Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca2+-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension–Ca2+ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca2+ activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca2+-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca2+ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.

Transient “deafness” accompanies auditory development during metamorphosis from tadpole to frog

During metamorphosis, ranid frogs shift from a purely aquatic to a partly terrestrial lifestyle. The central auditory system undergoes functional and neuroanatomical reorganization in parallel with the development of new sound conduction pathways adapted for the detection of airborne sounds. Neural responses to sounds can be recorded from the auditory midbrain of tadpoles shortly after hatching, with higher rates of synchronous neural activity and lower sharpness of tuning than observed in postmetamorphic animals. Shortly before the onset of metamorphic climax, there is a brief “deaf” period during which no auditory activity can be evoked from the midbrain, and a loss of connectivity is observed between medullary and midbrain auditory nuclei. During the final stages of metamorphic development, auditory function and neural connectivity are restored. The acoustic communication system of the adult frog emerges from these periods of anatomical and physiological plasticity during metamorphosis.

Correlation of α-Fetoprotein Expression in Normal Hepatocytes during Development with Tyrosine Phosphorylation and Insulin Receptor Expression

The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the α-fetoprotein-producing (AFP+) and AFP-nonproducing (AFP−) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP+ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP+ clones and likewise for adult hepatocytes and AFP− clones. The tyrosine phosphorylation of several proteins, including the β-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, and ras-guanosine triphosphatase-activating protein, was observed in AFP+ clones, whereas the same proteins were not phosphorylated in AFP− clones. We also observed that fetal hepatocytes and the AFP+ clones express 4 times more of the insulin receptor β-subunit compared with adult hepatocytes and AFP− clones and, accordingly, that these AFP+ clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP+ clones was higher than that measured in cells of AFP− clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP+ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.

Biogenesis of Polarized Epithelial Cells During Kidney Development In Situ: Roles of E-Cadherin–mediated Cell–Cell Adhesion and Membrane Cytoskeleton Organization

Organization of proteins into structurally and functionally distinct plasma membrane domains is an essential characteristic of polarized epithelial cells. Based on studies with cultured kidney cells, we have hypothesized that a mechanism for restricting Na/K-ATPase to the basal-lateral membrane involves E-cadherin–mediated cell–cell adhesion and integration of Na/K-ATPase into the Triton X-100–insoluble ankyrin- and spectrin-based membrane cytoskeleton. In this study, we examined the relevance of these in vitro observations to the generation of epithelial cell polarity in vivo during mouse kidney development. Using differential detergent extraction, immunoblotting, and immunofluorescence histochemistry, we demonstrate the following. First, expression of the 220-kDa splice variant of ankyrin-3 correlates with the development of resistance to Triton X-100 extraction for Na/K-ATPase, E-cadherin, and catenins and precedes maximal accumulation of Na/K-ATPase. Second, expression of the 190-kDa slice variant of ankyrin-3 correlates with maximal accumulation of Na/K-ATPase. Third, Na/K-ATPase, ankyrin-3, and fodrin specifically colocalize at the basal-lateral plasma membrane of all epithelial cells in which they are expressed and during all stages of nephrogenesis. Fourth, the relative immunofluorescence staining intensities of Na/K-ATPase, ankyrin-3, and fodrin become more similar during development until they are essentially identical in adult kidney. Thus, renal epithelial cells in vivo regulate the accumulation of E-cadherin–mediated adherens junctions, the membrane cytoskeleton, and Na/K-ATPase through sequential protein expression and assembly on the basal-lateral membrane. These results are consistent with a mechanism in which generation and maintenance of polarized distributions of these proteins in vivo and in vitro involve cell–cell adhesion, assembly of the membrane cytoskeleton complex, and concomitant integration and retention of Na/K-ATPase in this complex.

Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction

Previous studies have shown that proinflammatory cytokines, such as tumor necrosis factor (TNF), are expressed after acute hemodynamic overloading and myocardial ischemia/infarction. To define the role of TNF in the setting of ischemia/infarction, we performed a series of acute coronary artery occlusions in mice lacking one or both TNF receptors. Left ventricular infarct size was assessed at 24 h after acute coronary occlusion by triphenyltetrazolium chloride (TTC) staining in wild-type (both TNF receptors present) and mice lacking either the type 1 (TNFR1), type 2 (TNFR2), or both TNF receptors (TNFR1/TNFR2). Left ventricular infarct size as assessed by TTC staining was significantly greater (P < 0.005) in the TNFR1/TNFR2-deficient mice (77.2% ± 15.3%) when compared with either wild-type mice (46.8% ± 19.4%) or TNFR1-deficient (47.9% ± 10.6%) or TNFR2-deficient (41.6% ± 16.5%) mice. Examination of the extent of necrosis in wild-type and TNFR1/TNFR2-deficient mice by anti-myosin Ab staining demonstrated no significant difference between groups; however, the peak frequency and extent of apoptosis were accelerated in the TNFR1/TNFR2-deficient mice when compared with the wild-type mice. The increase in apoptosis in the TNFR1/TNFR2-deficient mice did not appear to be secondary to a selective up-regulation of the Fas ligand/receptor system in these mice. These data suggest that TNF signaling gives rise to one or more cytoprotective signals that prevent and/or delay the development of cardiac myocyte apoptosis after acute ischemic injury.

Expression of mouse telomerase catalytic subunit in embryos and adult tissues

Telomerase is a ribonucleoprotein complex that elongates telomeres, allowing the stable maintenance of chromosomes during multiple cell divisions. Here, we describe the isolation and characterization of the catalytic subunit of mouse telomerase, mTERT (mouse telomerase reverse transcriptase), an essential protein component of the telomerase complex. During embryonic development, mTERT mRNA is abundantly expressed in the whole embryo, especially in regions of intense proliferation. We found that the mTERT mRNA expression in both embryonic and adult tissues is independent of the essential RNA component of telomerase, mTR, and therefore, of the formation of active telomerase complexes. mTERT protein is present exclusively in tissues with telomerase activity, such as testis, spleen, and thymus. mTERT protein is barely detectable in the thymus of mTR−/− mice, suggesting that mTERT protein stability in this tissue may depend on the actual assembly of active telomerase complexes. Finally, we found that mouse and human telomerase catalytic subunit is located in the cell nucleus, and its localization is not regulated during cell cycle progression.

Identification of two novel transmembrane γ-carboxyglutamic acid proteins expressed broadly in fetal and adult tissues

The proline-rich γ-carboxyglutamic acid (Gla) proteins (PRGPs) 1 and 2 are the founding members of a family of vitamin K-dependent single-pass integral membrane proteins characterized by an extracellular amino terminal domain of approximately 45 amino acids that is rich in Gla. The intracellular carboxyl terminal region of these two proteins contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover. In this report, we describe the cloning of the cDNAs for two additional human transmembrane Gla proteins (TMG) of 20–24 kDa named TMG3 and TMG4. These two proteins possess extracellular Gla domains with 13 or 9 potential Gla residues, respectively, followed by membrane-spanning hydrophobic regions and cytoplasmic carboxyl terminal regions that contain PPXY motifs. This emerging family of integral membrane Gla proteins includes proline-rich Gla protein (PRGP) 1, PRGP2, TMG3, and TMG4, all of which are characterized by broad and variable distribution in both fetal and adult tissues. Members of this family can be grouped into two subclasses on the basis of their gene organization and amino acid sequence. These observations suggest novel physiological functions for vitamin K beyond its known role in the biosynthesis of proteins involved in blood coagulation and bone development. The identification and characterization of these proteins may allow a more complete understanding of the teratogenic consequences of exposure in utero to vitamin K antagonists, such as warfarin-based anticoagulants.

