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Cellular responses to biotic stress frequently involve signaling pathways that are conserved across eukaryotes. These pathways include the cytoskeleton, a proteinaceous network that senses external cues at the cell surface and signals to interior cellular components. During biotic stress, dynamic cytoskeletal rearrangements serve as a platform from which early immune-associated processes are organized and activated. Bacterial pathogens of plants and animals use proteins called type III effectors (T3Es) to interfere with host immune signaling, thereby promoting virulence. We previously found that RipU, a T3E from the soilborne phytobacterial pathogen Ralstonia solanacearum, co-localizes with the plant cytoskeleton. Here, we show that RipU from R. solanacearum K60 (RipUK60) associated with and altered the organization of both the actin and microtubule cytoskeleton. We found that pharmacological disruption of the tomato (Solanum lycopersicum) cytoskeleton promoted R. solanacearum K60 colonization. Importantly, tomato plants inoculated with R. solanacearum K60 lacking RipUK60 (ΔripUK60) had reduced wilting symptoms and significantly reduced root colonization when compared to plants inoculated with wild-type R. solanacearum K60. Collectively, our data suggest that R. solanacearum K60 uses the type III effector RipUK60 to remodel cytoskeletal organization, thereby promoting pathogen virulence.

Abstract Exocytosis plays an essential role in delivering proteins, lipids, and cell wall polysaccharides to the plasma membrane and extracellular spaces. Accurate secretion through exocytosis is key to normal plant development as well as responses to biotic and abiotic stresses. During exocytosis, an octameric protein complex named the exocyst facilitates the tethering of secretory vesicles to the plasma membrane. Despite some understanding of molecular and cellular aspects of exocyst function obtained through reverse genetics and direct interaction assays, knowledge about upstream modulators and genetic interactors remains limited. Traditional genetic screens encounter practical issues in exocyst subunit mutant backgrounds, such as lethality of certain knockout mutants and/or potential redundancy of EXO70 homologs. To address these challenges, this study leverages the tunable and reversible nature of chemical genetics, employing Endosidin2 (ES2)—a synthetic inhibitor of EXO70—for a large‐scale chemical genetic mutant screen in Arabidopsis. This approach led to the identification of 70 ES2‐hypersensitive mutants, named es2s. Through a whole‐genome sequencing‐based mapping strategy, 14 nonallelic es2s mutants were mapped and the candidate mutations reported here. In addition, T‐DNA insertion lines were tested as alternative alleles to identify causal mutations. We found that T‐DNA insertion alleles for DCP5, VAS1/ISS1, ArgJ, and MEF11 were hypersensitive to ES2 for root growth inhibition. This research not only offers new genetic resources for systematically identifying molecular players interacting with the exocyst in Arabidopsis but also enhances understanding of the regulation of exocytosis.

To sustain normal growth and allow rapid responses to environmental cues, plants alter the plasma membrane protein composition under different conditions presumably by regulation of delivery, stability, and internalization. Exocytosis is a conserved cellular process that delivers proteins and lipids to the plasma membrane or extracellular space in eukaryotes. The octameric exocyst complex contributes to exocytosis by tethering secretory vesicles to the correct site for membrane fusion; however, whether the exocyst complex acts universally for all secretory vesicle cargo or just for specialized subsets used during polarized growth and trafficking is currently unknown. In addition to its role in exocytosis, the exocyst complex is also known to participate in membrane recycling and autophagy. Using a previously identified small molecule inhibitor of the plant exocyst complex subunit EXO70A1, Endosidin2 (ES2), combined with a plasma membrane enrichment method and quantitative proteomic analysis, we examined the composition of plasma membrane proteins in the root of Arabidopsis seedlings, after inhibition of the ES2-targetted exocyst complex, and verified our findings by live imaging of GFP-tagged plasma membrane proteins in root epidermal cells. The abundance of 145 plasma membrane proteins was significantly reduced following short-term ES2 treatments and these likely represent candidate cargo proteins of exocyst-mediated trafficking. Gene Ontology analysis showed that these proteins play diverse functions in cell growth, cell wall biosynthesis, hormone signaling, stress response, membrane transport, and nutrient uptake. Additionally, we quantified the effect of ES2 on the spatial distribution of EXO70A1 with live-cell imaging. Our results indicate that the plant exocyst complex mediates constitutive dynamic transport of subsets of plasma membrane proteins during normal root growth.

