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PubMed2006

Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies.

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells.

Algorithms↗

PubMed1991

Expression of v-src in embryonic neural retina alters cell adhesion, inhibits histogenesis, and prevents induction of glutamine synthetase.

Using Rous sarcoma virus as the vector, v-src or c-src genes were introduced into 6-day chicken embryo retina tissue in organ culture and their effects on retina development were investigated. Overexpression of c-src in many of the cells had no noticeable effect on retina development. In contrast, infection with v-src resulted in abnormal histogenesis and inhibition of differentiation. Although only a portion of the cells in infected tissue expressed the oncogene and displayed the transformation phenotype, the other cells were also hindered from becoming normally positioned and organized. Therefore, presence of oncogene-transformed cells within the tissue hindered organization and development of adjacent nontransformed cells. Failure of normal cell relationships impeded induction by cortisol of glutamine synthetase in Muller glia, which requires contact associations of the glia cells with neurons. The transformed cells tended to assemble into chaotic clusters, suggesting that their adhesiveness and contact affinities had become altered. This was confirmed by aggregation experiments with dissociated cells which showed that adhesiveness of transformed cells was greatly reduced and that they had lost the ability to cohere with nontransformed cells. In binary mixtures of transformed and nontransformed cells, the two sorted out into separate aggregates. Transformed cells formed loose clusters devoid of tissue architecture; aggregates of nontransformed cells became organized into retinotypic structures, and glutamine synthetase was inducible. Our findings suggest that the mechanisms of cell adhesion and cell affinities are a key target of v-src activity in infected cells and that modification of the cell surface may be a leading factor in other cellular changes characteristic of the v-src transformation phenotype.

Animals↗

PubMed2001

The constraint based decomposition (CBD) training architecture.

The Constraint Based Decomposition (CBD) is a constructive neural network technique that builds a three or four layer network, has guaranteed convergence and can deal with binary, n-ary, class labeled and real-value problems. CBD is shown to be able to solve complicated problems in a simple, fast and reliable manner. The technique is further enhanced by two modifications (locking detection and redundancy elimination) which address the training speed and the efficiency of the internal representation built by the network. The redundancy elimination aims at building more compact architectures while the locking detection aims at improving the training speed. The computational cost of the redundancy elimination is negligible and this enhancement can be used for any problem. However, the computational cost of the locking detection is exponential in the number of dimensions and should only be used in low dimensional spaces. The experimental results show the performance of the algorithm presented in a series of classical benchmark problems including the 2-spiral problem and the Iris, Wine, Glass, Lenses, Ionosphere, Lung cancer, Pima Indians, Bupa, TicTacToe, Balance and Zoo data sets from the UCI machine learning repository. CBD's generalization accuracy is compared with that of C4.5, C4.5 with rules, incremental decision trees, oblique classifiers, linear machine decision trees, CN2, learning vector quantization (LVQ), backpropagation, nearest neighbor, Q* and radial basis functions (RBFs). CBD provides the second best average accuracy on the problems tested as well as the best reliability (the lowest standard deviation).

Algorithms↗

PubMed1987

Morphology and development of Cowdria ruminantium in Amblyomma ticks.

The morphology and development of Cowdria ruminantium have been studied in Amblyomma hebraeum and A. variegatum. Colonies of C. ruminantium have so far been demonstrated microscopically in gut, salivary gland cells, haemocytes and malphighian tubules of infected Amblyomma ticks. Colonies in gut cells were seen in both unfed and feeding ticks but colonies in salivary gland acini were observed only in nymphs that had fed for 4 days. Although the predominant type seen in both tick stages was the reticulated form that appeared to divide by binary fission, electron dense forms were also present. The latter are similar to those forms documented in endothelial cells of the vertebrate host as well as in cell culture. The presence of colonies of C. ruminantium in salivary glands of feeding ticks, along with the demonstration of different morphologic forms of the organism, suggests that a developmental cycle of the organism occurs in its invertebrate host. It is thought that organisms first infect and develop within gut cells. From there subsequent stages continue their development in haemolymph and salivary glands and are then transferred to the vertebrate host during tick feeding. Further studies are needed to completely understand the development of C. ruminantium in ticks and its subsequent transmission by these parasites.

