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Tachycardiomyopathy (TCM) is a largely reversible form of non-ischemic heart failure. The underlying mechanism are, however, still today poorly understood. Recent data indicate distinct changes in mitochondrial distribution in these patients, compared to other non-ischemic cardiomyopathies.This study investigated underlying mechanisms in mitochondrial dynamics in endomyocardial biopsy samples (EMB) from patients with TCM and compared them to patients with dilated cardiomyopathy (DCM), which show similar clinical features.

Germany

Oxidative modifications of low-density lipoprotein (ox-LDL) play a key role in initial steps of atheroprogression possibly via specific scavenger receptors on inflammatory and endothelial cells. Amongst others, CD68 might play a crucial role in this leading to fatty streak formation.

Germany

Hepatocellular carcinoma (HCC) represents the second most common cause of cancer-related deaths worldwide, not least due to its high chemoresistance. The long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1), localised in nuclear paraspeckles, has been shown to enhance chemoresistance in several cancer types. Since data on NEAT1 in HCC chemosensitivity are completely lacking and chemoresistance is linked to poor prognosis, we aimed to study NEAT1 expression in HCC chemoresistance and its link to HCC prognosis.

Germany

CXCL14, a secreted chemokine peptide that promotes obesity-induced insulin resistance, is expressed by islets, but its effects on islet function are unknown. The aim of this study was to determine the role of CXCL14 in β-cells and investigate how it transduces these effects.

Germany

Endothelial cells exposed to the Random Positioning Machine (RPM) reveal three different phenotypes. They grow as a two-dimensional monolayer and form three-dimensional (3D) structures such as spheroids and tubular constructs. As part of the ESA-SPHEROIDS project we want to understand how endothelial cells (ECs) react and adapt to long-term microgravity.

Germany

Recently, we have demonstrated that episodic hypoxia occurs in kidneys of mice challenged repetitively with the immunosuppressant cyclosporine A (CsA), in analogy to humans on CsA treatment. However, the molecular consequences of episodic hypoxia remain poorly defined, as is its impact on cell survival. Here, we systematically study cell response to episodic, as compared to single course hypoxia.

Germany

Fabry disease (FD) is a lysosomal storage disorder characterized by impaired alpha-galactosidase A (α-Gal A) enzyme activity due to mutations in the GLA gene. While virtually all tissues are affected, renal damage is particularly critical for the patients' outcome. Currently, powerful diagnostic tools and in vivo research models to study FD in the kidney are lacking, which is a major obstacle for further improvements in diagnosis and therapy. The present study focuses on the effects of enzyme replacement therapy on a previously established podocyte cell culture model of Fabry disease.

Germany

Adipocyte hypertrophy in obesity is associated with inflammation and adipose tissue fibrosis which both contribute to metabolic diseases. Mechanisms regulating lipid droplet expansion are poorly understood. Knock down of the scaffold protein beta 2 syntrophin (SNTB2) increases lipid droplet size of 3T3-L1 adipocytes and the physiological relevance of SNTB2 in adipose tissue morphology and metabolic health was analyzed herein.

Germany

The two-pore-domain potassium channel TASK-1 regulates atrial action potential duration. Due to the atrium-specific expression of TASK-1 in the human heart and the functional upregulation of TASK-1 currents in atrial fibrillation (AF), TASK-1 represents a promising target for the treatment of AF. Therefore, detailed knowledge of the molecular determinants of TASK-1 inhibition may help to identify new drugs for the future therapy of AF. In the current study, the molecular determinants of TASK-1 inhibition by the potent and antiarrhythmic compound A293 (AVE1231) were studied in detail.

Germany

Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice.

Germany

Amyotrophic lateral sclerosis (ALS) is the most common degenerative motor neuron disease in humans. However, the pathogenesis of ALS is not yet understood. The wobbler mouse is considered as an animal model for the sporadic form of ALS due to its spontaneous mutation in the Vps54 gene. Due to transactivation of NDRG2 by p53, this tumor suppressor might play a functional role in stress induced cell death in wobbler mice as well as ALS patients. Furthermore, deregulated microRNAs are often related to neurodegenerative diseases. Thus, the NDRG2 linked miR-375-3p was of interest for this study.

Germany

Hydrophobic bile salts, such as glycochenodeoxycholate (GCDC) can trigger hepatocyte apoptosis, which is prevented by tauroursodesoxycholate (TUDC), but the effects of GCDC and TUDC on sinusoidal bile salt uptake via the Na⁺-taurocholate transporting polypeptide (Ntcp) are unclear.

Germany

Label="BACKGROUND/AIMS" NlmCategory="OBJECTIVE">The transient receptor potential cation channel subfamily C member 6 (TRPC6) is a Ca²⁺-permeable nonselective cation channel and has received recent attention because of its capability to promote chronic kidney disease (CKD). The aims of this study were (i) to examine whether deletion of TRPC6 impacts on renal fibrosis and inflammatory cell infiltration in an early CKD model of unilateral ureter obstruction (UUO) in mice; and (ii) whether TRPC6-deficiency as well as UUO affect the regulation of TRPC expression in murine kidneys.

Germany

Infections with Coxsackievirus B3 and other members of the enterovirus genus are a common reason for myocarditis and sudden cardiac death in modern society. Despite intensive scientific efforts to cure enterovirus infections, there is still no standardized treatment option. The complexity of Coxsackievirus B3´s effects on the host cell make well defined studies on this topic very challenging. However, recent publications report newly found effects of CVB3´s structural and non-structural proteins on infected cells. For the first time, the viral capsid protein VP1 was shown to have direct influence on the viral life-cycle. By shortening the G0 and the G2 phase and simultaneously prolonging the G1 and G1-S phase, the translation of viral proteins is enhanced and the production of viable CVB3 particles is promoted. Coxsackievirus B3´s viroporin, protein 2B, was recently studied in more detail as well. Structural and physiological analyses identified two hydrophilic α-helices in the structure of 2B, enabling it to insert into cellular membranes of host cells. As main target of 2B the endoplasmatic reticulum was identified. The insertion of 2B into the ER membranes leads to an uncontrolled calcium outflow into the cytoplasm. Additional insertion of 2B into the cell membrane leads to host cell destabilization and in the end to release of viral progeny. The importance of the Coxsackievirus B3´s proteases 2A and 3C in pathogenicity is observed since years. Recently, DAP5 and eIf4G were identified as new cleavage targets for protease 2A. Cleavage of DAP-5 into DAP5-N and DAP5-C changes the gene expression of the host cell and promotes cell death. Additionally, protease 3C targets and cleaves procaspase 8 promoting the mitochondrial apoptosis pathway and cell death. Recent studies identified significant effects of CVB3 on mitochondria of infected cells. Mouse cardiomyocytes showed decreased activities of respiratory chain complexes I-III and changed transcription of important subunits of the complexes I-IV. A disrupted energy metabolism may be one of the main causes of cardiac insufficiency and death in CVB3 infected patients. In addition to a modified energy metabolism, CVB3 affects cardiac ion channels, KCNQ1 in particular. SGK1, which is an important mediator in KCNQ1 membrane insertions, is highly upregulated during CVB3 infections. This results in an increased insertion of KCNQ1 into the cell membrane of cardiac cells. Under stress conditions, this KCNQ1 overshoot may lead to a disturbed cardiac action potential and therefore to sudden cardiac death, as it is often observed in CVB3 infected persons.

Germany

Conflicting results about vestibular function in progressive supranuclear palsy (PSP) prompted a systematic examination of the semicircular canal function, otolith function, and postural stability.

Germany

The availability of truly maturated cardiomyocytic subtypes is a major prerequisite for cardiovascular cell replacement therapies. Pluripotent stem cells provide a suitable source for the development of new strategies to overcome enormous hurdles such as yield, purity and safety of in vitro generated cells.

Germany

Eryptosis, the suicidal death of red blood cells (RBCs), is characterized by phosphatidylserine (PS) exposure at the cell surface. It can be catalysed by a variety of abnormal conditions and diseases. Until now, the many questions surrounding the physiology and pathophysiology of eryptosis have not been sufficiently answered. Recently, we demonstrated IgM and IgA autoantibodies (aab) to induce PS exposure on circulating RBCs of patients with autoimmune haemolytic anaemia (AIHA). However, it remained unclear how these aab lead to eryptosis.

Germany

Adenosine release and connexin (Cx) hemichannel activity are enhanced in the respiratory epithelium during pathophysiological events such as inflammation. We analysed the interplay between Cx channels and adenosine signalling in human respiratory airway epithelium using the Calu-3 cell line as a model.

Germany

Perturbations in the expression of microRNAs (miRNAs) and their maturing machinery components such as Dicer have been previously described for basal cell carcinoma (BCC). However, the mutational status of Dicer in BCC is unclear. Further, the sclerodermiform subtype of BCC (sBCC) has not been previously investigated regarding its methylation profile or its smallRNA expression profile via RNA sequencing. We conducted this study to investigate the mutational status of Dicer in BCC.

Germany

The ability of urinary biomarkers to complement established clinical risk prediction models for postoperative adverse kidney events is unclear. We assessed the effect of urinary biomarkers linked to suspected pathogenesis of cardiac surgery-induced acute kidney injury (AKI) on the performance of the Cleveland Score, a risk assessment model for postoperative adverse kidney events.

Germany

MLK4 (KIAA1804) is the second most frequently mutated kinase in microsatellite stable (MSS) colorectal carcinomas (CRC). This molecule is known to regulate different physiological cellular processes, including cell cycle, senescence and apoptosis, and mechanistic evidence has been provided that MLK4 plays a role in carcinogenesis. However, whether this kinase exerts a tumor suppressive role or an oncogenic function is still an object of debate. This study aims to elucidate the role of MLK4 in the pathogenesis of CRC by investigating human tumor specimens.