Neutrophil migration and activation are essential for defense against pathogens. However, this process may also lead to collateral tissue injury. We used microRNA overexpression as a platform and discovered protein-coding genes that regulate neutrophil migration. Here we show that miR-99 decreased the chemotaxis of zebrafish neutrophils and human neutrophil-like cells. In zebrafish neutrophils, miR-99 directly targets the transcriptional factor RAR-related orphan receptor alpha (roraa). Inhibiting RORα, but not the closely related RORγ, reduced chemotaxis of zebrafish and primary human neutrophils without causing cell death, and increased susceptibility of zebrafish to bacterial infection. Expressing a dominant-negative form of Rorα or disrupting the roraa locus specifically in zebrafish neutrophils reduced cell migration. At the transcriptional level, RORα regulates transmembrane signaling receptor activity and protein phosphorylation pathways. Our results, therefore, reveal previously unknown functions of miR-99 and RORα in regulating neutrophil migration and anti-microbial defense.

Neutrophils are fast-moving cells essential for host immune functions. Although they primarily rely on glycolysis for ATP, isolated primary human neutrophils depend on mitochondrial membrane potential for chemotaxis. However, it is not known whether mitochondria regulate neutrophil motility in vivo, and the underlying molecular mechanisms remain obscure. Here, we visualized mitochondria in an interconnected network that localizes to the front and rear of migrating neutrophils using a novel transgenic zebrafish line. To disrupt mitochondrial function genetically, we established a gateway system harboring the CRISPR/Cas9 elements for tissue-specific knockout. In a transgenic line, neutrophil-specific disruption of mitochondrial DNA polymerase, polg, significantly reduced the velocity of neutrophil interstitial migration. In addition, inhibiting the mitochondrial electron transport chain or the enzymes that reduce mitochondrial reactive oxygen species also inhibited neutrophil motility. The reduced cell motility that resulted from neutrophil-specific knockout of sod1 was rescued with sod1 mRNA overexpression, or by treating with scavengers of reactive oxygen species. Together, our work has provided the first in vivo evidence that mitochondria regulate neutrophil motility, as well as tools for the functional characterization of mitochondria-related genes in neutrophils and insights into immune deficiency seen in patients with primary mitochondrial disorders. This article has an associated First Person interview with the first author of the paper.

Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.

The actin cytoskeleton plays a key role in the plant morphogenesis and is involved in polar cell growth, movement of subcellular organelles, cell division, and plant defense. Organization of actin cytoskeleton undergoes dynamic remodeling in response to internal developmental cues and diverse environmental signals. This dynamic behavior is regulated by numerous actin-binding proteins that integrate various signaling pathways. Production of the signaling lipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid affects the activity and subcellular distribution of several actin-binding proteins, and typically correlates with increased actin polymerization. Here we review current knowledge of the inter-regulatory dynamics between signaling phospholipids and the actin cytoskeleton in plant cells.

Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.

The actin cytoskeleton is a dynamic structure that coordinates numerous fundamental processes in eukaryotic cells. Dozens of actin-binding proteins are known to be involved in the regulation of actin filament organization or turnover and many of these are stimulus-response regulators of phospholipid signaling. One of these proteins is the heterodimeric actin-capping protein (CP) which binds the barbed end of actin filaments with high affinity and inhibits both addition and loss of actin monomers at this end. The ability of CP to bind filaments is regulated by signaling phospholipids, which inhibit the activity of CP; however, the exact mechanism of this regulation and the residues on CP responsible for lipid interactions is not fully resolved. Here, we focus on the interaction of CP with two signaling phospholipids, phosphatidic acid (PA) and phosphatidylinositol (4,5)-bisphosphate (PIP(2)). Using different methods of computational biology such as homology modeling, molecular docking and coarse-grained molecular dynamics, we uncovered specific modes of high affinity interaction between membranes containing PA/phosphatidylcholine (PC) and plant CP, as well as between PIP(2)/PC and animal CP. In particular, we identified differences in the binding of membrane lipids by animal and plant CP, explaining previously published experimental results. Furthermore, we pinpoint the critical importance of the C-terminal part of plant CPα subunit for CP-membrane interactions. We prepared a GST-fusion protein for the C-terminal domain of plant α subunit and verified this hypothesis with lipid-binding assays in vitro.

