Total synthesis of the α-subunit of human glycoprotein hormones: toward fully synthetic homogeneous human follicle-stimulating hormone

Described herein is the first total chemical synthesis of the unique α-subunit of the human glycoprotein hormone (α-hGPH). Unlike the biologically derived glycoprotein hormones, which are isolated as highly complex mixtures of glycoforms, α-hGPH obtained by chemical synthesis contains discrete homogeneous glycoforms. Two such systems have been prepared. One contains the disaccharide chitobiose at the natural N-glycosylation sites. The other contains dodecamer oligosaccharides at these same sites. The dodecamer sugar is a consensus sequence incorporating the key features associated with human glycoproteins.     

Why is autophagy important in human diseases?

The process of macroautophagy (referred to hereafter as autophagy), is generally characterized by the prominent formation of autophagic vesicles in the cytoplasm. In the past decades, studies of autophagy have been vastly expanded. As an essential process to maintain cellular homeostasis and functions, autophagy is responsible for the lysosome-mediated degradation of damaged proteins and organelles, and thus misregulation of autophagy can result in a variety of pathological conditions in human beings. Although our understanding of regulatory pathways that control autophagy is still limited, an increasing number of studies have shed light on the importance of autophagy in a wide range of physiological processes and human diseases. The goal of the reviews in the current issue is to provide a general overview of current knowledge on autophagy. The machinery and regulation of autophagy were outlined with special attention to its role in diabetes, neurodegenerative disorders, infectious diseases and cancer.     

Synthesis of N-glycyl-β-glycopyranosylamines, human milk fucooligosaccharide derivatives

The action of ammonium carbamate in aqueous methanol in the presence of NH₃ on three-, penta-, hexa-, octa-, and nonasaccharides of human milk and on decasaccharide (N-glycan from human immunoglobulin (IgG)) containing one or two a-l-fucose residues led to the corresponding β-glycopyranosylamines. After their N-acylation upon treatment with N-hydroxysuccinimide ester of N-Boc-glycine or N-Boc-glycine anhydride (Boc is the tert-butyloxycarbonyl) with subsequent removal of the Boc group, N-glycyl-β-glycopyranosylamines were obtained in up to 75% yield.     

Antimicrobial activity of human β-defensins against lactic acid bacteria

In this study, we evaluated the antimicrobial activity of human β-defensin-1 (hBD-1), human β-defensin-2 (hBD-2) and human β-defensin-3 (hBD-3) against three internationally common probiotic strains of lactic acid bacterium. Our results indicated that hBD-1, hBD-2 and hBD-3 at the range of 0.08–10 μg/mL do not have obvious antimicrobial activity against these strains. Viability of Bifidobacterium longum JDM301 (B. longum JDM301), Bifidobacterium lactis HN019 (B. lactis HN019) and Lactobacillus rhamnosus GG (LGG) were still very high even at concentration of 10 μg hBD/mL. Then, we explored the mechanism of resistance by using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to inhibit efflux pumps. In the presence of CCCP, hBD-1, hBD-2 and hBD-3 exhibited enhanced antibacterial effect against B. longum JDM301 and B. lactis HN019, but not against LGG. Efflux pumps in B. longum JDM301 and B. lactis HN019 may partly contribute to their resistance to hBD-1, hBD-2, and hBD-3.     

