Antiparallel β-sheet architecture in Iowa-mutant β-amyloid fibrils

Wild-type, full-length (40- and 42-residue) amyloid β-peptide (Aβ) fibrils have been shown by a variety of magnetic resonance techniques to contain cross-β structures in which the β-sheets have an in-register parallel supramolecular organization. In contrast, recent studies of fibrils formed in vitro by the Asp23-to-Asn mutant of 40-residue Aβ (D23N-Aβ(1-40)), which is associated with early onset neurodegeneration, indicate that D23N-Aβ(1-40) fibrils can contain either parallel or antiparallel β-sheets. We report a protocol for producing structurally pure antiparallel D23N-Aβ(1-40) fibril samples and a series of solid state nuclear magnetic resonance and electron microscopy measurements that lead to a specific model for the antiparallel D23N-Aβ(1-40) fibril structure. This model reveals how both parallel and antiparallel cross-β structures can be constructed from similar peptide monomer conformations and stabilized by similar sets of interactions, primarily hydrophobic in nature. We find that antiparallel D23N-Aβ(1-40) fibrils are thermodynamically metastable with respect to conversion to parallel structures, propagate less efficiently than parallel fibrils in seeded fibril growth, and therefore must nucleate more efficiently than parallel fibrils in order to be observable. Experiments in neuronal cell cultures indicate that both antiparallel and parallel D23N-Aβ(1-40) fibrils are cytotoxic. Thus, our antiparallel D23N-Aβ(1-40) fibril model represents a specific "toxic intermediate" in the aggregation process of a disease-associated Aβ mutant.     

Human β-glucuronidase

Our purpose is to develop a standard method for preparing the bile for -glucuronidase determination by removal of bile acids and conjugated bilirubin which interfere with its activity. The bile acids and conjugated bilirubin in their purified solutions and in the diluted gallbladder biles could be extracted completely with cholestyramine in powder form or tetrahexylammonium chloride (THAC) in chloroform or ethyl acetate. The enzyme was, however, partially precipitated with cholestyramine and denatured by chloroform but not by ethyl acetate. A standard procedure, therefore, includes extraction of the diluted gallbladder bile with THAC in ethyl acetate, followed by determination of the maximal velocity (V _max ) of the enzyme by a kinetic method employing phenolphthalein glucuronide as the substrate. The average V _max of -glucuronidase in the 20 normal gallbladder biles was 165±86 nmol/min/ml (mean±SD), a 23.5- fold increase over the activity before extraction. The measured activity represented the true activity of the enzyme in the bile for recovery of activity of the enzyme added to the bile was practically complete.     

Evaluating β-turn mimics as β-sheet folding nucleators

β-Turns are common conformations that enable proteins to adopt globular structures, and their formation is often rate limiting for folding. β-Turn mimics, molecules that replace the i + 1 and i + 2 amino acid residues of a β-turn, are envisioned to act as folding nucleators by preorganizing the pendant polypeptide chains, thereby lowering the activation barrier for β-sheet formation. However, the crucial kinetic experiments to demonstrate that β-turn mimics can act as strong nucleators in the context of a cooperatively folding protein have not been reported. We have incorporated 6 β-turn mimics simulating varied β-turn types in place of 2 residues in an engineered β-turn 1 or β-bulge turn 1 of the Pin 1 WW domain, a three-stranded β-sheet protein. We present 2 lines of kinetic evidence that the inclusion of β-turn mimics alters β-sheet folding rates, enabling us to classify β-turn mimics into 3 categories: those that are weak nucleators but permit Pin WW folding, native-like nucleators, and strong nucleators. Strong nucleators accelerate folding relative to WW domains incorporating all α-amino acid sequences. A solution NMR structure reveals that the native Pin WW β-sheet structure is retained upon incorporating a strong E-olefin nucleator. These β-turn mimics can now be used to interrogate protein folding transition state structures and the 2 kinetic analyses presented can be used to assess the nucleation capacity of other β-turn mimics.     