Nocardia are opportunistic human pathogens that can cause a range of debilitating and difficult to treat infections of the lungs, brain, skin, and soft tissues. Despite their close relationship to the well-known secondary metabolite-producing genus, Streptomyces , comparatively few natural products are known from the Nocardia , and even less is known about their involvement in the pathogenesis. Here, we combine chemistry, genomics, and molecular microbiology to reveal the production of terpenomycin, a new cytotoxic and antifungal polyene from a human pathogenic Nocardia terpenica isolate. We unveil the polyketide synthase (PKS) responsible for terpenomycin biosynthesis and show that it combines several unusual features, including "split", skipped, and iteratively used modules, and the use of the unusual extender unit methoxymalonate as a starter unit. To link genes to molecules, we constructed a transposon mutant library in N. terpenica , identifying a terpenomycin-null mutant with an inactivated terpenomycin PKS. Our findings show that the neglected actinomycetes have an unappreciated capacity for the production of bioactive molecules with unique biosynthetic pathways waiting to be uncovered and highlights these organisms as producers of diverse natural products.

Identification of peptides mediating protein-protein interaction (PPI) is crucial for understanding the function of interlinked proteins in cellular processes and amino acid-associated diseases. Traditional PPI assays are laborious, involving the generation of many truncated proteins. SPOT peptide assay allows high-throughput detection of domains essential for PPI by synthesizing several hundred peptides on a cellulose membrane. Here, we present a rapid SPOT peptide protocol for identifying the binding motifs, which mediate interaction between the chromatin remodeling factors BAF155/BAF170 and the epigenetic factor Kdm6b. For complete details on the use and execution of this protocol, please refer to Narayanan et al. (2015)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The axon initial segment (AIS) is a critical microdomain for action potential initiation and implicated in the regulation of neuronal excitability during activity-dependent plasticity. While structural AIS plasticity has been suggested to fine-tune neuronal activity when network states change, whether it acts in vivo as a homeostatic regulatory mechanism in behaviorally relevant contexts remains poorly understood. Using the mouse whisker-to-barrel pathway as a model system in combination with immunofluorescence, confocal analysis and electrophysiological recordings, we observed bidirectional AIS plasticity in cortical pyramidal neurons. Furthermore, we find that structural and functional AIS remodeling occurs in distinct temporal domains: Long-term sensory deprivation elicits an AIS length increase, accompanied with an increase in neuronal excitability, while sensory enrichment results in a rapid AIS shortening, accompanied by a decrease in action potential generation. Our findings highlight a central role of the AIS in the homeostatic regulation of neuronal input-output relations.

Heterocyst-forming cyanobacteria are organized as multicellular filaments of tightly interacting, functionally specialized cells. N 2 -fixing heterocysts differentiate from vegetative cells under nitrogen limitation in a semi-regular pattern along the filament. Diazotrophic growth requires metabolite exchange between neighboring cells within the filament. This exchange occurs via cell-cell junction complexes that span the gap between the plasma membranes and thereby cross the septal peptidoglycan through an array of uniform nanopores formed by AmiC-type cell wall hydrolases. We investigated how the lytic hydrolase AmiC1 (Alr0092) from Anabaena sp. PCC 7120, whose activity needs to be tightly controlled to avoid cell lysis, is regulated by the LytM factor Alr3353. Inactivation of alr3353 resulted in significantly fewer nanopores and as a consequence, a lower rate of fluorescent tracer exchange between cells. The mutant was not able to grow with N 2 as sole nitrogen source, although heterocysts were formed. Alr3353 localized mainly to fully developed intercellular septa of vegetative cells. The purified protein bound to peptidoglycan and enhanced the hydrolytic activity of AmiC1 in vitro. Our data show that the LytM factor Alr3353 regulates nanopore formation and cell-cell communication by directly interacting with AmiC1., (© 2018 John Wiley & Sons Ltd.)

We describe a structured inquiry laboratory exercise that examines transcriptional regulation of the NOS2 gene under conditions that simulate the inflammatory response in macrophages. Using quantitative PCR and the comparative CT method, students are able determine whether transcriptional activation of NOS2 occurs and to what degree. The exercise is aimed at second year undergraduates who possess basic knowledge of gene expression events. It requires only 4-5 hr of dedicated laboratory time and focuses on use of the primary literature, data analysis, and interpretation. Importantly, this exercise provides a mechanism to introduce the concept of differential gene expression and provides a starting point for development of more complex guided or open inquiry projects for students moving into upper level molecular biology, immunology, and biochemistry course work., (© 2015 The International Union of Biochemistry and Molecular Biology.)

