Lethal and Demographic Impact of Chlorpyrifos and Spinosad on the Ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae).

The appropriate use of biological agents and chemical compounds is necessary to establish successful integrated pest management (IPM) programs. Thus, the off-target effects of pesticides on biological control agents are essential considerations of IPM. In this study, the effects of lethal and sublethal concentrations of chlorpyrifos and spinosad on the demographic parameters of Habrobracon hebetor (Say) (Hymenoptera: Braconidae) were assessed. Bioassays were carried out on immature and adult stages by using dipping and contact exposure of dry pesticide residue on an inert material, respectively. The lethal concentration (LC)50 values of chlorpyrifos and spinosad were 3.69 and 151.37 ppm, respectively, on the larval stage and 1.75 and 117.37 ppm, respectively, on adults. Hazard quotient (HQ) values for chlorpyrifos and spinosad were 400 and 2.2, respectively, on the larval stage and 857.14 and 2.84, respectively, on adults. A low lethal concentration (LC30) was used to assess the sublethal effects of both pesticides on the surviving females. In each treatment, 25 survivors were randomly selected and transferred into 6-cm Petri dishes. Adults were provided daily with last instars of Anagasta kuehniella (Zeller) as a host until all of the females died. The number of eggs laid, percent of larvae hatched, longevity, and sex ratio were recorded. Stable population growth parameters were estimated by the Jackknife method. In control, chlorpyrifos, and spinosad treatments, the intrinsic rates of increase (r m) values were 0.23, 0.10, and 0.21, respectively. The results of this study suggest a relative compatibility between spinosad use and H. hebetor. Finally, further studies should be conducted under natural conditions to verify the compatibility of spinosad with H. hebetor in IPM programs.

Myocyte-specific enhancer factor 2 and thyroid hormone receptor associate and synergistically activate the alpha-cardiac myosin heavy-chain gene.

The muscle-specific regulatory region of the alpha-cardiac myosin heavy-chain (MHC) gene contains the thyroid hormone response element (TRE) and two A/T-rich DNA sequences, designated A/T1 and A/T2, the putative myocyte-specific enhancer factor 2 (MEF2) binding sites. We investigated the roles of the TRE and MEF2 binding sites and the potential interaction between thyroid hormone receptor (TR) and MEF2 proteins regulating the alpha-MHC promoter. Deletion mutation analysis indicated that both the A/T2 motif and TRE were required for muscle-specific expression of the alpha-MHC gene. The alpha-MHC enhancer containing both the A/T2 motif and TRE was synergistically activated by coexpression of MEF2 and TR in nonmuscle cells, whereas neither factor by itself activated the alpha-MHC reporters. The reporter construct containing the A/T2 sequence and the TRE linked to a heterologous promoter also showed synergistic activation by coexpression of MEF2 and TR in nonmuscle cells. Moreover, protein binding assays demonstrated that MEF2 and TR specifically bound to one another in vitro and in vivo. The MADS domain of MEF2 and the DNA-binding domain of TR were necessary and sufficient to mediate their physical interaction. Our results suggest that the members of the MADS family (MEF2) and steroid receptor superfamily (TR) interact with one another to synergistically activate the alpha-cardiac MHC gene expression.

Determination of the consensus binding site for MEF2 expressed in muscle and brain reveals tissue-specific sequence constraints.

The myocyte-specific enhancer factor-2 (MEF2) proteins are expressed in the three major types of muscle (skeletal, cardiac, and smooth) and function as transcriptional activators of muscle-specific and growth factor-regulated genes through binding to a canonical A/T-rich cis-element. Although MEF2 proteins are also expressed in brain, MEF2-regulated muscle-specific gene products are not detected in this tissue. To gain insight into the regulation of MEF2 function in vivo, we have selected its optimal DNA targets from a library of degenerate oligonucleotides using anti-MEF2A antibodies and cell extracts from skeletal muscle, heart, and brain. The consensus binding site in these three tissues contains an indistinguishable core motif, 5'-CT(A/t)(a/t)AAATAG-3'. However, the optimal target for MEF2 expressed in the brain shows additional sequence constraints (5'-TGTTACT(A/t)(a/t)AAATAGA(A/t)-3') that are not observed in the sequences selected with skeletal and cardiac muscle extracts. Thus, differences in DNA binding preferences of MEF2 proteins in muscle and brain may contribute to tissue-specific gene expression during myogenesis and neurogenesis.

