Telocytes in human isolated atrial amyloidosis: ultrastructural remodelling

The human heart can be frequently affected by an organ-limited amyloidosis called isolated atrial amyloidosis (IAA). IAA is a frequent histopathological finding in patients with long-standing atrial fibrillation (AF). The aim of this paper was to investigate the ultrastructure of cardiomyocytes and telocytes in patients with AF and IAA. Human atrial biopsies were obtained from 37 patients undergoing cardiac surgery, 23 having AF (62%). Small fragments were harvested from the left and right atrial appendages and from the atrial sleeves of pulmonary veins and processed for electron microscopy (EM). Additional fragments were paraffin embedded for Congo-red staining. The EM examination certified that 17 patients had IAA and 82% of them had AF. EM showed that amyloid deposits, composed of characteristic 10-nm-thick filaments were strictly extra-cellular. Although, under light microscope some amyloid deposits seemed to be located within the cardiomyocyte cytoplasm, EM showed that these deposits are actually located in interstitial recesses. Moreover, EM revealed that telopodes, the long and slender processes of telocytes, usually surround the amyloid deposits limiting their spreading into the interstitium. Our results come to endorse the presumptive association of AF and IAA, and show the exclusive, extracellular localization of amyloid fibrils. The particular connection of telopodes with amyloid deposits suggests their involvement in isolated atrial amyloidosis and AF pathogenesis.     

Neurohormonal and cytokine fluctuations following transcatheter closure for an atrial septal defect

Introduction

Inflammation and neurohormonal activation are considered to be involved in the development of earlier and/or later complications in congenital heart disease patients, even after a successful repair of the lesion. It is not yet clarified what is the role of the therapeutic interventions in the occurrence of such a response and how it could be associated with possible postoperative complications.

Aim

We sought to assess the inflammatory and neurohormonal response to transcatheter closure of secundum type atrial septal defects (ASD) over a six-month follow-up period. We also evaluated the association between the respective markers and catheterization data as well as echocardiographic measurements.

Methods

Plasma concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), N-terminal-proatrial natriuretic peptide (NT-proANP) and N-terminal-probrain natriuretic peptide (NT-proBNP) were assessed and echocardiographic measurements were performed in twenty-eight patients with atrial septal defect prior to, and at the first, second and sixth months post transcatheter closure. Thirty-three age-matched healthy volunteers were also enrolled.

Results

IL-6 plasma levels, although higher preoperatively, [physical logarithm (ln) IL-6: 3.37 ± 0.66 vs 2.92 ± 0.44 pg/ml, p = 0.015], reached control levels postoperatively, at the end of the third month, whereas TNF-α and IL-10 were not influenced by the procedure. NT-proANP levels were elevated preoperatively compared to the control group (ln NT-proANP 3.78 ± 0.572 vs 3.48 ± 0.30, p = 0.031), with a further significant increase during the 1st month (ln NT-proANP 3.78 ± 0.572 vs 4.2 ± 0.42, p = 0.006), following the pattern of the left atrial volume enlargement, and remained high even 6 months after the procedure .On the other hand, the initially normal concentrations of NT-proBNP, after a transient significant increase during the first month postoperatively (ln NT-proBNP 3.56 ± 0.94 vs 4.58 ± 0.91, p < 0.0001) returned to the controls’ levels at the end of the third month. Preoperative concentrations of NT-proANP positively correlated with NT-proBNP concentrations and pulmonary to systemic flow ratio (Qp/Qs).

Conclusions

Transcatheter closure could improve, on a mid- term basis, the inflammatory process but natriuretic peptides’ secretion continues in parallel with left atrial volume increase. Further follow up is required to determine the long-term progress of the inflammatory and neurohormonal response to the procedure.     

