Differentially expressed soluble proteins in aortic cells from atherosclerosis-susceptible and resistant pigeons.

Soluble proteins in aortic smooth muscle cells cultured from atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer pigeons were extracted and separated on 2-dimensional electrophoresis gels. Spots were analyzed with Phoretix software and compared between the 2 breeds. Proteins differentially expressed were arrayed on a map, plotting molecular weight against isoelectric point. Eight discrete zones were identified, 5 that included only proteins unique to susceptible cells and 3 that included proteins unique to resistant cells. Of the 88 differentially expressed proteins from susceptible cells, 41 were located in unique zones, whereas 29 of 82 differentially expressed proteins from resistant cells were in unique zones. Selected proteins from susceptibility, and resistance zones were annotated by peptide mass fragments, molecular weights, isoelectric points, and correspondence with genes differentially expressed between cells from the 2 breeds. Some of the annotated proteins (such as smooth muscle myosin phosphatase, myosin heavy chain, fatty acid-binding protein, ribophorin, heat shock protein, and tumor necrosis factor alpha-inducing factor) corresponded to the current hypotheses to explain atherogenesis. In addition, the unique electrophoretic migration zones of proteins associated with susceptibility or resistance should prove useful as a diagnostic tool in clinical settings where species or phenotypes, or both, susceptible or resistant to atherosclerosis can be identified.

Early-onset type 2 diabetes in obese white subjects is characterised by a marked defect in beta cell insulin secretion, severe insulin resistance and a lack of response to aerobic exercise training.

AIMS/HYPOTHESIS: Early-onset type 2 diabetes is associated with marked visceral obesity and extreme insulin resistance, but its pathogenesis and response to treatment are not completely understood. We studied physical fitness, whole-body and hepatic glucose turnover, and insulin secretion in young obese Irish subjects before and after 3 months of aerobic exercise training. We hypothesised that exercise alone, with stable diet, should improve insulin sensitivity. MATERIALS AND METHODS: Anthropometric parameters and maximum volume of oxygen utilisation (VO(2max)) were measured in 13 subjects with type 2 diabetes and 18 non-diabetic control subjects, matched for age and BMI. Insulin sensitivity and hepatic glucose turnover were measured using the hyperinsulinaemic-euglycaemic clamp. Insulin secretion was assessed from an OGTT and a modified intravenous glucose tolerance test. Some subjects (seven type 2 diabetic, 14 non-diabetic control subjects) then completed a 12-week supervised aerobic exercise programme. All measurements were repeated on completion of the exercise programme. RESULTS: Type 2 diabetic subjects had higher WHR, systolic blood pressure and triacylglycerols than non-diabetic control subjects. They were significantly more insulin-resistant as measured both by the clamp and oral glucose insulin sensitivity. They also displayed marked defects in insulin secretion in response to oral and intravenous glucose challenges. Exercise intervention had no significant effect on whole-body or hepatic insulin sensitivity or insulin secretion. VO(2max) increased significantly in the non-diabetic control subjects, but not in the type 2 diabetic subjects after exercise training. CONCLUSIONS/INTERPRETATION: Young obese subjects with type 2 diabetes are severely insulin-resistant with marked loss of beta cell function compared with control subjects matched for age and obesity. Neither group responded metabolically to aerobic exercise intervention.

Performance-related pay in health care.

OBJECTIVE: Performance-related pay (PRP) has been widely extended within the British public sector in the last 15-20 years, mostly because of pressure from central government. Its penetration in the National Health Service (NHS) has not so far been very deep but it has been sufficient to permit preliminary judgements on its likely impact. METHODS: Review of published accounts of the extent of use of PRP in the NHS and its impact, plus two case studies. RESULTS: There have been few rigorous studies reported. PRP has been introduced for a variety of reasons: an incentive to motivate staff; to enhance staff recruitment and retention; to signal a change in organizational culture; to control staff costs; to reduce the power of trades unions; to reinforce staff development policies. Very few NHS provider organizations have implemented PRP. Despite this, senior managers see real merit in it in improving staff performance and delivering a clear message about the importance of organizational performance. Employees are much more skeptical, seeing PRP as having no effect or being detrimental. CONCLUSIONS: PRP has had, at most, only a very modest beneficial impact in the British NHS. In the absence of better evidence, it would be sensible for government to continue to encourage local initiatives rather than propose a mandatory national scheme. It would also be prudent to subject local schemes to rigorous evaluation.

