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PubMed2006

Reciprocal leaf and root expression of AtAmt1.1 and root architectural changes in response to nitrogen starvation.

Nitrogen is an essential macronutrient for plant growth and survival. Here, the temporal and spatial sensing of nitrogen starvation is analyzed in Arabidopsis (Arabidopsis thaliana). The promoter for the high-affinity ammonium transporter, AtAmt1.1, is shown to be a valid indicator for nitrogen status in leaves and roots. An AtAmt1.1-Gal4 transgene using three 5x upstream activating sequence-driven reporters (luciferase, green fluorescent protein, and beta-glucuronidase) facilitated in vivo profiling at the whole-plant and cellular levels. The effects of nitrogen supply, light duration, light intensity, and carbon on the expression of the AtAmt1.1 gene in the roots and aerial tissues are reported. Under nitrogen starvation, high expression is observed in the roots and, under nitrogen-sufficient conditions, high expression is observed in the leaves. This reciprocal regulation of AtAmt1.1 was confirmed by quantitative reverse transcription-polymerase chain reaction, which was also used to quantitate expression of the five other Amt genes in Arabidopsis. Although some of these show tissue specificity (roots or leaves), none exhibit reciprocal regulation like the AtAmt1.1-encoded high-affinity transporter. This robust reciprocal expression suggests that Arabidopsis undergoes rapid resource reallocation in plants grown under different nitrogen supply regimens. Ultimately, nitrogen starvation-mediated reallocation results in root architectural restructuring. We describe the precise timing and cellular aspects of this nitrogen limitation response.

Arabidopsis↗
PubMed1994

Peptide mapping of subtilisins as a practical tool for locating protein sequence errors during extensive protein engineering projects.

Protein engineering based on rational design is an iterative process of sequential amino acid residue replacements. This requires a rapid and sensitive method for checking the protein sequence after each round of mutagenesis. As shown with subtilisin BL, acid treatment followed by urea denaturation renders the enzyme degradable by trypsin within 10 min. Separation of the peptides by reversed-phase HPLC produces a map that differentiates even the most conservative alteration on peptides as large as 48 amino acid residues. The method was used to uncover erroneous mutations; to determine the concentration of active protease relative to an internal standard of known specific activity; to measure the rate of oxidation of methionine-216 in the oxyanion hole of subtilisin BL; and to document that under these conditions no other methionine in the molecule is oxidized by hydrogen peroxide.

Amino Acid Sequence↗
PubMed2003

Genetic engineering of an allosterically based glucose indicator protein for continuous glucose monitoring by fluorescence resonance energy transfer.

Real-time monitoring of blood glucose could vastly reduce a number of the long-term complications associated with diabetes. In this article, we present a novel approach that relies on a glucose-binding protein engineered such that a 20% reduction in fluorescence due to the fluorescence resonance energy transfer occurs as a result of glucose binding. This change in fluorescence provides a signal for the optical detection of glucose. The novel glucose indicator protein (GIP) was created by fusing two fluorescent reporter proteins (green fluorescent proteins) to each end of an Escherichia coli glucose-binding protein in such a manner that the spatial separation between the fluorescent moieties changes when glucose binds, thus generating a distinct optical signal that can be used for glucose detection. By placing the GIP within a dialysis hollow fiber sensor, a microsensor has been developed for continuous monitoring of glucose. The sensor had a response time to sudden glucose changes within 100 s and was reversible. The sensor was shown to have an optional range on the order of 10 microM of glucose.

Amino Acid Sequence↗
PubMed2005

Detection of rampant nucleotide reversion at the origin of DNA replication of porcine circovirus type 1.

