Secondary prevention in diabetic and nondiabetic coronary heart disease patients: Insights from the German subset of the hospital arm of the EUROASPIRE IV and V surveys.

BACKGROUND: Patients with coronary heart disease (CHD) with and without diabetes mellitus have an increased risk of recurrent events requiring multifactorial secondary prevention of cardiovascular risk factors. We compared prevalences of cardiovascular risk factors and its determinants including lifestyle, pharmacotherapy and diabetes mellitus among patients with chronic CHD examined within the fourth and fifth EUROASPIRE surveys (EA-IV, 2012-13; and EA-V, 2016-17) in Germany. METHODS: The EA initiative iteratively conducts European-wide multicenter surveys investigating the quality of secondary prevention in chronic CHD patients aged 18 to 79 years. The data collection in Germany was performed during a comprehensive baseline visit at study centers in Würzburg (EA-IV, EA-V), Halle (EA-V), and Tübingen (EA-V). RESULTS: 384 EA-V participants (median age 69.0 years, 81.3% male) and 536 EA-IV participants (median age 68.7 years, 82.3% male) were examined. Comparing EA-IV and EA-V, no relevant differences in risk factor prevalence and lifestyle changes were observed with the exception of lower LDL cholesterol levels in EA-V. Prevalence of unrecognized diabetes was significantly lower in EA-V as compared to EA-IV (11.8% vs. 19.6%) while the proportion of prediabetes was similarly high in the remaining population (62.1% vs. 61.0%). CONCLUSION: Between 2012 and 2017, a modest decrease in LDL cholesterol levels was observed, while no differences in blood pressure control and body weight were apparent in chronic CHD patients in Germany. Although the prevalence of unrecognized diabetes decreased in the later study period, the proportion of normoglycemic patients was low. As pharmacotherapy appeared fairly well implemented, stronger efforts towards lifestyle interventions, mental health programs and cardiac rehabilitation might help to improve risk factor profiles in chronic CHD patients.

Long-term breeding progress of yield, yield-related, and disease resistance traits in five cereal crops of German variety trials.

KEY MESSAGE: Considerable breeding progress in cereal and disease resistances, but not in stem stability was found. Ageing effects decreased yield and increased disease susceptibility indicating that new varieties are constantly needed. Plant breeding and improved crop management generated considerable progress in cereal performance over the last decades. Climate change, as well as the political and social demand for more environmentally friendly production, require ongoing breeding progress. This study quantified long-term trends for breeding progress and ageing effects of yield, yield-related traits, and disease resistance traits from German variety trials for five cereal crops with a broad spectrum of genotypes. The varieties were grown over a wide range of environmental conditions during 1988-2019 under two intensity levels, without (I1) and with (I2) fungicides and growth regulators. Breeding progress regarding yield increase was the highest in winter barley followed by winter rye hybrid and the lowest in winter rye population varieties. Yield gaps between I2 and I1 widened for barleys, while they shrank for the other crops. A notable decrease in stem stability became apparent in I1 in most crops, while for diseases generally a decrasing susceptibility was found, especially for mildew, brown rust, scald, and dwarf leaf rust. The reduction in disease susceptibility in I2 (treated) was considerably higher than in I1. Our results revealed that yield performance and disease resistance of varieties were subject to considerable ageing effects, reducing yield and increasing disease susceptibility. Nevertheless, we quantified notable achievements in breeding progress for most disease resistances. This study indicated an urgent and continues need for new improved varieties, not only to combat ageing effects and generate higher yield potential, but also to offset future reduction in plant protection intensity.

Breeding progress of disease resistance and impact of disease severity under natural infections in winter wheat variety trials.