Overexpression of Gs alpha protein in the hearts of transgenic mice.

Alterations in beta-adrenergic receptor-Gs-adenylyl cyclase coupling underlie the reduced catecholamine responsiveness that is a hallmark of human and animal models of heart failure. To study the effect of altered expression of Gs alpha, we overexpressed the short isoform of Gs alpha in the hearts of transgenic mice, using a rat alpha-myosin heavy chain promoter. Gs alpha mRNA levels were increased selectively in the hearts of transgenic mice, with a level 38 times the control. Despite this marked increase in mRNA, Western blotting identified only a 2.8-fold increase in the content of the Gs alpha short isoform, whereas Gs activity was increased by 88%. The discrepancy between Gs alpha mRNA and Gs alpha protein levels suggests that the membrane content of Gs alpha is posttranscriptionally regulated. The steady-state adenylyl cyclase catalytic activity was not altered under either basal or stimulated conditions (GTP + isoproterenol, GTP gamma S, NaF, or forskolin). However, progress curve studies did show a significant decrease in the lag period necessary for GppNHp to stimulate adenylyl cyclase activity. Furthermore, the relative number of beta-adrenergic receptors binding agonist with high affinity was significantly increased. Our data demonstrate that a relatively small increase in the amount of the coupling protein Gs alpha can modify the rate of catalyst activation and the formation of agonist high affinity receptors.

Cardiac myocyte terminal differentiation. Potential for cardiac regeneration.

The exact mechanism of terminal differentiation in cardiac myocytes is currently unknown. Studies in the skeletal muscle system provided a model where muscle lineage termination gene directly interacts with Rb to produce and maintain the terminally differentiated state. This interaction provided the critical components for the lock in cell cycle arrest in skeletal muscle cell. Cardiac muscle appears on the surface very similar to skeletal muscle especially since they share large numbers of structural and contractile proteins. However, it is clear that cardiac muscle cells are distinct biologically at the regulatory level. First and foremost, differentiation and capacity for hyperplasia (mitosis) is not mutually exclusive, in that the heart being the first functional organ embryologically is able to grow via cell division until shortly after birth. Thereafter further growth is provided by hypertrophy. In skeletal muscle, these two processes, differentiation and ability to undergo mitosis, appear to be mutually exclusive. Second, cardiac muscles have not been shown to express any of the skeletal muscle determination basic helix loop helix factors like myoD or any proteins that are functionally similar. Third, heterokaryons of cardiac myocytes and fibroblasts reveal a lack of dominance of the cardiac muscle phenotype. This is distinctly different in skeletal muscle, whose phenotype is dominant which provided a platform to identify the skeletal muscle determination gene, myoD. Although various basic helix loop helix proteins and homeobox genes have been identified in cardiac myocytes, their function remains to be elucidated. At this time no cardiac determination gene has been identified. Despite these differences, we have shown that the biology of pocket proteins Rb and P107 is similar in skeletal and cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the myogenic lineage by MEF2A, a factor that induces and cooperates with MyoD.

Muscle enhancer factor-2A (MEF2A), a member of the MADS family, induced myogenic development when ectopically expressed in clones of nonmuscle cells of human clones, a function previously limited to the muscle basic helix-loop-helix (bHLH) proteins. During myogenesis, MEF2A and bHLH proteins cooperatively activate skeletal muscle genes and physically interact through the MADS domain of MEF2A and the three myogenic amino acids of the muscle bHLH proteins. Thus, skeletal myogenesis is mediated by two distinct families of mutually inducible and interactive muscle transcription factors, either of which can initiate the developmental cascade.

Identification of thyroid hormone receptor isoforms in thyrotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus.