Mutational analysis was conducted to investigate the involvement of the "loop-sequence" (which is flanked by a pair of 11-nucleotide inverted repeats) at the origin of DNA replication of porcine circovirus type 1 with respect to viral protein synthesis, DNA self-replication and progeny virus production. The results demonstrated that an octanucleotide (A1G2T3A4T5T6A7C8) embedded in the loop is essential for viral DNA replication. Similar to previous work with porcine circovirus type 2, this octanucleotide can be further condensed to an essential core element represented by AxTAxTAC. After transfection, mutations introduced into the positions indicated by x (positions 2 and 5) were retained in the progeny viruses, while mutations engineered into the positions specified by the indicated nucleotides either did not yield any progeny virus (positions 6, 7, and 8) or they reverted back to wild-type nucleotide to generate infectious progeny viruses (positions 1, 3, and 4). In comparison to porcine circovirus type 2, porcine circovirus type 1 mutant genomes with perturbed octanucleotide sequences exhibited higher propensity to revert to wild-type under similar experimental conditions. The rate and frequency at which some of the nucleotide reversions occurred suggest that base complementarity may not be the governing factor for nucleotide incorporation at the porcine circovirus origin of DNA replication, and that the Rep-associated protein(s) may play a critical role in this process.

Animals↗
PubMed2001

The use of GABA(A) receptors expressed in neural precursor cells for cell-based assays.

GABA(A) receptors are known targets for certain classes of environmental neurotoxins and pharmaceutical compounds. Since few neural cell lines express functional GABA(A) receptors, the capacity to rapidly screen for compounds that affect GABA(A) receptor function is presently limited. Previous work has demonstrated that rat neural precursor cells express functional GABA(A) receptors that can be monitored via Ca(2+) imaging. This study examined GABA(A) receptor subunit expression to determine whether GABA(A) receptor function and its interactions with neurotoxins is preserved after passaging. Neural precursor cells isolated from embryonic day 13 rat brain were expanded in serum-free medium containing basic fibroblast growth factor and passaged three times. Reverse transcription-polymerase chain reaction analysis demonstrated early expression of abundant mRNAs encoding various GABA(A) receptor subunits. Ca(2+) imaging showed that the highly proliferating precursor cells in passaged cultures maintained expression of functional GABA(A) receptors. In addition, we showed that trimethylolpropane phosphate, a neurotoxin generated during partial pyrolysis of a synthetic ester turbine engine lubricant, potently inhibited muscimol (GABA(A) receptor agonist) but not depolarization-induced cytosolic Ca(2+) increase. The findings of this study suggest that neural precursor cells may be well suited for the evaluation of certain environmental neurotoxins with convulsant activity. The potential use of neural precursor cells in high-throughput screens for compounds acting on GABA(A) receptors is discussed.

Animals↗
PubMed1992

Behavior of large nucleic acids in reversed-phase high-performance liquid chromatography.

Large nucleic acids can be separated by reversed-phase high-performance liquid chromatography. Analysis shows that the retention time depends not only on the chain length but also on the base composition and the secondary structure of the molecule. A model is proposed to interpret their behavior. This model, called "multiple-point interaction theory" is based on the observation that macromolecules are flexible and very large compared to the hydrophobic phase (octadecylsilane) of the column. It explains the behavior of large nucleic acids in terms of an equilibrium of the macromolecule between the two phases through a multiple-point attachment to the chromatographic matrix, the parameters of the equilibrium being both the hydrophobicity of the base and the number of attachment points. This model fits the experimental data and can be applied to all types of flexible macromolecules, especially proteins and nucleic acids, when they are chromatographed on reversed-phase columns. The model is used to explain the separation of nucleic acids of importance in molecular biology.

Chromatography, High Pressure Liquid↗
PubMed1989

Isolation of recombinant hirudin by preparative high-performance liquid chromatography.

The purification of recombinant hirudin variant 2-Lys47 (rHV2-Lys47), produced by a genetically engineered yeast strain, is described. rHV2-Lys47 expressed and secreted into the culture medium was the starting material for the purification process of hirudin from the culture broth after cell harvesting by centrifugation. Initial purification of the product by preparative reversed-phase high-performance liquid chromatography (HPLC) using step-gradient elution, followed by precipitation of rHV2-Lys47 in the presence of acetone, removed most of the contaminants from the culture medium. The pure product was obtained by successive preparative anion-exchange and reversed-phase HPLC on silica based stationary phases. Characterization of the final product by analytical HPLC, isoelectric focusing gel electrophoresis, quantitative amino acid composition and sequence analysis did not reveal any contaminants. Liquid secondary ion mass spectrometry was used to confirm its primary structure. The isolated product was tested in an inhibition assay of human alpha-thrombin and proved to be fully active.