Animals↗

PubMed1997

Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control.

Animals↗

PubMed2005

Precocious metamorphosis in transgenic silkworms overexpressing juvenile hormone esterase.

Insect growth and development are intricately regulated by the titers of juvenile hormones (JHs) and ecdysteroids (and/or their metabolites) in the insect hemolymph. Hydrolysis of the methyl ester of JH by a JH-specific esterase (JHE) is a key pathway for the degradation of JH. Here, we generate transgenic silkworm strains that overexpress JHE by using the binary GAL4/UAS system. Overexpression of JHE from the embryonic stage resulted in larval-pupal metamorphosis after the third stadium, two stadia earlier than that observed in wild-type insects. This precocious metamorphosis suggests that JHs are not critical for normal development of embryo or larva before the second molt in Lepidoptera (moths and butterflies). Our transgenic approach allowed us to dissect the function of key physiological events that occur from embryogenesis. Until now, these types of studies were possible only in later larval stadia by using physical techniques such as allatectomy or the application of JH analogues. We believe that our system will allow further pioneering studies in insect physiology.

Animals↗

PubMedDate not supplied

Ties in proximity and clustering compounds.

Hierarchical clustering algorithms such as Wards or complete-link are commonly used in compound selection and diversity analysis. Many such applications utilize binary representations of chemical structures, such as MACCS keys or Daylight fingerprints, and dissimilarity measures, such as the Euclidean or the Soergel measure. However, hierarchical clustering algorithms can generate ambiguous results owing to what is known in the cluster analysis literature as the ties in proximity problem, i.e., compounds or clusters of compounds that are equidistant from a compound or cluster in a given collection. Ambiguous ties can occur when clustering only a few hundred compounds, and the larger the number of compounds to be clustered, the greater the chance for significant ambiguity. Namely, as the number of "ties in proximity" increases relative to the total number of proximities, the possibility of ambiguity also increases. To ensure that there are no ambiguous ties, we show by a probabilistic argument that the number of compounds needs to be less than 2(n 1/4), where n is the total number of proximities, and the measure used to generate the proximities creates a uniform distribution without statistically preferred values. The common measures do not produce uniformly distributed proximities, but rather statistically preferred values that tend to increase the number of ties in proximity. Hence, the number of possible proximities and the distribution of statistically preferred values of a similarity measure, given a bit vector representation of a specific length, are directly related to the number of ties in proximities for a given data set. We explore the ties in proximity problem, using a number of chemical collections with varying degrees of diversity, given several common similarity measures and clustering algorithms. Our results are consistent with our probabilistic argument and show that this problem is significant for relatively small compound sets.

Journal Article↗

PubMed2006

Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression.

Selective genetic manipulation of neuronal function in vivo requires techniques for targeting gene expression to specific cells. Existing systems accomplish this using the promoters of endogenous genes to drive expression of transgenes directly in cells of interest or, in "binary" systems, to drive expression of a transcription factor or recombinase that subsequently activates the expression of other transgenes. All such techniques are constrained by the limited specificity of the available promoters. We introduce here a combinatorial system in which the DNA-binding (DBD) and transcription-activation (AD) domains of a transcription factor are independently targeted using two different promoters. The domains heterodimerize to become transcriptionally competent and thus drive transgene expression only at the intersection of the expression patterns of the two promoters. We use this system to dissect a neuronal network in Drosophila by selectively targeting expression of the cell death gene reaper to subsets of neurons within the network.

Animals↗

PubMed1999

Crystal engineering using bisphenols: interwoven ladders, sheet and framework structures in the binary adducts of 4,4'-sulfonyldiphenol with pyrazine (2/1), 4,4'-bipyridyl (1/1), trans-1,2-bis(4-pyridyl)ethene (1/1), 1,2-bis(4-pyridyl)ethane (1/1) and 4,4'-trimethylenedipyridine (1/1), and in 4,4'-sulfonyldiphenol-4,4'-trimethylenedipiperidine-water (2/2/1).