Computational combinatorial ligand design: Application to human α-thrombin

Summary A new method is presented for computer-aided ligand design by combinatorial selection of fragments that bind favorably to a macromolecular target of known three-dimensional structure. Firstly, the multiple-copy simultaneous-search procedure (MCSS) is used to exhaustively search for optimal positions and orientations of functional groups on the surface of the macromolecule (enzyme or receptor fragment). The MCSS minima are then sorted according to an approximated binding free energy, whose solvation component is expressed as a sum of separate electrostatic and nonpolar contributions. The electrostatic solvation energy is calculated by the numerical solution of the linearized Poisson-Boltzmann equation, while the nonpolar contribution to the binding free energy is assumed to be proportional to the loss in solvent-accessible surface area. The program developed for computational combinatorial ligand design (CCLD) allows the fast and automatic generation of a multitude of highly diverse compounds, by connecting in a combinatorial fashion the functional groups in their minimized positions. The fragments are linked as two atoms may be either fused, or connected by a covalent bond or a small linker unit. To avoid the combinatorial explosion problem, pruning of the growing ligand is performed according to the average value of the approximated binding free energy of its fragments. The method is illustrated here by constructing candidate ligands for the active site of human -thrombin. The MCSS minima with favorable binding free energy reproduce the interaction patterns of known inhibitors. Starting from these fragments, CCLD generates a set of compounds that are closely related to high-affinity thrombin inhibitors. In addition, putative ligands with novel binding motifs are suggested. Probable implications of the MCSS-CCLD approach for the evolving scenario of drug discovery are discussed.     

The molecular basis of pharmacological chaperoning in human α-galactosidase

Fabry disease patients show a deficiency in the activity of the lysosomal enzyme α-galactosidase (α-GAL or α-Gal A). One proposed treatment for Fabry disease is pharmacological chaperone therapy, where a small molecule stabilizes the α-GAL protein, leading to increased enzymatic activity. Using enzyme kinetics, tryptophan fluorescence, circular dichroism, and proteolysis assays, we show that the pharmacological chaperones 1-deoxygalactonojirimycin (DGJ) and galactose stabilize the human α-GAL glycoprotein. Crystal structures of complexes of α-GAL and chaperones explain the molecular basis for the higher potency of DGJ over galactose. Using site-directed mutagenesis, we show the higher potency of DGJ results from an ionic interaction with D170. We propose that protonation of D170 in acidic conditions leads to weaker binding of DGJ. The results establish a biochemical basis for pharmacological chaperone therapy applicable to other protein misfolding diseases.     

A new system for TNF-α quantification in human blood samples

Full-length human TNF-α and its truncated derivative in which 18 N-terminal amino acid residues were replaced with T7-tag were obtained as inclusion bodies as a result of expression of artificial genes in E. coli cells. The purification and refolding procedures for the recombinant proteins were developed, and biological activity of the resultant proteins was demonstrated. Polyclonal antibodies against TNF-α were obtained. A sandwich ELISA test system for TNF-α quantification (sensitivity, 100 pg/mL) was constructed. It was shown that this system is applicable for detecting TNF-α elevation in human blood.     

Determination of periodontal pathogens in human oral cavityEI北大核心CSCD

The simultaneous detection of Porphyromonas gingivalis （PG），Tannerela forsythia （TF），and Treponema dentinarum（TD）based on multiplex polymerase chain reaction（PCR）and capillary electrophoresis （CE），and the effect of sampling position were investigated. Results demonstrated that there was a linear relationship between migration time and DNA length when DNA fragments were separated by capillary electrophoresis，and the correlation coefficient was 0.9976. In 0.5% hydroxyethyl cellulose （HEC），the PCR products of these periodontal pathogens could be separated within 1.6 min. When sampling at different positions in the periodontal pocket，the PCR products were also different. When analyzing PCR products by capillary electrophoresis，the maximum relative deviation of the peak time was 7.4%. Based on fluorescence intensity，it could be inferred that the concentrations of PG，TD and TF in the gingival fluid were 5.13×10-11 ，7.89×10-11 and 4.87×10-11 ng/µμL，respectively. © 2023, Youke Publishing Co.,Ltd. All rights reserved.     