KEY MESSAGE: Breeding progress of resistance to fungal wheat diseases and impact of disease severity on yield reduction in long-term variety trials under natural infection were estimated by mixed linear regression models. This study aimed at quantifying breeding progress achieved in resistance breeding towards varieties with higher yield and lower susceptibility for 6 major diseases, as well as estimating decreasing yields and increasing disease susceptibility of varieties due to ageing effects during the period 1983-2019. A further aim was the prediction of disease-related yield reductions during 2005-2019 by mixed linear regression models using disease severity scores as covariates. For yield and all diseases, overall progress of the fully treated intensity (I2) was considerably higher than for the intensity without fungicides and growth regulators (I1). The disease severity level was considerably reduced during the study period for mildew (MLD), tan spot (DTR) and Septoria nodorum blotch (ear) (SNB) and to a lesser extent for brown (leaf) rust (BNR) and Septoria tritici blotch (STB), however, not for yellow/stripe rust (YLR). Ageing effects increased susceptibility of varieties strongly for BNR and MLD, but were comparatively weak for SNB and DTR. Considerable yield reductions under high disease severity were predicted for STB (-6.6%), BNR (-6.5%) and yellow rust (YLR, -5.8%), but lower reductions for the other diseases. The reduction for resistant vs. highly susceptible varieties under high severity conditions was about halved for BNR and YLR, providing evidence of resistance breeding progress. The empirical evidence on the functional relations between disease severity, variety susceptibility and yield reductions based on a large-scale multiple-disease field trial data set in German winter wheat is an important contribution to the ongoing discussion on fungicide use and its environmental impact.

Neural precursor cells possess multiple p53-dependent apoptotic pathways.

Neural precursor cells (NPCs) are markedly sensitive to apoptotic insults. p53-Dependent transcriptional activation of proapoptotic genes has been hypothesized to regulate NPC death in response to DNA damage. Recent studies of non-NPCs have also indicated that p53 may directly interact with Bcl-2 molecules and thereby regulate death independently of transcription. The contribution of transcription-independent p53 activation in NPC death has not been characterized. In this study, we found that apoptosis caused by chemotherapeutic agents in NPCs required p53 expression and new macromolecular synthesis. In contrast, NPC death induced by staurosporine, a broad kinase inhibitor, is regulated by p53 in the absence of macromolecular synthesis. The apoptosis effector molecules Bax and Bak, Apaf-1, and caspase-9 were shown to be downstream of p53 in both pathways. These findings indicate that p53 is in a unique position to regulate at least two distinct signaling portals that activate the intrinsic apoptotic death pathway in NPCs.

MatInspector and beyond: promoter analysis based on transcription factor binding sites.

MOTIVATION: Promoter analysis is an essential step on the way to identify regulatory networks. A prerequisite for successful promoter analysis is the prediction of potential transcription factor binding sites (TFBS) with reasonable accuracy. The next steps in promoter analysis can be tackled only with reliable predictions, e.g. finding phylogenetically conserved patterns or identifying higher order combinations of sites in promoters of co-regulated genes. RESULTS: We present a new version of the program MatInspector that identifies TFBS in nucleotide sequences using a large library of weight matrices. By introducing a matrix family concept, optimized thresholds, and comparative analysis, the enhanced program produces concise results avoiding redundant and false-positive matches. We describe a number of programs based on MatInspector allowing in-depth promoter analysis (DiAlignTF, FrameWorker) and targeted design of regulatory sequences (SequenceShaper).

Leaf-rust resistance in rye (Secale cereale L.). 2. Genetic analysis and mapping of resistance genes Pr3, Pr4, and Pr5.

Three dominant resistance genes, Pr3, Pr4, and Pr5, were identified by genetic analysis of resistance to leaf rust in rye (Puccinia recondita f. sp. secalis). Each of the three genes confers resistance to a broad scale of single-pustule isolates (SPIs), but differences could be observed for specific Pr gene/SPI combinations. Resistance conferred by the three genes was effective in both detached-leaf tests carried out on seedlings and in field tests of adult plants. Molecular marker analysis mapped Pr3 to the centromeric region of rye chromosome arm 1RS, whereas Pr4 and Pr5 were assigned to the centromeric region of 1RL. Chromosomal localization and reaction patterns to specific SPIs provide evidence that the three Pr genes represent distinct and novel leaf-rust resistance genes in rye. The contributions of these genes to resistance breeding in rye and wheat are discussed.

Ethanol-induced neuronal apoptosis in vivo requires BAX in the developing mouse brain.