The structures of six hydrogen-bonded adducts of 4,4'-sulfonyldiphenol with heteroaromatic amines have been determined. In 4,4'-sulfonyldiphenol-pyrazine (2/1) the pyrazine molecules lie across centres of inversion. The bisphenol molecules are linked into C(8) chains parallel to [100] by means of O-H.O=S hydrogen bonds, and antiparallel pairs of these chains are cross-linked by the pyrazine molecules, via O-H.N hydrogen bonds, to form molecular ladders containing R(6)(6)(50) rings between the rungs of the ladders. Each ladder is interwoven with two neighbouring ladders, thus producing a continuous two-dimensional sheet. The structure of 4,4'-sulfonyldiphenol-4,4'-bipyridyl (1/1) consists of spiral C(2)(2)(21) chains parallel to [010] containing alternating bisphenol and bipyridyl molecules linked by O-H.N hydrogen bonds: these chains are linked by two types of C-H.O hydrogen bonds which form C(5) chains along [001] and C(2)(2)(10) chains along [101], thus generating two interconnected nets characterized in the one case by a chequerboard pattern of R(6)(6)(44) and R(6)(6)(52) rings, and in the other by a single type of R(6)(6)(46) ring. 4,4'-Sulfonyldiphenol-trans-1,2-bis(4-pyridyl)ethene (1/1) [systematic name: 4,4'-sulfonyldiphenol-trans-4,4'-vinylenedipyridine (1/1)] and 4,4'-sulfonyldiphenol-1,2-bis(4-pyridyl)ethane (1/1) [systematic name: 4,4'-sulfonyldiphenol-trans-4,4'-ethylenedipyridine (1/1)] are isomorphous: the 1,2-bis(4-pyridyl)ethene component exhibits orientational disorder, corresponding approximately to a 180 degrees rotation of ca 23% of the molecules about the N.N vector; in each compound the structure is built from C(2)(2)(23) chains of alternating bisphenol and bis(pyridyl) molecules connected by O-H.N hydrogen bonds, running parallel to [112] and generated by translation. The [112] chains are linked by C-H.O hydrogen bonds which generate C(2)(2)(12) chains parallel to [101], so forming a two-dimensional net built from R(6)(6)(50) rings. The structure of 4,4'-sulfonyldiphenol-4,4'-trimethylenedipyridine (1/1) consists of C(2)(2)(24) chains parallel to [100] generated by translation and consisting of alternating bisphenol and bis(pyridyl) molecules linked by O-H.N hydrogen bonds. Pairs of such chains are coiled together to form double helices, and pairs of such double helices, of opposite hand, are linked together by paired C-H.O hydrogen bonds in R(2)(2)(10) rings to form pairs of interwoven ladders in which the C(2)(2)(24) chains form the uprights and the R(2)(2)(10) rings form the rungs, between which are R(6)(6)(50) rings: an R(2)(2)(10) ring belonging to one ladder lies at the centre of an R(6)(6)(50) ring belonging to the other. 4,4'-Sulfonyldiphenol-4,4'-trimethylenedipiperidine-water (2/2/1) is a salt, 2C(13)H(27)N(2)(+).2C(12)H(9)O(4)S(-).H(2)O, containing two independent singly protonated diamine cations, two independent bisphenolate anions, and neutral water molecules. The two independent diamine cations are linked by N-H.N hydrogen bonds into C(2)(2)(24) chains running parallel to [001] and generated by translation, and each type of bisphenolate anion forms an independent spiral C(12) chain, also parallel to [001]. The three types of chain are linked by the water molecules: the two types of bisphenolate chain are linked by water molecules acting as double donors in O-H.O(-) hydrogen bonds in a C(6)(4)(32) chain parallel to [100], thus generating a two-dimensional net built from R(8)(6)(56) rings; the diamine chains are linked to these nets by means of N-H.O hydrogen bonds in which the water molecules act as acceptors and further hydrogen bonds, of N-H.O(-) and N-H.O=S types, link these two-dimensional nets into a continuous three-dimensional framework.