Germany

We have previously shown that inhibition of the mitochondrial Kv1.3 channel results in an initial mitochondrial hyperpolarization and a release of oxygen radicals that mediate mitochondrial depolarization, cytochrome c release and death. Here, we investigated whether inhibition of Kv1.3 channels can also induce cellular resistance mechanisms that counteract the induction of cell death under certain conditions.

Germany

Pulmonary infections with Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus) are of utmost clinical relevance in patients with cystic fibrosis, chronic obstructive pulmonary disease, after trauma and burn, upon ventilation or in immuno-compromised patients. Many P. aeruginosa and S. aureus strains are resistant to many known antibiotics and it is very difficult or often impossible to eradicate the pathogens in patient´s lungs. We have recently shown that the sphingoid base sphingosine very efficiently kills many pathogens, including for instance P. aeruginosa, S. aureus or Acinetobacter baumannii, in vitro. In vivo experiments of our group on cystic fibrosis mice indicated that inhalation of sphingosine prevents or eliminates existing acute or chronic pneumonia with P. aeruginosa or S. aureus in these mice. We also demonstrated that sphingosine is safe to use for inhalation up to high doses, at least in mice. To facilitate development of sphingosine to an anti-bactericidal drug that can be used in humans for inhalation, safety data on non-rodents, larger animals are absolutely required.

Germany

It is well established that oxidative stress and inflammation are common pathogenic features of retinal degenerative diseases. ITH12674 is a novel compound that induces the transcription factor Nrf2; in so doing, the molecule exhibits anti-inflammatory, and antioxidant properties, and affords neuroprotection in rat cortical neurons subjected to oxidative stress. We here tested the hypothesis that ITH12674 could slow the retinal degeneration that causes blindness in rd10 mice, a model of retinitis pigmentosa.

Germany

Label="BACKGROUND/AIMS" NlmCategory="OBJECTIVE">Still in 1999 the first hints were published for the pharmacoresistant Ca_v2.3 calcium channel to be involved in the generation of epileptic seizures, as transcripts of alpha1E (Ca_v2.3) and alpha1G (Ca_v3.1) are changed in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Consecutively, the seizure susceptibility of mice lacking Ca_v2.3 was analyzed in great detail by using 4-aminopyridine, pentylene-tetrazol, N-methyl-D-aspartate and kainic acid to induce experimentally convulsive seizures. Further, γ-hydroxybutyrolactone was used for the induction of non-convulsive absence seizures. For all substances tested, Ca_v2.3-competent mice differed from their knockout counterparts in the sense that for convulsive seizures the deletion of the pharmacoresistant channel was beneficial for the outcome during experimentally induced seizures [1]. The antiepileptic drug lamotrigine reduces seizure activity in Ca_v2.3-competent but increases it in Ca_v2.3-deficient mice. In vivo, Ca_v2.3 must be under tight control by endogenous trace metal cations (Zn²⁺ and Cu²⁺). The dyshomeostasis of either of them, especially of Cu²⁺, may alter the regulation of Ca_v2.3 severely and its activity for Ca²⁺ conductance, and thus may change hippocampal and neocortical signaling to hypo- or hyperexcitation.

Germany

The cardiac current IKs is carried by the KCNQ1/KCNE1-channel complex. Genetic aberrations that affect the activity of KCNQ1/KCNE1 can lead to the Long QT Syndrome 1 and 5 and, thereby, to a predisposition to sudden cardiac death. This might be prevented by pharmacological modulation of KCNQ1/KCNE1. The prototypic KCNQ1/KCNE1 activator 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) represents a candidate drug. Here, we study the mechanism of DIDS action on KCNQ1/KCNE1.

Germany

This study aimed to establish a precise and well-defined working model, assessing pharmaceutical effects on vascular smooth muscle cell monolayer in-vitro. It describes various analysis techniques to determine the most suitable to measure the biomechanical impact of vasoactive agents by using CellDrum technology.

Germany

Alpha-1 antitrypsin deficiency (AATD) is a systemic disorder affecting mainly the lung and the liver and is caused by mutations in <i>SERPINA1</i>, the AAT gene. A homozygous "Pi*Z" mutation (Pi*ZZ genotype) may cause liver fibrosis on its own independently of pulmonary AATD manifestation, while heterozygous Pi*Z carriage (Pi*MZ genotype) is considered a strong risk factor for development of liver cirrhosis in patients with concomitant liver disease such as alcoholic and non-alcoholic liver disease. In Pi*ZZ homozygotes, liver disease constitutes the second leading cause of death and is highly heterogeneous. About 35% of Pi*ZZ individuals display significant liver fibrosis on biopsy (i.e., fibrosis stage &#8805; 2 on scale 0-4). Among non-invasive methods for liver fibrosis assessment, liver stiffness measurement (LSM) via vibration-controlled transient elastography (VCTE) has been most widely evaluated. Based on these data, Pi*ZZ adults have 20x increased odds of developing advanced liver fibrosis (i.e., fibrosis stage &#8805; 3) than adults without AAT mutation. Risk factors for accelerated fibrosis progression are male sex, age &#8805; 50 years, alcohol misuse, obesity, diabetes mellitus, or metabolic syndrome. Unlike VCTE, other ultrasound- and magnetic resonance-based elastography methods have been assessed in small cohorts of Pi*ZZ individuals and remain to be comprehensively validated. Among blood-based fibrosis tests, AST-to-platelet ratio index (APRI) correlates moderately with histologic fibrosis stage and LSM. Given APRI's wide availability, it can be used for risk stratification as an adjunct to LSM or when LSM is not at hand. Despite recent efforts, AATD-related liver disease, especially for genotypes other than Pi*ZZ, remains greatly understudied. AATD individuals should be offered liver biochemistry, liver ultrasound, and non-invasive fibrosis assessment at the time of diagnosis to detect potential complications and for proper risk stratification. If signs of AATD-related liver disease occur (i.e., pathologic fibrosis test or repeatedly elevated liver enzymes), patients should be referred to a health care center specialized in AATD-related liver disease and be screened for potentially treatable comorbidities. To exclude the latter, they may need a liver biopsy. Moreover, every health care provider of an AATD individual should be aware of the potential liver manifestation, counsel their patient on modifiable hepatic risk factors, and offer them regular liver check-ups.

Germany

Trace amines (TA) are small organic compounds that have neuromodulator activity due to their interaction with some neuron-related receptors, such as trace amine associated receptors (TAARs), α2-adrenergic receptor (α2-AR) and ß-adrenergic receptor (ß-AR). However, there is little information on whether TA and dopamine (DOP) can interact with other adrenergic receptors (ARs) such as the mammalian α1-AR and the bacterial counterpart QseC, which is involved in quorum sensing of some Gram-negative pathogens. The aim of this study was to investigate the interaction of TA and DOP with α1-AR and QseC.

Germany

Label="BACKGROUND/AIMS" NlmCategory="OBJECTIVE">The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca²⁺ influx into pancreatic beta cells by the opening of voltage-gated Ca²⁺ channels. Transient Receptor Potential Melastatin 3 proteins build Ca²⁺ permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca²⁺ influx and increased insulin release. Therefore, we hypothesized that in beta cells TRPM3 channels may contribute to pregnenolone sulfate- as well as to glucose-induced insulin release.

Germany

The NMDA receptor plays a key role in the pathogenesis of neurodegenerative disorders including Alzheimer's and Huntington's disease, as well as depression and drug or alcohol dependence. Due to its participation in these pathologies, the development of selective modulators for this ion channel is a promising strategy for rational drug therapy. The prototypical negative allosteric modulator ifenprodil inhibits selectively GluN2B subunit containing NMDA receptors. It was conformationally restricted as 2-methyl-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol, which showed high GluN2B affinity and inhibitory activity. For a better understanding of the relevance of the functional groups and structural elements, the substituents of this 3-benzazepine were removed successively (deconstruction). Then, additional structural elements were introduced (reconstruction) with the aim to analyze, which additional modifications were tolerated by the GluN2B receptor.

Germany

Although ion channels are crucial in many physiological processes and constitute an important class of drug targets, much is still unclear about their function and possible malfunctions that lead to diseases. In recent years, computational methods have evolved into important and invaluable approaches for studying ion channels and their functions. This is mainly due to their demanding mechanism of action where a static picture of an ion channel structure is often insufficient to fully understand the underlying mechanism. Therefore, the use of computational methods is as important as chemical-biological based experimental methods for a better understanding of ion channels. This review provides an overview on a variety of computational methods and software specific to the field of ion-channels. Artificial intelligence (or more precisely machine learning) approaches are applied for the sequence-based prediction of ion channel family, or topology of the transmembrane region. In case sufficient data on ion channel modulators is available, these methods can also be applied for quantitative structureactivity relationship (QSAR) analysis. Molecular dynamics (MD) simulations combined with computational molecular design methods such as docking can be used for analysing the function of ion channels including ion conductance, different conformational states, binding sites and ligand interactions, and the influence of mutations on their function. In the absence of a three-dimensional protein structure, homology modelling can be applied to create a model of your ion channel structure of interest. Besides highlighting a wide range of successful applications, we will also provide a basic introduction to the most important computational methods and discuss best practices to get a rough idea of possible applications and risks.