Quantitation ofE. coli protein impurities in recombinant human interferon-γ

A multiple antigen ELISA for E. coli proteins (ECPs) that may be present in purified recombinant human interferon-gamma (rIFN-gamma) was developed. SDS-PAGE and Western blotting analyses showed that the assay antibodies reacted with a wide spectrum of ECPs in the standard and with ECPs in a production run. In spike recovery studies, rIFN-gamma at concentrations of 0.05 mg/mL and higher augmented the immunoreactivity of the ECPs in the standard curve (1.3-40.0 ng ECPs/mL) by approx 50%. To determine ECP content in purified rIFN-gamma, 0.2 mg/mL of rIFN-gamma was added to the standard curve diluent to compensate for enhanced immunoreactivity. The assay was precise (interassay precision of ECP controls < or = 4.1 %CV) and accurate with recoveries of 111-115% of expected for ECPs (15-40 ng/mL) spiked into purified rIFN-gamma (1 mg/mL). Linearity of dilution for ECPs spiked into rIFN-gamma was obtained (r = 0.999). Moreover, linearity of dilution was obtained for ECPs in "in-process" samples, demonstrating the required condition of antibody excess for this type of multiple antigen ELISA. ECPs were not detectable in several purified lots of rIFN-gamma. Therefore, these lots contained < 1.3 ppm ECPs.     

Liquid chromatography–mass spectrometric determination of plasmalogens in human plasma

A new liquid chromatography–mass spectrometry (LC/MS) method has been developed for the quantitative analysis of plasmalogens in human plasma using a nonendogenous plasmalogen (1-O-1′-(Z)-tricosenyl-2-oleoyl-rac-glycero-3-phosphocholine, PLS 23:0/18:1) as an internal standard. 1-O-1′-(Z)-Tricosenyl glyceryl ether was prepared by reacting lithioalkoxyallyl with 1-iodoeicosane as the key intermediate in the formation of PLS 23:0/18:1. In LC/MS analyses, PLS 23:0/18:1 generated significant fragment ions in positive and negative modes. In positive ion mode, the [M+H]⁺ of PLS 23:0/18:1 yielded unique fragments with cleavages at the sn-1 and sn-2 positions of the glycerol backbone. In negative ion mode, the [M+CH₃COO]⁻ of PLS 23:0/18:1 resulted in characteristic fragmentation at the sn-2 and sn-3 positions. 1-O-1′-(Z)-Hexadecenyl-2-linoleoyl-rac-glycero-3-phosphocholine (PLS 16:0/18:2) and 2-arachidonoyl-O-1′-(Z)-hexadecenyl-rac-glycero-3-phosphocholine (PLS 16:0/20:4) were chemically synthesized as PLS 23:0/18:1. The calibration curves obtained for PLS 16:0/18:2 and PLS 16:0/20:4 were linear throughout the calibration range (0.04–1.60 pmol). The LOD (S/N = 5:1) was 0.008 pmol and the LOQ (S/N = 6:1) was 0.01 pmol for both PLS 16:0/18:2 and PLS 16:0/20:4. Plasma concentrations of PLS 16:0/18:2 and PLS 16:0/20:4 were 4.0 ± 1.3 μM and 3.5 ± 1.2 μM (mean ± SD), respectively, in five healthy volunteers.     

Total synthesis of human urotension-Ⅱ by microwave-assisted solid phase method

Human urotension-Ⅱwas synthesized efficiently on Wang resin under microwave irradiation using Fmoc/tBu orthogonal protection strategy.Disulphide bridge was formed on solid phase with the irradiation of microwave,then the whole peptide was cleaved from the resin.The purity of crude peptide cyclized under microwave irradiation was higher than that under room temperature.     

Antifungal activity of silver nanoparticle-encapsulated β-cyclodextrin against human opportunistic pathogens

Silver nanoparticles were synthesised by reducing silver acetate with a long-chain aliphatic amine. β-Cyclodextrin (CD)-stabilised silver nanoparticles were successfully synthesised and characterised by the UV–vis spectroscopy and scanning electron microscopy analysis. This system was examined for their antifungal activity against opportunistic human pathogens such as Aspergillus fumigatus, Mucor ramosissimus and Chrysosporium species. This study clearly demonstrates that the present system is a powerful antifungal agent against human opportunistic pathogenic fungi.     