Journal Article↗

PubMed2004

Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity.

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.

Cell Line↗

PubMed1991

Requirements for cell surface expression of the human TCR/CD3 complex in non-T cells.

The T-cell antigen receptor (TCR) consists of a glycoprotein heterodimer (alpha/beta or gamma/delta) which is non-covalently associated with at least four or five invariant polypeptides (CD3 gamma, delta, epsilon, zeta and eta). In T-cell variants lacking TCR alpha, beta or zeta, it has been shown that incomplete TCR/CD3 complexes are retained within the cell. To examine requirements for cell surface expression of TCR/CD3, we transfected COS monkey kidney cells with cDNAs encoding TCR alpha, beta and CD3 gamma, delta, epsilon and zeta. We report that cell surface appearance of TCR/CD3 on COS cells requires coordinate expression of all six proteins. In the absence of the zeta chain, subcomplexes comprising from two to five chains were readily demonstrable in COS cells, but they failed to reach the cell surface or to acquire N-linked oligosaccharide side chains indicating failure to reach the medial Golgi. Pulse-chase metabolic labelling of transfected COS cells showed that three chains (CD3 gamma, CD3 epsilon, and zeta) were stable while three (TCR alpha, TCR beta and CD3 delta) were rapidly degraded. In two- and three-chain co-transfections specific intracellular subcomplexes were formed between TCR alpha and CD3 gamma, TCR alpha and CD3 delta, or TCR beta and CD3 epsilon. Binary subcomplexes having at least one stable chain (CD3 epsilon - TCR beta) were stable while one formed by two unstable chains (TCR alpha - CD3 delta) was still degraded. Assembly of the TCR/CD3 complex in COS cells thus appears centered around the metabolically stable CD3 gamma and CD3 epsilon proteins. Site-specific mutations of the negatively-charged transmembrane amino acid of residues of the CD3 chains to alanines served to either abolish (for TCR alpha - CD3 delta and TCR beta - CD3 epsilon) or diminish (for TCR alpha -CD3 gamma) these TCR-CD3 interactions. These mutations had no effect, however, on CD3-CD3 interactions or upon synthesis, metabolism, or intracellular distributions of the CD3 proteins. The transmembrane domains of CD3 gamma, delta, and epsilon thus appear to play a major role in associations of CD3 with TCR chains.

Animals↗

PubMed1994

Creation and phenotypic analysis of alpha-lactalbumin-deficient mice.

alpha-Lactalbumin is an abundant milk-specific calcium metalloprotein which has an evolutionary relationship to lysozyme. It modifies the substrate specificity of a Golgi galactosyltransferase by forming the lactose synthetase binary complex. Lactose, together with other sugars and diffusible ions, is responsible for the osmotic pressure of milk. To assess the involvement of alpha-lactalbumin in lactogenesis, alpha-lactalbumin-deficient mice were created by disrupting the gene by homologous recombination in embryonic stem cells. Homozygous mutant mice are viable and fertile but females cannot feed their offspring. They produce a highly viscous milk that pups appear to be unable to remove from the mammary gland. This milk is rich in fat and protein and is devoid of alpha-lactalbumin and lactose. The phenotype of heterozygous mice was found to be intermediate, with a 40% decrease in alpha-lactalbumin but only a 10-20% decrease in the lactose content of their milk compared with wild-type animals. These results emphasize the key function of alpha-lactalbumin in lactogenesis and open new opportunities to manipulate milk composition.