Germany

The family of two-pore domain potassium (K_2P) channels is critically involved in central cellular functions such as ion homeostasis, cell development, and excitability. K_2P channels are widely expressed in different human cell types and organs. It is therefore not surprising that aberrant expression and function of K_2P channels are related to a spectrum of human diseases, including cancer, autoimmune, CNS, cardiovascular, and urinary tract disorders. Despite homologies in structure, expression, and stimulus, the functional diversity of K_2P channels leads to heterogeneous influences on human diseases. The role of individual K_2P channels in different disorders depends on expression patterns and modulation in cellular functions. However, an imbalance of potassium homeostasis and action potentials contributes to most disease pathologies. In this review, we provide an overview of current knowledge on the role of K_2P channels in human diseases. We look at altered channel expression and function, the potential underlying molecular mechanisms, and prospective research directions in the field of K_2P channels.

Germany

Potassium channels of the tandem of two-pore-domain (K_2P) family were among the last potassium channels cloned. However, recent progress in understanding their physiological relevance and molecular pharmacology revealed their therapeutic potential and thus these channels evolved as major drug targets against a large variety of diseases. However, after the initial cloning of the fifteen family members there was a lack of potent and/or selective modulators. By now a large variety of K_2P channel modulators (activators and blockers) have been described, especially for TASK-1, TASK-3, TREK-1, TREK2, TRAAK and TRESK channels. Recently obtained crystal structures of K_2P channels, alanine scanning approaches to map drug binding sites, in silico experiments with molecular dynamics simulations (MDs) combined with electrophysiological studies to reveal the mechanism of channel inhibition/activation, yielded a good understanding of the molecular pharmacology of these channels. Besides summarizing drugs that were identified to modulate K_2P channels, the main focus of this article is on describing the differential binding sites and mechanisms of channel modulation that are utilized by the different K_2P channel blockers and activators.

Germany

Podocytes are lost in most glomerular diseases, leading to glomerulosclerosis and progressive kidney disease. It is generally assumed, that podocytes are exposed to the filtration flow and thus to significant shear forces driving their detachment from the glomerular basement membrane (GBM). In this context, foot process effacement has been proposed as potential adaptive response to increase adhesion of podocytes to the GBM.

Germany

During an immune response, type I interferon (IFN-I) signaling induces a wide range of changes, including those which are required to overcome viral infection and those which suppress cytotoxic T cells to avoid immunopathology. During certain bacterial infections, IFN-I signaling exerts largely detrimental effects. Although the IFN-I family of proteins all share one common receptor, biologic responses to signaling vary depending on IFN-I subtype. Here, we asked if one IFN-I subtype dominates the pro-bacterial effect of IFN-I signaling and found that control of Listeria monocytogenes (L.m.) infection is more strongly suppressed by IFN-β than IFN-α.

Germany

The K_ca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca²⁺ concentration. These K⁺ channels are widely expressed in many organs and involved in many pathologies. In particular, K_ca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of K_ca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of K_ca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using K_ca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to K_ca3.1 channels in order to provide a deeper understanding of K_ca3.1 channel pharmacology. Alltogether, K_ca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.

Germany

The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca²⁺ release-activated Ca²⁺, transient receptor potential, P2X, volume-regulated anion and K⁺ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.

Germany

Podocyte differentiation is essential for proper blood filtration in the kidney. It is well known that transcription factors play an essential role to maintain the differentiation of podocytes. The present study is focused on the basic helix-loop-helix (bHLH) transcription factor Tcf21 (Pod1) which is essential for the development of podocytes in vivo. Since parietal epithelial cells (PECs) are still under debate to be progenitor cells which can differentiate into podocytes, we wanted to find out whether the expression of Tcf21 induces a transition of PECs into podocytes.

Germany

Neanderthals, although well adapted to local environments, were rapidly replaced by anatomically modern humans (AMH) for unknown reasons. Genetic information on Neanderthals is limited restricting applicability of standard population genetics.

Germany

Protein homeostasis strongly depends on the targeted and selective removal of unneeded or flawed proteins, of protein aggregates, and of damaged or excess organelles by the two main intracellular degradative systems, namely the ubiquitin proteasomal system (UPS) and the autophagosomal lysosomal system. Despite representing completely distinct mechanisms of degradation, which underlie differing regulatory mechanisms, growing evidence suggests that the UPS and autophagy strongly interact especially in situations of overwhelming and impairment, and that both are involved in podocyte proteostasis and in the pathogenesis of podocyte injury. The differential impact of autophagy and the UPS on podocyte biology and on podocyte disease development and progression is not understood. Recent advances in understanding the role of the UPS and autophagy in podocyte biology are reviewed here.

Germany

Pituitary pars intermedia dysfunction (PPID), a neurodegenerative disease leading to reduced dopamine production, is a common disease in aged horses. The treatment is based on administration of the dopamine agonist pergolide. This drug has been related to valvular fibrosis in humans, but the cardiovascular effect of this drug has not yet been investigated in horses.

Germany

Oxidative stress and infections by Pseudomonas aeruginosa (P. aeruginosa) are prominent in lungs of patients suffering from cystic fibrosis (CF).

Germany

Viral diseases are a major threat to modern society and the global health system. It is therefore of utter relevance to understand the way viruses affect the host as a basis to find new treatment solutions. The understanding of viral myocarditis (VMC) is incomplete and effective treatment options are lacking. This review will discuss the mechanism, effects, and treatment options of the most frequent myocarditis-causing viruses namely enteroviruses such as Coxsackievirus B3 (CVB3) and Parvovirus B19 (PVB19) on the human heart. Thereby, we focus on: 1. Viral entry: CVB3 use Coxsackievirus-Adenovirus-Receptor (CAR) and Decay Accelerating Factor (DAF) to enter cardiac myocytes while PVB19 use the receptor globoside (Gb4) to enter cardiac endothelial cells. 2. Immune system responses: The innate immune system mediated by activated cardiac toll-like receptors (TLRs) worsen inflammation in CVB3-infected mouse hearts. Different types of cells of the adaptive immune system are recruited to the site of inflammation that have either protective or adverse effects during VMC. 3. Autophagy: CVB3 evades autophagosomal degradation and misuses the autophasomal pathway for viral replication and release. 4. Viral replication sites: CVB3 promotes the formation of double membrane vesicles (DMVs), which it uses as replication sites. PVB19 uses the host cell nucleus as the replication site and uses the host cell DNA replication system. 5. Cell cycle manipulation: CVB3 attenuates the cell cycle at the G1/S phase, which promotes viral transcription and replication. PVB19 exerts cell cycle arrest in the S phase using its viral endonuclease activity. 6. Regulation of apoptosis: Enteroviruses prevent apoptosis during early stages of infection and promote cell death during later stages by using the viral proteases 2A and 3C, and viroporin 2B. PVB19 promotes apoptosis using the non-structural proteins NS1 and the 11 kDa protein. 7. Energy metabolism: Dysregulation of respiratory chain complex expression, activity and ROS production may be altered in CVB3- and PVB19-mediated myocarditis. 8. Ion channel modulation: CVB3-expression was indicated to alter calcium and potassium currents in Xenopus laevis oocytes and rodent cardiomyocytes. The phospholipase 2-like activity of PVB19 may alter several calcium, potassium and sodium channels. By understanding the general pathophysiological mechanisms of well-studied myocarditis-linked viruses, we might be provided with a guideline to handle other less-studied human viruses.

Germany

Viruses have been widely used to treat cancer for many years and they achieved tremendous success in clinical trials with outstanding results, which has led to the foundation of companies that develop recombinant viruses for a better tumor treatment. Even though there has been a great progress in the field of viral tumor immunotherapy, until now only one virus, the oncolytic virus talimogene laherparepvec (TVEC), a genetically modified herpes simplex virus type 1 (T-VEC), has been approved by the FDA for cancer treatment. Although oncolytic viruses showed progress in certain cancer types and patient populations but they have yet shown limited efficacy when it comes to solid tumors. Only recently it was demonstrated that the immune stimulatory aspect of oncolytic viruses can strongly contribute to their anti-tumoral activity. One specific example in this context are arenaviruses, which have been shown to be non-cytopathic in nature lead to the massive immune activation within the tumor resulting in strong anti-tumoral activity. This strong immune activation might be also linked to their noncytopathic features, as their immune stimulatory potential is not self-limiting as is the case for oncolytic viruses due to their fast eradication by anti-viral immune effects. Because of this strong immune activation, arenaviruses appear superior to oncolytic viruses when it comes to potent and long-lasting anti-tumor effects in a broad variety of tumor types. Currently one of the most promising therapeutics which has turned to be very much beneficial for the treatment of different cancer types is represented by antibodies targeting checkpoint inhibitors such as PD-1/PD-L-1. In this review, we will summarize anti-tumoral effects of arenaviruses, and will discuss their potential to be combined with checkpoint inhibitors for a more efficient tumor treatment, which further emphasizes that arenavirus therapy as a viroimmunotherapy can be an efficient tool for the better clearance of tumors.

Germany

For adult men with congenital heart disease (ACHD), data on erectile dysfunction (ED) is limited. We aimed to assess the frequency of ED, its role in patient-physician communication and to identify parameters predicting ED.

Germany

A method of isolation of alpha-1-antitrypsin (alpha-1-AT) in good yield from normal human plasma is described. A key step was affinity chromatography employing an antiserum which had been depleted of alpha-1-AT antibodies. The final preparations were homogeneous by immunological and physicochemical criteria. The specific activity of the purified alpha-1-AT was 0.363 mg of active bovine trypsin inhibited per 1.0 mg of inhibitor. Polyacrylamide gel patterns at both alkaline and acid pH of highly pure preparations frequently, but not invariably, showed multiple hands. Molecular weight studies by sedimentation equilibrium ultracentrifugation in aqueous buffer and in 6 M guanidine as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis suggest that alpha-1-AT is a single polypeptide chain having a molecular weight of 49,500. Other physical and chemical properties of the inhibitor are described. A limited N-terminal sequence (Glu-Asp-Pro-Gln-Gly-Asx-Ala-Ala) was obtained. It was found that alpha-1-AT easily forms polymers and higher aggregates when exposed to denaturing agents such as 8 M urea and 6 M guanidine. The results suggest that aggregation is determined by both covalent and noncovalent forces.