Negative genetic correlation for adult fitness between sexes reveals ontogenetic conflict in Drosophila

Because of their distinctive roles in reproduction, females and males are selected toward different optimal phenotypes. Ontogenetic conflict between the sexes arises when homologous traits are selected in different directions. The evolution of sexual dimorphism by sex-limited gene expression alleviates this problem. However, because the majority of genes are not sex-limited, the potential for substantial conflict may remain. Here we assess the degree of ontogenetic conflict in the fruit-fly, Drosophila melanogaster, by cloning 40 haploid genomes and measuring their Darwinian fitness in both sexes. The intersexual genetic correlations for juvenile viability, adult reproductive success, and total fitness were used to gauge potential conflict during development. First, as juveniles, where the fitness objectives of the two sexes appear to be similar, survival was strongly positively correlated across sexes. Second, after adult maturation, where gender roles diverge, a significant negative correlation for reproductive success was found. Finally, because of counterbalancing correlations in the juvenile and adult components, no intersexual correlation for total fitness was found. Highly significant genotype-by-gender interaction variance was measured for both adult and total fitness. These results demonstrate strong intersexual discord during development because of the expression of sexually antagonistic variation.

Profilin I is essential for cell survival and cell division in early mouse development

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1ko) allele in mice. Homozygous pfn1ko/ko mice are not viable. Pfn1ko/ko embryos died as early as the two-cell stage, and no pfn1ko/ko blastocysts were detectable. Adult pfn1ko/wt mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1ko/wt embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.

Changes in global gene expression patterns during development and maturation of the rat kidney

We set out to define patterns of gene expression during kidney organogenesis by using high-density DNA array technology. Expression analysis of 8,740 rat genes revealed five discrete patterns or groups of gene expression during nephrogenesis. Group 1 consisted of genes with very high expression in the early embryonic kidney, many with roles in protein translation and DNA replication. Group 2 consisted of genes that peaked in midembryogenesis and contained many transcripts specifying proteins of the extracellular matrix. Many additional transcripts allied with groups 1 and 2 had known or proposed roles in kidney development and included LIM1, POD1, GFRA1, WT1, BCL2, Homeobox protein A11, timeless, pleiotrophin, HGF, HNF3, BMP4, TGF-α, TGF-β2, IGF-II, met, FGF7, BMP4, and ganglioside-GD3. Group 3 consisted of transcripts that peaked in the neonatal period and contained a number of retrotransposon RNAs. Group 4 contained genes that steadily increased in relative expression levels throughout development, including many genes involved in energy metabolism and transport. Group 5 consisted of genes with relatively low levels of expression throughout embryogenesis but with markedly higher levels in the adult kidney; this group included a heterogeneous mix of transporters, detoxification enzymes, and oxidative stress genes. The data suggest that the embryonic kidney is committed to cellular proliferation and morphogenesis early on, followed sequentially by extracellular matrix deposition and acquisition of markers of terminal differentiation. The neonatal burst of retrotransposon mRNA was unexpected and may play a role in a stress response associated with birth. Custom analytical tools were developed including “The Equalizer” and “eBlot,” which contain improved methods for data normalization, significance testing, and data mining.

The copper transporter CTR1 provides an essential function in mammalian embryonic development

Copper serves as an essential cofactor for a variety of proteins in all living organisms. Previously, we described a human gene (CTR1;SLC31A1) that encodes a high-affinity copper-uptake protein and hypothesized that this protein is required for copper delivery to mammalian cells. Here, we test this hypothesis by inactivating the Ctr1 gene in mice by targeted mutagenesis. We observe early embryonic lethality in homozygous mutant embryos and a deficiency in copper uptake in the brains of heterozygous animals. Ctr1−/− embryos can be recovered at E8.5 but are severely developmentally retarded and morphologically abnormal. Histological analysis reveals discontinuities and variable thickness in the basement membrane of the embryonic region and an imperfect Reichert's membrane, features that are likely due to lack of activity in the collagen cross-linking cupro-enzyme lysyl oxidase. A collapsed embryonic cavity, the absence of an allantois, retarded mesodermal migration, and increased cell death are also apparent. In the brains of heterozygous adult mice, which at 16 months are phenotypically normal, copper is reduced to approximately half compared with control littermates, implicating CTR1 as the required port for copper entry into at least this organ. A study of the spatial and temporal expression pattern of Ctr1 during mouse development and adulthood further shows that CTR1 is ubiquitously transcribed with highest expression observed in the specialized epithelia of the choroid plexus and renal tubules and in connective tissues of the eye, ovary, and testes. We conclude that CTR1 is the primary avenue for copper uptake in mammalian cells.