TRH gene expression in hypophysiotropic neurons of the hypothalamic paraventricular nucleus (PVN) is under regulation by thyroid hormone circulating in the bloodstream. To determine whether thyroid hormone could exert effects directly on TRH-producing neurons in the PVN, the presence of thyroid hormone receptors (TR) in these neurons was determined by double labeling immunocytochemical techniques, using specific antiserum to each of the functional TRs, TR alpha 1, TR beta 1, and TR beta 2, followed by antiserum to prepro-TRH-(25-50) as a marker for TRH neurons. In addition, the presence of the TR variant, TR alpha 2, was sought in these cells. Immunoreactive TR alpha 1 and TR beta 2 were found in the greatest percentage of TRH neurons in the PVN (91.1 +/- 2.5% and 83.8 +/- 2.1%) and intensely stained the nucleus. Immunoreactive TR beta 1 was also found in the majority of TRH neurons, but stained PVN cells only lightly compared to the other TRs. TR alpha 2 was found to coexist in only a minority of TRH neurons in the PVN and also lightly immunostained the nucleus compared to its more intense labeling in other regions of the brain. We conclude that hypophysiotropic TRH neurons contain functional TRs, and therefore, these neurons could be directly influenced by thyroid hormone. The relative paucity of TR alpha 2 in these cells could contribute to the selectivity of this population of TRH neurons to the effects of circulating levels of thyroid hormone.

Reversal of terminal differentiation mediated by p107 in Rb-/- muscle cells.

The terminal differentiation of mammalian muscle cells requires the tumor suppressor retinoblastoma protein (Rb). Unlike their wild-type counterparts, multinucleated myotubes from mouse cells deficient in Rb (Rb-/-) were induced by serum to re-enter the cell cycle. Development of the myogenic phenotype in Rb-/- cells correlated with increased expression of p107, which interacted with myogenic transcription factors. Serum-induced cell cycle reentry, on the other hand, correlated with decreased p107 expression. Thus, although p107 could substitute for Rb as a cofactor for differentiation, it could not maintain the terminally differentiated state in Rb-/- myotubes.

A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeo domain of the cut homeo proteins.

The recently cloned Clox (Cut-like homeo box) and CDP (CCAAT displacement protein), two mammalian counterparts of the Drosophila Cut homeo protein, correspond to alternatively spliced products of the same gene (mClox, for mammalian Cut-like homeo box). Although these proteins reportedly bind to apparently unrelated DNA sequences, we show by in vitro selection of optimal binding sites that both Clox and CDP have the same preferred DNA-binding specificity. The palindromic consensus target sequence, 5'-(t/a)(a/t)tATCGATTAt(t/c)(t/g)(t/a)-3', contains a bona fide homeo domain binding motif (ATTA). In addition, 37% of the in vitro-selected sequences have a CCAAT box, the canonical target for members of the family of CCAAT-binding factors. A characteristic feature of the cut homeo proteins is the presence of three evolutionarily conserved 73-amino-acid repeats of unknown function, the so-called cut repeats. We present evidence that the cut repeat II binds to mClox consensus targets independently of the DNA-binding activity of the homeo domain. In vitro selection of binding sites shows that the optimal targets for the cut repeat II contain one or more CCAAT boxes and, like the homeo domain, an ATTA core. These results indicate that the DNA-binding activity of the second cut repeat can account for the suggested role of CDP mClox as CCAAT displacement protein, a putative repressor of gene expression. We also report that the mClox homeo domain and cut repeat II interact in vitro in the absence of DNA. This interaction, which greatly enhances the DNA-binding activity of the binary complex, is specific to the cut homeo proteins. No cooperativity was observed between the cut repeat II and the homeo domains of Oct-1 and Gtx. Furthermore, the Drosophila cut repeat II, which does not appear to bind to DNA, also enhances the DNA-binding activity of the mClox homeo domain. Thus, the bifunctional cut repeat II, which defines a new family of bipartite DNA-binding proteins, is likely to play an important role in the function of the cut homeo proteins.

Basic mechanisms of cardiac gene expression.