US

A method for assessing the binding of 3H-labeled prostaglandin E1 ([3H]PGE1) to cell membranes has been developed and used to study the interaction of [3H]PGE1 with membranes from cultured mammalian cells. Receptor sites were identified by correlation of the potency of a series of compounds to compete for [3H]PGE1 binding sites and to stimulate adenylate cyclase activity, by correlation of rates of binding and change in enzyme activity, and by the correspondence of [3H]PGE1-binding activity with the presence or absence of PGE1-sensitive adenylate cyclase in several clones. In clone B82, a murine L-cell, [3H]PGE1 binds with an activation energy of 14 kcal/mol to a class of sites with an affinity of 0.5 X 10(8) M-1 and a capacity of 150 fmol/mg of protein. Concentration dependence of adenylate cyclase activation by PGE1 (KD =30 nM) and kinetic analysis of [3H]PGE1 binding (k1 = 4 X 10(6) liters/mol/min, k-1 0.15/min) verify this affinity. Concentration dependence and specificity of binding and activation of adenylate cyclases in neuroblastoma clone N4TG1 and N18TG2 substantiate the method. In several clones that lack PGE1-responsive adenylate cyclase, no specific [3H]PGE1 binding is detectable.

US

The acyl-carrier protein (ACP) of Escherichia coli is a protein of molecular weight 8847 with a 4'-phosphopanthetheine prosthetic group. ACP functions (via the SH of the prosthetic group) as a coenzyme in the synthesis of fatty acids and complex lipids. We report proton nuclear magnetic resonance (NMR) studies of the structure of ACP under various experimental conditions. The motion of the fatty acyl chain of acyl-ACP has been investigated by 19FNMR studies of difluorotetradecanoyl-ACP. 31PNMR studies of the prosthetic group phosphorus of ACP and acyl-ACP are also reported. We make the following conclusions: (1) the structure of ACP is stabilized by surface charge, and (2) the fatty acid residue of acyl-ACP does not move freely and seems immobilized by an interaction with the protein moiety.

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The axial projection of the glutamine synthetase molecule has been reconstructed from electron micrographs of a stained preparation by using a new method of correlation search and averaging. The average over 50 individual molecules appears as a radial pattern with sixfold symmetry. The handedness evident in the average is attributed to nonuniformity of the negative stain.

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A simple, reproducible and quantitative method for evaluating certain non-specific immunological inhibitors in a variety of biological fluids is described. Human lymphocytes were stimulated with PHA in the presence of colchicine. Phytohaemagglutinin stimulated a large percentage of cells and colchicine's selective blockage of mitosis limited the stimulated cells to one S phase. These conditions effectively established a maximum amount of DNA synthesis within each culture. Quantification of suppression was then achieved by measuring a decrease from this maximum. The PHA-colchicine assay was successfully used to quantify inhibition by normal plasma, normal mouse sera, mouse neonate sera, murine Ehrlich's and sarcoma I ascitic fluids and an immunoregulatory alpha-globulin peptide preparation. Because of the ability to obtain a specific inhibitory activity for the suppressive factors, this assay was particularly suited for following the isolation of inhibitors during the fractionation of suppressive substances from complex fluids.

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Benzodiazepine derivatives are the most commonly prescribed anti-anxiety agents in clinical practice. Six benzodiazepine anxiolytics are now available in the United States. Additional drugs are used in other parts of the world, and many others are in various stages of clinical testing. All these benzodiazepine derivatives have similar neuropharmacological properties--they reduce anxiety, produce sedation and sleep, have anticonvulsant effects, and can produce muscle relaxation. Faced with this bewildering array of drugs from the same class which are very similar in intrinsic effects upon the brain, the clinician may well ask how best to make a rational choice among the available derivatives. Despite neuropharmacological similarities, there are differences among benzodiazepines in patterns of absorption, distribution, and elimination by the human body. These pharmacokinetic differences may in turn lead to apparent differences in clinical action. This review summarizes pertinent pharmacokinetic characteristics of benzodiazepine anti-anxiety agents.

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The awareness of extrapyramidal reactions during initiation of neuroleptic treatment was studied in 14 patients. Only one patient spontaneously identified the presence of dystonia. The other 13, including 3 who had experienced extrapyramidal reactions during previous hospitalizations, did not fully identify the presence of symptoms, although several had vague discomfort. There was marked variability in acknowledgement of symptoms in response to prompting by staff members. The findings are similar to reports of agnosia for hemiparesis after parietal lobe injury and also to descriptions of agnosia in animals caused by destruction of dopaminergic neurons. Since extrapyramidal reactions represent blockade of dopaminergic neurotransmission, patients' inability to perceive the reactions may represent evidence for catecholaminergic modulation of sensory perception.

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Neuroendocrine studies that examine the changes in serum prolactin levels following intramuscular (im) neuroleptics have usually monitored prolactin levels before and for 90 minutes to 3 hours after neuroleptic injection. Recent studies have suggested that this may be an inadequate period of time. In the present study, six male and four female psychiatric inpatients, who had not received neuroleptic medication for at least 1 week before the study began, received an injection of chlorpromazine (CPZ) 25 mg im; serum prolactin levels were monitored for 6 hours after injection. Peak serum prolactin levels occurred at 60 minutes in one subject, 90 minutes in three subjects, 120 minutes in two subjects, 180 minutes in three subjects, and 240 minutes in one subject. Area under the serum prolactin curve at 2 hours and area under the curve at 3 hours after CPZ injection were found to be good predictors (r = 0.86; r = 0.95, respectively) of 6-hour area under the curve. Two-hour studies should therefore not be considered inadequate; however, a 3-hour study length results in more precise characterization of prolactin response to im CPZ.

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Chlorpromazine and thioridazine are widely used antipsychotic agents that are extensively metabolized. Parent compounds and metabolites have diverse pharmacologic activities, and differences in patterns of metabolism may explain differences in therapeutic and side effects from individual to individual. Radioreceptor assays were used to determine the neuroleptic, antimuscarinic, and anti-alpha-noradrenergic potency of chlorpromazine, thioridazine, and their metabolites. The results indicate that these metabolites show a wide range of potencies. The spectrum of activity of a metabolite may be quite different from that of its parent compound. The clinical relevance of these findings to individual differences in drug response is discussed. The combined use of radioreceptor assays and chemical assays in future clinical research is proposed.

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We have isolated temperature resistant revertants from temperature sensitive E. coli strains containing either a thermolabile glutaminyl-tRNA synthetase or leucyl-tRNA synthetase. Among the revertants which still contained the thermolabile leucyl-tRNA synthetase we found two classes of regulatory mutants (leuX and leu Y) which have elevated levels of this enzyme. The leuX mutation specifies an operator-promoter region adjacent to the structural gene (leuS) for the enzyme. The leuY gene maps away from the leuS gene and codes for a protein. Using these mutants we demonstrated that the levels of leucyl-tRNA are related to the derepression of the leucine and isoleucine-valine operons. Among the revertants which still contained the thermolabile glutaminyl-tRNA synthetase were characterized three classes of mutants, glnT, glnU, and glnR. The glnT and glnU mutants contain elevated levels of tRNAgln, while the glnR mutant possesses elevated levels of glutaminyl-tRNA synthetase. The level of glutamine synthetase, the enzyme responsible for the formation of glutamine, is also derepressed in the glnT and glnR mutants.

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As a function of buffer pH, [125I]-insulin binding to rat mammary cells, rat adipocytes, or membranes prepared therefrom, at 4 degrees or 20 degrees C, showed 2 peaks in different buffers. Specific insulin binding at the pH 7.7. peak (100 +/- 11%) was lower than at pH 8.8 (140 +/- 17%) with no change in nonspecific binding. Although insulin stimulation of glucose uptake into fat cells was highest at pH 7.5, this response was also seen at pH 8.6. Scatchard affinity profiles, or in the kinetics of dissociation. Insulin degradation (< 10%) and binding to insulin antibody were similar over the pH range of 7 to 9.

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Bicarbonate was found to stimulate ATP breakdown by rabbit or cat ciliary body-iris homogenates. Maximum HCO3- stimulation of ATPase with Tris-Hepes buffer occured at pH 8.0. Acid pH and chloride ions in the media reduced the activity of the HCO3--stimulated ATPase. The Km for ATP was 0.55 mmolar and for HCO3-, 20 mmlar. HCO3- ATPase was not inhibited by acetazolamide added to in vitro. It is postulated that ATPase represents the linkage step of energy donor mechanism and active CT secretion in acid aqueous humors (human, cat.) or HCO3- secretion in alkaline aqueous humor (rabbit, guinea pig). Inhibition of Cl- or HCO3- secretion by acetazolamide results from decreased intracellular HCO3- levels which, in turn, reduces the stimulation of the HCO3- ATPase.

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The relative contribution of the renin-angiotensin system, adenocorticotrophic hormone (ACTH) and plasma electrolytes in the response of plasma aldosterone to 30 minutes of 65 degrees head-up tilt was assessed in 10 essential hypertensive patients. Studies were carried out before and during acute blockade of renin release by propranolol, ACTH suppression by dexamethasone and combined renin and ACTH blockade. In control studies orthostasis induced significant increases only in plasma renin activity and aldosterone. In contrast, when the renin response to tilt was acutely suppressed by propranolol administration, the aldosterone response was nonetheless maintained but now appeared to be under ACTH control, since concurrent increases in cortisol were observed. During ACTH suppression aldosterone increased during tilt and so did renin. However, during combined ACTH and renin blockade aldosterone failed to increase during tilt. These studies suggest that the aldosterone secretory response to head-up tilt is normally mediated by the renin-angiotensin system but, when the renin response is suppressed, an ACTH response is elicited which assumes a backup role. However, when these two systems are blocked other factors appear unable to respond during tilt to support a normal aldosterone response.