The β3-Adrenergic System and β3-Adrenergic Agonists

Reviews in Endocrine and Metabolic Disorders -     

Sicherheit von β-Blockern

β-blockers clearly prolong life in patients with heart failure and after myocardial infarction in all controlled prospective trials. Nevertheless, many colleagues and even more patients fear unwanted effects like low blood pressure, bradycardia, increased bronchial resistance, or erectile dysfunction. There are a limited number of serious side effects, which have to be recognized. In spite of these, we should encourage our patients, who profit from the beneficial actions of β-blockers, to take them regularly. Some controversial aspects of β-blocker therapy are discussed in the light of newer studies.     

Dominantly Inherited β-Thalassemia

Dominantly inherited β-thalassemia (thal) or “inclusion body β-thalassemias” are heterogeneous at the molecular level and are due to mutations at or near the β-globin gene locus. Many of these involve mutations of exon 3 of the β-globin gene. They include frameshifts, premature chain termination (nonsense) mutations, and complex rearrangements that lead to the synthesis of truncated or elongated and highly unstable β-globin gene products. The resulting β chain variants are very unstable, and in many cases, the products of the dominantly inherited β-thal are not detectable. Hematological and clinical observations made in several families with comparable forms of β-thal and with certain highly unstable hemoglobin (Hb) variants, have indicated a striking overlap; many subjects with detectable unstable Hb variants and with a dominant type of β-thal without a detectable abnormal Hb, have similar phenotypes. Here, a review of dominantly inherited β-thal is given, and new examples of hyperunstable Hbs (Hb Stara Zagora and Hb Jambol) are presented.

The first example is a hyperunstable variant named Hb Stara Zagora that was found in a 2-year-old Bulgarian boy. The abnormal Hb is associated with severe hemolytic anemia as a consequence of its hyper instability. The anemia was noticed at the age of 2 months and since then he has been on a regular monthly blood transfusion regimen. Hemoglobin analysis revealed no abnormalities, except the presence of inclusion bodies. Sequencing of the β-globin gene revealed a heterozygosity for a 6 bp deletion (?TGGCTA) at codons 137 (the second and third bp), 138 and 139 (the first bp), thus forming a new codon at position 139 (GAT). This event eliminates three amino acids (Val-Ala-Asn) and introduces a new residue (Asp). It creates a new restriction site for HphI. The parents and his twin brother had no history of hemolysis. The paternity of the child was confirmed by DNA analysis.

The second example is a new hyperunstable variant named Hb Jambol, found as a de novo mutation in a 2-year-old Bulgarian girl with severe hemolytic anemia. The mutation was detected through RNA/DNA analysis. It represents a complex genomic rearrangement involving an insertion of 23 nucleotides (nts) after IVS-II-535, a deletion of 310 nts extending from IVS-II-550 to the first nt of codon 108, and an insertion of 28 nts at the deletion junctions (derived from inverted sequence between nts +3707 and +3734 3′ to the β-globin gene termination codon). At the protein level, this mutation leads to a deletion of four amino acid residues (Leu-Leu-Glu-Asn) at positions 105, 106, 107 and 108, and an insertion of nine residues (Val-Pro-Ser-Val-Thr-Leu-Phe-Phe-Asp) at the same location, creating an abnormal elongated β chain of 151 amino acid residues. The parents had no history of hemolysis. The paternity of the child was confirmed by DNA analysis.     

