An exon that prevents transport of a mature mRNA

In Caenorhabditis elegans, pre-mRNA for the essential splicing factor U2AF65 sometimes is spliced to produce an RNA that includes an extra 216-bp internal exon, exon 3. Inclusion of exon 3 inserts an in-frame stop codon, yet this RNA is not subject to SMG-mediated RNA surveillance. To test whether exon 3 causes RNA to remain nuclear and thereby escape decay, we inserted it into the 3′ untranslated region of a gfp reporter gene. Although exon 3 did not affect accumulation or processing of the mRNA, it dramatically suppressed expression of green fluorescent protein (GFP). We showed by in situ hybridization that exon 3-containing gfp RNA is retained in the nucleus. Intriguingly, exon 3 contains 10 matches to the 8-bp 3′ splice-site consensus. We hypothesized that U2AF might recognize this octamer and thereby prevent export. This idea is supported by RNA interference experiments in which reduced levels of U2AF resulted in a small burst of gfp expression.

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Overexpression of human copper,zinc-superoxide dismutase (SOD1) prevents postischemic injury

Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper,zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.

Expression of a β-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice

Heart failure is accompanied by severely impaired β-adrenergic receptor (βAR) function, which includes loss of βAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of βAR function is agonist-stimulated receptor phosphorylation by the βAR kinase (βARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in βAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of βARK1 or the β2AR were mated into a genetic model of murine heart failure (MLP−/−). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP−/− and MLP−/−/β2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP−/−/βARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP−/−/βARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP−/− mice but less than controls. Importantly, heightened βAR desensitization in the MLP−/− mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the βARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal βAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit βARK1 as a novel mode of therapy.

Aurintricarboxylic acid prevents GLUR2 mRNA down-regulation and delayed neurodegeneration in hippocampal CA1 neurons of gerbil after global ischemia

Aurintricarboxylic acid (ATA), an inhibitor of endonuclease activity and other protein–nucleic acid interactions, blocks apoptosis in several cell types and prevents delayed death of hippocampal pyramidal CA1 neurons induced by transient global ischemia. Global ischemia in rats and gerbils induces down-regulation of GluR2 mRNA and increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced Ca2+ influx in CA1 before neurodegeneration. This result and neuroprotection by antagonists of AMPA receptors suggests that formation of AMPA receptors lacking GluR2, and therefore Ca2+ permeable, leads to excessive Ca2+ influx in response to endogenous glutamate; the resulting delayed neuronal death in CA1 exhibits many characteristics of apoptosis. In this study, we examined the effects of ATA on expression of mRNAs encoding glutamate receptor subunits in gerbil hippocampus after global ischemia. Administration of ATA by injection into the right cerebral ventricle 1 h before (but not 6 h after) bilateral carotid occlusion prevented the ischemia-induced decrease in GluR2 mRNA expression and the delayed neurodegeneration. These findings suggest that ATA is neuroprotective in ischemia by blocking the transcriptional changes leading to down-regulation of GluR2, rather than by simply blocking endonucleases, which presumably act later after Ca2+ influx initiates apoptosis. Maintaining formation of Ca2+ impermeable, GluR2 containing AMPA receptors could prevent delayed death of CA1 neurons after transient global ischemia, and block of GluR2 down-regulation may provide a further strategy for neuroprotection.

Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses

The rice blast fungus, Magnaporthe grisea, generates enormous turgor pressure within a specialized cell called the appressorium to breach the surface of host plant cells. Here, we show that a mitogen-activated protein kinase, Mps1, is essential for appressorium penetration. Mps1 is 85% similar to yeast Slt2 mitogen-activated protein kinase and can rescue the thermosensitive growth of slt2 null mutants. The mps1–1Δ mutants of M. grisea have some phenotypes in common with slt2 mutants of yeast, including sensitivity to cell-wall-digesting enzymes, but display additional phenotypes, including reduced sporulation and fertility. Interestingly, mps1–1Δ mutants are completely nonpathogenic because of the inability of appressoria to penetrate plant cell surfaces, suggesting that penetration requires remodeling of the appressorium wall through an Mps1-dependent signaling pathway. Although mps1–1Δ mutants are unable to cause disease, they are able to trigger early plant-cell defense responses, including the accumulation of autofluorescent compounds and the rearrangement of the actin cytoskeleton. We conclude that MPS1 is essential for pathogen penetration; however, penetration is not required for induction of some plant defense responses.

Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons in vivo. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood–brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons in vitro. To test the efficacy of the Ad-mediated GDNF gene transfer in vivo, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-β-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-β-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.