γ-Secretase inhibitors repress thymocyte development

A major therapeutic target in the search for a cure to the devastating Alzheimer's disease is γ-secretase. This activity resides in a multiprotein enzyme complex responsible for the generation of Aβ42 peptides, precipitates of which are thought to cause the disease. γ-Secretase is also a critical component of the Notch signal transduction pathway; Notch signals regulate development and differentiation of adult self-renewing cells. This has led to the hypothesis that therapeutic inhibition of γ-secretase may interfere with Notch-related processes in adults, most alarmingly in hematopoiesis. Here, we show that application of γ-secretase inhibitors to fetal thymus organ cultures interferes with T cell development in a manner consistent with loss or reduction of Notch1 function. Progression from an immature CD4−/CD8− state to an intermediate CD4+/CD8+ double-positive state was repressed. Furthermore, treatment beginning later at the double-positive stage specifically inhibited CD8+ single-positive maturation but did not affect CD4+ single-positive cells. These results demonstrate that pharmacological γ-secretase inhibition recapitulates Notch1 loss in a vertebrate tissue and present a system in which rapid evaluation of γ-secretase-targeted pharmaceuticals for their ability to inhibit Notch activity can be performed in a relevant context.

Cloning of a Tobacco Apoplasmic Invertase Inhibitor1 : Proof of Function of the Recombinant Protein and Expression Analysis during Plant Development

Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.

Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1.

The X chromosome-linked transcription factor GATA-1 is expressed specifically in erythroid, mast, megakaryocyte, and eosinophil lineages, as well as in hematopoietic progenitors. Prior studies revealed that gene-disrupted GATA-1- embryonic stem cells give rise to adult (or definitive) erythroid precursors arrested at the proerythroblast stage in vitro and fail to contribute to adult red blood cells in chimeric mice but did not clarify a role in embryonic (or yolk sac derived) erythroid cells. To examine the consequences of GATA-1 loss on embryonic erythropoiesis in vivo, we inactivated the GATA-1 locus in embryonic stem cells by gene targeting and transmitted the mutated allele through the mouse germ line. Male GATA-1- embryos die between embryonic day 10.5 and 11.5 (E10.5-E11.5) of gestation. At E9.5, GATA-1- embryos exhibit extreme pallor yet contain embryonic erythroid cells arrested at an early proerythroblast-like stage of their development. Embryos stain weakly with benzidine reagent, and yolk sac cells express globin RNAs, indicating globin gene activation in the absence of GATA-1. Female heterozygotes (GATA-1+/-) are born pale due to random inactivation of the X chromosome bearing the normal allele. However, these mice recover during the neonatal period, presumably as a result of in vivo selection for progenitors able to express GATA-1. Our findings conclusively establish the essential role for GATA-1 in erythropoiesis within the context of the intact developing mouse and further demonstrate that the block to cellular maturation is similar in GATA-1- embryonic and definitive erythroid precursors. Moreover, the recovery of GATA-1+/- mice from anemia seen at birth provides evidence indicating a role for GATA-1 at the hematopoietic progenitor cell level.

Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.

We describe the construction of a safe, replication-defective and efficient lentiviral vector suitable for in vivo gene delivery. The reverse transcription of the vector was found to be a rate-limiting step; therefore, promoting the reaction inside the vector particles before delivery significantly enhanced the efficiency of gene transfer. After injection into the brain of adult rats, sustained long-term expression of the transgene was obtained in the absence of detectable pathology. A high proportion of the neurons in the areas surrounding the injection sites of the vector expressed the transduced beta-galactosidase gene. This pattern was invariant in animals sacrificed several months after a single administration of the vector. Transduction occurs by integration of the vector genome, as it was abolished by a single amino acid substitution in the catalytic site of the integrase protein incorporated in the vector. Development of clinically acceptable derivatives of the lentiviral vector may thus enable the sustained delivery of significant amounts of a therapeutic gene product in a wide variety of somatic tissues.