Effects of lithospermic acid on hIAPP aggregation and amyloid-induced cytotoxicity by multiple analytical methods

The abnormal aggregation of human islet amyloid polypeptide (hIAPP) is a crucial pathogenic factor associated with type 2 diabetes (T2D). The development of effective inhibitors to prevent hIAPP aggregation is a common therapeutic strategy against T2D. Lithospermic acid (LA) is a natural compound with diversified biological activities. In this study, electrospray ionization coupled with ion mobility–mass spectrometry, thioflavin T fluorescence assay, Congo red binding assay, Nile red fluorescence assay, circular dichroism spectroscopy, transmission electron microscopy, cell toxicity, lactate dehydrogenase assay (LDH) assay and molecular docking were combined to explore the influence of LA on hIAPP aggregation. Results showed that LA had favorable binding affinity to hIAPP and formed hIAPP–LA complexes, which could alter the relative abundance of the compact and extended conformers and promoted the transition of extended structures to compact conformers. LA also displayed strong inhibitory actions on fibrillation and potential protective effects against hIAPP-induced cell toxicity. Therefore, the obtained results were useful to understand the possible inhibitory mechanism of LA on hIAPP aggregation and provided valuable reference for the screening of potent amyloid inhibitors.     

Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone

The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the − NH − groups has been replaced by − CH₂−) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.     

The enhancing effect of homocysteine thiolactone on insulin fibrillation and cytotoxicity of insulin fibril

In the current study both structural alteration and fibrillation of insulin were studied in the presence of homocysteine thiolactone (HCTL). The spectroscopic studies revealed that HCTL increases rate of insulin unfolding, giving rise to the appearance of solvent-exposed hydrophobic regions and induces a transition from α-helix into predominantly β-sheet structures. Thioflavin-T fluorescence studies revealed that HCTL markedly enhanced the quantity of insulin fibril formation in both agitating and non-agitating systems. Also gel electrophoresis results suggest that HCTL accelerates the process of formation of high molecular weight insulin aggregates. Moreover, insulin fibrils obtained in the presence of HCTL and those collected earlier in the pathway of insulin fibrillation displayed improved cytotoxicity against cancer cells. The enhancement of insulin fibril formation with elevated cytotoxic properties as occurred in the presence of HCTL, may suggest this homocysteine derivative as a possible contributing factor in the pathology of insulin fibrils.     

Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone

The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the -NH- groups has been replaced by -CH(2)-) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.     

Highly sensitive fluorescent detection of trypsin based on BSA-stabilized gold nanoclusters

In this study, fluorescent metal nanoclusters are presented as novel probes for sensitive detection of protease for the first time. The sensing mechanism is based on trypsin digestion of the protein template of BSA-stabilized Au nanoclusters. The decrease in fluorescence intensity of BSA-Au nanoclusters caused by trypsin allows the sensitive detection of trypsin in the range of 0.01-100 μg/mL. The detection limit for trypsin is 2 ng/mL (86 pM) at a signal-to-noise ratio of 3. The present nanosensor for trypsin detection possesses red emission, excellent biocompatibility, high selectivity, and good stability. In addition, we demonstrated the application of the present approach in real urine samples, which suggested its potential for diagnostic purposes.     

Further studies of the ability of xyloglucan oligosaccharides to inhibit auxin-stimulated growth

The structural features required for xyloglucan oligosaccharides to inhibit 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stem segments have been investigated. A nonasaccharide (XG9) containing one fucosyl-galactosyl side chain and an undecasaccharide (XG11) containing two fucosyl-galactosyl side chains were purified from endo-β-1,4-glucanase-treated xyloglucan, which had been isolated from soluble extracellular polysaccharides of suspension-cultured sycamore (Acerpseudoplatanus) cells and tested in the pea stem bioassay. A novel octasaccharide (XG8′) was prepared by treatment of XG9 with a xyloglucan oligosaccharide-specific α-xylosidase from pea seedlings. XG8′ was characterized and tested for its ability to inhibit auxin-induced growth. All three oligosaccharides, at a concentration of 0.1 microgram per milliliter, inhibited 2,4-dichlorophenoxyacetic acid-stimulated growth of pea stem segments. XG11 inhibited the growth to a greater extent than did XG9. Chemically synthesized nona- and pentasaccharides (XG9, XG5) inhibited 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stems to the same extent as the same oligosaccharides isolated from xyloglucan. A chemically synthesized structurally related heptasaccharide that lacked a fucosyl-galactosyl side chain did not, unlike the identical heptasaccharide isolated from xyloglucan, significantly inhibit 2,4-dichlorophenoxyacetic acid-stimulated growth.     

Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains

Gold nanoparticles modified with nuclear localization peptides were synthesized and evaluated for their subcellular distribution in HeLa human cervical epithelium cells, 3T3/NIH murine fibroblastoma cells, and HepG2 human hepatocarcinoma cells. Video-enhanced color differential interference contrast microscopy and transmission electron microscopy indicated that transport of nanoparticles into the cytoplasm and nucleus depends on peptide sequence and cell line. Recently, the ability of certain peptides, called protein transduction domains (PTDs), to transclocate cell and nuclear membranes in a receptor- and temperature-independent manner has been questioned (see for example, Lundberg, M.; Wikstrom, S.; Johansson, M. (2003) Mol. Ther. 8, 143-150). We have evaluated the cellular trajectory of gold nanoparticles carrying the PTD from HIV Tat protein. Our observations were that (1) the conjugates did not enter the nucleus of 3T3/NIH or HepG2 cells, and (2) cellular uptake of Tat PTD peptide-gold nanoparticle conjugates was temperature dependent, suggesting an endosomal pathway of uptake. Gold nanoparticles modified with the adenovirus nuclear localization signal and the integrin binding domain also entered cells via an energy-dependent mechanism, but in contrast to the Tat PTD, these signals triggered nuclear uptake of nanoparticles in HeLa and HepG2 cell lines.     

Benzylisoquinoline compounds inhibit the ability of calmodulin to activate cyclic nucleotide phosphodiesterase

Benzylisoquinoline compounds antagonised the ability of calmodulin (CaM) to stimulate the activity of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE). This 'anti-CaM' activity was related to the hydrophobicity of the non-polar terminal region of the antagonist molecule. Antagonistic potency increased with the increase of hydrophobicity; the anti-CaM activity did not change when the polar terminus was a tertiary amine or quarternary amine. The anti-CaM potency was greater for bisbenzylisoquinoline compounds than for monobenzylisoquinoline compounds. Among the bisbenzylisoquinoline compounds anti-CaM pathway was: D3 greater than D2 berbamine greater than daurisoline greater than dauricine. Compound D3, which exhibited an IC50 value of 2.8 microM, was one of the most potent calmodulin antagonists, among benzylisoquinoline compounds, so far reported.     

The ability of salts to inhibit the reaction between periodate anions and ovotransferrin.

N1-Acetylspermidine is not detectable in rat heart, but its content greatly increases after a single injection of isoprenaline (10 mg/kg), reaching a concentration of about 10 nmol/g of tissue 4 h after the treatment. Part of the accumulated N1-acetylspermidine was split to putrescine. Isoprenaline also caused an increase of N1-acetylspermidine in the spleen, where its concentration increased 3.5-fold 6 h after the catecholamine. The accumulation of N1-acetylspermidine was dependent on the dose of isoprenaline in both the heart and the spleen, and was strongly inhibited by beta-antagonists and inhibitors of protein synthesis.     

The ability of microfilariae to evade in vitro cell-mediated cytotoxicity

When microfilariae (Mf) of Dirofilaria immitis, both uterine and systemic, were incubated in an in vitro cytotoxicity assay with neutrophils and sera from dogs with occult infections, some Mf remained free of adherent cells and consequently evaded cytotoxicity. The ability to evade cytotoxicity could not be related to the age of the Mf, and host albumin was not detected on any Mf, either uterine or systemic. However, it was shown that some Mf failed to bind IgM, IgG and C3 when incubated with occult sera. It is suggested that the ability of some Mf to evade serum-dependent, neutrophil-mediated cytotoxicity in vitro was related to differences in their antigenicity.     