Although the physiological properties of the myocardium and their dynamic character have been the focus of intense research during the past three decades, the biochemical and molecular correlates underlying cardiac development and performance have, until recently, remained poorly understood. The development of modern biology has provided the necessary tools to undertake the study of the mechanisms involved in cardiac development and to understand the basis for important clinical and experimental problems in cardiovascular physiology. Most of the gene encoding contractile proteins have been cloned and characterized. The availability of molecular probes and the ability to introduce genes into individual cell types and tissues of living animals are the most important breakthroughs of molecular and cell biology. This permits not only analysis of basic mechanisms of gene expression but has also significant practical applications for gene therapy. It is now possible to analyse the role of different regulatory gene sequences and identify their corresponding transactive factors. In addition direct gene injection makes it possible to study gene expression in a natural context, under conditions that are physiologically relevant and controllable.

A fourth human MEF2 transcription factor, hMEF2D, is an early marker of the myogenic lineage.

The transition from multipotent mesodermal precursor to committed myoblast and its differentiation into a mature myocyte involve molecular events that enable the cell to activate muscle-specific genes. Among the participants in this process is the myocyte-specific enhancer factor 2 (MEF2) family of tissue-restricted transcription factors. These factors, which share a highly conserved DNA-binding domain including a MADS box, are essential for the expression of multiple muscle genes with cognate target MEF2 sites in cis. We report here a new human MEF2 factor, hMEF2D, which is unique among the members of this family in that it is present not only in myotubes but also in undifferentiated myoblasts, even before the appearance of myogenin. hMEF2D comprises several alternatively spliced products of a single gene, one of which is the human homolog of the Xenopus SRF-related factor SL-1. Like its relatives, cloned hMEF2D is capable of activating transcription via sequence-specific binding to the MEF2 site, recapitulating endogenous tissue-specific MEF2 activity. Indeed, while MEF2D mRNAs are ubiquitous, the protein is highly restricted to those cell types that contain this activity, implicating posttranscriptional mechanisms in the regulation of MEF2D expression. Alternative splicing may be important in this process: two alternative MEF2D domains, at least one of which is specifically included during myogenic differentiation, also correlate precisely with endogenous MEF2 activity. These findings provide compelling evidence that MEF2D is an integral link in the regulatory network for muscle gene expression. Its presence in undifferentiated myoblasts further suggests that it may be a mediator of commitment in the myogenic lineage.

Gene injection into canine myocardium as a useful model for studying gene expression in the heart of large mammals.

We have investigated the regulated expression of genes injected into the heart of large mammals in situ. Reporter constructs using the chloramphenicol acetyltransferase gene under the control of muscle-specific beta-myosin heavy chain (beta-MHC) or promiscuous (mouse sarcoma virus) promoters were injected into the canine myocardium. There was a linear dose-response relation between the level of gene expression and the quantity of plasmid DNA injected between 10 and 200 micrograms per injection site. The level of reporter gene expression did not correlate with the amount of injury imposed on the cardiac tissue. There was no regional variation in expression of injected reporter genes throughout the left ventricular wall. By use of both the mouse sarcoma virus and a muscle-specific beta-MHC promoter, reporter gene expression was one to two orders of magnitude greater in the heart than in skeletal muscle. Expression in the left ventricle was threefold higher than in the right ventricle. Chloramphenicol acetyltransferase activity was detected at 3, 7, 14, and 21 days after injection, with maximal expression at 7 days after injection. Statistical analysis of coinjection experiments revealed that coinjection of a second gene construct (Rous sarcoma virus-luciferase) is useful in the control of transfection efficiency in vivo. Furthermore, using reporter constructs containing serial deletions of the 5' flanking region of the beta-MHC gene, we performed a series of experiments that demonstrate the utility of this model in mapping promoter regions and identifying important regulatory gene sequences in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation.

The experiments reported here document that the tumor suppressor retinoblastoma protein (pRB) plays an important role in the production and maintenance of the terminally differentiated phenotype of muscle cells. We show that pRB inactivation, through either phosphorylation, binding to T antigen, or genetic alteration, inhibits myogenesis. Moreover, inactivation of pRB in terminally differentiated cells allows them to reenter the cell cycle. In addition to its involvement in the myogenic activities of MyoD, pRB is also required for the cell growth-inhibitory activity of this myogenic factor. We also show that pRB and MyoD directly bind to each other, both in vivo and in vitro, through a region that involves the pocket and the basic-helix-loop-helix domains, respectively. All the results obtained are consistent with the proposal that the effects of MyoD on the cell cycle and of pRB on the myogenic pathway result from the direct binding of the two molecules.