Chromatography, High Pressure Liquid↗
PubMed2004

Trans-splicing repair of CD40 ligand deficiency results in naturally regulated correction of a mouse model of hyper-IgM X-linked immunodeficiency.

X-linked immunodeficiency with hyper-IgM (HIGM1), characterized by failure of immunoglobulin isotype switching, is caused by mutations of the CD40 ligand (CD40L), which is normally expressed on activated CD4(+) T cells. As constitutive expression of CD40L induces lymphomas, we corrected the mutation while preserving the natural regulation of CD40L using pre-mRNA trans-splicing. Bone marrow from mice lacking CD40L was modified with a lentivirus trans-splicer encoding the normal CD40L exons 2-5 and was administered to syngenic CD40L-knockout mice. Recipient mice had corrected CD40L mRNA, antigen-specific IgG1 responses to keyhole limpet hemocyanin immunization, regulated CD4(+) T-cell CD40L expression after CD3 stimulation in primary and secondary transplanted mice, attenuation of Pneumocystis carinii pneumonia, and no evidence of lymphoproliferative disease over 1 year. Thus, HIGM1 can be corrected by CD40L trans-splicing, leading to functional correction of the genetic defect without the adverse consequences of unregulated expression of the CD40L gene.

Animals↗
PubMed2005

Genetic engineering of human embryonic stem cells with lentiviral vectors.

Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

Cell Differentiation↗
PubMed1983

Improved methods for structure probing in large RNAs: a rapid 'heterologous' sequencing approach is coupled to the direct mapping of nuclease accessible sites. Application to the 5' terminal domain of eukaryotic 28S rRNA.

We have developed a combined approach for probing native structures in large RNAs. In the first method, after digestion with a structure specific nuclease, accessible sites are mapped at sequence resolution along the entire RNA molecule which is used as a template for the reverse transcriptase elongation of a 5' end labelled selected primer (coding strand of a small restriction fragment of the cloned gene). This method circumvents any prior end-labelling of RNA, a technique with major limitations for large RNAs. In the second approach, a rapid "heterologous" sequencing can be easily applied to definite domains of an RNA molecule in a variety of species (or individuals), without additional DNA cloning nor end-labelling of RNA. By taking advantage of the presence of evolutionary conserved tracts within an RNA sequence, it allows a rapid analysis of RNA folding patterns in terms of phylogenetic comparisons : when located within such a conserved tract, selected restriction fragments from a cloned gene can be used as heterologous primers for sequencing the upstream divergent region in RNAs of other species by currently available technology, i.e. reverse transcriptase elongation in the presence of chain terminator dideoxynucleotides.

Animals↗
PubMed1999

An indicator cell assay for T-cell tropic, macrophage-tropic, and primary isolates of HIV-1 based on green fluorescent protein.