重度心力衰竭患者血清RDW、CPP、NT-proANP的临床意义及其与预后相关性分析

摘要 目的：探讨重度心力衰竭患者血清红细胞分布宽度（RDW）、和肽素（CPP）、氨基末端 A 型利钠肽（NT-proANP）的临床意义及其与预后相关性。方法：选取我院2020年1月与到2022年12月收治的98例心力衰竭患者作为研究对象,将所有患者应用Killip分级进行分组,Ⅰ级16例,Ⅱ级23例,Ⅲ级例21,Ⅳ级38例,并选取同期来我院体检的50名健康志愿者作为对照组,对比五组患者血清RDW、CPP、NT-proANP表达水平,分析RDW、CPP、NT-proANP与重度心力衰竭的相关性。随后将Ⅲ级与Ⅳ级重度心力衰竭的59例患者依照其预后情况分为死亡组（n=21）和存活组（n=38）,对比两组患者临床一般情况与血清RDW、CPP、NT-proANP表达水平,并分析血清RDW、CPP、NT-proANP对重度心力衰竭的预后预测价值。结果：五组受检者血清RDW、CPP、NT-proANP水平对比差异显著,Ⅳ级组明显高于Ⅲ级、Ⅱ级、Ⅰ级和对照组（P＜0.05）;Spearman相关分析结果显示：血清RDW、CPP、NT-proANP与重度心力衰竭呈正相关（P＜0.05）;曲线下面积(AUC)从依次为RDW (0.688)、CPP（0.667）、NT-proANP（0.656）、三者联合（0.671）。RDW诊断灵敏度为67.61 %,特异度为66.85 %,CPP诊断灵敏度为60.03 %,特异度为67.53%,NT-proANP诊断灵敏度为61.24 %,特异度为66.53 %,三者联合诊断灵敏度为74.58 %,特异度为86.32 %;存活组与死亡组患者Killip分级、合并陈旧性心肌梗死、RDW、CPP、NT-proANP水平对比差异显著（P＜0.05）;logistic回归分析结果表明：RDW、CPP、NT-proANP为重度心力衰竭预后的独立预测指标（P＜0.05）。结论：血清RDW、CPP、NT-proANP与重度心力衰竭具有明显相关性,其对于重度心力衰竭的诊断临界值分别为17.58 %、1772.62 pg/mL、1.12 nmol/mL。同时三者为重度心力衰竭预后不良的独立影响因素。     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).     

Comparative study of cardiac electrophysiological effects of atrial natriuretic peptide

The effects of atrial natriuretic peptide (ANP) on action potential characteristics were studied in various (human, rabbit, guinea-pig) atrial and guinea-pig right ventricular papillary muscles. ANP (1–100 nM) did not modify the resting membrane potential nor the maximum rate of depolarization phase (V_max). Up to 10 nM, ANP dose-dependently decreased the action potential amplitude both in guinea-pig atrial and ventricular muscles, but it did not affect this parameter in the other atrial preparations. ANP caused a dose-dependent, marked decrease of action potential duration (APD) in practically every cardiac preparation studied (exception of guinea-pig left atrium). The strongest effect on APD can be observed in human atrial and guinea-pig ventricular fibers. The K⁺ channel blocker 4-aminopyridine (1 mM) and the ATP-dependent K⁺ channel inhibitor glibenclamide (10Nl) prevented the effect of ANP on APD in both ventricular atrial preparations. ANP prevented the appearance of isoprenaline (0.5 M) induced slow AP in K⁺ depolarized myocardium. The present data suggest that ANP may inhibit the slow inward Ca²⁺ channel activity and facilitate the K⁺ channel activity.     

Effects of eel atrial natriuretic peptide on NaCl and water transport across the intestine of the seawater eel