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Pulmonary mixed function oxidations are altered by in vivo exposure to paraquat or ozone. The cytochrome P-450-mediated benzphetamine N-demethylase activity was inhibited by low doses of both oxidants (paraquat, 20 mg/kg, ip; ozone, 1 ppm, 24 hrs). The effect of paraquat did not appear until 4 days after treatment and was still apparent after 2 weeks. The inhibition following exposure to ozone appeared immediately (1 day) and had resolved by one week. Cytochrome b5-mediated lipid desaturation was unaffected by the paraquat treatment and was stimulated by ozone. These alterations in pulmonary mixed function oxidase activities could not be explained by microsomal lipid peroxidation. The concentrations of cytochromes P450 and b5 also did not accurately reflect the altered enzymatic activities. The sites of the paraquat- and ozone-mediated alterations are still unknown.

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The circadian rhythms of sucrase, maltase, isomaltase, trehalase, lactase, gamma-glutamyltransferase, leucylnaphthylamide hydrolyzing activity, alkaline phosphatase and monosaccharide transport were assessed in each fifth of the small intestine of the rat in order to determine if an entire enzyme or transport system population responded in a similar manner or if there were regional differences. Animals were maintained under a light-dark cycle and fed from 1400-1800, EST for 7 days. Functional activities were assessed every 4 h for 24 h, inclusively. Quantitative, and in a few instances, qualitative differences in different areas of the intestine were found for all functions. There were portions of the lactase and alkaline phosphatase populations which displayed no rhythmicity in activity. When rhythmicity was observed there were differences in the activity patterns along the intestine for all functions. Thus, the rhythm patterns obtained from homogenates of the entire small intestine are a composite of the patterns in regions of high average activity. Also, there appears to be a reasonable amount of local control of the various functions.

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The fluorescence parameters of ethenoadenosine derivatives are influenced by metal cations and pH, as summarized here. The pH profile of ethenoadenosine determined by fluorescence intensity gives a normal titration curve and is not affected by ionic strength. In contrast, the pH titration curves of etheno-ATP, etheno ADP, and etheno AMP depend upon ionic strength. At high ionic strength normal curves are obtained, whereas at low ionic strength anomalies are obtained; this suggests that the phosphates can interact with the ring, possibly by hydrogen binding to the ring nitrogens. The room temperature fluorescence of ethenoadenosine occurs from the base form, although excitation of either the acid or base forms can contribute to the emission. This result can be explained if the excited state pK is lower than the ground state pK, and if deprotonation occurs within the time scale of the excited state. At low pH values the fluorescence lifetime of the base form is dependent upon the buffer concentration, indicating that the reverse reaction, protonation, occurs. The affinity constants for the binding of metals to the ethenoadenosine phosphates resemble those for the corresponding adenosine phosphates. Ni(II) and Co(II) are more effective than Mn(II) in quenching the fluorescence of ethenoadenosine phosphates; this result is predicted by Förster's theory for energy transfer based upon the overlap between donor emission spectrum and acceptor absorption spectrum. The diamagnetic ions Mg(II), Ca(II), and Zn(II) do not appear to affect the fluorescence of the ethenoadenosine phosphates directly, but rather to affect the conformation of the molecule, thereby affecting the quantum yield.

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A quasi-short-circuit (tunable voltage clamp) measurement method with microsecond time resolution was applied to a bacteriorhodopsin model membrane formed by a novel interfacial technique. A new component (B1) of the displacement photocurrent was recorded: it has no detectable latency at an instrumental time constant of 1.5 museconds, and persists at 5 degrees C. In addition, a slower component (B2) of opposite polarity inhibited by low temperature (5 degrees C) and low pH (pH = 3.0) was recorded. The technique is very sensitive for the study of fast capacitative photoresponses in model membranes, and allows the detection of charge displacements in bacteriorhodopsin associated with distinct stages of the photochemical transformation.

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The effects of low pH, and of alkaline earth cations, were examined on calcium uptake by pinched-off nerve terminals (synaptosomes). This uptake appears to be mediated by voltage-sensitive Ca channels (J. Physiol. 247:617, 1975). Ca uptake was measured in low (5 mM) or high (77 mM) potassium media. The extra uptake promoted by depolarizing (K-rich) media was almost maximal at pH 7.5, and decreased as the pH was lowered. Data relating depolarization-induced 45Ca uptake to pH fit a titration curve with a pKa approximately 6. Experiments in which Ca concentration and pH were both varied indicated that Ca2+ and H+ compete for a common binding site. Inhibition of depolarization-induced 45Ca uptake by the alkaline earth cations was studied to determine the apparent binding sequence for these cations in the Ca channels: Ca greater than Sr greater than Ba greater than Mg. This sequence resembles that observed for block of Ca channels in other preparations. The apparent binding sequence of the alkaline earth cations and the apparent pKa (approximately 6) of the Ca-binding site indicate that the Ca channel is a "high field strength" system. Protonation of a Ca channel binding site could explain the inhibitory effect of low pH on Ca-dependent neurotransmitter release (cf. Del Castillo et al., J. Cell. Comp. Physiol. 59:35, 1962).

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Lipid bilayer permeation coefficients for the neutral maleic acid molecule and the maleate monoanion have been determined by proton magnetic resonance techniques. Phosphatiydylcholine-cholesterol (2:1) unilamellar vesicles were prepared having an initial maleate anion concentration gradient stabilized by coupling to an impermeant potassium counterion. The coupling was released by addition of valinomycin, and the time evolution of external pH, internal pH, and maleate concentration followed using nuclear magnetic resonance areas and chemical shifts. Transport rate equations were numerically integrated to fit the date, yielding best fit permeation coefficients of 4 X 10(-9) and 4 X 10(-5) cm/s for maleate monoanion and maleic acid, respectively.

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The viscoelastic behavior of rat 9L cellular DNA was studied as a function of the detergent used for lysis, the pH and duration of lysis, and gamma ray dose. For nondenaturing lysis conditions, a model of the DNA was proposed to account for the effects of these agents on the viscoelastic retardation time. It was concluded that these agents affect the hydrodynamic radius of the DNA rather than its molecular weight. For denaturing lysis conditions, molecular weights calculated from the relaxation time were consistent with those calculated from alkaline sucrose sedimentation profiles.

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The Hodgkin-Huxley kinetic parameters, alpha h and beta h, which govern the rate of recovery from and development of sodium channel inactivation, respectively, have been measured as a function of membrane potential and external pH using a three-pulse protocol. alpha h but not beta h is substantially accelerated by reducing external pH from 7.4 to 6.4. The alpha h vs. voltage curve appears to be selectively shifted in the depolarizing direction by approximately 12 mV for this pH change, giving an apparent, h infinity curve shift of approximately 6 mV in the same direction (less inactivation).

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Recent evidence suggests that of calcitonin (CT) and parathyroid hormone (PTH) is controlled by factors other than the ambient serum calcium concentration. We studied the effects of infusions of four neuroendocrine modulators upon CT and PTH levels: isoproterenol (beta-adrenergic agonist), methoxamine (alpha adrenergic agonist), prostaglandin E2, and somatostatin. Isoproterenol was a consistent secretagogue for both hormones. Maximal CT increments during isoproterenol infusion in normal subjects were 13 +/- 2 pg/ml (mean +/- SEM, n = 6, P less than 0.001; basal, 26 +/- 5). Maximal increments in PTH were 113 +/- 22 pg/ml (P less than 0.01, n = 6; basal, 430 +/- 11). Infusions of methoxamine increased CT by 13 +/- 5 pg/ml (n = 5, P less than 0.05; basal, 43 +/- 13), but had no effect on PTH. The means of the maximal CT increments during isoproterenol (21 +/- 8 pg/ml) and methoxamine infusion (28 +/- 11 pg/ml) were not statistically different from those achieved by acute elevations of serum calcium levels within the physiological range (41 +/- 23 pg/ml). Infusions of somatostatin and prostaglandin E2 had no or only transient effects on basal or stimulated CT or PTH levels. Our data suggest that adrenergic input modulates CT and PTH secretion in humans independently of changes in serum calcium.

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In human placental explants cultured in vitro, dopamine inhibited human chorionic somatomammotropin (hCS) secretion into the culture media. In the control flasks, the level of hCS secretion was 130.5 +/- 7.8 micrograms/g tissue (n = 6). When 1 mM dopamine was added, hCS levels decreased to 80.2 +/- 11.5 micrograms/g tissue (P less than 0.01). Dopamine (5 and 10 mM) further lowered hCS levels. In contrast, 1 mM pimozide enhanced hCS secretion by 2-fold as compared to control levels (248.2 +/- 44.8 vs. 130.5 +/- 7.8, P less than 0.02). The simultaneous addition of dopamine did not alter the stimulatory effect of pimozide on hCS secretion. In separate experiments, arginine (1 and 5 mM) and somatostatin (1 microgram/ml culture media) did not alter hCS secretion from placental explants. These results suggest that hCS secretion is modulated by dopaminergic receptors.