This laboratory module simulates the process used by working scientists to ask and answer a question of biological interest. Instructors facilitate acquisition of knowledge using a comprehensive, inquiry-based approach in which students learn theory, hypothesis development, experimental design, and data interpretation and presentation. Using inflammation in macrophages as a model system, students perform a series of molecular biology techniques to address the biological question: "Does stimulus 'X' induce inflammation?" To ask this question, macrophage cells are treated with putative inflammatory mediators and then assayed for evidence of inflammatory response. Students become familiar with their assigned mediator and the relationship between their mediator and inflammation by conducting literature searches, then using this information to generate hypotheses which address the effect of their mediator on induction of inflammation. The cellular and molecular approaches used to test their hypotheses include transfection and luciferase reporter assay, immunoblot, fluorescence microscopy, enzyme-linked immunosorbent assay, and quantitative PCR. Quantitative and qualitative reasoning skills are developed through data analysis and demonstrated by successful completion of post-lab worksheets and the generation and oral presentation of a scientific poster. Learning objective assessment relies on four instruments: pre-lab quizzes, post-lab worksheets, poster presentation, and posttest. Within three cohorts (n = 85) more than 95% of our students successfully achieved the learning objectives.

Optogenetics has rapidly become a standard method in neuroscience research. Although significant progress has been made in the development of molecular tools, refined techniques for combined light delivery and recording in vivo are still lacking. For example, simultaneous intracellular recording and light stimulation have only been possible by using two separate positioning systems. To overcome this limitation, we have developed a glass pipette holder which contains an additional port for the insertion of an optical fiber into the pipette. This device, which we called "optopatcher" allows whole cell patch-clamp recording simultaneously with direct projection of light from the recording pipette. The holder spares the use of an additional manipulator and, importantly, enables accurate, stable and reproducible illumination. In addition, replacement of standard pipettes is done as easily as with the available commercial holders. Here we used the optopatcher in vivo to record the membrane potential of neurons from different cortical layers in the motor cortex of transgenic mice expressing channelrhodopsin-2 under the Thy1 promoter. We demonstrate the utility of the optopatcher by recording LFP and intracellular responses to light stimulation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPδ are known to participate in the regulation of many genes associated with inflammation. However, little is known about the activation and function of C/EBPβ and -δ in inflammatory responses elicited by Fcγ receptor (FcγR) activation. Here we show that C/EBPβ and -δ activation are induced in IgG immune complex (IC)-treated macrophages. The increased expression of C/EBPβ and -δ occurred at both mRNA and protein levels. Furthermore, induction of C/EBPβ and -δ was mediated, to a large extent, by activating FcγRs. Using siRNA-mediated knockdown as well as macrophages deficient for C/EBPβ and/or -δ, we demonstrate that C/EBPβ and -δ play a critical role in the production of TNF-α, MIP-2, and MIP-1α in IgG IC-stimulated macrophages. Moreover, both ERK1/2 and p38 MAPK are involved in C/EBP induction and TNF-α, MIP-2, and MIP-1α production induced by IgG IC. We provide the evidence that C5a regulates IgG IC-induced inflammatory responses by enhancing ERK1/2 and p38 MAPK activities as well as C/EBPβ and -δ activities. Collectively, these data suggest that C/EBPβ and -δ are key regulators for FcγR-mediated induction of cytokines and chemokines in macrophages. Furthermore, C/EBPs may play an important regulatory role in IC-associated inflammatory responses.

The prevalence of obesity, an established epidemiologic risk factor for many chronic diseases including cancer, has been steadily increasing in the US over several decades. The mechanisms used to regulate energy balance and adiposity and the relationship of these factors to cancer are not completely understood. Here we have used knockout mice to examine the roles of the transcription factors CCAAT/enhancer-binding protein (C/EBP) beta and C/EBPdelta in regulating body composition and systemic levels of hormones such as insulin-like growth factor-1 (IGF-1), leptin and insulin that mediate energy balance. Dual-energy X-ray absorptiometry showed that C/EBPbeta, either directly or indirectly, modulated body weight, fat content and bone density in both males and females, while the effect of C/EBPdelta was minor and only affected adiposity and body weight in female animals. Levels of IGF-1, leptin and insulin in the serum were decreased in both male and female C/EBPbeta(-/-) mice, and C/EBPbeta was associated with their promoters in vivo. Moreover, colon adenocarcinoma cells displayed reduced tumorigenic potential when transplanted into C/EBPbeta-deficient animals, especially males. Thus, C/EBPbeta contributes to endocrine expression of IGF-1, leptin and insulin, which modulate energy balance and can contribute to cancer progression by creating a favorable environment for tumor cell proliferation and survival.