Quantitation of HIV-1 in blood is now widely used by clinicians to manage antiviral therapy. Current methods to detect viral RNA are expensive, have slow turnaround times, and do not directly quantitate infectious particles. Indicator cell assay (ICA) methods for titering HIV-1 rely on the activation of HIV-1 long terminal repeat (LTR)-driven expression of a reporter gene by the viral tat gene product, which is expressed early in the course of infection. The Aequorea victoriana green fluorescent protein (GFP) has proven to be a useful reporter gene for detecting tat-mediated HIV-LTR activation. A general approach to developing a clinically useful ICA required a method of introducing the LTR-GFP expression cassette into various HIV1-infectable cell lines. The LTR-GFP expression cassette was inserted into the LXSN retrovector in a reverse orientation with respect to transcription from the 5' LTR. In cells transduced by the RH5 retrovector, GFP expression was tightly dependent on expression of HIV-1 tat. The PM1 human T-cell line was transduced with RH5 and was further engineered to express the CCR5 HIV-1 CD4 coreceptor constitutively. The resulting cell line, D5-R5, was susceptible to infection by primary HIV-1 strains, macrophage-tropic (M-tropic) and T-cell tropic (T-tropic) laboratory strains, and syncytium-inducing (SI) and and non-SI (NSI) variants. Four days after HIV-1 infection of the indicator cells, GFP expression was detected and quantitated by fluorescence activated cell sorter (FACS), without any false-positive signals. This GFP-based ICA method is of potential use in clinical management of HIV-1, especially in the detection and recovery of drug-resistant virus and the direct determination of antiviral drug sensitivities.

Cell Line↗
PubMed2004

Evolution of the HIV-1 envelope glycoproteins with a disulfide bond between gp120 and gp41.

BACKGROUND: We previously described the construction of an HIV-1 envelope glycoprotein complex (Env) that is stabilized by an engineered intermolecular disulfide bond (SOS) between gp120 and gp41. The modified Env protein antigenically mimics the functional wild-type Env complex. Here, we explore the effects of the covalent gp120 - gp41 interaction on virus replication and evolution. RESULTS: An HIV-1 molecular clone containing the SOS Env gene was only minimally replication competent, suggesting that the engineered disulfide bond substantially impaired Env function. However, virus evolution occurred in cell culture infections, and it eventually always led to elimination of the intermolecular disulfide bond. In the course of these evolution studies, we identified additional and unusual second-site reversions within gp41. CONCLUSIONS: These evolution paths highlight residues that play an important role in the interaction between gp120 and gp41. Furthermore, our results suggest that a covalent gp120 - gp41 interaction is incompatible with HIV-1 Env function, probably because this impedes conformational changes that are necessary for fusion to occur, which may involve the complete dissociation of gp120 from gp41.

Amino Acid Substitution↗
PubMed2004

Regulation of glucose transporter expression in cardiac myocytes: p38 MAPK is a strong inducer of GLUT4.

OBJECTIVE: In vivo differentiation of cardiac myocytes is associated with downregulation of the glucose transporter isoform GLUT1 and upregulation of the isoform GLUT4. Adult rat cardiomyocytes in primary culture undergo spontaneous dedifferentiation, followed by spreading and partial redifferentiation, which can be influenced by growth factors. We used this model to study the signaling mechanisms modifying the expression of GLUT4 in cardiac myocytes. RESULTS: Adult rat cardiomyocytes in primary culture exhibited spontaneous upregulation of GLUT1 and downregulation of GLUT4, suggesting resumption of a fetal program of GLUT gene expression. Treatment with IGF-1 and, to a minor extent, FGF-2 resulted in restored expression of GLUT4 protein and mRNA. Activation of p38 MAPK mediated the increased expression of GLUT4 in response to IGF-1. Transient transfection experiments in neonatal cardiac myocytes confirmed that p38 MAPK could activate the glut4 promoter. Electrophoretic mobility shift assay in adult rat cardiomyocytes and transient transfection experiments in neonatal cardiac myocytes indicated that MEF2 was the main transcription factor transducing the effect of p38 MAPK activation on the glut4 promoter. CONCLUSION: Spontaneous dedifferentiation of adult rat cardiomyocytes in vitro is associated with downregulation of GLUT4, which can be reversed by treatment with IGF-1. The effect of IGF-1 is mediated by the p38 MAPK/MEF2 axis, which is a strong inducer of GLUT4 expression.

Animals↗
PubMed2003

Characterisation of Toxoplasma gondii engineered to express mouse interferon-gamma.