A single episode of ethanol intoxication triggers widespread apoptotic neurodegeneration in the infant rat or mouse brain. The cell death process occurs over a 6-16 h period following ethanol administration, is accompanied by a robust display of caspase-3 enzyme activation, and meets ultrastructural criteria for apoptosis. Two apoptotic pathways (intrinsic and extrinsic) have been described, either of which may culminate in the activation of caspase-3. The intrinsic pathway is regulated by Bax and Bcl-XL and involves Bax-induced mitochondrial dysfunction and release of cytochrome c as antecedent events leading to caspase-3 activation. Activation of caspase-8 is a key event preceding caspase-3 activation in the extrinsic pathway. In the present study, following ethanol administration to infant mice, we found no change in activated caspase-8, which suggests that the extrinsic pathway is not involved in ethanol-induced apoptosis. We also found that ethanol triggers robust caspase-3 activation and apoptotic neurodegeneration in C57BL/6 wildtype mice, but induces neither phenomenon in homozygous Bax-deficient mice. Therefore, it appears that ethanol-induced neuroapoptosis is an intrinsic pathway-mediated phenomenon involving Bax-induced disruption of mitochondrial membranes and cytochrome c release as early events leading to caspase-3 activation.

Leaf-rust resistance in rye (Secale cereale L.). 1. Genetic analysis and mapping of resistance genes Pr1 and Pr2.

Genetic analysis of resistance to leaf rust in rye (Puccinia recondita f. sp. secalis) led to the identification of two dominant resistance genes, Pr1 and Pr2. Both genes proved to be effective against a local leaf-rust population as well as a subset of single-pustule isolates (SPIs) the latter of which comprised SPIs with very high virulence complexity. Resistance conferred by Pr1 and Pr2 was expressed in detached-leaf tests of seedlings as well as in field tests of adult plants. Molecular marker analysis allowed us to map Pr1 in the proximal part of rye chromosome 6RL, whereas Pr2 was assigned to the distal part of chromosome 7RL. These results are discussed in view of homoeology relationships among Triticeae. A proposal is submitted for the designation of resistance genes to rye leaf rust which would avoid interference with existing gene-symboling in respect to wheat leaf-rust resistances introgressed from rye into wheat or triticale.

p53 deficiency fails to prevent increased programmed cell death in the Bcl-X(L)-deficient nervous system.

Bcl-X(L) mice display a similar neurodevelopmental phenotype as rb, DNA ligase IV, and XRCC4 mutant embryos, suggesting that endogenous Bcl-X(L) expression may protect immature neurons from death caused by DNA damage and/or cell cycle dysregulation. To test this hypothesis, we generated bcl-x/p53 double mutants and examined neuronal cell death in vivo and in vitro. Bcl-X(L)-deficient primary telencephalic neuron cultures were highly susceptible to the apoptotic effects of cytosine arabinoside (AraC), a known genotoxic agent. In contrast, neurons lacking p53, or both Bcl-X(L) and p53, were markedly, and equivalently, resistant to AraC-induced caspase-3 activation and death in vitro indicating that Bcl-X(L) lies downstream of p53 in DNA damage-induced neuronal death. Despite the ability of p53 deficiency to protect Bcl-X(L)-deficient neurons from DNA damage-induced apoptosis in vitro, p53 deficiency had no effect on the increased caspase-3 activation and neuronal cell death observed in the developing Bcl-X(L)-deficient nervous system. These findings suggest that Bcl-X(L) expression in the developing nervous system critically regulates neuronal responsiveness to an apoptotic stimulus other than inadequate DNA repair or cell cycle abnormalities.

Chloroquine-induced neuronal cell death is p53 and Bcl-2 family-dependent but caspase-independent.

Chloroquine is a lysosomotropic agent that causes marked changes in intracellular protein processing and trafficking and extensive autophagic vacuole formation. Chloroquine may be cytotoxic and has been used as a model of lysosomal-dependent cell death. Recent studies indicate that autophagic cell death may involve Bcl-2 family members and share some features with caspase-dependent apoptotic death. To determine the molecular pathway of chloroquine-induced neuronal cell death, we examined the effects of chloroquine on primary telencephalic neuronal cultures derived from mice with targeted gene disruptions in p53, and various caspase and bcl-2 family members. In wild-type neurons, chloroquine produced concentration- and time-dependent accumulation of autophagosomes, caspase-3 activation, and cell death. Cell death was inhibited by 3-methyladenine, an inhibitor of autophagic vacuole formation, but not by Boc-Asp-FMK (BAF), a broad caspase inhibitor. Targeted gene disruptions of p53 and bax inhibited and bcl-x potentiated chloroquine-induced neuron death. Caspase-9- and caspase-3-deficient neurons were not protected from chloroquine cytotoxicity. These studies indicate that chloroquine activates a regulated cell death pathway that partially overlaps with the apoptotic cascade.