We present a novel database system for organizing and selecting quantitative experimental data on single neurons and neuronal microcircuitry that has proven useful for reference-keeping, experimental planning and computational modelling. Building on our previous experience with large neuroscientific databases, the system takes into account the diversity and method-dependence of single cell and microcircuitry data and provides tools for entering and retrieving published data without a priori interpretation or summarizing. Data representation is based on the framework suggested by biophysical theory and enables flexible combinations of data on membrane conductances, ionic and synaptic currents, morphology, connectivity and firing patterns. Innovative tools have been implemented for data retrieval with optional relaxation of search criteria along the conceptual dimensions of brain region, cortical layer, cell type and subcellular compartment. The relaxation procedures help to overcome the traditional trade-off between exact, non-interpreted data representation in the original nomenclature and convenient data retrieval. We demonstrate the use of these tools for the construction, tuning and validation of a multicompartmental model of a layer V pyramidal cell from the rat barrel cortex. CoCoDat is freely available at . Its application is scalable from offline use by individual researchers via local laboratory networks to a federation of distributed web sites in platform-independent XML format using Axiope tools.

The tryptic FAD-peptide carrying the flavin in 8alpha-(N3)histidyl linkage as natural hapten was isolated by HPLC from the bacterial enzyme 6-hydroxy-d-nicotine oxidase. The same flavin protein linkage is found in the mitochondrial succinate dehydrogenase flavoprotein subunit, the predominant flavoprotein with covalently bound FAD in mitochondria of cardiomyocytes. Peripheral blood mononuclear cells (PBMC) were isolated from four patients with acute myocarditis, seven patients with dilated cardiomyopathy (DCM) and from four healthy control individuals. The response of PBMC to the FAD-peptide was evaluated by measuring proliferation ([3H]-dThd incorporation) and cytokine secretion [interferon (IFN)-gamma]. PBMC from all patients with acute myocarditis showed positive responses to the FAD-peptide, in contrast to PBMC from patients with DCM or control individuals. Following the recovery of the patients from the acute inflammation of the heart, PBMC no longer exhibited a proliferation response to the FAD-peptide. A chemically synthesized FAD-free peptide with identical amino acid sequence induced no response of PBMC. The results are consistent with a recall response by activated T cells, specific for the normally cryptic mitochondrial flavin-hapten, which may be liberated following cardiomyocyte destruction during the inflammation of the heart.

The role of mitochondrial proteins as antigens to antibodies of anti-M7 sera was analysed by flavin fluorescence, one- and two-dimensional Western blots and blue native gel electrophoresis. Flavin fluorescence of succinate dehydrogenase (SucDH, complex II of the respiratory chain) of rat liver inner mitochondrial membranes correlated with the immunoreactivity of a representative anti-M7 myocarditis serum. Antigens of isolated bovine heart mitochondria reacting with antibodies of myocarditis serum on two-dimensional Western blots were identified by MALDI-TOF and NanoESI mass spectrometry as myosin heavy chain beta and as dihydrolipoamide dehydrogenase of the mitochondrial 2-oxoacid dehydrogenase complexes. The SucDH-flavoprotein was not resolved as a discrete protein spot on two-dimensional polyacrylamide gels. However, separation of the rat liver inner mitochondrial membrane complexes by blue native gel electrophoresis followed by Western blotting, and Western blots of purified Escherichia coli SucDH complex revealed that anti-M7 sera contained antibodies directed against the SucDH-flavoprotein subunit.

The effects of digitoxin and/or diuretic agents were investigated in patients with congestive heart failure (CHF) in sinus rhythm with respect to changes in hemodynamic parameters, cardiac dimensions, and bicycle ergometric exercise capacity. In a randomized, double-blind study 16 male patients with CHF NYHA class II and III received a placebo for 1 week (baseline) and then were randomly allocated, double blind, to take either digitoxin (digitalis group, DI: N = 8) or trichlormethiazide/amiloride (diuretic group, DG: N = 8) for 3 weeks (VP I). The patients who were first treated with digitoxin received the diuretic agent for a further 3 weeks and vice versa (VP II). At baseline and after VP I and II, a physical examination, two-dimensional echocardiography, and bicycle ergometry were performed. Heart rate (HR), systolic (BPs), and diastolic (BPd) blood pressure at rest, and BPs and 50 watts, were not significantly changed during the observation period. HR at 50 watts was decreased in DI (11.5 +/- 10.1 beats/min.) after VP I and II, but not in DG. BPd was significantly reduced after VP II in DI (8.2 +/- 4.6 mmHg) and in DG (9.3 +/- 8.9 mmHg). DI presents at baseline significantly higher end-diastolic (LVEDV) and end-systolic (LVESV) left ventricular dimensions, whereas left atrial diameter (LA) and stroke volume (SV) and ejection fraction (LVEF) were not significantly different. After VP I, a significantly decreased LA was found in DI, but not in DG. After VP II, all cardiac dimensions were significantly reduced compared with the baseline in DI, whereas in DG only a decrease in LVESV was found. SV was significantly increased in DI, but not in DG after VP I, SV and LVEF were significantly improved in DI and in DG after VP II. Exercise capacity did not change significantly in DI and DG. Digitoxin in combination with trichlormethiazide/amiloride is effective in reducing primarily enlarged left atrial and left ventricular dimensions, and is sufficient to improve the impaired systolic left ventricular function in CHF of NYHA class II and III in sinus rhythm. However, a significant increase in exercise capacity was not found. Treatment with digitoxin seems to be more relevant as a monotherapy with trichlormethiazide/amiloride.