PKC β1和PKC β2在早期胃癌中的表达

目的:观察PKCβ1和PKCβ2在早期胃癌癌组织及其癌周不典型增生和肠上皮化生中的表达.方法:对42例早期胃癌胃全切标本的癌组织、癌旁不典型增生、肠上皮化生及正常胃黏膜进行PKCβ1和PKCβ2免疫组织化学染色.结果:正常胃黏膜上皮颈部散在PKCβ1阳性细胞,黏膜表面上皮细胞及幽门腺PKCβ1和PKCβ2阴性;胃底腺壁细胞和主细胞PKCβ1和PKCβ2呈强阳性.胃黏膜内淋巴滤泡中心区PKCβ1强阳性,PKCβ2阴性;相反,淋巴滤泡帽带区PKCβ1阴性,而PKCβ2强阳性.胃癌、癌旁不典型增生、肠上皮化生PKCβ1的表达明显增强,而PKCβ2的表达无明显增强,肠型胃癌PKCβ1的表达明显高于弥漫型.PKC β1的表达与早期胃癌的浸润深度无关.结论:PKC β1在胃黏膜腺上皮颈部和淋巴滤泡中心的强阳性表达提示PKC β1可能与细胞的增生活性有关;胃癌及癌前病变中PKC β1的表达增强显示PKC β1参与了胃癌的癌变过程,并可能在癌变早期发生改变.     

Susceptibility of new β-lactams to the expanded-spectrum β-lactamase CTX-1

Summary Forty-three clinical isolates of enterobacteria were selected for the production of the new plasmid-mediated expanded-spectrum -lactamase CTX-1. The geometric means of MICs were ranged as follows: ticarcillin, >4096 mg/l; ticarcillin + clavulanic acid (2 mg/l), 64–87 mg/l; LY 163892, 8.0–69.1 mg/l; cefotaxime, 5.7–26.4 mg/l; temocillin, 8.0–21.8 mg/l; Ro 158074, 4.0–18.7 mg/l aztreonam, 1.0–14.4 mg/l and BMY 28142, 1.4–2.8 mg/l. Moxalactam, imipenem and CM 40876 were resistant to hydrolysis and MICs were lower than 2.0 mg/l. A high protective effect on cefotaxime (MIC0.5 mg/l) was obtained by sulbactam (4 mg/l).Escherichia coli transconjugants from each species showed similar levels of MICs.

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Empfindlichkeit neuer -Laktame gegenüber der Breitspektrum -Laktamase CTX-1

Zusammenfassung 43 Isolate von Enterobakterien wurden im Hinblick auf die Bildung der neuen, plasmidkodierten Breitspektrum--Laktamase CTX-1 ausgewählt. Die geometrischen Mittel der MHK-Werte lagen in folgenden Bereichen: Ticarcillin >4096 mg/l; Ticarcillin plus Clavulansäure (2 mg/l) 64–87 mg/l; LY 163892, 8,0–69,1 mg/l; Cefotaxim 5,7–26,4 mg/l; Temocillin, 8,0–21,8 mg/l; Ro 158074, 4,0–18,7 mg/l; Aztreonam, 1,0–14,4 mg/l und BMY 28142, 1,4–2,8 mg/l. Moxalactam, Imipenem und CM 40876 wurden nicht hydrolysiert, die MHK-Werte lagen unter 2,0 mg/l. Sulbactam (4 mg/l) hatte eine hohe protektive Wirkung auf Cefotaxim (MHK0,5 mg/l).Escherichia coli-Transkonjuganten von jeder Spezies wiesen ähnliche MHK-Werte auf.

     

Neue β‑Laktam-Antibiotika und β‑Laktamase-Inhibitoren gegen multiresistente Gram-negative Erreger

Background

The worldwide spread of multidrug-resistant Gram-negative bacteria (MDR-GN) continues. Treatment options for infections caused by MDR-GN remain scarce and only few new substances are currently in clinical phase II/III studies or have already been granted market approval.

Objectives

To provide an overview about current data on new β?lactam antibiotics and β?lactamase inhibitor combinations, respectively. New macrolides, ketolides and aminoglycosides are not addressed.

Materials and methods

Selective literature research regarding published data on ceftazidim/avibactam, ceftolozan/tazobactam, imipenem/cilastatin?+?relebactam, meropenem/vaborbactam, aztreonam/avibactam and cefiderocol, as well as registered trials.

Results

The development of new antimicrobials for the treatment of MDR-GN infections offers new options for attending physicians. β?Lactamase producers are inhibited by these new substances, though with varying efficacy; however, there are still no adequate treatment options for metallo-β-lactamase (MBL) producers.