Neural precursor cell apoptosis and glial tumorigenesis following transplacental ethyl-nitrosourea exposure.

Neural precursor cells (NPCs) populate the embryonic ventricular zone and persist in the subependymal zone of the adult brain. We hypothesized that hereditary and/or acquired mutations in apoptosis-associated genes, such as p53 and caspases, may protect NPCs from DNA damage-induced death and predispose them to subsequent neoplastic transformation. To test this hypothesis, we exposed NPCs from wild-type and targeted gene-disrupted mouse embryos (p53, caspase-9, caspase-3, and bax mutants) to ethyl-nitrosourea (ENU), a known DNA mutagen and neural carcinogen, and measured NPC viability. We found that ENU produced caspase-3 activation and apoptotic NPC death 6-24 h after administration both in vivo and in vitro. This effect was critically dependent on p53 and caspase-9 expression. The long-term effect of intrauterine ENU exposure was examined in control and p53-deficient mice. High grade glial tumors were found in 60% of p53(-/-) young adult mice exposed to ENU on gestational day 12.5 but not in p53(+/-) or p53(+/+) littermates or in untreated p53-deficient mice. All the tumors were located supratentorially and possessed strong immunoreactivity for glial fibrillary acidic protein and the anti-apoptotic molecule Bcl-X(L). These results suggest that intrauterine exposure of NPCs to certain DNA damaging agents may synergistically interact with specific genetic abnormalities (e.g. p53 deficiency) to produce glial neoplasms in the adult brain.

Mcl-1 deficiency results in peri-implantation embryonic lethality.

We disrupted the Mcl-1 locus in murine ES cells to determine the developmental roles of this Bcl-2 family member. Deletion of Mcl-1 resulted in peri-implantation embryonic lethality. Mcl-1(-/-) embryos do not implant in utero, but could be recovered at E3.5-4.0. Null blastocysts failed to hatch or attach in vitro, indicating a trophectoderm defect, although the inner cell mass could grow in culture. Of note, Mcl-1(-/-) blastocysts showed no evidence of increased apoptosis, but exhibited a delay in maturation beyond the precompaction stage. This model indicates that Mcl-1 is essential for preimplantation development and implantation, and suggests that it has a function beyond regulating apoptosis.

Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis.

The protein Bid is a participant in the pathway that leads to cell death (apoptosis), mediating the release of cytochrome c from mitochondria in response to signals from 'death' receptors known as TNFR1/Fas on the cell surface. It is a member of the proapoptotic Bcd-2 family and is activated as a result of its cleavage by caspase 8, one of a family of proteolytic cell-death proteins. To investigate the role of Bid in vivo, we have generated mice deficient for Bid. We find that when these mice are injected with an antibody directed against Fas, they nearly all survive, whereas wild-type mice die from hepatocellular apoptosis and haemorrhagic necrosis. About half of the Bid-deficient animals had no apparent liver injury and showed no evidence of activation of the effector caspases 3 and 7, although the initiator caspase 8 had been activated. Other Bid-deficient mice survived with only moderate damage: all three caspases (8 and 37) were activated but their cell nuclei were intact and no mitochondrial cytochrome c was released. We also investigated the effects of Bid deficiency in cultured cells treated with anti-Fas antibody (hepatocytes and thymocytes) or with TNFalpha. (fibroblasts). In these Bid-/- cells, mitochondrial dysfunction was delayed, cytochrome c was not released, effector caspase activity was reduced and the cleavage of apoptosis substrates was altered. This loss-of-function model indicates that Bid is a critical substrate in vivo for signalling by death-receptor agonists, which mediates a mitochondrial amplification loop that is essential for the apoptosis of selected cells.

Distal regulatory elements control MRF4 gene expression in early and late myogenic cell populations.

