis to a/a or alpha/alpha to mate. Here we have used a mouse model for systemic infection to test the hypothesis that a/alpha strains predominate in nature because they have a competitive advantage over a/a and alpha/alpha offspring in colonizing hosts. Single-strain injection experiments revealed that a/alpha strains were far more virulent than either their a/a or alpha/alpha offspring. When equal numbers of parent a/alpha and offspring a/a or alpha/alpha cells were co-injected, a/alpha always exhibited a competitive advantage at the time of extreme host morbidity or death. When equal numbers of an engineered a/a/alpha2 strain and its isogenic a/a parent strain were co-injected, the a/a/alpha2 strain exhibited a competitive advantage at the time of host morbidity or death, suggesting that the genotype of the mating-type (MTL) locus, not associated genes on chromosome 5, provides a competitive advantage. We therefore propose that heterozygosity at the MTL locus not only represses white-opaque switching and genes involved in the mating process, but also affects virulence, providing a competitive advantage to the a/alpha genotype that conserves the mating system of C. albicans in nature.

Animals↗
PubMed2005

Tumor vaccine based on cell surface expression of DcR3/TR6.

DcR3/TR6, a secreted protein belonging to the TNF receptor superfamily, interacts with lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entrance mediator (LIGHT), Fas ligand (FasL), and TL1A, all members of the TNF superfamily. Solid-phase TR6 can trigger reverse signaling of LIGHT and FasL expressed on T cells, and lead to T cell costimulation. In this study, we engineered tumor cells to express cell surface TR6 and used these cells as a tumor vaccine. We demonstrated that mastocytoma P815 cells expressing surface TR6 (TR6-P815) effectively augmented the T cells response in vitro and ex vivo in terms of proliferation, as well as IL-2 and IFN-gamma secretion. TR6-P815 cells had reduced tumorigenicity compared with parental P815 cells. When inactivated TR6-P815 cells were employed as a vaccine, they protected the mice from challenge with live parental P815 cells, and eliminated established P815 tumors. The cell surface TR6-based tumor vaccine was also effective against low antigenicity tumors, such as B16 melanoma; co-administration of bacillus Calmette-Guérin further enhanced the vaccine's efficacy. Thus, cell surface TR6 expression is a useful addition to our tumor vaccine arsenal.

Animals↗
PubMed2000

[The modulation of type I collagen and its receptor system on biological characteristics of osteoblasts].

This paper addresses the question whether type I collagen-receptor system is necessary for the functional activity of osteoblasts. Through blocking type I collagen-receptor system by type I collagen antibody or integrin alpha 2 beta 1 antibody, the proliferation of osteoblasts was studied by cell count, the apoptosis was studied by flow cytometry, and the mRNA expressions of type I collagen, integrin alpha 2 beta 1 and osteocalcin were also detected by RT-PCR techniques. When type I collagen-receptor system was blocked, osteoblasts showed high apoptosis rate, lower growth kinetics and weak mRNA expression of type I collagen, integrin alpha 2 beta 1 and osteocalcin; the blocking effect was reversible. It suggests that type I collagen-receptor system is necessary for osteoblastic phenotype. The biomaterials for bone tissue engineering should be constructed according to the extracellular matrix of osteoblasts, which supply normal extracellular environment for osteoblasts.

Apoptosis↗
PubMed2006

Common plantain. A collection of expressed sequence tags from vascular tissue and a simple and efficient transformation method.

The vascular tissue of higher plants consists of specialized cells that differ from all other cells with respect to their shape and size, their organellar composition, their extracellular matrix, the type of their plasmodesmata, and their physiological functions. Intact and pure vascular tissue can be isolated easily and rapidly from leaf blades of common plantain (Plantago major), a plant that has been used repeatedly for molecular studies of phloem transport. Here, we present a transcriptome analysis based on 5,900 expressed sequence tags (ESTs) and 3,247 independent mRNAs from the Plantago vasculature. The vascular specificity of these ESTs was confirmed by the identification of well-known phloem or xylem marker genes. Moreover, reverse transcription-polymerase chain reaction, macroarray, and northern analyses revealed genes and metabolic pathways that had previously not been described to be vascular specific. Moreover, common plantain transformation was established and used to confirm the vascular specificity of a Plantago promoter-beta-glucuronidase construct in transgenic Plantago plants. Eventually, the applicability and usefulness of the obtained data were also demonstrated for other plant species. Reporter gene constructs generated with promoters from Arabidopsis (Arabidopsis thaliana) homologs of newly identified Plantago vascular ESTs revealed vascular specificity of these genes in Arabidopsis as well. The presented vascular ESTs and the newly developed transformation system represent an important tool for future studies of functional genomics in the common plantain vasculature.

Arabidopsis↗
PubMed2007

Comparison of fresh and predegenerated muscle-vein-combined guides for the repair of rat median nerve.

Over the last 10 years, we have investigated a particular type of bioengineered nerve guide, the muscle-vein-combined tube, which is made by filling a vein with skeletal muscle. In our previous studies we have always used fresh skeletal muscle to fill vein conduits. In the present study we compared the use of fresh and predegenerated (freeze-thawed) skeletal muscle for muscle-vein-combined nerve guides. In this study, a 10-mm-long rat median nerve defect was repaired using either type of nerve guide. The samples were analyzed 5 and 30 days after surgery by light and electron microscopy. In addition, reverse transcription polymerase chain reaction (RT-PCR) was carried out to investigate the expression of mRNAs coding for glial markers, as well as glial growth factor (NRG1) and its receptors (erbB2 and erbB3). Results showed differences between the two types of nerve guides at postoperative day 5; however, no difference was detected at day 30 suggesting that both types of tissue-engineered conduit are effective for repairing peripheral nerve defects in this experimental model.