Molecular mechanisms of cardiac gene expression.

Although the physiological properties of the myocardium and their dynamic character have been the focus of intense research during the past three decades, the biochemical and molecular correlates underlying cardiac development and performance have, until recently, remained poorly understood. The development of modern cellular and molecular biology has provided the necessary tools to undertake the study of the mechanisms involved in cardiac development and to understand the basis for important clinical and experimental problems in cardiovascular physiology. Most of the gene encoding contractile proteins have been cloned and characterized. The availability of molecular probes and the ability to introduce genes into individual cell types and tissues of living animals, are the most important breakthroughs of molecular and cell biology. This permits not only to analyze basic mechanisms of gene expression but has also significant practical applications for gene therapy. It is now possible to analyze the role of different regulatory gene sequences and identify their corresponding trans-active factors. In addition, direct gene injection makes it possible to study gene expression in a natural context, under conditions that are physiologically relevant and controllable.

The D domain of the thyroid hormone receptor alpha 1 specifies positive and negative transcriptional regulation functions.

Four structural domains are characteristic of the members of the thyroid/steroid receptor superfamily. Of these, the A/B and D domains are the least conserved. We have investigated the role of two clusters of positively charged amino acids within the D domain of the thyroid hormone receptor alpha 1 (TR alpha 1). The sequences Lys134-Arg-Lys and Arg188-Arg-Lys, individually or together, were substituted to the neutral residues TIT in three mutants named alpha 1-1, alpha 1-2, and alpha 1-3, respectively. Subcellular localization of transiently transfected wild-type and mutated TRs was monitored by immunostaining, using a TR alpha 1-specific antibody. The wild-type and the alpha 1-2 TRs were detected exclusively in the nucleus, in the presence or absence of thyroid hormone. In contrast, the alpha 1-1 and alpha 1-3 mutants accumulated in both cytoplasm and nucleus, underscoring the importance of the Lys134-Arg-Lys residues for correct nuclear targeting. More importantly, although the mutants had unimpaired DNA- and hormone-binding activities, all three had lost positive and negative transcriptional regulatory functions. Thus, transactivation and repression functions can be entirely dissociated from the other properties of the receptor. In addition, substitution of either one of the positively charged amino acid clusters was sufficient to convert the native TR alpha 1 into a dominant, thyroid hormone-dependent receptor antagonist. These observations, which underline the functional relevance of the D domain for TR alpha 1 function, may also have implications for the autosomal dominant syndrome of generalized resistance to thyroid hormone.

Clox, a mammalian homeobox gene related to Drosophila cut, encodes DNA-binding regulatory proteins differentially expressed during development.

We report the isolation of a cDNA encoding a mammalian homeoprotein related to the Drosophila cut gene product, called Clox, for Cut like homeobox. In addition to the homeodomain, three 73-amino acid repeats, the so-called cut repeats, are also conserved between Cut and the mammalian counterpart described here. This conservation suggests that the cut repeat motif may define a new class of homeoproteins. Both cloned and endogenous Clox proteins are nuclear DNA-binding proteins with very similar sequence specificity. Western blot analysis revealed several distinct Clox protein species in a variety of tissues and cell types. The relative abundance of these proteins is regulated during mouse development and cell differentiation in culture. Interestingly, approximately 180-190 x 10(3) M(r) Clox proteins predominate in early embryos and are upregulated in committed myoblasts and chondrocytes, but downregulated upon terminal differentiation. Clox DNA-binding activity is correlated with the abundance of these proteins. In contrast, larger Clox protein species (approximately 230-250 x 10(3) M(r)) are detected mainly in adult tissues and in terminally differentiated cells. Cotransfection experiments show that Clox proteins can function as repressors of tissue-specific gene transcription. Thus, Clox, like their Drosophila counterparts, are candidate regulators of cell-fate specification in diverse differentiation programs.