Comparisons of the results of angiographic and pathologic-anatomic estimations of the degree of coronary artery stenosis are rare and the findings contradictory. In the present study, postmortem examinations were carried out on 26 hearts with coronary artery disease. Quantitative planimetric measurements of 203 coronary artery cross-sections were performed and compared with the results of postmortem coronary angiograms. Using a score rating scale, a highly significant difference (P < 0.001) between the angiographic and morphologic findings was detected, with the degree of stenosis and underestimated angiographically in 96 of the 203 sections (47%), overestimated in 18 (9%), and correctly estimated in 89 (44%). The tendency to underestimation was equal in all of the coronary arteries (left anterior descending, circumflex, and right coronary arteries); there were no statistically significant differences (P < 0.20, P < 0.30, P < 0.10). The frequency of angiographic underestimation of the degree of morphologic stenosis--especially in critical stenoses of more than 60%)--has important implications in the assessment of whether coronary artery surgery is warranted.

Endocarditis was suspected in a 66-year-old man who had enterococcal septicaemia. Thrombotic involvement of the mitral, aortic and tricuspid valves by the endocarditis was demonstrated by echocardiography, the diagnosis and localisation of the endocarditic deposits being confirmed at necropsy, the patient's condition making operative treatment impossible. The characteristic echocardiographic finding was of abnormal echos from the valves with largely normal valve movements. Atrial myxoma and artefacts had to be considered in the differential diagnosis.

Percutaneous transluminal balloon-catheter dilatation successfully removed the stenosis in three out of four patients with obstruction of a subclavian artery. The arm oscillogram became normal and after an average of four months later the three patients remain without symptoms. The method is particularly suitable, as an alternative to operative intervention, if the stenosis is in the proximal part of the subclavian artery.

Echocardiograms, radiological heart volume and datas by treadmill-exercis of 50 endurance athlets were studied. There was a highly significant correlation between radiological heart volume and left ventricular dimension as well as left ventricular volume including stroke volume. Other good relations were found between echocardiographic measurements and maximal oxygen uptake. These findings of left ventricular echocardiographic dimensions compared with heart volume and oxygen uptake support the value of this method and permit its use in the analysis of physiological and pathologic changes.

The long-term involution of physiological cardiomegaly and cardiac hypertrophy. Med. Sci. Sports Exerc., Vol. 21, No. 3, pp. 244-249, 1989. Forty-five former athletes in endurance disciplines, primarily Olympic medalists and World Cup, European Cup, and German champions, for whom results of an exercise ECG and radiological heart volume measurement were available from their active competitive phase, were examined. The study protocol included clinical examination, laboratory controls, resting and exercise ECG, determination of cardiac volume, and one- and two-dimensional echocardiographic examination. Of the 45 former athletes contacted, 38 appeared for examination. Of these, four presented with heart disease (two with infarction, one with aortic stenosis, and one with arrhythmia). The remaining 34 were divided into groups of still active (more than 300 kcal.wk-1) and inactive (less than 300 kcal.wk-1) athletes. The interval between the first and second examination averaged 23 yr. The active former athletes showed a weight increase of 5.2% (P less than 0.01) and a reduction of 14% in ergometric performance (P less than 0.02). The inactive group had a marked weight increase of 17.4% (P less than 0.001); the ergometric performance was lowered by 20% (P less than 0.001). The absolute heart size had decreased in the active group by 6.1% (NS) and the relative heart size by 10.7% (P less than 0.005); the corresponding values in the inactive group were 4.5% (NS) and 18% (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

In 19 of more than 130 patients treated with percutaneous transluminal angioplasty (PTA), obstructions of the distal popliteal artery and the lower leg arteries encouraged us to extend the treatment to this vascular region. Two weeks after PTA the arteries were patent in 16 of the 19 cases (84%). These results are comparable with those of vascular surgery, but are gained with less injury to the patient and a shorter hospitalization time.