Expression and isolation of antimicrobial small molecules from soil DNA libraries.

Natural products have been a critically important source of clinically relevant small molecule therapeutics. However, the discovery rate of novel structural classes of antimicrobial molecules has declined. Recently, increasing evidence has shown that the number of species cultivated from soil represents less than 1% of the total population, opening up the exciting possibility that these uncultured species may provide a large untapped pool from which novel natural products can be discovered. We have constructed and expressed in E. coli a BAC (bacterial artificial chromosome) library containing genomic fragments of DNA (5-120kb) isolated directly from soil organisms (S-DNA). Screening of the library resulted in the identification of several antimicrobial activities expressed by different recombinant clones. One clone (mg1.1) has been partially characterized and found to express several small molecules related to and including indirubin. These results show that genes involved in natural product synthesis can be cloned directly from S-DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.

A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. One strategy to treat GVHD is to equip donor T cells with a conditional suicide mechanism that can be triggered when GVHD occurs. The herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir system used clinically has several limitations, including immunogenicity and cell cycle dependence. An alternative switch based on chemically inducible apoptosis was designed and evaluated. A chimeric human protein was expressed comprising an extracellular marker (DeltaLNGFR), the Fas intracellular domain, and 2 copies of an FK506-binding protein (FKBP). Primary human T lymphocytes retrovirally transduced with this construct could be purified to homogeneity using immunomagnetic beads. Genetic integrity of the construct was ensured by redesigning repetitive sequences. Transduced T cells behaved indistinguishably from untransduced cells, retaining the ability to mount a specific antiallogeneic immune response. However, they rapidly underwent apoptosis with the addition of subnanomolar concentrations of AP1903, a bivalent "dimerizer" drug that binds FKBP and induces Fas cross-linking. A single 2-hour treatment eliminated approximately 80% of T cells, and multiple exposures induced further apoptosis. T cells were eliminated regardless of their proliferation state, suggesting that the AP1903/Fas system, which contains only human components, is a promising alternative to HSV-tk for treating GVHD.

Preparation of RNA from tissues and cells.

Most procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA. The first basic protocol describes hot phenol extraction of RNA; the method eliminates or minimizes DNA contamination by the shearing of DNA. The second basic protocol allows rapid preparation of total cytoplasmic RNA by using a nonionic detergent to lyse the plasma membrane, leaving the nuclei intact. The nuclei and hence the bulk of the cellular DNA are then removed with a simple brief centrifugation. A guanidinium thiocyanate protocol describes the separation of RNA from other cellular macromolecules in a guanidinium lysate using a CsCl step gradient. A protocol is also provided for isolation of poly(A(+)) mRNAs from total RNA.

Preparation of bacterial RNA.

Procedures for isolating RNA from bacteria involve disruption of the cells, followed by steps to separate the RNA from contaminating DNA and protein. Lysis strategies differ in the protocols presented in this unit, including chemical degradation of gram-negative cell walls using sucrose/detergent or lysozyme, and sonication to break open gram-positive cell walls. Combinations of enzymatic degradation, organic extraction, and alcohol or salt precipitation are employed in the procedures to isolate the RNA from other cellular components, and various inhibitors of ribonuclease activity (diethylpyrocarbonate, vanadyl-ribonucleoside complex, and aurintricarboxylic acid) are described. If extremely high-quality RNA is required (e.g., for gene expression studies), instructions are provided for CsCl step-gradient centrifugation to remove all traces of contaminating DNA.

Ribonuclease protection assay.

Sequence-specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter. The plasmid is cleaved with a restriction enzyme, and the plasmid DNA is transcribed with bacteriophage RNA polymerase, which efficiently transcribes the cloned sequence into a discrete RNA species of known specific activity and high abundance. The RNA is purified by removal of the DNA template, protein, and the unincorporated label. Alternatively, the probe is purified by gel electrophoresis, as described in a support protocol. The probe RNA is hybridized to sample RNAs and the hybridization reactions are treated with ribonuclease to remove free probe, leaving intact fragments of probe annealed to homologous sequences in the sample RNA. These fragments are analyzed by electrophoresis on a sequencing gel and the presence of the target mRNA is revealed by the appearance of an appropriately sized fragment of the probe.

Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms.

Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.

A ligand-reversible dimerization system for controlling protein-protein interactions.

Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains are fused to a signaling domain of interest, allowing crosslinking to be initiated by addition of a bivalent FKBP ligand. In the course of protein engineering studies on human FKBP, we discovered that a single point mutation in the ligand-binding site (Phe-36 --> Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analytical ultracentrifugation, and x-ray crystallographic studies show that the mutant (F(M)) forms discrete homodimers with micromolar affinity that can be completely dissociated within minutes by addition of monomeric synthetic ligands. These unexpected properties form the basis for a "reverse dimerization" regulatory system involving F(M) fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have used this strategy to rapidly and reversibly aggregate fusion proteins in different cellular compartments, and to provide an off switch for transcription. Reiterated F(M) domains should be generally useful as conditional aggregation domains (CADs) to control intracellular events where rapid, reversible dissolution of interactions is required. Our results also suggest that dimerization is a latent property of the FKBP fold: the crystal structure reveals a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dramatic change in quaternary structure.

Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum.

A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

Improving management of atrial fibrillation and anticoagulation in a community hospital.

BACKGROUND: Clinical trials have established the safety and efficacy of warfarin anticoagulation for stroke prevention in patients with atrial fibrillation. Other studies have documented patterns of underutilization and suboptimal warfarin therapy; physician underuse of warfarin may reflect the demands associated with monitoring the drug's effects. BASELINE STUDY: At Carney Hospital, a 230-bed acute care community teaching hospital in Boston, a retrospective chart review indicated that between July 1, 1995, and June 30, 1996, of 465 patients admitted with atrial fibrillation, 209 (45%) patients were discharged with warfarin therapy: 198 were receiving warfarin at admission, and 11 began therapy during hospitalization. Analysis of the admission international normalized ratios (INRs) indicated that a minority of patients on warfarin were safely anticoagulated at the time of admission. DESIGNING THE INTERVENTION: An anticoagulation clinic was established in fall 1997 to increase utilization of warfarin, standardize anticoagulation practices, and minimize physician time and effort needed to ensure safe anticoagulation. In early 1998 monitoring of hospitalized patients with chronic atrial fibrillation began. RESULTS: The proportion of patients receiving warfarin therapy at admission increased from 46% in February-May 1998 to 63% in April-June 1999. Between October 1997 and July 1998, 49.1% of the 2,738 patient visits to the anticoagulation clinic showed an INR in the desired range. For the 2,238 visits during January through August 1999, 53.7% of the INRs were in the desired range. DISCUSSION: Establishment of a clinic to oversee warfarin therapy and dissemination of indications for anticoagulation in patients with atrial fibrillation were followed by increases in the frequency of warfarin use in hospital patients and the incidence of safe therapy in ambulatory patients.

Proteasome-mediated degradation of transcriptional activators correlates with activation domain potency in vivo.

We show that the intracellular concentration of transcriptional activator proteins is regulated by the proteasome-mediated protein degradation pathway. The rate of degradation of activators by proteasomes correlates with activation domain potency in vivo. Mutations either in the activation domain residues involved in target protein interaction or in the DNA-binding domain residues essential for DNA binding abolish the transcriptional activation function in vivo and render the activator resistant to degradation by proteasomes. Finally, using a rapamycin-regulated gene expression system, we show that recruiting activation domains to DNA-bound receptor proteins greatly enhanced the rate of degradation of reconstituted activators. These observations suggest that in mammalian cells efficient recruitment of activator-target protein complexes to the promoter means that they are subjected to rapid degradation by proteasomes. We propose that proteasome-mediated control of the intracellular levels of transcriptional activators could play an important role in the regulation of gene expression.

Cell surface tagging and a suicide mechanism in a single chimeric human protein.