Animals↗

PubMed1993

Avian 3-hydroxy-3-methylglutaryl-CoA synthase. Characterization of a recombinant cholesterogenic isozyme and demonstration of the requirement for a sulfhydryl functionality in formation of the acetyl-enzyme reaction intermediate.

cDNA encoding avian liver hydroxymethylglutaryl-CoA synthase has been cloned into a pET vector, and the resulting expression plasmid has been used to transform Escherichia coli BL21 (DE3). Heterologous expression of hydroxymethylglutaryl-CoA synthase occurs upon growth of this bacterial strain in the presence of isopropyl-1-thio-beta-D-galactopyranoside, with the target enzyme representing over 20% of total cellular protein. Recombinant enzyme is soluble and stable in crude E. coli extracts, facilitating its isolation in homogeneous form. With respect to specific activity, acylation stoichiometry, Km,Ac-CoA, and binding of a spin-labeled substrate analog, the recombinant enzyme is equivalent to avian enzyme, suggesting its utility for mechanistic and structural studies. Our earlier prediction that this avian cDNA encodes the cholesterogenic cytosolic isozyme is supported by a series of experimental observations. Upon SDS-polyacrylamide gel electrophoresis, the recombinant synthase exhibits mobility in agreement with the 57.6-kDa deduced molecular mass, which exceeds the 53-kDa estimate and experimental observation for the ketogenic mitochondrial isozyme. Activity of the recombinant synthase is stimulated by Mg2+, as predicted for the cholesterogenic cytosolic isozyme and in contrast to the inhibition observed for the mitochondrial isozyme. Although antibody prepared against avian mitochondrial synthase effectively detects both avian mitochondrial and recombinant synthases on Western blots, antibody prepared against rodent cytosolic synthase discriminates between the two proteins, sensitively detecting recombinant enzyme while reacting poorly with authentic mitochondrial enzyme. Directed mutagenesis of the recombinant synthase has been performed to produce a C129S variant, in which the sulfhydryl previously implicated in formation of the acetyl-S-enzyme reaction intermediate is replaced by a hydroxyl group. EPR measurements on the binary C129S-spin-labeled acyl-CoA complex demonstrate that the mutant's substrate binding site is unperturbed in comparison with wild-type protein. These data illustrate the utility of spin-labeled substrate analogs as tools to stringently evaluate the structural integrity of engineered proteins. C129S is catalytically inactive (10(5)-fold decrease in kcat) despite retaining the ability to form noncovalent complexes with acetyl-CoA or a spin-labeled acetyl-CoA analog. The demonstrated failure of C129S to form a covalent acyl-O-enzyme species accounts for these observations; data derived from experiments performed with a C129G mutant confirm this conclusion. These results distinguish hydroxymethylglutaryl-CoA synthase from beta-ketoacyl thiolase.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyl Coenzyme A↗

PubMed2005

Generation of variability by in vivo recombination of halves of a (beta/alpha)8 barrel protein.

Similar to what has been achieved with nucleic acids, directed evolution of proteins would be greatly facilitated by the availability of large libraries and efficient selection methods. So far, host cell transformation efficiency has been a bottleneck, practically limiting libraries to sizes less than 10(9). One way to circumvent this problem has been implemented with antibody systems, where contribution to the binding site is provided by two different polypeptides (light and heavy chains). The central concept is the construction of binary systems in which the gene from the two chains are separated by a cre-lox recombinase recognition site, packaged in a phage, and subsequently introduced, by multiple infection, into a recombinase expressing cell [Sblattero D, Bradbury A. Nat Biotechnol 2000;18(1):75-80]. Here, we describe the development of a system which applies the same concept to a single-domain enzyme, the cytoplasmic (beta/alpha)8 barrel protein phosphoribosyl anthranilate isomerase (PRAI) from E. coli. For that purpose, we identified the site at which a loop containing the recognition sequence for cre-lox recombinase could be inserted yielding a functional enzyme. We evaluated the effect of this insertion on the capability of the engineered gene to complement a trp F-E. coli strain and the efficiency of the system to recover the original sequence from an abundance of non-functional mutant genes.

Aldose-Ketose Isomerases↗

PubMed2002

A reinvestigation of brindley's gland exocrine compounds of Rhodnius prolixus (Hemiptera: Reduviidae).