This report describes the echocardiographic features seen in 6 patients with valvular vegetations as documented by autopsy or surgery. 4 mitral, 3 aortic and 1 tricuspid valves were investigated using M-mode echoscan(n = 8). Characteristic findings observed were non-uniform thickening of the typical valve pattern in sytole and diastole without restriction of valve motion. In 7 cases the exact echocardiographic localization of valvular vegetation was confirmed in situ, whereas localization of vegetation in tricuspid valve disease was uncomplete.

In order to document the possible influence of stress limiting factors (STLF: chest pain, ST-segment depression) on exercise-induced blood lactate increase in CHD patients (post myocardial infarction) 88 males were examined in a stepwise bicycle stress test. Blood lactate samples were drawn at the end of each stress step. The patients were divided into 2 groups without (n = 45) and with (n = 43) STLF, higher blood lactate concentrations, however, were only observed in relation to the exercise intensity reached at the end of the stress test. Neither the symptom chest pain nor the degree of ST-depression showed a significant influence on the physical performance capacity and the lactate concentration at the anaerobic threshold. Nevertheless, a physical performance capacity above 1 W/kg seems to be necessary to reach the anaerobic threshold level. When the lactate measurement is used in CHD patients, the influence of age on the maximum lactate concentrations must be considered.

Radiological heart volume estimations and echocardiographic studies were performed on 30 male and 15 female middle distance athletes at rest, during supine ergometric exercise at 10 and 100 Watt and 1 minute after excerise. During exercise, there is an increase in cardiac output, in the small hearts of 6%, in the large hearts of 16%, due to a small enddiastolic increase and an associated decrease in the endsystolic left ventricular diameter. At rest and at 50 Watt, the cardiac output, corrected for body surface area, was practically the same for all volunteers, at 100 Watt the volunteers with the largest hearts had the highest cardiac output. This is interpreted as evidence for increased cardio-circulatory reserves. The contractility parameters at the various exercise grades varied dependant on heart size; when related to heart rate the variations were practically abolished. The results suggest that variations in the dynamics of small and large hearts are not primarily due to intrinsic factors but rather related to extrinsic factors such as sympathetic drive expressed by the changes in heart rate.

In this paper evidence is presented that in few patients with clear cut myocardial infarction as judged by physical signs, ECG and laboratory findings, the arteriography and morphology still may indicate healthy, unaffected coronary arteris. In those cases first of all the differential diagnosis has to include a silent myocarditis by whatever origin. This assumption may also be correct, if there are no or only few signs for this disease. Extensive diagnostic precedures should be performed under those circumstances.

Cytophotometric measurements of the DNA content of human heart muscle nuclei were performed on normal and congenitally malformed hearts of children. Congenital malformations of the heart lead to an early increase of the ploidy pattern as a sign of myocardial overload. This increase is significant. It starts, at the earliest, after the 3rd week of life. The degree of polyploidization of the muscle nuclei in malformed hearts is dependent on the type of overload. Pressure load produces a stronger shift to higher ploidy values than does volume load. The polyploidization is not dependent on either the heart weight at autopsy or the age of the child. It may be concluded that increasing functional requirement is the main cause of physiological and pathological polyploidization in hearts of children.

Using one- and two-dimensional echocardiographic parameters, left ventricular function and dimensions were investigated in 11 patients with chronic heart failure (NYHA stages II-III) and in 10 normal subjects after administration of low-dose digitoxin (0.07 mg). Tests were performed before the begin of therapy, on the 3rd day following rapid saturation, and on the 15th day under maintenance therapy. There was no significant decrease either of heart volume as assessed by X-ray or of enddiastolic volume measured echocardiographically. However--predominantly in patients--a marked decrease in endsystolic diameter (p less than 0.01) and an increase in posterior wall motion amplitude (p less than 0.05) was observed resulting in increased stroke volume, shortening fraction (p less than 0.001). Early diastolic left ventricular filling speed also increased significantly in both groups (p less than 0.01). Changes in these parameters were more pronounced in patients than in normal subjects. In both groups the effects were achieved soon after rapid saturation, increasing slightly during the period of chronic administration of low-dose digitoxin. Parallel to the changes in echocardiographic parameters, a noticeable clinical improvement occurred among patients. No side effects were observed as serum digitoxin levels were in the therapeutic range. It may be concluded that low-dose digitoxin can be employed to increase cardiac contractility in patients with heart failure.