Many therapeutic uses of gene-modified cells could benefit from inclusion of a surface marker for immunoselecting transduced cells. Another desired feature is a failsafe mechanism to ablate engineered cells if required. We describe here a system that combines a cell surface tag and an inducible apoptosis mechanism in a single protein. Spencer et al. (Curr. Biol. 1996;6:839-847) described an inducible cell suicide gene containing a myristoylation sequence, the human protein FKBP12, and the intracellular domain of Fas. Cells expressing this protein apoptose on treatment with a cell-permeable chemical dimerizing agent that binds two FKBP domains and cross-links the chimeric Fas proteins. We modified this system by anchoring a Fas-FKBP construct to the membrane with the extracellular and transmembrane domains of the low-affinity nerve growth factor receptor (LNGFR), thereby uniting cell surface tagging with the inducible apoptosis mechanism. Cells retrovirally transduced with this construct apoptosed on exposure to a chemical dimerizer, AP1903 (Clackson et al., Proc. Natl. Acad. Sci. U.S.A. 1998;95:10437-10442). The LNGFR-tagged construct showed an unpredicted clear advantage over the myristoylation-anchored construct in its efficiency of signaling in HT1080 cells. This linked marker and failsafe mechanism may have particularly attractive safety properties for gene therapy. The use of gene-modified cells in basic research and clinical studies is enhanced by the use of a selectable surface marker for immunoselection of transduced cells. Another desired feature for gene and cell therapies is an inducible suicide system to eliminate transduced cells when necessary. Spencer et al. (Curr. Biol. 1996;6:839-847) described a potential failsafe mechanism whereby exposure of cells to a chemical dimerizing agent activates the Fas-mediated apoptotic pathway. In this system, the intracellular signaling domain of Fas is linked to one or more copies of the human protein FKBP12. Treatment of engineered cells with a cell-permeable chemical dimerizing agent that simultaneously binds to two FKBP domains cross-links the chimeric Fas protein and induces apoptosis. Here, we modify the system by anchoring a Fas-FKBP construct to the membrane with the extracellular domain of the low-affinity nerve growth factor receptor (LNGFR), to unite cell surface tagging of transduced cells with the inducible apoptosis mechanism. Cells retrovirally transduced with this construct undergo apoptosis on exposure to a chemical dimerizer, AP1903. A linked marker and failsafe mechanism may have particularly attractive safety properties for gene therapy.

A general strategy to enhance the potency of chimeric transcriptional activators.

Efforts to increase the potency of transcriptional activators are generally unsuccessful because poor expression of activators in mammalian cells limits their delivery to target promoters. Here we report that the effectiveness of chimeric activators can be dramatically improved by expressing them as noncovalent tetrameric bundles. Bundled activation domains are much more effective at activating a reporter gene than simple monomeric activators, presumably because, at similar expression levels, up to 4 times as many the activation domains are delivered to the target promoter. These bundled activation domains are also more effective than proteins in which activation domains are tandemly reiterated in the same polypeptide chain, because such proteins are very poorly expressed and therefore not delivered effectively. These observations suggest that there is a threshold number of activation domains that must be bound to a promoter for activation, above which promoter activity is simply a function of the number of activators bound. We show that bundling can be exploited practically to enhance the sensitivity of mammalian two-hybrid assays, enabling detection of weak interactions or those between poorly expressed proteins. Bundling also dramatically improves the performance of a small-molecule-regulated gene expression system when the expression level of regulatory protein is limiting, a situation that may be encountered in gene therapy applications.

Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers.

The four members of the ErbB family of receptor tyrosine kinases are involved in a complex array of combinatorial interactions involving homo- and heterodimers. Since most cell types express more than one member of the ErbB family, it is difficult to distinguish the biological activities of different homo- and heterodimers. Here we describe a method for inducing homo- or heterodimerization of ErbB receptors by using synthetic ligands without interference from the endogenous receptors. ErbB receptor chimeras containing synthetic ligand binding domains (FK506-binding protein [FKBP] or FKBP-rapamycin-binding domain [FRB]) were homodimerized with the bivalent FKBP ligand AP1510 and heterodimerized with the bifunctional FKBP-FRB ligand rapamycin. AP1510 treatment induced tyrosine phosphorylation of ErbB1 and ErbB2 homodimers and recruitment of Src homology 2 domain-containing proteins (Shc and Grb2). In addition, ErbB1 and ErbB2 homodimers activated downstream signaling pathways leading to Erk2 and Akt phosphorylation. However, only ErbB1 homodimers were internalized upon AP1510 stimulation, and only ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to form foci; however, cells expressing ErbB2 homodimers displayed a five- to sevenfold higher focus-forming ability. Using rapamycin-inducible heterodimerization we show that c-Cbl is unable to associate with ErbB1 in a ErbB1-ErbB2 heterodimer most likely because ErbB2 is unable to phosphorylate the c-Cbl binding site on ErbB1. Thus, we demonstrate that ErbB1 and ErbB2 homodimers differ in their abilities to transform fibroblasts and provide evidence for differential signaling by ErbB homodimers and heterodimers. These observations also validate the use of synthetic ligands to study the signaling and biological specificity of selected ErbB dimers in any cell type.