Inhibition of the electrostatic interaction between β-amyloid peptide and membranes prevents β-amyloid-induced toxicity

The accumulation of β-amyloid peptides (Aβ) into senile plaques is one of the hallmarks of Alzheimer disease. Aggregated Aβ is toxic to cells in culture and this has been considered to be the cause of neurodegeneration that occurs in the Alzheimer disease brain. The discovery of compounds that prevent Aβ toxicity may lead to a better understanding of the processes involved and ultimately to possible therapeutic drugs. Low nanomolar concentrations of Aβ1-42 and the toxic fragment Aβ25-35 have been demonstrated to render cells more sensitive to subsequent insults as manifested by an increased sensitivity to formazan crystals following MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction. Formation of the toxic β-sheet conformation by Aβ peptides is increased by negatively charged membranes. Here we demonstrate that phloretin and exifone, dipolar compounds that decrease the effective negative charge of membranes, prevent association of Aβ1-40 and Aβ25-35 to negatively charged lipid vesicles and Aβ induced cell toxicity. These results suggest that Aβ toxicity is mediated through a nonspecific physicochemical interaction with cell membranes.

Expression of adenoviral E3 transgenes in β cells prevents autoimmune diabetes

The adenovirus (Ad) genome contains immunoregulatory and cytokine inhibitory genes that are presumed to function in facilitating acute infection or in establishing persistence in vivo. Some of these genes are clustered in early region 3 (E3), which contains a 19-kDa glycoprotein (gp19) that inhibits the transport of selected class I major histocompatibility complex (MHC) molecules out of the endoplasmic reticulum. In addition, the E3 region contains three protein inhibitors of the cytolytic function of tumor necrosis factor α (TNF-α). Because type I autoimmune diabetes destroys islets by mechanisms that involve class I MHC and TNF-α, we investigated whether the entire cassette of Ad E3 genes might prevent the onset of diabetes in a well studied lymphocytic choriomeningitis viral (LCMV) murine model of virus-induced autoimmune diabetes. In this model, a LCMV polypeptide (either glycoprotein or nucleoprotein) expressed as a transgene in the islets is a target for autoimmune destruction of β cells after LCMV infection. In this scenario the LCMV-induced immune response is directed not only against the virus but also against the LCMV transgenes expressed in the β cells. Our experiments demonstrated a very efficient prevention of this LCMV-triggered diabetes by the Ad E3 genes. This resulted from the inhibition of target cell recognition by a fully competent and LCMV-primed immune system. Unlike the results from the β-2 microglobulin gene deletion experiments, our approach shows that selective regulation at the level of the target cell is sufficient to prevent autoimmune diabetes without disrupting the function of the systemic immune response. Although the Ad genes in these experiments were provided as transgenes, recent experiments may permit the introduction of such genes through the use of viral vectors. Although the decrease in class I MHC in islets by Ad genes was demonstrated in these in vivo studies, the relative importance of this process and the control of TNF-α cytolysis must await further genetic dissection of the introduced Ad genes.

Long-term suppression of Leydig cell steroidogenesis prevents Leydig cell aging

Male aging is accompanied by reduced testosterone production by the Leydig cells, the testosterone-producing cells of the testis. The mechanism by which this occurs is unknown. Based on the observations that reactive oxygen is capable of damaging components of the steroidogenic pathway and that reactive oxygen is produced during steroidogenesis itself, we hypothesized that long-term suppression of steroidogenesis might inhibit or prevent age-related deficits in Leydig cell testosterone production. To test this, we administered contraceptive doses of testosterone to groups of young (3 months old) and middle-aged (13 months old) Brown Norway rats via Silastic implants to suppress endogenous Leydig cell testosterone production. After 8 months, the implants were removed, which rapidly (days) restores the ability of the previously suppressed Leydig cells to produce testosterone. Two months after removing the implants, when the rats of the two groups were 13 and 23 months of age, respectively, the Leydig cells in both cases were found to produce testosterone at the high levels of young Leydig cells, whereas significantly lower levels were produced by the 23-month-old controls. Thus, by placing the Leydig cells in a state of steroidogenic “hibernation,” the reductions in Leydig cell testosterone production that invariably accompany aging did not occur. If hormonal contraception in the human functions the same way, the adverse consequences of reduced testosterone in later life (osteoporosis, reduced muscle mass, reduced libido, mood swings, etc.) might be delayed or prevented.

The Malthusian parameter of ascents: What prevents the exponential increase of one’s ancestors?

The reason that the indefinite exponential increase in the number of one’s ancestors does not take place is found in the law of sibling interference, which can be expressed by the following simple equation:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\begin{matrix}{\mathit{N}}_{{\mathit{n}}} \enskip & \\ {\mathit{{\blacksquare}}} \enskip & \\ {\mathit{ASZ}} \enskip & \end{matrix} {\mathrm{\hspace{.167em}{\times}\hspace{.167em}2\hspace{.167em}=\hspace{.167em}}}{\mathit{N_{n+1},}}\end{equation*}\end{document} where Nn is the number of ancestors in the nth generation, ASZ is the average sibling size of these ancestors, and Nn+1 is the number of ancestors in the next older generation (n + 1). Accordingly, the exponential increase in the number of one’s ancestors is an initial anomaly that occurs while ASZ remains at 1. Once ASZ begins to exceed 1, the rate of increase in the number of ancestors is progressively curtailed, falling further and further behind the exponential increase rate. Eventually, ASZ reaches 2, and at that point, the number of ancestors stops increasing for two generations. These two generations, named AN SA and AN SA + 1, are the most critical in the ancestry, for one’s ancestors at that point come to represent all the progeny-produced adults of the entire ancestral population. Thereafter, the fate of one’s ancestors becomes the fate of the entire population. If the population to which one belongs is a successful, slowly expanding one, the number of ancestors would slowly decline as you move toward the remote past. This is because ABZ would exceed 2. Only when ABZ is less than 2 would the number of ancestors increase beyond the AN SA and AN SA + 1 generations. Since the above is an indication of a failing population on the way to extinction, there had to be the previous AN SA involving a far greater number of individuals for such a population. Simulations indicated that for a member of a continuously successful population, the AN SA ancestors might have numbered as many as 5.2 million, the AN SA generation being the 28th generation in the past. However, because of the law of increasingly irrelevant remote ancestors, only a very small fraction of the AN SA ancestors would have left genetic traces in the genome of each descendant of today.