Recent studies have shown the feasibility of using Toxoplasma gondii as an expression system for heterologous protein. For better understanding of the mechanism of interferon-gamma (IFN-gamma) dependent immunity to T. gondii, the parasites were stably transfected with IFN-gamma gene, under control of the GRA1 promoter. Immunofluorescence analyses showed that recombinant mouse IFN-gamma localised to discrete punctuate structures consistent with dense granules and secreted into the vacuolar space. The production of IFN-gamma was detectable in both extracellular parasites and the parasite-infected cells. Growth of the recombinant parasites was inhibited in the mouse macrophage cell line (J774A.1 cells), but not in monkey kidney adherent fibroblasts (Vero cells), demonstrating the species-specificity of IFN-gamma. Addition of anti-mouse IFN-gamma antibody resulted in growth recovery of the recombinant parasites, suggesting that IFN-gamma, secreted from the parasitised host cells across the parasitophorous vacuole membrane, acted in a paracrine manner. Reverse transcription (RT)-PCR analysis revealed significant expression of inducible nitric oxide synthase mRNA and high levels of nitric oxide production in recombinant parasite-infected J774A.1 cells. A competitive inhibitor of the L-arginine-dependent effector pathway, N(G)-monomethyl-L-arginine, inhibited the reduction of recombinant parasite growth in J774A.1 cells. Taken together, our data suggest that the T. gondii expression system may provide a new tool for cytokine gene expression and that parasites engineered to express a cytokine gene may be rationally designed for use in studies on immune responses to T. gondii.

Animals↗
PubMed2003

Reversal of diabetes in the rat by injection of hematopoietic stem cells infected with recombinant adeno-associated virus containing the preproinsulin II gene.

AIM: To study the effect of injecting hematopoietic stem cells containing the preproinsulin gene II (rI2) via recombinant adeno-associated virus (rAAV) into normal and streptozotocin-diabetic rats. METHODS: rI2 was transfected into rat hematopoietic stem cells using rAAV vector. Stem cells were injected by intravenous route into normal and STZ-induced diabetic rats to study blood sugar and expression of rI2 in various tissues. The pLP-1 recombinant plasmid containing rI2 (vLP-1) was engineered as previously described. Bone marrow from female Wistar-Furth rats was enriched for stem cells by using plastic adherence and monoclonal antirat CD3 and CD45 RA to deplete T and B cells. The remaining cells were exposed to vLP-1 (multiplicity of infection MOI =50:1 or 100:1) for 2 h. Approximately ten million exposed stem cells were injected by intravenous route into each animal; there were four groups: normal animals at MOI 50:1 (group 1) or MOI 100:1 (group 2); group 3 animals (n = 9) were streptozotocin-induced diabetic animals at MOI 100:1. Animals that showed reversal of diabetes from group 3 were sacrificed for study of gene expression at weeks 1, 2, and 6, respectively. Control diabetic animals did not receive stem cells or virus constituted group 4. Expression of rI2 was analyzed by RT-PCR and Southern analyses. RESULTS: Despite introduction of insulin gene, groups 1 and 2 had blood sugar concentrations that remained within normal levels, while 3 of 9 animals in group 3 showed reversal of diabetes; using RT-PCR,group 1 expressed rI2 in liver, spleen, thymus, brain, and heart at week 1 only. In group 2, rI2 was seen in the thymus up to 6 weeks; in diabetic animals (group 3) rI2 was seen in liver, bone marrow, spleen, thymus, and peripheral blood lymphocytes at week 2 and in thymus and lymphocytes at week 6. CONCLUSIONS: We have shown that (1) rAAV is a useful vector for transferring rI2 into rat hematopoietic stem cells; (2) normal animals remained euglycemic after injection of stem cells containing rI2 despite identification in various tissues suggesting autoregulation, and (3) short-term reversal of diabetes was achieved in some animals by injection of stem cells containing rI2.

Animals↗
PubMed1998

Infection of ducklings with virus particles containing linear double-stranded duck hepatitis B virus DNA: illegitimate replication and reversion.