Long-term regulated expression of growth hormone in mice after intramuscular gene transfer.

Effective delivery of secreted proteins by gene therapy will require a vector that directs stable delivery of a transgene and a regulatory system that permits pharmacologic control over the level and kinetics of therapeutic protein expression. We previously described a regulatory system that enables transcription of a target gene to be controlled by rapamycin, an orally bioavailable drug. Here we demonstrate in vivo regulation of gene expression after intramuscular injection of two separate adenovirus or adeno-associated virus (AAV) vectors, one encoding an inducible human growth hormone (hGH) target gene, and the other a bipartite rapamycin-regulated transcription factor. Upon delivery of either vector system into immunodeficient mice, basal plasma hGH expression was undetectable and was induced to high levels after administration of rapamycin. The precise level and duration of hGH expression could be controlled by the rapamycin dosing regimen. Equivalent profiles of induction were observed after repeated administration of single doses of rapamycin over many months. AAV conferred stable expression of regulated hGH in both immunocompetent and immunodeficient mice, whereas adenovirus-directed hGH expression quickly extinguished in immunocompetent animals. These studies demonstrate that the rapamycin-based regulatory system, delivered intramuscularly by AAV, fulfills many of the conditions necessary for the safe and effective delivery of therapeutic proteins by gene therapy.

Distinct mechanisms of activation of Stat1 and Stat3 by platelet-derived growth factor receptor in a cell-free system.

Ligand-dependent activation of the platelet-derived growth factor receptor (PDGFR) in fibroblasts in culture leads to the activation of the JAK family of protein-tyrosine kinases and of the transcription factors Stat1 and Stat3. To determine the biochemical mechanism of STAT activation by PDGFR, we devised a cell-free system composed of a membrane fraction from cells overexpressing PDGFR. When supplemented with crude cytosol, the membrane fraction supported PDGF- and ATP-dependent activation of both Stat1 and Stat3. However, the extent of Stat3 activation differed depending on the source of the cytosolic fraction. Using purified recombinant STAT proteins produced in Escherichia coli, we found that Stat1 could be activated by immunopurified PDGFR and showed no additional requirement for membrane- or cytosol-derived proteins. In contrast, activation of Stat3 exhibited a strong requirement for the cytosolic fraction. The activity present in the cytosolic fraction could be depleted with antibodies to JAK proteins. We conclude that the mechanisms of activation of Stat1 and Stat3 by PDGFR are distinct. Stat1 activation appears to result from a direct interaction with the receptor, whereas Stat3 activation additionally requires JAK proteins.

Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer.

Stable delivery of a therapeutic protein under pharmacologic control was achieved through in vivo somatic gene transfer. This system was based on the expression of two chimeric, human-derived proteins that were reconstituted by rapamycin into a transcription factor complex. A mixture of two adeno-associated virus vectors, one expressing the transcription factor chimeras and one containing erythropoietin (Epo) under the control of a promoter responsive to the transcription factor, was injected into skeletal muscle of immune-competent mice. Administration of rapamycin resulted in 200-fold induction of plasma Epo. Stable engraftment of this humanized system in immune-competent mice was achieved for 6 months with similar results for at least 3 months in a rhesus monkey.

Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway.

In vertebrates, many cytokines and growth factors have been identified as activators of the JAK/STAT signaling pathway. In Drosophila, JAK and STAT molecules have been isolated, but no ligands or receptors capable of activating the pathway have been described. We have characterized the unpaired (upd) gene, which displays the same distinctive embryonic mutant defects as mutations in the Drosophila JAK (hopscotch) and STAT (stat92E) genes. Upd is a secreted protein, associated with the extracellular matrix, that activates the JAK pathway. We propose that Upd is a ligand that relies on JAK signaling to stimulate transcription of pair-rule genes in a segmentally restricted manner in the early Drosophila embryo.