Conclusions

Clinical data are still indifferent and come from heterogeneous patient collectives. Direct comparisons with established treatment strategies, such as the “last-resort use” of polymyxins are hardly possible. Cases of early development of resistance have already been described. Finally, the importance of toxicity and optimal dosing—in organ failure or organ replacement procedures such as dialysis—remain unclear.

     

Riboflavin-responsive defects of β-oxidation

The key reaction in the β-oxidation of fatty acids is the acyl-CoA dehydrogenation, catalyzed by short chain, medium chain, and long chain acyl-CoA dehydrogenases. Acyl-CoA dehydrogenation reactions are also involved in the metabolism of the branched chain amino acids, where isovaleryl-CoA and 2-methylbutyryl-CoA dehydrogenases are involved and in the metabolism of lysine, 5-hydroxylysine and tryptophan, where glutaryl-CoA dehydrogenase functions. In all of these dehydrogenation systems reducing equivalents are transported to the main respiratory chain by electron transfer flavoprotein (ETF) and electron transfer flavoprotein dehydrogenase (ETFDH), which are common to all the dehydrogenation systems. The acyl-CoA dehydrogenation enzymes are dependent on flavin adenine dinucleotide (FAD) as coenzyme, for which riboflavin is the precursor. Patients with multiple acyl-CoA dehydrogenation deficiencies have been found in whom the defect has been located to ETF and/or ETFDH. A few patients with multiple acyl-CoA dehydrogenation deficiencies have been described, in whom no defects in acyl-CoA dehydrogenases, ETF or ETFDH have been found but who respond clinically and biochemically to pharmacological doses of riboflavin. This indicates a defect related to the metabolism of FAD. An uptake defect of riboflavin or a synthesis defect of FAD from riboflavin have been excluded byin vivo andin vitro studies. A mitochondrial transport defect of FAD or a defect in the binding FAD to ETF and/or ETFDH remains possible.     

Antiarrhythmische Therapie mit β-Rezeptor-Antagonisten

β-Blockers are an essential component of medical therapy in patients with ischemic heart disease or cardiac dysfunction of any genesis. They have an effect at the level of the sinus and the atrioventricular node, as well as on the atrial and ventricular refractory period of the myocardium. Overall, there are complicated antifibrillatory effects which are involved in the reduction of morbidity and mortality of this the therapy. According to the guidelines, it is important to uptitrate to highest tolerated dose. In patients with atrial fibrillation, antiadrenergic therapy should be the first line treatment; if well tolerated, then β-blockers alone or as a combination with an antiarrhythmic drug is preferable. Future prospective studies on the antiarrhythmic effects in this therapeutic area should include comparisons of different α - and β-selective active substances. Increasing knowledge of the differential therapy with the available active substances including intravenously applicable short-acting β-blockers, e.g., in intensive care therapy - should distinguish the different therapeutic effects.     

Influence of β2-adrenoceptor gene polymorphisms on β2-adrenoceptor expression in human lung

BackgroundThe aim of the present study was to establish whether polymorphisms, especially those within the promoter region, of the β₂-adrenoceptor gene (ADRB2) influence β₂-adrenoceptor expression in human lung.MethodsThe density of β-adrenoceptors in human lung tissue (n = 88) was determined by saturation binding using the radioligand, iodinated cyanopindolol. Discrimination of β₁- and β₂-adrenoceptors was determined using the highly selective β₁-adrenoceptor antagonist, CGP20712A. Genotype was determined at 5 positions of ADRB2 previously reported as polymorphic. Potential influences of single nucleotide polymorphisms (SNPs) within the promoter region (?367, ?47) and coding block (46, 79, 491) of ADRB2 on β₂-adrenoceptor expression were investigated.ResultsThe density of β₂-adrenoceptors was variable among the 88 lung preparations studied ranging from 17 to 177 fmol/mg protein (mean ± S.E.M., 72 ± 4 fmol/mg protein). There was no influence of genotype on β₂-adrenoceptor expression for any of the polymorphisms studied except at position 491. The polymorphism at position 491C > T, leading to a change from thr to ile at amino acid 164, is uncommon. Preparations genotyped as heterozygous (126 ± 15 fmol/mg protein; n = 5) expressed significantly (P = 0.0005) higher levels of β₂-adrenoceptor than those that were homozygous (69 ± 4 fmol/mg protein; n = 83).ConclusionWith the exception of position 491, these data indicate that polymorphisms of ADRB2 do not influence β₂-adrenoceptor expression in human lung.     