Ratiometric optical detection of pyrophosphate based on aggregation-caused dual-signal response of gold nanoclusters

Sensing of pyrophosphate ion (PPi) has received much attention due to the strong demand for clinical diagnostics. Here, based on gold nanoclusters (Au NCs), a ratiometric optical detection method for PPi is developed by simultaneously detecting the dual signals of fluorescence (FL) and second-order scattering (SOS). The PPi is detected by inhibiting the formation of aggregates of Fe³⁺ with Au NCs. Binding of Fe³⁺ to Au NCs causes aggregation of Au NCs, which leads to fluorescence quenching and scattering increasing. The presence of PPi can competitively bind Fe³⁺ to re-disperse the Au NCs and finally recover the fluorescence and reduce the scattering signal. The designed PPi sensor shows a high sensitivity with a linear range 5–50 μM and a detection limit of 1.2 μM. In addition, the assay has excellent selectivity for PPi, which makes its application in real biological samples extremely valuable.     

PKC-dependent phosphorylation may regulate the ability of connexin43 to inhibit DNA synthesis

Phosphorylation affects several biological functions of connexin43 (Cx43), although its role on Cx43-mediated inhibition of DNA synthesis is not known. Previous studies showed increased Cx43 phosphorylation on serine in response to growth factor stimulation of cardiomyocytes, mediated by protein kinase C-epsilon (PKCepsilon). Here we report that activation of PKCepsilon is also necessary for stimulation of cardiomyocyte DNA synthesis and mitosis. We have investigated the participation of specific serine residues that are putative PKC targets in producing phosphorylated Cx43 species and also in regulating DNA synthesis in cardiomyocytes. Interference with the PKC signaling system and/or the phosphorylation of specific amino-acids of Cx43 may allow regulation of the mitogenic response.     

Daurisoline derivatives inhibit the ability of calmodulin to stimulate cyclic nucleotide phosphodiesterase activity

Daurisoline alkaloid derivatives were found to be potent calmodulin (CaM) antagonists. The ability of daurisoline derivatives to attenuate the stimulatory effect on calmodulin activated cyclic nucleotides phosphodiesterase (CaM-PDE) was studied. These compounds did not inhibit the basal activity of this enzyme. The hydrophobicity of these compounds was related to their inhibitory potency. It is suggested that such drugs bind directly to calmodulin in a Ca2(+)-dependent fashion, as indicated by their ability to change calmodulin fluorescence.     

Trace detection of picloram using an electrochemical immunosensor based on three-dimensional gold nanoclusters

Picloram, a herbicide widely used for broadleaf weed control, is persistent and mobile in soil and water with adverse health and environmental effects. It is important to develop a sensitive method for accurate detection of trace picloram in the environment. In this article, a type of ordered three-dimensional (3D) gold (Au) nanoclusters obtained by two-step electrodeposition using the spatial obstruction/direction of the polycarbonate membrane is reported. Bovine serum albumin (BSA)-picloram was immobilized on the 3D Au nanoclusters by self-assembly, and then competitive immunoreaction with picloram antibody and target picloram was executed. The horseradish peroxidase (HRP)-labeled secondary antibody was applied for enzyme-amplified amperometric measurement. The electrodeposited Au nanoclusters built direct electrical contact and immobilization interface with protein molecules without postmodification and positioning. Under the optimal conditions, the linear range for picloram determination was 0.001-10 μg/ml with a correlation coefficient of 0.996. The detection and quantification limits were 5.0 × 10⁻⁴ and 0.0021 μg/ml, respectively. Picloram concentrations in peach and excess sludge supernatant extracts were tested by the proposed immunosensor, which exhibited good precision, sensitivity, selectivity, and storage stability.     

Synthesis of C-linked triazole-containing AFGP analogues and their ability to inhibit ice recrystallization

C-Linked antifreeze glycoprotein (C-AFGP) analogues have been shown to have potent ice recrystallization inhibition (IRI) activity. However, the lengthy synthesis of these compounds is not amenable to large-scale preparation for the many commercial, industrial, and medical applications that exist. This paper describes the synthesis of triazole-containing AFGPs using a convergent solid-phase synthesis (SPS) approach in which multiple carbohydrate derivatives are coupled to a resin-bound synthetic peptide in a single step. Modified "Click" conditions using dry DMF as solvent with catalytic Cu(II), sodium ascorbate, and microwave radiation afforded the synthesis of AFGP analogues 9-12 in 16-54% isolated yield. Compound 9 demonstrated no IRI activity, while compounds 10, 11, and 12 were moderate inhibitors of ice recrystallization. These results suggest that, while the triazole group is a structural mimetic of an amide bond, the amide bond in C-AFGP analogue 3 is an essential structural feature necessary for potent IRI activity.