Summary Eel atrial natriuretic peptide inhibited the serosa-negative transepithelial potential difference and short-circuit current, accompanied by a decrease in NaCl and water absorption across the seawater eel intestine. Similar effects were obtained after treatment with N-terminally truncated eel atrial natriuretic peptide (5–27), indicating that N-terminal amino acids are not essential for the action of eel atrial natriuretic peptide. Although mammalian atrial natriuretic peptides also inhibited the short-circuit current, a 100-fold higher concentration was reuired to obtain the same effect as with eel atrial natriuretic peptide, indicating that eel atrial natriuretic peptide is 100 times as potent in eel intestine as the mammalian atrial natriuretic peptides. Similarly, in mammalian atrial natriuretic peptide, the four N-terminal amino acids had no significant effects. However, when the C-terminal tyrosine was removed, the potency of rat atrial natriuretic peptide was lowered. Compared with the effects of acetylcholine, serotonin and histamine, eel atrial natriuretic peptide was the most potent inhibitor, with 100% inhibition at 10^-7 M; 50% inhibition was obtained at 10^-2 M in acetylcholine, and 30% inhibition in serotonin (10^-5 M) and histamine (10^-3 M). These inhibitory effects of eel atrial natriuretic peptide were not diminished even in the presence of tetradoxin, and were mimicked by 8-bromoguanosine 3,5-cyclic monophosphate. Based on these results, structure-activity relationships of eel atrial natriuretic peptide and a possible mechanism of action of eel atrial natriuretic peptide are discussed.Abbreviations 8BrcGMP 8-bromoguanosine 3,5-cyclic monophosphate - eANP eel atrial natriuretic peptide - hANP human atrial natriuretic peptide - 5-HT 5-hydroxytryptamine creatine sulphate - I _sc short-circuit current - PD transepithelial potential difference - rANP rat atrial natriuretic peptide - R _t tissue resistance - TTX tetrodotoxin     

N-Terminal amino acid sequence analysis indicates that isolated atrial amyloid is derived from atrial natriuretic peptide

Isolated atrial amyloid, the most frequent senile cardiac amyloid type, was chemically analysed. Amyloid fibrils obtained from a patient (NIP) were extracted and the predominant lowmolecular-weight polypeptide (approximately 3.5 kDa, designated ASc₂ NIP) was isolated by size exclusion high performance liquid chromatography in 60% formic acid. N-Terminal amino acid sequence analysis of this polypeptide was identical to that of the atrial natriuretic peptide α-hANP for the first 12 residues determined.     

Syntheses and biological activities of atrial natriuretic polypeptide analogs

Recently several peptides with natriuretic and diuretic potencies were isolated from human and rat atrial extract, and the precursors of the peptides were sequenced. Of the peptides, -human and rat atrial natriuretic polypeptides (-hANP, -rANP), consisting of 28 amino acids, are thought to be essential to the potency and to play an important role in the blood pressure regulation system. The amino acid sequence of -hANP is different from that of -rANP only at the position 12 (isoleucine in -rANP). In the present study, we synthesized ANPs and their analogs using a new deprotection procedure based on the concept of push-pull mechanism. Using the synthetic ANP analog, we also developed a radioimmunoassay for -ANP and examined the structure-activity relationship. Synthetic -hANP caused potent, rapid, and short-acting increases in Na⁺ and Cl^– excretion, and also an increase in urine flow and K⁺ excretion of lesser magnitude, when injected into rat. Also, we synthesized a cyclic part of -hANP, -ANP(7–23)-NH₂. Since this peptide had a little diuretic and natriuretic potency, we attempted to synthesize a chemically stable -hANP analog. We considered that the disulfide bond would be equivalent to propylene with regard to interatomic distance and employed 8-aminocaprylic acid instead of cystine. This cyclic peptide, named cyclonatrin-54, had a somewhat higher potency than -hANP(7–23)-NH₂ for diuresis and natriuresis, as expected. Furthermore, using a synthetic intermediate of cyclonatrin-54, we prepared a linear ANP analog, -hANP(8–22), Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly. This linear 15-amino acid peptide had a dose-dependent natriuretic and diuretic activity, but no hypotensive effect. It was surprising that a linear peptide exhibited a potent natriuretic activity. For the first time, a linear peptide has been prepared that has substantial natriuretic and diuretic potency. We synthesized some analogs of this 15-amino acid peptide and investigated the structure-activity relationship.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Infrared spectroscopic study of the metal-coordination structures of calcium-binding proteins