MRF4 is a muscle-specific transcription factor that belongs to a family of basic helix-loop-helix proteins known as the myogenic regulatory factors (MRFs). In vitro studies have shown that expression of the MRF4 gene is controlled by a proximal promoter element (-336 to +71) that binds the muscle-specific transcription factors MEF2 and myogenin to activate transcription. To examine further the regulatory elements necessary for endogenous MRF4 gene expression during development, transgenic mice were generated that contained either a proximal MRF4 promoter-LacZ reporter gene (-336 MRF4-nLacZ) or a MRF4-LacZ reporter gene containing 8.5 kb of 5' flanking sequence (-8500 MRF4-nLacZ). Characterization of individual transgenic mouse lines throughout development revealed that expression of both transgenes is restricted to skeletal muscle tissue. However, unlike previous in vitro data, the proximal promoter transgene exhibits only limited transcriptional activity at all developmental time points, whereas the -8500 MRF4-nLacZ lines fully recapitulate the later developmental expression patterns and exhibit transcription in myotomal cells during somitic differentiation. Tissue culture analysis of myogenic cells isolated from E12.5, E16.5, and adults confirmed that the -8500 MRF4-nLacZ transgene is expressed in greater than 90% of the myotubes for all myogenic populations. These results indicate that 8.5 kb of MRF4 5' flanking sequence contains all the regulatory elements necessary for late MRF4 expression and that at least some of these elements lie upstream of the -336 proximal promoter. It is also likely that distant upstream regulatory sequences control early somitic MRF4 expression. These findings, coupled with previous in vitro studies, suggest that the early and late developmental expression patterns of the MRF4 gene are controlled by distinct sets of regulatory elements.

Maturation of the acetylcholine receptor in skeletal muscle: regulation of the AChR gamma-to-epsilon switch.

During the development of the mammalian neuromuscular junction, acetylcholine receptors (AChRs) become localized to the postsynaptic muscle membrane. As this process nears completion, the fetal form of the receptor, containing a gamma subunit (composition alpha 2 beta gamma delta) is gradually replaced by an epsilon subunit-containing adult form (alpha 2 beta epsilon delta). To understand how this transition is controlled, we compared the expression and regulation of the AChR gamma and epsilon subunits in developing, adult, and cultured muscles. Immunostaining with subunit-specific antibodies showed that replacement of gamma subunit- by epsilon subunit-containing AChRs occurs largely during the first postnatal week in fast-twitch muscles, and occurs homogeneously throughout individual endplates. In the slow-twitch soleus, however, this transition is delayed, and in the multiply innervated slow fibers of extraocular muscle, gamma subunit expression persists into adulthood. The transcriptional bases of the AChR subunit transition, and of these intermuscular variations, were demonstrated in mice bearing transgenes containing promoter elements from the AChR gamma and epsilon subunit genes, each coupled to a nuclear-localized beta-galactosidase (nlacZ) reporter. We show that transgene expression is stimulated by the nerve-derived inducer of AChR expression, ARIA, in myotubes cultured from gamma-nlacZ as well as epsilon-nlacZ mice. However, the expression of gamma-nlacZ, but not epsilon-nlacZ, is increased by treatment of myotubes with TTX, and the ARIA sensitivity of gamma-nlacZ is dependent on the electrical state of the myotube. Thus, the promoters of the gamma and epsilon subunit genes may integrate ARIA- and activity-dependent signals in different ways to generate their complementary patterns of expression.

Overexpression of myogenin in muscles of transgenic mice: interaction with Id-1, negative crossregulation of myogenic factors, and induction of extrasynaptic acetylcholine receptor expression.