A chemical, electrophysiological and behavioral study was carried out to analyze the volatile compounds of the Brindley's gland of Rhodnius prolixus Stål. Six compounds were found in the Brindley's gland of males and females: acetic, isobutyric, caproic acids and three compounds not identified. The mass spectra of the unidentified compounds have the appearance of a propionate ester, a butyrate ester and a valerate ester. There was no difference in the concentration of these compounds in the glands of males and females. The major component was isobutyric acid. The electroantennographic (EAG) evaluation of isobutyric, acetic, and caproic acids elicited weak responses. With both sexes, the EAG responses for the acids were no different from the control response at any of the doses evaluated (1, 10, 100, and 500 microg). The behavioral response of males and females to acetic, isobutyric, and caproic acids at 1, 5, and 10 microg and binary and tertiary mixtures of the three compounds was evaluated in a Y-olfactometer. Males showed preference for acetic acid at a dose of 1 microg, but not at 5 or 10 microg. Females showed preference for this compound at the dose of 5 microg. Males showed preference for isobutyric acid at 1 and 5 microg, but not at 10 microg. Females did not show any preference for isobutyric acid. Males were attracted to caproic acid at the dose of 1 and 10 microg. Females were attracted to the caproic acid. Males, but not females, preferred the mixture of acetic and isobutyric acids over control. Neither males nor females showed preference for the mixture of acetic and caproic acids or hexane control. Females, but not males, were attracted to the mixture of the isobutyric and caproic acids. Only males showed a positive response for the tertiary mixture of the acids.

Acetates↗

PubMed1991

Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements.

We have developed a binary transgenic system that activates an otherwise silent transgene in the progeny of a simple genetic cross. The system consists of two types of transgenic mouse strains, targets and transactivators. A target strain bears a transgene controlled by yeast regulatory sequences (UAS) that respond only to the yeast transcriptional activator GAL4. A transactivator strain expresses an active GAL4 gene that can be driven by any selected promoter. The current paradigm uses the murine growth factor int-2 cDNA as the target gene and the GAL4 gene driven by the mouse mammary tumor virus long terminal repeat as the transactivator. Both target and transactivator strains are phenotypically normal. By contrast, the bigenic offspring of these two strains express high levels of the target int-2 gene in each organ expressing the GAL4 transactivator. They also display a characteristic dominant int-2 phenotype that consists of epithelial hyperplasia in mammary and salivary glands, as well as prostatic and epididymal hypertrophy, which results in male sterility.

Animals↗

PubMed2004

GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells.

The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes.

Adenoviridae↗

PubMed2004

The binary interacting network of the conserved oligomeric Golgi tethering complex.

Several recent studies have revealed the existence of a conserved oligomeric Golgi (COG) complex consisting of several novel proteins as well as known Golgi proteins that were identified by independent approaches. The mammalian COG complex contains eight subunits: COG1/LdlBp, COG2/LdlCp, COG3/Sec34, COG4/Cod1, COG5/GTC-90/Cod4, COG6/Cod2, COG7, and COG8/Dor1. COG1, COG2, and COG7 seem structurally unique to mammalian cells, whereas the other five subunits are structurally conserved in yeast, which also contains three other unique proteins (COG1/Sec36p/Cod3p, COG2/Sec35p, and COG7/Cod5p). We report here the network of intermolecular interactions of the COG complex, revealed by in vitro translation and co-immunoprecipitation approaches. Our results suggest that COG4 serves as a core component of the complex by interacting directly with COG1, COG2, COG5, and COG7. COG3 is incorporated by its direct interaction with COG1 and COG2, whereas COG6 and COG8 do not interact with any individual subunit. Incorporation of COG6 into the complex depends on the concerted interaction of both COG5 and COG7, whereas optimal incorporation of COG8 depends on the concerted interaction of COG5, COG6, and COG7. Because COG4 (together with COG1, COG2, and COG3) is among the four essential genes of the COG complex in yeast, this molecular network highlights the structural basis for a crucial role of COG4 in the assembly/function of the complex. A model for the assembly of the COG complex is presented.

Animals↗