Carboxylate (COO⁻) groups can coordinate to metal ions in of the following four modes: ‘unidentate’, ‘bidentate’, ‘bridging’ and ‘pseudo-bridging’ modes. COO⁻ stretching frequencies provide information about the coordination modes of COO⁻ groups to metal ions. We review the Fourier-transform infrared spectroscopy (FTIR) of side-chain COO⁻ groups of Ca²⁺-binding proteins: pike parvalbumin pI 4.10, bovine calmodulin and Akazara scallop troponin C. FTIR spectroscopy of Akazara scallop troponin C has demonstrated that the coordination structure of Mg²⁺ is distinctly different from that of Ca²⁺ in the Ca²⁺-binding site. The assignments of the COO⁻ antisymmetric stretch have been ensured on the basis of the spectra of calcium-binding peptide analogues. The downshift of the COO⁻ antisymmetric stretching mode from 1565 cm^-1 to 1555-1540 cm⁻¹ upon Ca²⁺ binding is a commonly observed feature of FTIR spectra for EF-hand proteins.     

Ganglioside-induced amyloid formation by human islet amyloid polypeptide in lipid rafts

Human islet amyloid polypeptide (hIAPP) is the primary component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus. However, it is unknown how amyloid fibrils are formed in vivo. In this study, we demonstrate that gangliosides play an essential role in the formation of amyloid deposits by hIAPP on plasma membranes. Amyloid fibrils accumulated in ganglioside- and cholesterol-rich microscopic domains (‘lipid rafts’). The depletion of gangliosides or cholesterol significantly reduced the amount of amyloid deposited. These results clearly showed that the formation of amyloid fibrils was mediated by gangliosides in lipid rafts.     

Receptor-specific ligands distinguish natriuretic peptide receptors-A and -C in primate tissues

Systemic clearance of atrial natriuretic peptide (ANP) is in part due to neutral endopeptidase (NEP) proteolysis and natriuretic peptide receptor-C (NPR-C) mediated endocytosis. Biological responses to ANP are primarily mediated by the membrane guanylyl cyclase-A/natriuretic peptide receptor-A (NPR-A). Analogs of ANP selective for NPR-A and/or resistant to NEP may have increased activity in those tissues where NPR-C and NEP are coexpressed with NPR-A. The analog of ANP termed vANP; [(R3D, G9T, R11S, M12L, G16R)ANP] is selective for human NPR-A with at least 10,000 fold reduction in affinity for human NPR-C. We report that rat NPR-A is insensitive to 10 nM vANP, demonstrating the limitations of this species in evaluating human therapeutic candidates. As an alternative approach we tested the binding and potency of receptor-selective and NEP-resistant ANP analogs in rhesus monkey tissues. Competition binding studies with a simplified version of vANP, sANP [(G9T, R11S, G16R)rANP], in rhesus monkey kidney and lung membrane preparations shows displacement of ¹²⁵I-ANP from only a fraction of the total ANP receptor population, 30 and 85%, respectively. The remaining ANP binding sites can be occupied with the NPR-C selective ligand cANP(4-23). These data strongly suggest that only two classes of ANP receptor are present in these membrane preparations, NPR-A and NPR-C. The NEP resistant sANP derivative called sANP(TAPR) was 8 fold more potent (ED₅₀ = 0.6 nM) than rANP (ED₅₀ = SnM) in stimulating cGMP production in the lung membrane preparation. Our results demonstrate that the rhesus monkey natriuretic peptide receptors reflect the pharmacology of the human receptors, and that this species may be suitable to determine the role of NPR-C and NEP in peptide clearance and attenuating functional responses.     