Animals↗
PubMed2002

Metabolic engineering of xanthophyll content in tomato fruits.

Ripe tomato fruits accumulate significant amounts of the linear carotene lycopene, but only trace amounts of xanthophylls (oxygenated carotenoids). We overexpressed the lycopene beta-cyclase (b-Lcy) and beta-carotene hydroxylase (b-Chy) genes under the control of the fruit-specific Pds promoter. Transgene and protein expression was followed through semi-quantitative reverse transcription-PCR, Western blotting, and enzyme assays. Fruits of the transformants showed a significant increase of beta-carotene, beta-cryptoxanthin and zeaxanthin. The carotenoid composition of leaves remained unaltered. The transgenes and the phenotype are inherited in a dominant Mendelian fashion. This is the first example of successful metabolic engineering of xanthophyll content in tomato fruits.

Arabidopsis↗
PubMed2006

Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin.

Lactate, one of the major waste products in mammalian cell culture, can inhibit cell growth and affect cellular metabolism at high concentrations. To reduce lactate formation, lactate dehydrogenase-A (LDH-A), an enzyme catalyzing the conversion of glucose-derived pyruvate to lactate, was down-regulated by an expression vector of small interfering RNAs (siRNA) in recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Three clones expressing low levels of LDH-A, determined by reverse transcription-PCR and an enzyme activity test, were established in addition to a negative control cell line. LDH-A activities in the three clones were decreased by 75-89%, compared with that of the control CHO cell line, demonstrating that the effect of siRNA is more significant than that of other traditional methods such as homologous recombination (30%) and antisense mRNA (29%). The specific glucose consumption rates of the three clones were reduced to 54-87% when compared to the control cell line. Similarly, the specific lactate production rates were reduced to 45-79% of the control cell line level. In addition, reduction of LDH-A did not impair either cell proliferation or hTPO productivity. Taken together, these results show that the lactate formation rate in rCHO cell culture can be efficiently reduced through the down-regulation of LDH via siRNA.

Animals↗
PubMed2003

Human arteries engineered in vitro.

There is a pressing need to develop methods to engineer small-calibre arteries for bypass surgery. We hypothesized that the rate-limiting step that has thwarted previous attempts to engineer such vessels from non-neonatal tissues is the limited proliferative capacity of smooth muscle cells (SMCs), which are the main cellular component of these vessels. Ectopic expression of the human telomerase reverse transcriptase subunit (hTERT) has been shown recently to extend the lifespan of certain human cells. We therefore introduced hTERT into human SMCs and found that the resulting cells proliferated far beyond their normal lifespan but retained characteristics of normal control SMCs. Importantly, using these non-neonatal SMCs, we were able to engineer mechanically robust human vessels, a crucial step towards creating arteries of clinical value for bypass surgery.

Aorta↗
PubMed2006

Chitinase 3-like-1 exacerbates intestinal inflammation by enhancing bacterial adhesion and invasion in colonic epithelial cells.

BACKGROUND & AIMS: Dysregulated host/microbial interactions appear to play a central role in the development of inflammatory bowel disease (IBD). However, molecular events leading to the dysregulation have not yet been defined fully. Studies were designed to characterize a key molecule that is involved in the dysregulation. METHODS: Colonic mucosal RNA from C57BL/6 mice on days 4 and 8 with administration of 4% dextran sulfate sodium for 5 days were subjected to DNA microarray analysis. Chitinase 3-like-1 (CHI3L1) messenger RNA and protein expressions were examined by reverse-transcription polymerase chain reaction and immunohistochemistry. A gentamicin protection assay of Salmonella typhimurium was performed using epithelial cell lines that are engineered genetically to overexpress or lack mouse CHI3L1. To examine the functional role of CHI3L1 in vivo, anti-CHI3L1 antibody was administered into the dextran sulfate sodium colitis model. RESULTS: Microarray analysis identified that CHI3L1 is up-regulated specifically in inflamed mucosa. The expression of CHI3L1 protein clearly was detectable in lamina propria and colonic epithelial cells (CECs) in several murine colitis models and ulcerative colitis and Crohn's disease patients but absent in normal controls. The gentamicin protection assays using intracellular bacteria showed that CHI3L1 is required for the enhancement of adhesion and internalization of these bacteria in CEC. In vivo neutralization experiments showed that CHI3L1 contributes to the facilitation of bacterial invasion into the intestinal mucosa and the development of acute colitis. CONCLUSIONS: CHI3L1 plays a pathogenic role in colitis, presumably by enhancing the adhesion and invasion of bacteria on/into CEC. Inhibition of CHI3L1 activity would be a novel therapeutic approach for IBD.

Animals↗
PubMed1999

Control of erythropoietin secretion by doxycycline or mifepristone in mice bearing polymer-encapsulated engineered cells.

Cell encapsulation offers a safe and manufacturable method for the systemic delivery of therapeutic proteins from genetically engineered cells. However, control of dose delivery remains a major issue with regard to clinical application. We generated populations of immortalized murine NIH 3T3 fibroblasts that secrete mouse erythropoietin (Epo) in response to stimulation by doxycycline or mifepristone. Engineered cells were introduced into AN69 hollow fibers, which were implanted in the peritoneal cavity or recipient mice. Animals receiving doxycycline or mifepristone showed stable polyhemia and increased serum Epo concentrations over a 6-month observation period, whereas animals not receiving the inducer drug had normal hematocrits. Epo secretion could be switched on and off, depending on the presence of doxycycline in the drinking water. In contrast, polyhemia was hardly reversible after subcutaneous injections of mifepristone. These data show that a permanent and regulated systemic delivery of a therapeutic protein can be obtained by the in vivo implantation of engineered allogeneic cells immunoprotected in membrane polymers.

3T3 Cells↗