Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.

Induction of insulin and islet amyloid polypeptide production in pancreatic islet glucagonoma cells by insulin promoter factor 1.

Insulin promoter factor 1 (IPF1), a member of the homeodomain protein family, serves an early role in pancreas formation, as evidenced by the lack of pancreas formation in mice carrying a targeted disruption of the IPF1 gene [Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. (1994) Nature (London) 371, 606-609]. In adults, IPF1 expression is restricted to the beta-cells in the islets of Langerhans. We report here that IPF1 induces expression of a subset of beta-cell-specific genes (insulin and islet amyloid polypeptide) when ectopically expressed in clones of transformed pancreatic islet alpha-cells. In contrast, expression of IPF1 in rat embryo fibroblasts factor failed to induce insulin and islet amyloid polypeptide expression. This is most likely due to the lack of at least one other essential insulin gene transcription factor, the basic helix-loop-helix protein Beta 2/NeuroD, which is expressed in both alpha- and beta-cells. We conclude that IPF1 is a potent transcriptional activator of endogenous insulin genes in non-beta islet cells, which suggests an important role of IPF1 in beta-cell maturation.

Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus.

The Agrobacterium tumefaciens VirB7 lipoprotein contributes to the stabilization of VirB proteins during biogenesis of the putative T-complex transport apparatus. Here, we report that stabilization of VirB7 itself is correlated with its ability to form disulfide cross-linked homodimers via a reactive Cys-24 residue. Three types of beta-mercaptoethanol-dissociable complexes were visualized with VirB7 and/or a VirB7::PhoA41 fusion protein: (i) a 9-kDa complex corresponding in size to a VirB7 homodimer, (ii) a 54-kDa complex corresponding in size to a VirB7/VirB7::PhoA41 mixed dimer, and (iii) a 102-kDa complex corresponding to a VirB7::PhoA41 homodimer. A VirB7C24S mutant protein was immunologically undetectable, whereas the corresponding VirB7C24S::PhoA41 derivative accumulated to detectable levels but failed to form dissociable homodimers or mixed dimers with wild-type VirB7. We further report that VirB7-dependent stabilization of VirB9 is correlated with the ability of these two proteins to dimerize via formation of a disulfide bridge between reactive Cys-24 and Cys-262 residues, respectively. Two types of dissociable complexes were visualized: (i) a 36-kDa complex corresponding in size to a VirB7/VirB9 heterodimer and (ii) an 84-kDa complex corresponding in size to a VirB7/VirB9::PhoA293 heterodimer. A VirB9C262S mutant protein was immunologically undetectable, whereas the corresponding VirB9C262S::PhoA293 derivative accumulated to detectable levels but failed to form dissociable heterodimers with wild-type VirB7. Taken together, these results support a model in which the formation of disulfide cross-linked VirB7 dimers represent critical early steps in the biogenesis of the T-complex transport apparatus.

Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.

Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects.

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.

Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus.

Using genetically engineered glomerular mesangial cells, an in vivo gene transfer approach was developed that specifically targets the renal glomerulus. By combining this system with a tetracycline (Tc)-responsive promoter, the present study aimed to create a reversible on/off system for site-specific in vivo control of exogenous gene activity within the glomerulus. In the Tc regulatory system, a Tc-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription by binding to a tTA-responsive promoter located in a response plasmid. Tc inhibits this tTA-dependent transactivation via its affinity for tTA. In double-transfected cells, therefore, the activity of a transgene can be controlled by Tc. Cultured rat mesangial cells were cotransfected with a regulator plasmid and a response plasmid that introduces a beta-galactosidase gene. In vitro, stable double-transfectant MtTAG cells exhibited no beta-galactosidase activity in the presence of Tc. However, following withdrawal of Tc from culture media, expression of beta-galactosidase was induced within 24 h. When Tc was again added, the expression was rapidly resuppressed. Low concentrations of Tc were sufficient to maintain the silent state of tTA-dependent promoter. MtTAG cells were then transferred into the rat glomeruli via renal artery injection. In the isolated chimeric glomeruli, expression of beta-galactosidase was induced ex vivo in the absence of Tc, whereas it was repressed in its presence. When Tc-pretreated MtTAG cells were transferred into the glomeruli of untreated rats, beta-galactosidase expression was induced in vivo within 3 days. Oral administration of Tc dramatically suppressed this induction. These data demonstrate the feasibility of using mesangial cell vectors combined with the Tc regulatory system for site-specific in vivo control of exogenous gene expression in the glomerulus.

Expression of galectin-3 modulates T-cell growth and apoptosis.