Indole-3-acetic acid concentration and ethylene evolution during early fruit development in peach

Ethylene evolution was measured from greenhouse-grown Jerseyglo peach fruits beginning 29 days after anthesis. Indole-3-acetic acid (IAA) levels were measured in the pericarp and seed tissues of individual fruits on a single shoot when variable ethylene evolution was noted. Despite hand-pollinating all flowers on the same day, variability within the shoot existed in fruit fresh weight, IAA levels, and ethylene evolution. Seed IAA concentration increased as fruit and seed fresh weight increased and ranged from 106 to 1572 ng. g^–1. As pericarp fresh weight increased, IAA levels in this tissue decreased. Ethylene evolution rates ranged from 0.21 to 1.07 nl. g.^–1 h^–1 and were not correlated with IAA concentration in seed, pericarp, or the whole fruit. High rates of ethylene evolution from the whole fruit occurred prior to increased IAA concentration in the seed.Fruits were excised from field-grown Redskin peach trees beginning 40 days after full bloom. Fruits from field sampled shoots appeared to be more physiologically advanced than the greenhouse-grown Jerseyglo fruits. Pericarp IAA concentration was low, ranging from 2.8 to 6.5 ng. g^–1. Seed concentrations accounted for 75% of the IAA found in the fruit and ranged from 239 to 1042 ng. g^–1. As with greenhouse-grown samples, whole fruit IAA concentration tended to decrease as fruits increased in fresh weight.     

Immunolocalization of atrial natriuretic peptide in mast cells of human and rat pericardium

It is known that various heart disorders are accompanied by an elevated level of atrial natriuretic peptide (ANP), a regulator of cardiovascular homeostasis, in the pericardial fluid. Which cells produce ANP in the pericardial cavity is unclear. Using immunoelectron microscopy, we examined ANP localization in human and rat pericardium. ANP-immunobinding material was found in granules of mast cells (MC) localized in pericardial connective tissue. In rat pericardium, the average MC size is 6.5 × 12.5 μm and the MC density is about 50 cells per 1 mm² section area. For the human pericardium, these parameters are 9.1 × 13.6 μm and 10 cells per 1 mm², respectively. The results show that MCs are probably implicated in the pericardial endocrine function and in controlling the ANP level in the pericardial cavity.     

Identification and partial characterization of immunoreactive and bioactive atrial natriuretic peptide from eel heart

Summary Cardiocytes positive for human atrial natriurectic peptide (hANP) were identified histochemically in the eel atrium, but they were not found in the ventricule. Secretory granules were frequently observed in atrial cardiocytes by electron microscopy, but the number of such granules was quite small in the ventricle. Immunogold cytochemistry revealed that immunoreactive ANP (IR-ANP) in atrial cardiocytes was localized in these granules. In spite of poor immunostaining of the eel ventricle, an acid extract of the ventricle contained 25±4 ng·g tissue^-1 (n=9) of IR-ANP when the level of IR-ANP was measured by radioimmunoassay (RIA) for hANP. This level was one eight of that measured in atrial extracts (203±13 ng·g tissue^-1, n=9). Plasma contained 116.7±18.6 pg·ml^-1 (n=9) of IR-ANP. An extract of eel hearts decreased arterial pressure in eels and quail as did hANP. The level of ANP in the extract, as measured by an eel vasodepressor bioassay, was much greater than that measured by RIA for hANP. The immunoreactive and bioactive ANP in the heart extract are identical since the vasodepressor activity disappeared after IR-ANP was absorbed by excess antibodies raised against hANP. Chromatography on Sephadex G-75 generated a major peak of IR-ANP at a position that corresponded to a molecular weight of 14 kD and minor peaks at 3–7 kD from both plasma and heart extract. Reverse phase HPLC of plasma and heart extract generated several peaks of IR-ANP at positions more hydrophilic than those of mammalian ANPs. These results show that eel hearts contain immunoreactive and bioactive ANPs which are distinctly different from hANP. These ANPs are synthesized both in the atrium and in the ventricle, and they are secreted into the circulation mostly in the larger molecular form. The atrial ANP may be stored in the granules and secreted upon exposure of eels to certain stimuli, but the ventricular ANP may be secreted constitutively into the circulation without prior storage in the granules.Abbreviations ANP atrial natriuretic peptide - BSA bovine serum albumin - IR-ANP immunoreactive ANP - PBS phosphate-buffered saline - RIA radioimmunoassay     

Continuos intravenous infusion of atrial natriuretic peptide (ANP) prevented liver fibrosis in rat