To investigate the role of myogenin in regulating acetylcholine receptor expression in adult muscle, this muscle-specific basic helix-loop-helix transcription factor was overexpressed in transgenic mice by using regulatory elements conferring strong expression confined to differentiated postmitotic muscle fibers. Many of the transgenic mice died during the first postnatal week, but those that survived into adulthood displayed normal muscle histology, gross morphology, and motor behavior. The mRNA levels of all five acetylcholine receptor subunits (alpha, beta, gamma, delta, and epsilon) were, however, elevated. Also, the level of receptor protein was increased and high levels of receptors were present throughout the extrasynaptic surface membrane of the muscle fibers. Thus, elevated levels of myogenin are apparently sufficient to induce acetylcholine supersensitivity in normally innervated muscle of adult mice. The high neonatal mortality rate of the mice overexpressing myogenin hindered the propagation of a stable line. In an attempt to increase survival, myogenin overexpressers were mated with a line of transgenic mice overexpressing Id-1, a negative regulator that interacts with the basic helix-loop-helix family of transcription factors. The Id-1 transgene apparently worked as a second site suppressor and abolished the high rate of neonatal mortality. This effect indicates that Id-1 and myogenin interact directly or indirectly in these animals. Further study indicated that myogenin overexpression had no effect on the level of endogenous myogenin mRNA, while the levels of myoD and MRF4 mRNAs were reduced. Overexpression of the negative regulator Id-1 increased the mRNA levels of all the myogenic factors. These findings are consistent with a hypothesis suggesting that myogenic factors are influenced by mechanisms that maintain cellular homeostasis.

Proteins of the Xenopus laevis zinc finger multigene family as targets for CK II phosphorylation.

Zn finger proteins (ZFPs) of the C2/H2 type in Xenopus laevis are encoded by a multigene family comprising several hundred members. Based upon conserved sequence features outside the Zn finger region, ZFPs can be subdivided into distinct subfamilies. Two of such subfamilies are characterized by conserved, N-terminal amino acid sequences termed the FAX and the FAR Domain. Here we present data suggesting that the zinc finger proteins of the FAR-ZFP subfamily are targets for CK II mediated phosphorylation. Expression of these proteins during oogenesis coincides with CK II activity in unfertilized eggs. Additionally, we have found that XlcOF 7.1, a member of the FAX-ZFP subfamily, is also phosphorylated by CK II. The target sites for in vitro phosphorylation are localized within the conserved N-terminal domains but not within the Zn finger regions. However, amino acid sequence comparison revealed that individual phosphoacceptor sites are not generally conserved among all members of the respective ZFP subfamilies. The relevance of a potential CK II phosphorylation for the regulation of ZFP activity in vivo is discussed.

The FAR domain defines a new Xenopus laevis zinc finger protein subfamily with specific RNA homopolymer binding activity.

The zinc finger motif defines a large superfamily of nucleic acid binding proteins. Conserved amino acid sequence elements associated with structurally variant zinc finger clusters define subfamilies of zinc finger proteins (ZFPs). The FAR domain (Finger Associated Repeats) is a novel type of repeat element found at the amino-terminus in a subfamily of Xenopus laevis ZFPs. Northern blot analyses of three different members of the FAR subfamily (XFO 6, XFO 9-3 and XFG 68) revealed that each of these genes is transcribed during oogenesis, embryogenesis and in all investigated tissues of adult animals thereby indicating a ubiquitous distribution of transcripts. All FAR-ZFPs tested so far have specific RNA homopolymer binding activity; they associate preferentially with poly(U). The FAR repeats possess limited primary sequence homology with a sequence in the nucleolar shuttling protein NO38, within a region that contains a casein kinase II phosphorylation site.

Activin A induced expression of a fork head related gene in posterior chordamesoderm (notochord) of Xenopus laevis embryos.

A gene family encoding the fork head/HNF-3 domain has been identified in the South African clawed frog, Xenopus laevis. Screening of genomic DNA and gastrula stage derived cDNA libraries with a DNA fragment encoding the Drosophila fork head domain led to the isolation of a number of different clones encoding this motif. While one of the Xenopus fork head sequences, XFD-3, represents the Xenopus counterpart to rat HNF-3 beta, all other sequences encode novel types of fork head related proteins. Here we report on XFD-1, a DNA binding protein which can bind to the HNF-3 alpha target sequence. Analysis of temporal and spatial expression revealed that the gene is activated at blastula stage and that transcripts are localized in a rather thin stripe of cells invaginating the dorsal blastopore lip (organizer) during gastrulation. XFD-1 mRNA is localized within the notochord and, by the end of neurulation, is no longer detectable. In the animal cap assay the gene is activated by incubation with the vegetalizing factor (activin A) but not with bFGF.