Galectin-3 is a member (if a large family of beta-galactoside-binding animal lectins. It has been shown that the expression of galectin-3 is upregulated in proliferating cells, suggesting a possible role for this lectin in regulation of cell growth. Previously, we have shown that T cells infected with human T-cell leukemia virus type I express high levels of galectin-3, in contrast to uninfected cells, which do not express detectable amounts of this protein. In this study, we examined growth properties of human leukemia T cells transfected with galectin-3 cDNA, and thus constitutively overexpressing this lectin. Transfectants expressing galectin-3 displayed higher growth rates than control transfectants, which do not express this lectin. Furthermore, galectin-3 expression in these cells confers resistance to apoptosis induced by anti-Fas antibody and staurosporine. Galectin-3 was found to have significant sequence similarity with Bcl-2, a well-characterized suppressor of apoptosis. In particular, the lectin contains the NWGR motif that is highly conserved among members of the Bcl-2 family and shown to be critical for the apoptosis-suppressing activity. We further demonstrated that galectin-3 interacts with Bc1-2 in a lactose-inhibitable manner. We conclude that galectin-3 is a regulator of cell growth and apoptosis and it may function through a cell death inhibition pathway that involves Bcl-2.

Vitamin E prevents oxidative modification of brain and lymphocyte band 3 proteins during aging.

Antioxidants may play an important role in preventing free radical damage associated with aging by interfering directly in the generation of radicals or by scavenging them. We investigated the effects of a high vitamin E and/or a high beta-carotene diet on aging of the anion transporter, band 3, in lymphocytes and brain. The band 3 proteins function as anion transporters, acid base regulators, C02 transporters, and structural proteins that provide a framework for membrane lipids and that link the plasma membrane to the cytoskeleton. Senescent cell antigen (SCA), which terminates the life of cells, is a degradation product of band 3. This study was conducted as a double-blind study in which eight groups of middle-aged or old mice received either high levels of beta-carotene and/or vitamin E or standard levels of these supplements in their diets. Anion transport kinetic assays were performed on isolated splenic lymphocytes. Immunoreactivity of an antibody that recognizes aging changes in old band 3 preceding generation of SCA was used to quantitate aged band 3 in brain tissue. Results indicate that vitamin E prevented the observed age-related decline in anion transport by lymphocytes and the generation of aged band 3 leading to SCA formation. beta-Carotene had no significant effect on the results of either assay. Since increased aged band 3 and decreased anion transport are initial steps in band 3 aging, which culminates in the generation of SCA and cellular removal, vitamin E prevents or delays aging of band 3-related proteins in lymphocytes and brain.

Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

Analysis of metabolic pathways by the growth of cells in the presence of organic solvents.

A new approach to the analysis of metabolic pathways involving poorly water-soluble intermediates is proposed. It relies upon the ability of the hydrophobic intermediates formed by a sequence of intracellular reactions to cross the membrane(s) and partition between aqueous and organic phases, when cells are incubated in the presence of a nonpolar and nontoxic organic solvent. As a result of this thermodynamically driven efflux of the formed intermediates from the cell, they accumulate in the organic medium in sufficient quantities for GC-MS analysis and identification. This enables direct determination of the sequence of chemical reactions involved with no requirement for the isolation of each individual metabolite from a cell-free extract. The feasibility of the proposed methodology has been demonstrated by the elucidation of the biosynthesis of (R)-gamma-decalactone from (R)-ricinoleic acid catalyzed by the yeast Sporidiobolus ruinenii grown in the presence of decane. The corresponding 4-hydroxy-acid intermediates, formed in the course of beta-oxidation of (R)-ricinoleic acid, were simultaneously observed in a single experiment on the same chromatogram. Potential applications of this proposed methodology are briefly discussed.

Logarithm of odds (lods) for linkage in complex inheritance.

Lod scores provide a method to unify linkage tests based on identity by descent and identity in marker state while permitting selection of the most informative individuals through their disease-related phenotypes and markers in relatives. After parametric lods are reviewed, a nonparametric approach that depends on a single logistic parameter beta is introduced. Lods for parents tested or unknown are derived, multiple pairwise mapping is presented, and power is shown to be good even for moderately small values of beta. Comparison of parametric and nonparametric approaches (yet to be made) will provide for polygenes the efficiency and reliability that lod scores gave to mapping of major loci 40 years ago.

Production of transgenic dwarf surfclams, Mulinia lateralis, with pantropic retroviral vectors.

A pantropic pseudotyped retroviral vector containing the envelope protein of vesicular stomatitis virus was used as a gene transfer vector in the dwarf surfclam, Mulinia lateralis. These pantropic retroviral vectors have an extremely broad host cell range and can infect many nonmammalian species. Newly fertilized dwarf surfclam eggs were electroporated at 700 V in the presence of 1 x 10(4) colony-forming units of pantropic pseudotyped retroviral particles. Infection was well tolerated and did not affect the survival rate of the embryos. Gametes collected from P1 presumptive transgenic animals were analyzed for the presence of provirus by PCR, and in different experiments 13-33% of the gamete pools were positive for the transgene. Dot blot hybridization of DNA samples from the F1 offspring of two different crosses between infected P1 and wild-type individuals revealed that 28% and 31% of F1 offspring were transgenic, respectively. Southern blot analysis of DNA isolated from PCR-positive F1 animals confirmed integration of a single copy of the provirus into the host genome. Thus, the germ lines of these two P1 transgenic animals were mosaic for the transgene. Expression of beta-galactosidase encoded by the provirus was detected in transgenic but not control surfclam embryos. Pantropic pseudotyped retroviral vectors provide a useful method for the stable introduction of foreign genetic information into surfclams and may facilitate the introduction of desirable genetic traits into commercially important shellfish and crustaceans.