Coculturing diverse clonal populations prevents the early-stage neoplastic progression that occurs in the separate clones

Most human cancers are of monoclonal origin and display many genetic alterations. In an effort to determine whether clonal expansion itself could account for the large number of genetic alterations, we compared spontaneous transformation in cloned and uncloned populations of NIH 3T3 cells. We have reported that progressive transformation of these cells, which is driven by the stress of prolonged contact inhibition at confluence, occurs far more frequently in cultures of recent monoclonal origin than in their uncloned progenitors. In the present work we asked how coculturing six clones at early and late stages of progression would affect the dynamics of transformation in repeated rounds of confluence. When coculture started with clones in early stages of transformation, marked by light focus formation, there was a strong inhibition of the progression to the dense focus formation that occurred in separate cultures of the individual clones. In contrast, when coculture started after the individual clones had progressed to dense focus formation, there was selection of transformants from the clone producing the largest and densest foci. Mixing the cells of a single clone with a large excess of uncloned cells from a subline that was refractory to transformation markedly decreased the size of dense foci from clones in transit from light to dense focus formation, but had much less effect on foci from clones with an established capacity for dense focus formation. The major finding of protection against progression by coculturing clones in early stages of transformation suggests that the expansion of a rogue clone in vivo increasingly isolates many of its cells from genetically stabilizing interactions with surrounding clones. Such clonal isolation might account for the increase in mutation rates associated with the dysplasia in colorectal adenomas that signifies the transition between benign and malignant growth.

Blockade of type β transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat

We eliminated type β transforming growth factor (TGF-β) signaling by adenovirus-mediated local expression of a dominant-negative type II TGF-β receptor (AdCATβ-TR) in the liver of rats treated with dimethylnitrosamine, a model of persistent liver fibrosis. In rats that received a single application of AdCATβ-TR via the portal vein, liver fibrosis as assessed by histology and hydroxyproline content was markedly attenuated. All AdCATβ-TR-treated rats remained alive, and their serum levels of hyaluronic acid and transaminases remained at low levels, whereas all the AdCATβ-TR-untreated rats died of liver dysfunction. The results demonstrate that TGF-β does play a central role in liver fibrogenesis and indicate clearly in a persistent fibrosis model that prevention of fibrosis by anti-TGF-β intervention could be therapeutically useful.

Gβ5 prevents the RGS7-Gαo interaction through binding to a distinct Gγ-like domain found in RGS7 and other RGS proteins

The G protein β subunit Gβ5 deviates significantly from the other four members of Gβ-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gβ5 in vivo, we have isolated a native Gβ5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gβ5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gβ5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gβ5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gβ5 or anti-RGS7 antibodies. The specific Gβ5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Gγ subunits. Deletion of this domain prevents the RGS7-Gβ5 binding, although the interaction with Gα is retained. Substitution of the Gγ-like domain of RGS7 with a portion of Gγ1 changes its binding specificity from Gβ5 to Gβ1. The interaction of Gβ5 with RGS7 blocked the binding of RGS7 to the Gα subunit Gαo, indicating that Gβ5 is a specific RGS inhibitor.

Elongin BC complex prevents degradation of von Hippel-Lindau tumor suppressor gene products

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene causes the familial cancer syndrome, VHL disease, characterized by a predisposition to renal cell carcinoma and other tumor types. Loss of VHL gene function also is found in a majority of sporadic renal carcinomas. A preponderance of the tumor-disposing inherited missense mutations detected in VHL disease are within the elongin-binding domain of VHL. This region mediates the formation of a multiprotein VHL complex containing elongin B, elongin C, cul-2, and Rbx1. This VHL complex is thought to function as an E3 ubiquitin ligase. Here, we report that VHL proteins harboring mutations which disrupt elongin binding are unstable and rapidly degraded by the proteasome. In contrast, wild-type VHL proteins are directly stabilized by associating with both elongins B and C. In addition, elongins B and C are stabilized through their interactions with each other and VHL. Thus, the entire VHL/elongin complex is resistant to proteasomal degradation. Because the elongin-binding domain of VHL is frequently mutated in cancers, these results suggest that loss of elongin binding causes tumorigenesis by compromising VHL protein stability and/or potential VHL ubiquitination functions.