Neurofunctional imaging of β-cell dynamics

Here, we outline how islet cells use autocrine and paracrine 'circuits' of classical neurotransmitters and their corresponding receptors and transporters to communicate with vicinal β-cells to regulate glucose-stimulated insulin secretion. Many of these same circuits operate in the central nervous system and can be visualized by molecular imaging. We discuss how these techniques might be applied to measuring the dynamics of β-cell function in real time.     

The evolution of β2-agonists

β-agonists have been widely used in the treatment of asthma for many years. Although concerns have been expressed over their safety, based largely upon epidemics of increased mor tality in asthmatics associated with high doses of isoprenaline in the 1960s and fenoterolin the 1970s and 1980s, the specific β₂-agonists are vital drugs in asthma management. The short-acting β₂-agonists have an important prophylactic role in the prevention of exercise-induced bronchoconstriction, and are essential in the emergency treatment of severe asthma. However, little if any benefit seems to be derived from regular use of short-acting β₂-agonists and regular or frequent use can increase the:severity of the condition. The development of β₂-agonists with long-acting properties, such as salmeterol and formoterol, has provided advantages over short-acting β-agonists, such as prolonged bronchodilation, reduced day-and night-time symptoms and improved quality of sleep, and has reduced the requirement for short-acting β₂-agonists as relief medication. Both drugs are well tolerated and, when added to inhaled corticosteroids, produce greater improvement in lung function than increased steroid dose alone. Because of its rapid onset of action, formoterol also has the potential to be used for as-needed bronchodilator therapy in asthma.     

Glucocorticoid Receptor β: View II

There is a clear role for mechanisms that modulate glucocorticoid receptor (GR) function. The non-steroid-binding GRβ isoform has been proposed to play a role in this modulation but the published data are contradictory. The relative levels of this isoform appear to be low. Alternative mechanisms for the modulation of glucocorticoid action are described and contrasted with the proposed role for GRβ.     

Characterisation and expression of β1-, β2- and β3-adrenergic receptors in the fathead minnow (Pimephales promelas)

Complimentary DNAs for three beta-adrenergic receptors (βARs) were isolated and characterised in the fathead minnow. The encoded proteins of 402 (β₁AR), 397 (β₂AR) and 434 (β₃AR) amino acids were homologous to other vertebrate βARs, and displayed the characteristic seven transmembrane helices of G Protein-coupled receptors. Motifs and amino acids shown to be important for ligand binding were conserved in the fathead minnow receptors. Quantitative RT-PCR revealed the expression of all receptors to be highest in the heart and lowest in the ovary. However, the β₁AR was the predominant subtype in the heart (70%), and β₃AR the predominant subtype in the ovary (53%). In the brain, β₁AR expression was about 200-fold higher than that of β₂- and β₃AR, whereas in the liver, β₂AR expression was about 20-fold and 100-fold higher than β₃- and β₁AR expression, respectively. Receptor gene expression was modulated by exposure to propranolol (0.001-1 mg/L) for 21 days, but not in a consistent, concentration-related manner. These results show that the fathead minnow has a beta-adrenergic receptor repertoire similar to that of mammals, with the molecular signatures required for ligand binding. An exogenous ligand, the beta-blocker propranolol, is able to alter the expression profile of these receptors, although the functional relevance of such changes remains to be determined. Characterisation of the molecular targets for beta-blockers in fish will aid informed environmental risk assessments of these drugs, which are known to be present in the aquatic environment.