The literature about pathological septal motion in coronary artery disease is contradictory. Purpose of this study was to reveal the sensitivity and specificity of the echocardiography for diagnosis of stenoses and occlusions (S/O) of the left anterior descending artery (LAD). 50 patients with CAD were studied using coronary angiography and echocardiography. In 32 of the cases a critical stenosis (over 70% narrowing) of the LAD was present. History of old infarction was found in 80% of the patients. Angina pectoris was absent at the time of the investigation. It was proven that the sensitivity of the echocardiography for the diagnosis of coronary stenoses and for proximal and peripheral LAD-S/O is not high (44%-61%). 50 percent of all LAD-S/O and 61% of the proximal LAD-S/O have a pathological septal movement. In presence of a pathological septal motion, however, it may be concluded that there is a critical stenosis in one or more of the three main coronary arteries (predictive value 91%). The diagnosis of the localization of the occlusion by echocardiography is not possible. 1/3 of peripheral LAD-S/O also lead to a dyscinetic septum which mostly is found in proximal LAD-S/O. The number of false positive (pathological) echocardiographic septal dyscinetic motion is low (12%). The sensitivity of echocardiographic method for diagnose of coronary alteration is increased when using the differentiation between stenosis and occlusion of LAD. By that it can be revealed that full occlusions of the LAD lead in 80% to septal dyscinetic motion, whereas stenoses are only rarely combined with pathological septum (36%). It is concluded that not only the localization but predominantly the degree of the vessel-narrowing influences the septal movement. Perhaps these findings may help to declare the contradictory results in literature.

In the present study, 17 patients with angiographically proven dilative cardiomyopathy (CM) were investigated simultaneously by both invasive right heart catheter and 1D/2D echocardiography at rest (R) and during bicycle exercise (E) stress test. They were compared with 14 normal subjects (N). The echocardiographic contractility reserve was determined as an increase in the systolic pump function (shortening fraction-SF, ejection fraction - EF) and compared with pulmonary capillary wedge pressure during exercise. 14/17 echocardiograms of dilative cardiomyopathy and all echocardiograms of normal subjects at rest as well as during exercise could be accepted. In N, echocardiographic parameters of systolic function significantly increased during exercise, whereas dilative cardiomyopathy showed decreased contractility reserve with no increase in pump function (for CM patients, at rest SF: 21.3% +/- 8%; EF: 41.6% +/- 14%; exercise SF: 21.3 +/- 9%; EF: 40.7% +/- 15%). Hemodynamic investigation in CM showed no increase in stroke volume accompanied by a pathologic increase in pulmonary wedge pressure, ranging from x = 16.4 mmHg at rest, up to 30.0 mm Hg in exercise (p less than 0.001), whereas in normal subjects, no pathologic increase in pressure was present (from 10.3 mm Hg at rest to 13.7 mm Hg during exercise). There were closed relations between invasive and non-invasive data. The relative alteration of stroke volume during E showed in N and in CM good correlation with echocardiography (N + 19%, CM -2%) and invasive data (N + 18%, CM + 2%). FSe during stress and PCPm showed a closed inverse correlation during exercise (p less than 0.001; r = 0.8). The lower the contractility in echocardiogram, the higher PCPm was in exercise.

The left ventricular volume (EDV), the left ventricular total volume (TDV), and the stroke volume (SV) of 40 male untrained subjects and 68 endurance athletes were determined using one-dimensional echocardiography, two-dimensional echocardiography, and a combined method. The accuracy of the volume and stroke volume measurements was checked by comparing them to ergometrically determined maximum oxygen pulse (max O2-P) and to radiographic heart volume (HV). There was clear improvement in the EDV, TDV, and SV measurements when using two-dimensional echocardiography and the combined method in comparison to one-dimensional echocardiography. The best correlation to the max-O2-P was reached by TDV2 (combined method, r = 0.8738). This method includes the myocardium of the left ventricle similar to heart volume measurements. The relationship is as close as between HV and max O2-P (r = 0.8665). The method suggested here is sufficiently accurate to be used in performance diagnosis to determine the size of the left ventricular volume and to classify pathological size changes or those due to training.