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The zoological specificity of human thyroid-stimulating antibody (TSAb) that occurs in the blood in Graves' disease was examined by assessing its effect on the thyroid of the dog, guinea pig, calf, and mouse as well as that of man. With all but murine glands, thyroid stimulation was assayed by measuring the increase in the concentration of cAMP in the thyroid slices or fragments after 2 h of incubation in buffer containing TSAb. Effects on the thyroid of the mouse were monitored by the in vivo bioassay for LATS. Sera from 33 patients with Graves' disease were obtained and concentrates of TSAb were prepared by precipitation of IgG with 1.64 M (NH4)2SO4. These all stimulated the human thyroid, 13 were LATS-positive, and they variably affected the tissues of other species; of 27 tested, 14 stimulated the thyroid of the dog, 8 out of 23 stimulated the thyroid of the guinea pig, and 12 out of 16 stimulated the gland of the calf. The more potent the TSAb as assayed with human tissue, the more likely was it to stimulate other species of thyroid; however, frequent exceptions occurred. In a separate analysis of 35 LATS-positive preparations of TSAb, correlation between the responses in the LATS and human thyroid slice assays was statistically significant (P less than 0.001). The data are compatible with the view that stimulation by TSAb of nonhuman thyroids, including the murine as in the LATS bioassay, reflects cross-reaction of this immunoglobulin with an antigen that has sufficient similarity to the human molecule to be recognized by the human antibody.

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To determine if the adrenergic nervous system, and specifically tyrosine hydroxylase, plays a role in the extrathyroidal conversion of T4 to T3, normal male volunteers were treated with T4 and subsequently with T4 and alpha-methyl-p-tyrosine (alpha-MPT), an inhibitor of tyrosine hydroxylase, for 2 weeks. The mean serum T4 and T3 concentrations increased during T4 administration and remained at the same levels during combined T4 and alpha-MPT administration. Urinary vanillylmandelic acid excretion declined significantly during alpha-MPT administration. These results do not support the hypothesis that tyrosine hydroxylase is involved in extrathyroidal T3 production.

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Normal human plasma contains "inactive renin," whose ability to generate angiotensin I increases after exposure to pH 3.3. Big renin is a partially inactive enzyme of larger molecular weight, which is also activated at pH 3.3, and is found of pregnant women, and in amniotic fluid, but not in normal plasma. We have compared the effects of acid exposure and storage at 4 and -4 C on normal plasma and plasma containing big renin. The concentration of inactive renin in normal plasma was approximately equal to that of normal active renin, and its activity increased slowly on prolonged standing at -4 but not 4 C. In contrast, the activity of big renin increased by 50% as early as 1-3 days at 4 C and increased even more quickly at -4 C. Acid treatment of plasma containing big renin caused 4-10 times greater increase in active renin than similar treatment of normal plasma. During gel filtration, both cold-activated and previously acidified big renin coeluted with unactivated big renin. These data indicate that big renin is highly susceptible to cold or acid activation and that such activation of big renin does not result in a detectable decrease in its molecular weight of 60,000 daltons. Furthermore, acid and cold seem to activate the same pool of inactive renin in normal plasma. Although both normal and big renin are stable for long periods below -20 C, a serious overestimate of plasma renin activity can occur if plasma is stored just above its freezing point before assay.

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Plasma PRL was measured at 20-min intervals in six patients with Parkinson's disease under various treatment protocols. In addition, 24-h mean GH levels were measured. The results of these studies showed that two untreated patients with Parkinson's disease had normal 24-h mean PRL levels with the normal increase during sleep. During chronic treatment with L-dopa-carbidopa (Sinemet), the 24-h PRL level was 12.8 +/- 4.9 ng/ml (mean +/- SD) and there was persistence of augmented PRL secretion during sleep. The 24-h mean GH level ranged from 1.5-4.4 ng/ml, with a mean of 2.5 ng/ml. The addition of a dopamine agonist (Lergotrile mesylate) resulted in a significant (P less than 0.01) suppression of the 24-h mean PRL levels and abolition of the normal sleep augmentation after 2 weeks of therapy. This suppression was maintained in one patient who was restudied 4 months after the addition of dopamine agonist therapy to L-dopa-carbidopa. The 24-h mean GH levels did not change significantly after the addition of the dopamine agonist when compared to L-dopa-carbidopa alone. These results suggest a dichotomy between the PRL and GH responses to combined L-dopa-carbidopa and dopamine agonist therapy. In addition, the preservation of normal PRL regulation in the two untreated patients with Parkinson's disease suggests that dopaminergic neurons are not universally affected in this disorder.

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The effect of somatostatin (SRIF) on human chorionic somatomammotropin (hCS) secretion was studied in human placental explants cultured in vitro. In the experimental flasks, SRIF was added in a concentration of 10, 100, and 1000 ng/ml media; hCS levels measured by RIA were not different from those found in the control flasks. In separate experiments, we investigated the action of SRIF on hCG secretion by a human malignant choriocarcinoma cell line maintained in tissue culture. SRIF (1000 ng/ml) did not inhibit basal or dibutyryl cAMP-induced stimulation of hCG secretion. These results suggest that somatostatin does not suppress hCS or hCG release in vitro from normal or malignant trophoblast, respectively.

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The rise and subsequent return to basal of glucose production (Ra) during a constant glucagon infusion ("downregulation") has suggested to some workers that glucagon's effects are evanescent. To examine whether glucagon displays persistent biological activity even after downregulation, 6 healthy males received an 8 hour infusion of somatostatin and glucagon, with 3H-3-glucose to measure glucose turnover. Ra rose from 2.8 +/- 0.3 to 4.2 +/- 0.3 mg/kg . min at 90 minutes, returned to basal levels at 150 minutes, and remained at this level for the ensuing 330 minutes. Six additional subjects received an 8 hour somatostatin infusion, with glucagon administered concomitantly for the first 5 hours. Glucagon withdrawal at 5 hours produced an immediate decline in Ra from 1.8 +/- 0.2 to 0.9 +/- 0.2 mg/kg . min. Thus, even after downregulation the maintenance of basal Ra is dependent on circulating glucagon.

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We used a modification of the TSH radioreceptor assay to detect TSH-binding inhibition (TBI) activity in serum and serum fractions from normal subjects and patients with Graves' disease. TBI activity is present in normal IgG prepared by DEAE-Sephadex chromatography and in normal globulins prepared by precipitation at 1.6 M ammonium sulfate. Other normal serum proteins also had TBI activity when large concentrations were tested. Gel filtration chromatography and powder block electrophoresis were used to prepare fractions of normal and Graves' disease sera. In these fractions from normal serum. TBI activity was found in both gamma-globulin and alpha-globulin-albumin fractions electrophoretically and in both 7S and 4S peaks from gel filtration. TBI activity from Graves' disease patients' sera was similarly distributed, but relatively more TBI accompanied the electrophoretic gamma-globulins. Sepharose Protein-A and anti-IgG were used as immunoabsorbents to isolate and purify IgG from normal and Graves' disease sera. TBI activity in IgG was proportional to the IgG concentration, indicating that the TBI which migrates as a gamma-globulin electrophoretically is an IgG and thus may possibly be an antibody. Inhibitory activity found in normal serum globulins and the non-IgG fractions of both normal and abnormal sera seriously interferes with attempts to use the TSH radioreceptor assay to study the hypothesized anti-TSH, receptor antibody in the serum of patients with Graves' disease.

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Intensive laboratory investigation of patients with recurrent infections, and with infections with microbial species not usually considered to be pathogenic, have led to the identification of several defects in granulocyte function. The two functions of granulocytes which have received most attention in the past decade have been locomotion (especially response to chemotactic stimulation) and microbicidal activity. Defective granulocyte chemotaxis has been demonstrated in patients with clinical manifestations suggesting abnormalities related to vasoactive amines, i.e., patients with eczema and extreme IgE hyperimmunoglobulinemia. The depressed granulocyte chemotactic responsiveness found in these patients can be reproduced in vitro when histamine and beta adrenergic agents are incubated with control granulocytes. Since these compounds have been shown to increase levels of intracellular cyclic AMP in other cells, there appears to be an association between cyclic nucleotide metabolism and regulation of granulocyte locomotion. Defective granulocyte microbicidal activity is found in patients with chronic granulomatous disease and it has been shown that there is little increase in oxidative metabolism during phagocytosis by these cells. Methods for quantitating the oxidative metabolism of granulocytes and monocytes include oxygen uptake, reduction of nitroblue tetrazolium, formate oxidation, and chemiluminescence response during phagocytosis. Since products of oxygen metabolism, i.e., hydrogen peroxide, superoxide or singlet oxygen do not accumulate in granulocyte phagocytic vacuoles, intracellular microbes are not killed (except bacterial species that produce hydrogen peroxide). The biochemical basis for defective oxidative metabolism in granulocytes from patients with chronic granulomatous disease appears to be associated with abnormal nucleotide oxidase activity.

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The clinical effectiveness of published algorithms in correcting serum total calcium (CaT) for the effects of protein, albumin, and pH was tested. Corrected calcium (CaC) values obtained by 13 of these methods were compared with values of measured free calcium (CaF) in 55 samples from normal controls and 404 samples from patients with various disorders of calcium metabolism. Three criteria were used to compare either CaC or CaT with measured CaF: 1) the correlation coefficient, 2) the average absolute deviation from measured CaF of the values of CaF predicted by the linear regression of CaF on each CaC, and 3) the number of samples in which CaC or CaT gave a different impression of normality than measured CaF. Application of the 13 published algorithms produced varied results, but none produced substantially better agreement between CaC and CaF than was found between CaT and CaF. The application of additional algorithms derived by multiple linear regression using our data base gave slightly better results than any of the published algorithms, but many values of CaC remained which were disparate from the measured value of CaF. Correction of measured total calcium by using other concurrently obtained chemistry values does not seem to adequately predict calcium status as measured by free calcium.