Apoptosis of hemopoietic cells by the human granulocyte-macrophage colony-stimulating factor mutant E21R.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) binds to a high-affinity heterodimeric receptor composed of a specific alpha chain and a common beta chain (beta(c)), which is shared with the receptors for interleukins 3 and 5. Hemopoietic cell survival requires GM-CSF binding this high-affinity receptor. We have recently developed the GM-CSF mutant E21R, which selectively binds to the alpha chain and behaves as a competitive GM-CSF antagonist. We have now examined the role of E21R on the survival of hemopoietic cells and found that E21R causes apoptosis (programmed cell death) of normal and malignant cells directly in the absence of GM-CSF. The direct apoptotic effect of E21R occurred in a dose- and time-dependent manner. Apoptosis by E21R was dependent on cells expressing the high-affinity GM-CSF receptor and could be blocked by GM-CSF. Significantly, apoptosis of the cells occurred even in the presence of the survival factors granulocyte CSF and stem cell factor but was prevented by engagement of beta(c) with interleukin 3. The initiation of apoptosis required phosphorylation, transcriptional activity, and protein synthesis. These findings support a model whereby binding of E21R to the alpha chain leads to apoptosis, while beta(c) plays an important role in cell survival. This model may be applicable to other multimeric cytokine receptors and offers a novel approach for the treatment of human leukemia.

beta-Amyloid toxicity in organotypic hippocampal cultures: protection by EUK-8, a synthetic catalytic free radical scavenger.

Oxygen free radicals have been proposed to mediate amyloid peptide (beta-AP)-induced neurotoxicity. To test this hypothesis, we evaluated the effects of EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, on neuronal injury produced by beta-AP in organotypic hippocampal slice cultures. Cultures of equivalent postnatal day 35 (defined as mature) and 14 (defined as immature) were exposed to various concentrations of beta-AP (1-42 or 1-40) in the absence or presence of 25 microM EUK-8 for up to 72 hours. Neuronal injury was assessed by lactate dehydrogenase release and semiquantitative analysis of propidium iodide uptake at various times after the initiation of beta-AP exposure. Free radical production was inferred from the relative increase in dichlorofluorescein fluorescence, and the degree of lipid peroxidation was determined by assaying thiobarbituric acid-reactive substances. Treatment of mature cultures with beta-AP (50-250 microg/ml) in serum-free conditions resulted in a reproducible pattern of damage, causing a time-dependent increase in neuronal injury accompanied with formation of reactive oxygen species. However, immature cultures were entirely resistant to beta-AP-induced neurotoxicity and also demonstrated no dichlorofluorescein fluorescence or increased lipid peroxidation after beta-AP treatment. Moreover, mature slices exposed to beta-AP in the presence of 25 microM EUK-8 were significantly protected from beta-AP-induced neurotoxicity. EUK-8 also completely blocked beta-AP-induced free radical accumulation and lipid peroxidation. These results not only support a role for oxygen free radicals in beta-AP toxicity but also highlight the therapeutic potential of synthetic radical scavengers in Alzheimer disease.

Protection of nonobese diabetic mice from diabetes by intranasal or subcutaneous administration of insulin peptide B-(9-23).

The observation that overt type I diabetes is often preceded by the appearance of insulin autoantibodies and the reports that prophylactic administration of insulin to biobreeding diabetes-prone (BB-DP) rats, nonobese diabetic (NOD) mice, and human subjects results in protection from diabetes suggest that an immune response to insulin is involved in the process of beta cell destruction. We have recently reported that islet-infiltrating cells isolated from NOD mice are enriched for insulin-specific T cells, that insulin-specific T cell clones are capable of adoptive transfer of diabetes, and that epitopes present on residues 9-23 of the B chain appear to be dominant in this spontaneous response. In the experiments described in this report, the epitope specificity of 312 independently isolated insulin-specific T cell clones was determined and B-(9-23) was found to be dominant, with 93% of the clones exhibiting specificity toward this peptide and the remainder to an epitope on residues 7-21 of the A chain. On the basis of these observations, the effect of either subcutaneous or intranasal administration of B-(9-23) on the incidence of diabetes in NOD mice was determined. The results presented here indicate that both subcutaneous and intranasal administration of B-(9-23) resulted in a marked delay in the onset and a decrease in the incidence of diabetes relative to mice given the control peptide, tetanus toxin-(830-843). This protective effect is associated with reduced T-cell proliferative response to B-(9-23) in B-(9-23)-treated mice.