Human myocyte-specific enhancer factor 2 comprises a group of tissue-restricted MADS box transcription factors.

The MEF2 site is an essential element of muscle enhancers and promoters that is bound by a nuclear activity found, so far, only in muscle and required for tissue-specific transcription. We have cloned a group of transcription factors from human muscle that are responsible for this activity: They are present in muscle-specific DNA-binding complexes, have a target sequence specificity identical to that of the endogenous activity, and are MEF2 site-dependent transcriptional activators. These MEF2 proteins comprise several alternatively spliced isoforms from one gene and a related factor encoded by a second gene. All share a conserved amino-terminal DNA-binding domain that includes the MADS homology. MEF2 transcripts are ubiquitous but accumulate preferentially in skeletal muscle, heart, and brain. Specific alternatively spliced isoforms are restricted to these tissues, correlating exactly with the presence of endogenous MEF2 activity. Furthermore, MEF2 protein is detected only in skeletal and cardiac muscle nuclei and not in myoblast and nonmuscle cells. Thus, post-transcriptional regulation is important in the generation of tissue-specific MEF2 activity. Cardiac and smooth, as well as skeletal, muscles contain functionally saturating levels of MEF2 trans-activating factors that are absent in nonmuscle cells. Moreover, MEF2 is induced in nonmuscle cells by MyoD; however, MEF2 alone is insufficient to produce the full muscle phenotype. Implications for the molecular mechanisms of myogenesis are considered.

A MyoD1-independent muscle-specific enhancer controls the expression of the beta-myosin heavy chain gene in skeletal and cardiac muscle cells.

The beta-cardiac myosin heavy chain is the major contractile protein expressed in two sarcomeric muscles of distinct embryologic origins, the ventricular myocardium and slow twitch skeletal muscle. Characterization of the cis-acting regulatory sequences of the human and the rat beta-MHC genes established that their expression in these two muscle types is controlled, at least in part, by common mechanisms involving a muscle-specific enhancer. This enhancer consists of distinct but cooperative subelements that interact with muscle-specific nuclear proteins. In contrast to other muscle-specific enhancers, the beta-MHC gene enhancer is unresponsive, directly or indirectly, to the muscle lineage-determining and muscle gene-transactivating helix-loop-helix factors MyoD and myogenin. A MyoD-binding site in the rat beta-MHC promoter is not required for transcriptional activity in skeletal and cardiac cells, but is necessary for activation in 10T1/2 and CV1 cells transfected with MyoD. In addition, this element is absent from the human beta-MHC promoter. Thus, MyoD and MyoD-related processes are neither required nor sufficient for the expression of the beta-MHC gene either in cardiac or skeletal muscle cells. These observations provide evidence for the existence of myogenic regulatory programs that precede and/or differ from those governed by known myogenic helix-loop-helix transactivators.

A retinoic acid-responsive element in the apolipoprotein AI gene distinguishes between two different retinoic acid response pathways.

The gene coding for apolipoprotein AI, a plasma protein involved in the transport of cholesterol and other lipids in the plasma, is expressed predominantly in liver and intestine. Previous work in our laboratory has shown that hepatocyte-specific expression is determined by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located in the region -222 to -110 nucleotides upstream of the apolipoprotein AI gene transcription start site (+1). In this study, it was found that site A is a highly selective retinoic acid-responsive element (RARE) that responds preferentially to the recently identified retinoic acid receptor RXR alpha over the previously characterized retinoic acid receptors RAR alpha and RAR beta. Control experiments indicated that a RARE in the regulatory region of the laminin B1 gene responds preferentially to RAR alpha and RAR beta over RXR alpha, while a previously described palindromic thyroid hormone-responsive element responds similarly to all three of these receptors. Gel retardation experiments showed that the activity of these RAREs is concordant with receptor binding. These results indicate that different RAREs may play a fundamental role in defining distinctive retinoic acid cellular response pathways and suggest that retinoic acid response pathways mediated by RXR alpha play an important role in cholesterol and retinoid transport and metabolism.