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Four characteristics of culture medium that are important to embryo development and nutrition of the blastocyst have been discussed. An examination of several of the most commonly used media for embryo culture demonstrates many similarities among them. The milliosmolarities of the media range from the hypoosmotic optimums (256 milliosmols) demonstrated in several in vitro studies to the physiologic range (308 to 315 milliosmols). Media between these extremes generally allow good development. Low oxygen concentrations (5%) in the culture environment allow somewhat better development of early cleavage stages, but recent studies suggest the difference between development in 5 and 20% oxygen to be less than originally thought. The media most commonly employed for early embryo culture contain bicarbonate as the buffer, but maintenance of pH is probably not the most crucial role of the CO2-bicarbonate content of the media. Likewise, since 1965 almost all media used to culture embryos have used pyruvate as the primary energy source. This is particularly important when early stages, before blastocyst development, are cultured. The concentration used generally falls within the optimum range of 2.5 to 5.0 X 10(-4)M first reported. Although glucose is not oxidized well by the early cleavage stages, it is an important energy source for all blastocysts. Furthermore, glucose contributes more than any other carbon source, including amino acids, to protein formation. Much is yet to be learned concerning the nutrition of the blastocyst, but our knowledge has increased immensely during the last 15 years. Hopefully our progress will be at least as rapid in the coming decade.

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The kinetics of cell wall turnover in Bacillus subtilis have been examined in detail. After pulse labeling of the peptidoglycan with N-acetylglucosamine, the newly formed peptidoglycan is stable for approximately three-quarters of a generation and is then degraded by a process that follows first-order kinetics. Deprivation of an auxotroph of amino acids required for protein synthesis results in a cessation of turnover. If a period of amino acid starvation occurs during the lag phase of turnover, then the initiation of turnover is delayed for a period of time equivalent to the starvation period. During amino acid starvation, new cell wall peptidoglycan is synthesized and added to preexisting cell wall. This peptidoglycan after resumption of growth is also subject to degradation (turnover). It is suggested that cell wall turnover is dependent on cell growth and elongation. Several possible control mechanisms for cell wall autolytic enzymes are discussed in light of these observations.

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Earlier studies indicated that the gene of an ammonium-inducible glutamate dehydrogenase (GDH) was inducible throughout the cell cycle and was expressible shortly after replication early in the S-phase in synchronous Chlorella cells growing at a rate of 13% per h in the absence of inducer. In the present study, synchronous cells cultured at the same growth rate in the continuous presence of inducer accumulated this enzyme in a linear manner, with a positive rate change observed late instead of early in the S-phase. At a growth rate of 26% per h, the positive rate change appeared to be displaced to 1.5 h before the S-phase in the next cell cycle. With 2'-deoxyadenosine, an in vivo inhibitor of deoxyribonucleic acid (DNA) synthesis, the magnitude of the positive rate change was shown to be proportional to the relative increase in DNA in the previous cell cycle. Collectively, these data support the idea that expression of newly replicated genes of this enzyme can be delayed into the subsequent cell cycle in cells in the continuous presence of inducer. Studies with cycloheximide indicated that the inducible GDH and another GDH isozyme were stable in fully induced cells in the absence of protein synthesis. However, after ammonium was removed from the culture medium, the activity of the inducible GDH decreased rapidly in vivo, with a half-time of 5 to 10 min at 38.5 degrees C, whereas the rate of accumulation of the other GDH isozyme did not change. Addition of cycloheximide, at the time of inducer removal, prevented this loss in activity of the inducible GDH. The inability to rescue the activity of the inducible GDH, by readdition of ammonium during the deinduction period, indicates that this enzyme probably underwent irreversible inactivation and/or proteolytic degradation.

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The effect of different extraction procedures on the yields of water-soluble and water-insoluble glycogen fractions from a number of Saccharomyces strains was studied by using a specific method for glycogen determination. The similarity of the yields obtained by the different procedures showed that neither form of glycogen is an artifact, and variations in the relative amounts of glycogen in the two fractions during cell growth and in different yeast strains suggest that they represent different pools of storage material with specific roles in cell development and differentiation. A proportion of the water-insoluble glycogen fraction, solubilized by mechanical agitation, was shown to be strongly associated with a beta-glucan-like polysaccharide that may be a cell wall component.

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Enzymatic methyl ester formation in Escherichia coli ribosomal proteins was observed. Alkali lability of the methylated proteins and derivatization of the methyl groups as methyl esters of 3,5-dinitrobenzoate indicate the presence of protein methyl esters. The esterification reaction occurs predominantly on the 30S ribosomal subunit, with protein S3 as the major esterified protein. When the purified 30S subunit was used as the methyl acceptor, protein S9 was also found to be esterified. The enzyme responsible for the esterification of free carboxyl groups in proteins, protein methylase II (S-adenosyl-L-methionine:protein carboxyl methyltransferase, EC 2.1.1.24), was identified in E. coli Q13. This enzyme is extremely unstable when compared with that from mammalian origin. By molecular sieve chromatography, E. coli protein methylase II showed multiple peaks, with a major broad peak around 120,000 daltons and several minor peaks in the lower-molecular-weight region. Rechromatography of the major enzyme peak showed activities in several fractions that are much lower in molecular weight. The substrate specificity of the E. coli enzyme is similar to that of the mammalian enzyme. The Km value for S-adenosyl-L-methionine is 1.96 X 10(-6) M, and S-adenosyl-L-homocysteine was found to be a competitive inhibitor, with a Ki value of 1.75 X 10(-6) M.

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Preservation of donor tissue for human keratoplasty has been evaluated using modified M-K medium. The medium has been modified by changing the antibiotic and buffer and adding a pH indicator. These changes offered theoretical and practical advantages. There were no primary donor failures in 50 consecutive penetrating keratoplasties with an overall success rate of 92 percent. Clinical specular microscopy revealed good endothelial cell survival.

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The various neurohumoral and intrinsic factors that control the uteroplacental hemodynamics in health and disease and in responses to physiologic and pharmacologic stimuli have been reviewed. The following conclusions may be derived: We still need improvement in our methodology of monitoring uterine blood flow. The present methods, which have some reliability, are not easily applicable to human subjects and even in animals their use presents problems of accuracy and sensitivity with which the investigator must become familiar. The marked and progressive increase in uterine blood flow that occurs during pregnancy is caused by complex factors, some of which are hormonal and hemodynamic in nature. The increased vascularity of the pregnant uterus and the opening of the arterioles during the process of formation of the intervillous space are important factors that facilitate the increase in uterine blood flow. The increment seems to be totally derived from the increment in the cardiac output that occurs during pregnancy. There seems to be no redistribution among the regional blood flows of the body. In the anesthetized condition the blood flow to the uterus depends largely on the perfusing pressure; the critical closing pressure seems to be around the 40 mm Hg level. This linear flow-pressure relationship does not, however, apply to the unanesthetized condition. A rise or fall in the perfusing pressure in the conscious state may be accompanied by an increase or decrease in the uterine blood flow, depending on the underlying mechanisms. Factors that lead to alpha-adrenergic stimulation produce an increase in uterine vascular resistance and a decrease in flow, irrespective of the status of the perfusing pressure. beta-adrenergic stimulation may increase uterine blood flow either through their vasodilating action or through their myometrial relaxing effects. Hypertensive diseases are most often accompanied by a decrease in uterine blood flow, whereas hypoxic states may decrease the flow even though the arterial pressure may not change significantly. It is extremely risky to extrapolate from information obtained in the anesthetized animal to the unanesthetized, conscious animal. Likewise, data obtained from normotensive conditions may not hold true for the hypertensive or hypotensive states. This is of particular relevance when one is dealing with the effects of pharmacologic agents that act on the cardiovascular system. Uterine contractions, whether induced through spontaneous or oxytocin-induced labor, produce a decrease in uterine blood flow.(ABSTRACT TRUNCATED AT 400 WORDS)

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The reinforcing properties of etonitazene, both conditioned and unconditioned, were measured in rats that had received saline only by continuous intravenous infusion ("saline" group) and in two groups of rats that had been physically dependent on morphine to equal degrees (and presumably had developed equal degrees of tolerance to morphine): one by once daily passive intravenous injection of morphine ("injection" group) and the other by passive continuous intravenous morphine infusion at the same daily doses for approximately the same number of days ("infusion" group). Prior to passive saline and morphine administration, all rats were trained to press right- and left-sided levers for water reinforcement from 1600 to 0800 hrs to a not more than 60-40 split, and these and other measures ("baselines") were repeated after recovery from the early (acute) morphine-abstinence syndromes. Then etonitazene, 5 micrograms/ml, was substituted for water on the nonpreferred side and all measures were repeated from 1600 to 0800 hrs once every two weeks for 20 weeks (10 "relapse" tests). It was postulated that the daily cycles of morphine-abstinence and suppression of abstinence in the injection group only would generate latent interoceptively conditioned reinforcing properties of morphine because of conditioning of suppression of abstinence to the concomitant internal sensorial effects of morphine, which would persist after morphine withdrawal and be transferred to the internal effects of another opioid, etonitazene. It was found that across the first nine relapse tests, the injection group consumed significantly more etonitazene than the infusion group, while there were no significant differences in water consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

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Emergence of antibiotic resistance is related to the ease of mutation, to the extent of exchange of genetic information in bacteria by conjugation, transformation, and transduction, and to the large-scale use of antimicrobial agents in the biosphere. In addition to the development of resistance through chromosomal mutation and exchange of chromosomal genes among organisms, there is a more profound enlargement of the gene pool by the dissemination and amplification of plasmids. Two examples of the exchange of antibiotic resistance are analyzed: the transfer of plasmids from Bacteroides fragilis to Escherichia coli and the emergence of antibiotic-resistant strains of STreptococcus pneumoniae. Plasmids encoding antibiotic resistance in B. fragilis were transferred to E. coli by DNA-mediated transformation and conjugation. The beta-lactamase in the transformants and transconjugants displayed the same substrate specificity and electrophoretic mobility as the donor strain. The plasmid apparently was integrated rapidly into the chromosome of the recipient strain. Multiple antibiotic-resistant strains of S. pneumoniae were analyzed for plasmids, and none were detected. Furthermore, no evidence of linkage between the traits of multiple antibiotic resistance was observed. beta-Lactamase was not detected in the penicillin-resistant strains; therefore, it is likely that the resistance in these strains was chromosomal rather than plasmid-mediated. The range of genetic exchange and the use of Koch's postulates in determining the genetic mechanism of antibiotic resistance are illustrated and discussed.