Double-stranded linear DNA is synthesized as a minor viral DNA species by all hepadnaviruses. In a previous study (W. Yang and J. Summers, J. Virol. 69:4029-4036, 1995) we showed that virus particles containing linear DNA of the duck hepatitis B virus (DHBV) could initiate an infection of primary duck hepatocytes. In cells infected by linear DNA containing viruses the transcriptional template, covalently closed circular DNA, was formed by circularization of linear DNA by nonhomologous recombination between the two ends. This process was shown to result in viral DNA replication through multiple generations of linear DNA intermediates, a process we called illegitimate replication. In this study we showed that viruses containing linear DHBV DNA produced by engineered insertions in the r sequence, which encodes the 5' end of the pregenome, could infect hepatocytes in vivo, and these hepatocytes proceeded to carry out illegitimate replication. Nonhomologous recombination quickly produced revertants and partial revertants in which all or part of the insertion was deleted. One such partial revertant that replicated primarily through circular DNA intermediates, but which synthesized elevated levels of linear DNA, could be sustained for several days as the predominant genotype in vivo, but this mutant was eventually displaced by variants showing full reversion to legitimate replication and that synthesized normal low levels of linear DNA. Full revertants did not necessarily contain the wild-type r sequence. The results suggest that the linear DNA produced during DHBV infection initiates cycles of illegitimate replication by generating mutants with altered r sequences. Some r sequence mutants carry out a mixture of legitimate and illegitimate replication that can contribute to elevated production of linear DNA in individual cells.

Animals↗
PubMed2006

Tissue-engineered human vascular media produced in vitro by the self-assembly approach present functional properties similar to those of their native blood vessels.

We have developed a tissue-engineering approach for the production of a completely biological blood vessel from cultured human cells. In the present study, we took advantage of this tissue-engineering method to demonstrate that it can be used to reproduce the subtle differences in the expression of receptors present on the media of native human blood vessels. Indeed, a small percentage (3 of 18) of native human umbilical cord veins (HUCVs) responded to endothelin, the most powerful vasopressor agent known to date, via both endothelin A (ET(A)) and endothelin B (ET(B)) receptor activation. In contrast, most HUCVs tested responded to ET via ET(A) receptor activation only. Tissue-engineered vascular media (TEVM) were next reconstructed by using vascular smooth muscle cells (VSMCs) isolated and cultured from HUCVs expressing both ET(A) and ET(B) receptors to determine the functional integrity of our TEVM model. The reconstructed TEVM presents an endothelin response similar to that of respective HUCVs from which VSMCs were isolated. Reverse transcriptase polymerase chain reaction on TEVM reconstructed in vitro correlated these vasocontractile profiles by showing the presence of messenger RNA for both ET(A) and ET(B) receptors. Taken together with recently published results on TEVM expressing only ET(A) receptor, these results show that our reconstructed TEVM present a similar ET response profile as the blood vessel from which the VSMCs were isolated and cultured. These findings indicate that subtle differences, such as receptor expression, are preserved in the reconstructed tissue. Therefore, our TEVM offers a valuable human in vitro model with which to study the functionality of human blood vessels, such as their vasoactive response, or to perform pharmacologic studies.

Blood Vessels↗
PubMed2005

Magnetic resonance imaging of mouse spinal cord.

The feasibility of performing high-resolution in vivo MRI on mouse spinal cord (SC) at 9.4 T magnetic field strength is demonstrated. The MR properties of the cord tissue were measured and the characteristics of water diffusion in the SC were quantified. The data indicate that the differences in the proton density (PD) and transverse relaxation time between gray matter (GM) and white matter (WM) dominate the contrast seen on the mouse SC images at 9.4 T. However, on heavily T(2)-weighted images these differences result in a reversal of contrast. The diffusion of water in the cord is anisotropic, but the WM exhibits greater anisotropy and principal diffusivity than the GM. The quantitative data presented here should establish a standard for comparing similar measurements obtained from the SCs of genetically engineered mouse or mouse models of SC injury (SCI).

Algorithms↗