The atrial natriuretic peptide (ANP) are used as the acute heart failure treatment in clinical and reported the suppression of fibrosis in the heart, lung recently. The aim of this study was to analyze the suppressive effect of liver fibrosis about ANP. In vitro, rat hepatic stellate cell line (HSC-T6) were treated with ANP. In vivo, Wister rats were injected with dimethylnitrosamine (DMN) twice a week via intra-peritoneal for 4 weeks. ANP group was given by continuance intravenous dosage system used 24 h infusion pump for 3 weeks after 1 week of DMN administration. In vitro, ANP suppressed α-SMA expression and was inhibited the growth of HSC, and reduced the expression of type 1 procollagen, TIMP-1, -2 expression. In vivo, The ANP group showed lower serum AST, ALT, HA level. Liver fibrosis was suppressed by ANP. ANP also decreased gene expression of type 1 procollagen, TIMP-1, -2 and α-SMA, TGF-β1 expression. Our results showed that continuous ANP infusion has the specific capacity of inhibiting HSC activation and protecting hepatocytes and the useful capacity to suppress the liver fibrosis.     

Effect of PT-treatment on ANP-mediated inhibition of adenylate cyclase and amylase release in rat parotid gland

Effects of pertussis toxin (PT) treatment on atrial natriuretic peptide (ANP)-mediated inhibition of adenylate cyclase and amylase release were investigated in rat parotid gland. Adenylate cyclase activity stimulated by GTPS in PT-treated membranes was much larger than that in normal membranes. ANP dose-dependently inhibited adenylate cyclase stimulated by GTPS in control rat parotid membranes, however in membranes prepared from PT-injected (in vivo) rat parotid gland, ANP did not inhibit adenylate cyclase. ANP(10^–7M) inhibited cAMP accumulation stimulated by forskolin (10^–6M) in control rat parotid acinar cells by about 34%, however, in PT-treated cells, the inhibitory effect of ANP was attenuated completely. In control cells, amylase release stimulated by isoproterenol (10^–6M) and forskolin (10^–6M) were also depressed by ANP (10^–7M) by 27 and 30%, respectively. The inhibitory response of ANP on amylase release was completely attenuated by PT-treatment. Gi was detected as a ADP-ribosylated 41-KDa protein by incubation of parotid membranes with PT and [-³²P]NAD. In rat parotid gland, these results suggested that ANP mediates adenylate cyclase/cAMP system and consequently reduces amylase release through ANP-C receptor coupled to Gi. (Mol Cell Biochem)139: 53–58, 1994)     

Arsenic contamination of groundwater and prevalence of arsenical dermatosis in the Hetao plain area,Inner Mongolia,China

The present study determined cardiac chamber-specific alterations of the expression of the atrial and brain natriuretic peptide (ANP and BNP) genes with a small increase in age beyond adulthood and with systemic hypertension of intermediate duration. The expression distributions of these genes was determined using in situ hybridization in the right and left atria (RA and LA), and the right and left ventricles (RV and LV) in Wistar Kyoto rats (WKY) and age-matched Spontaneously Hypertensive rats (SHR) at ages 6 months (adult) and 8 months (advanced-age beyond adulthood).In all rat groups, both genes were expressed (ANP > BNP) in the LA and LV, and were not expressed in the RA and RV. The genes were expressed in the LA in all rat groups; the ANP, but not the BNP, expression increased with advancing age and with superimposed hypertension. They were expressed in the LV of the advanced-age WKY, adult and advanced-age SHR, but not in the adult WKY. The ANP mRNA labeling in the LA was diffuse and interspersed with dense accumulations, whereas BNP labeling was diffuse. The labeling of both genes in the form of sparse clusters was seen in the LV of the advanced-age SHR. Our study showed that ANP and BNP expression in left heart chambers increased with a small increase in age, with hypertension of intermediate duration, and with modest left ventricular hypertrophy. The chamber-specific expression distribution could be due to special groups of cardiac cells, or to local chamber-specific factors.     

Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's Aβ peptide

The oligomerization of Aβ peptide into amyloid fibrils is a hallmark of Alzheimer’s disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aβ and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aβ peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aβ fibrillation. The three histidine residues at positions 6, 13 and 14 of Aβ(1–40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.     

In Vitro Amyloidogenic Peptides of Galectin-7: POSSIBLE MECHANISM OF AMYLOIDOGENESIS OF PRIMARY LOCALIZED CUTANEOUS AMYLOIDOSIS

Pathogenesis of primary localized cutaneous amyloidosis (PLCA) is unclear, but pathogenic relationship to keratinocyte apoptosis has been implicated. We have previously identified galectin-7, actin, and cytokeratins as the major constituents of PLCA. Determination of the amyloidogenetic potential of these proteins by thioflavin T (ThT) method demonstrated that galectin-7 molecule incubated at pH 2.0 was capable of binding to the dye, but failed to form amyloid fibrils. When a series of galectin-7 fragments containing β-strand peptides were prepared to compare their amyloidogenesis, Ser³¹-Gln⁶⁷ and Arg¹²⁰-Phe¹³⁶ were aggregated to form amyloid fibrils at pH 2.0. The rates of aggregation of Ser³¹-Gln⁶⁷ and Arg¹²⁰-Phe¹³⁶ were dose-dependent with maximal ThT levels after 3 and 48 h, respectively. Their synthetic analogs, Phe³³-Lys⁶⁵ and Leu¹²¹-Arg¹³⁴, which are both putative tryptic peptides, showed comparable amyloidogenesis. The addition of sonicated fibrous form of Ser³¹-Gln⁶⁷ or Phe³³-Lys⁶⁵ to monomeric Ser³¹-Gln⁶⁷ or Phe³³-Lys⁶⁵ solution, respectively, resulted in an increased rate of aggregation and extension of amyloid fibrils. Amyloidogenic potentials of Ser³¹-Gln⁶⁷ and Phe³³-Lys⁶⁵ were inhibited by actin and cytokeratin fragments, whereas those of Arg¹²⁰-Phe¹³⁶ and Leu¹²¹-Arg¹³⁴ were enhanced in the presence of Gly⁸⁴-Arg¹¹³, a putative tryptic peptide of galectin-7. Degraded fragments of the galectin-7 molecule produced by limited trypsin digestion, formed amyloid fibrils after incubation at pH 2.0. These results suggest that the tryptic peptides of galectin-7 released at neutral pH, may lead to amyloid fibril formation of PLCA in the intracellular acidified conditions during keratinocyte apoptosis via regulation by the galectin-7 peptide as well as actin and cytokeratins.     

Cloning and functional expression of the bovine natriuretic peptide receptor-B (natriuretic factor R1c subtype)

We describe the isolation of a 3,276 base pair cDNA for the bovine natriuretic peptide receptor-B (NPR-B). Expression of this clone in Cos-P cells demonstrates that it encodes an agonist-dependent guanylyl cyclase. Porcine CNP stimulates the activity of this receptor up to 200-fold with an ED₅₀ of 12±2 nM, whereas brain natriuretic peptide C-type natriuretic peptide (CNP) and atrial natriuretic factor (ANF) are less efficacious. In addition, ligand binding studies indicate that this receptor exhibits the pharmacology appropriate for the bovine NPR-B. CNP binds to Cos-P cell membranes expressing this clone with a K_d of 13±1 pM, and natriuretic peptides compete for [¹²⁵I]-CNP binding with a rank order of pCNP>pBNP>rANF. Thus, the expressed receptor-guanylyl cyclase exhibits the expected pharmacological profile for ligand binding and cyclase activation of the bovine NPR-B receptor.Abbreviations BSA bovine serum albumin - dNTP deoxynucleotide triphosphate - SDS sodium dodecyl sulfate - DEAE-dextran diethylaminoethyl-dextran - EDTA ethylenediamine tetraacetic acid - Tris Tris(hydroxymethyl)aminomethane - DMSO dimethyl sulfoxide - RP-HPLC reverse phase-high performance liquid chromatography - AMV avian myeloblastosis virus - Arg arginine - Lys lysine