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1. After separation by SDS gel-chromatography, analysis of AEP-containing glycoproteins from M. senile, indicated 66% amino acids with 220 AEP res./1000 res. and 30% carbohydrate for high mol. wt (greater than 10(7) forms and 80% amino acids with 25-50 AEP res./1000 res. and 10% carbohydrate for low mol. wt (2-4 x 10(4) forms. 2. Uronic acids, sulfate, lipid, and sialic acids were absent. 3. Mild base digestion released AEP-hexosamine containing oligosaccharides and destroyed ser-thr residues in the high mol. wt components. 4. Phosphonoglycoproteins appear to be acidic connective tissue components with AEP linked to hexosamine containing oligosaccharide side chains.

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1. The adenosine deaminase has an approximate molecular weight of 130,000-140,000 and the composition of two polypeptide units (mol. wt about 68,000) is suggested, by means of SDS disc electrophoresis. 2. Both the alpha (Vm/Km) and beta (Vm) parameters were varied with pH and temperature. RSS (relative substrate specificity) adenosine and deoxyadenosine values for alpha and beta were 1.2 and 1.1, respectively. 3. Adenine, 2'-, 3', 5'-AMP, 5'-deoxyAMP, ADP and ATP were not deaminated by the enzyme. 4. Inhibition by Mg2+ was found in reaction with adenosine at pH 8 but not with deoxyadenosine at the same pH. Mn2+, which did not affect the reaction rate at pH 4 and 5, showed competitive inhibitory effects at pH 6, 7 and 8.

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1. 2,4-Dinitrophenol (2,4-DNP) in substrate level concentrations (200 microM-1 mM) temporarily inhibits H2 production by Tritrichomonas foetus and Trichomonas vaginalis as well as the accumulation of metronidazole, dependent on its reduction by the two trichomonad species and by Entamoeba invadens. 2. 2,4-DNP competes for the reducing equivalents which are necessary for H2 production or for the reduction of metronidazole, thereby inhibiting these processes. 2,4-DNP is reduced to 2-amino, 4-nitrophenol. 3. 2,4-DNP in concentrations up to 800 microM has no effect on the uptake of O2 by these organisms. 4. 2,4-DNP has some toxicity for T. foetus.

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1. Two peroxidases, differing in ionic character and substrate specificity, have been isolated from the tropical marine sponge Iotrochota birotulata. 2. Both peroxidases catalyze the oxidation of a number of substrates, and one peroxidase possesses a specificity similar to the terrestrial fungal enzyme chloroperoxidase. 3. Based on inhibition studies utilizing sodium azide, potassium cyanide and 8-hydroxyquinoline, it appears that the peroxidases from I. birotulata are haemoprotein complexes. 4. One peroxidase appears to possess subunit structure, and requires bound divalent metal cations for activity.

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1. The synthesis of gamma-glutamylhydroxamate from glutamate and hydroxylamine has been utilized as an approximation of glutamine synthetase activity in kidneys of rabbit, rat, dog, monkey and man. 2. Kidneys of rabbit contain glutamine synthetase in high activity; those of rat, in intermediate activity; and those of dog, monkey and man, in negligible activity. 3. No more enzyme is present in kidneys of the latter two species than in those of the dog, in which the enzyme is generally considered to be absent.

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1. An NADP-specific glutamate dehydrogenase (E.C. 1.4.1.4) of mitochondrial origin has been detected in M. senile, a sea anemone. 2. Substrate specificity and starch gel electrophoresis experiments indicated an absence of the NAD(P) glutamate dehydrogenase (E.C. 1.4.1.3). 3. This NADP specific GDH activity appears to be the sole GDH activity in species of the animal phylum Coelenterata.

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1. In vitro assay conditions have been defined for measurement of delta 9 desaturase activity in Tetrahymena pyriformis W. 2. The reaction depends on the presence of oxygen and a reduced pyridine nucleotide cofactor. FAD supports a low level of enzymatic activity. 3. Both stearyl-CoA and palmityl-CoA are acceptable substrates. Oleate formation is maximal at 30 degrees C. 4. Delta-9 desaturase activity appears to be localized in the microsomal fraction. Delta-6 and/or delta 12 desaturase activities have also been observed. 5. When the specificity of the delta 9 desaturase towards stearyl-CoA and palmityl-CoA was observed at 30 and 16 degrees C it was found that lowering the assay temperature did not affect specificity. Stearyl-CoA was more readily desaturated at both temperatures. 6. Exogenous oleyl-CoA and diisopropylfluorophosphate had little effect on delta 9 desaturase activity. However, cyanide strongly inhibited desaturation and a sensitivity to sulfhydryl-binding reagents has also been demonstrated.

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1. The P50 of Cancer Magister hemocyanin in hemolymph was 40.0 mm at 25 degrees C, nmax = 3.9; at 14 degrees C, 15.0 mm and 3.4; and at 10 degrees C. 10.2 mm and 3.3. 2. In the absence of Ca2+ and Mg2+, the P50 at 25 degrees C was in the range of 150 to 250 mm, depending upon pH. 3. Our results show that temperature variation and pH variation may be important factors in the physiological regulation of oxygen delivery in this species. 4. The automatic recording device of Imai et al. (1970) previously used for the study of hemoglobins, was found to be eminently suitable for the study of O2 affinities of hemocyanins.

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1. Human haptoglobin type 1-1, porcine haptoglobin, and equine haptoglobin were isolated and purified. 2. These haptoglobins were similar in polyacrylamide gel electrophoresis and in subunit structure but showed microheterogeneity in isoelectric focusing. 3. Isoelectric points of human haptoglobin as determined with photopolymerized gels were found to be 4.03-4.24, of porcine haptoglobin 4.0-4.30, and of horse haptoglobin 3.80-4.15, respectively. 4. Results obtained with chemically polymerized gels were 0.08-0.3 pH units higher. 5. Examined haptoglobins differed also in the ability of complex formation with hemoglobin, in sialic acid content and in antigenic specificity.

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1. Kinetic data for avian erythrocyte AMP-deaminase in lysate supernatants and 2000-fold purified enzyme were consistent with an allosteric model having four binding sites for substrate. 2. Relative to the purified enzyme, AMP-deaminase in lysate supernatants exhibited a greater S0.5 and enhanced sensitivity toward phytic acid, but was far less sensitive toward potassium ion. 3. In the absence of potassium chloride, the enzymatic activity in lysates exhibited hysteresis at subsaturating 5'-AMP. This response was modified reversibly by allosteric ligands. 4. It is concluded that the characteristics of avian RBC AMP-deaminase, as expressed in lysates, may reflect important intermolecular interactions and better represent the regulatory properties of this enzyme in erythrocytes.

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1. Crithidia fasciculata adapted to growth in the presence of 10(-5) M carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, maintained adenosine phosphate pools and an adenylate energy charge comparable to those of control cells. 2. CCCP-adapted cells in the presence of the uncoupler respire endogenous substrate at a greater rate than control cells and this effect of CCCP appears readily reversible. 3. CCCP-treated, adapted cells, supporting high endogenous respiration rates, were not responsive to added substrates which significantly stimulated the oxygen utilization of normal C. fasciculata. 4. CCCP-adapted cells, provided with [U-14C]-labeled proline, utilize this substrate at 67% the rate of control cells, but divide the isotopic label between CO2 and protein in a ratio identical to that of normal cells. 5. The transport of alanine and proline by adapted C. fasciculata was severely impaired, while the transport of tyrosine and leucine was unaffected.

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1. Some mechanisms of action of neuroleptics at cellular level are reviewed, mainly the effects on synaptic transmission and the effects on chromatin. 2. As regard to the effects at synaptic level, a brief review is presented on the available evidence in support of the currently prevailing dopamine hypothesis. 3. Studies carried out on the mechanisms and sites of action of neuroleptics on chromatin show that: a. Behavioral changes caused by psychotropic drugs in experimental animals are associated with chromatin alterations and induced macromolecular syntheses. b. Parkinsonian and possibly drug-induced extrapyramidal symptoms are associated with aberrations in protein synthesis. c. Destabilization under "stress" of the heterochromatin in schizophrenics seems to be due to histone modifications and is partly prevented by neuroleptic treatment.

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1. Central injection of a long lasting enkephalin analog (D-Ala2-leu-enkephalin-amide) produced a syndrome of stereotyped motor activity. 2. This activation syndrome was significantly reduced by two dopamine (DA) blockers (pimozide, fluspririlene) and by an agonist of inhibition-mediating DA receptors [(3,4-dihydroxyphenylamino)-2-imidazoline]. 3. Intact DA functioning is necessary for the occurrence of this enkephalin-mediated behavioral syndrome.

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