Aptamer sandwich assays: human α-thrombin detection using liposome enhancement

Fluorescent dye-encapsulating liposomes tagged with aptamers were developed and used as reporting signals in an aptamer-based sandwich assay. α-Thrombin was utilized as a prototypical analyte as two well-studied aptamers binding distinct epitopes are available to form a sandwich complex. Cholesteryl–TEG-modified aptamers were embedded into the liposomal lipid bilayer while the interior cavity of the liposomes encapsulated fluorescent sulforhodamine B dye. Such liposomes successfully formed a sandwich complex with α-thrombin and a microtiter plate immobilized aptamer, proving that aptamers retain their ability to fold when anchored to the liposome surface. Parameters studied included liposomal aptamer coverage, sandwich aptamer orientation, aptamer label orientation, aptamer spacer length and type, incubation buffer, and aptamer concentration. The optimized conditions found here in the fluorescence assay led to a limit of detection of 64 pM or 2.35 ng/mL, corresponding to 6.4 fmol or 235 pg, respectively, in a 100 μL volume. This is an order of magnitude lower than previous sandwich aptamer assays using the same sequences with lowest reported limits of detection of 0.45 nM. In addition, the assay was applied successfully to the detection of α-thrombin in human plasma. The success of this method in a standard microtiter plate format and the relatively facile functionalization of liposomes with aptamers suggest that this approach provides a versatile option for routine analytical applications.     

Restricted variation in the glycosylation of human α1-acid glycoprotein

Summary The glycosylation patterns of multiglycosylated proteins reflect both the cellular control of glycosyl transferase activity and the local control of the transfer event. Little information is available concerning these mechanisms. Changes in glycosylation occur in the disease state and provide a convenient way of examining the control mechanism(s) operating at individual glycosylation sites. Chromatographic methods have been applied to the human orosomucoid (OMD) isolated from seventeen different pathologies. Based on the distribution of the glycans at the individual sites it is clear that the major protein-glycan interactions that restrict glycosylation in normal OMD remain intact in the disease state in the presence of changing glycosyl transferase activity.     

2DIR spectroscopy of human amylin fibrils reflects stable β-sheet structure

The aggregation of human amylin to form amyloid contributes to islet β-cell dysfunction in type 2 diabetes. Studies of amyloid formation have been hindered by the low structural resolution or relatively modest time resolution of standard methods. Two-dimensional infrared (2DIR) spectroscopy, with its sensitivity to protein secondary structures and its intrinsic fast time resolution, is capable of capturing structural changes during the aggregation process. Moreover, isotope labeling enables the measurement of residue-specific information. The diagonal line widths of 2DIR spectra contain information about dynamics and structural heterogeneity of the system. We illustrate the power of a combined atomistic molecular dynamics simulation and theoretical and experimental 2DIR approach by analyzing the variation in diagonal line widths of individual amide I modes in a series of labeled samples of amylin amyloid fibrils. The theoretical and experimental 2DIR line widths suggest a "W" pattern, as a function of residue number. We show that large line widths result from substantial structural disorder and that this pattern is indicative of the stable secondary structure of the two β-sheet regions. This work provides a protocol for bridging MD simulation and 2DIR experiments for future aggregation studies.     

Analytical methods for selected emerging contaminants in human matrices—a review

Emerging contaminants are a broad category of chemicals, previously unknown or unrecognized as being of concern, but which, because of their potential health effects associated with human exposure, are under increasing scrutiny. To accurately measure their levels in biological matrices, specific and sensitive analytical methods have recently been developed. We have reviewed here the methods used for analysis of selected emerging organic contaminants, for example metabolites of organophosphate triesters, metabolites of new phthalates or phthalate substitutes, perchlorate, organic UV filters, and polycyclic siloxanes, in human matrices. Although the use of new techniques and approaches has been emphasized, we also acknowledge methods previously used for other contaminants and adapted for the emerging contaminants listed above. In all cases, chromatography and mass spectrometry were the techniques of choice, because of their selectivity and sensitivity for measurements at ng?g^?1 levels. Critical issues and challenges have been discussed, together with recommendations for further improvement in particular cases (e.g. metabolites of phthalates or their substitutes). In particular, the use of labeled internal standards, the availability of certified reference materials, and the need for interlaboratory comparison exercises are key aspects of further development of this field of research.

Virus rejection with two model human enteric viruses in membrane bioreactor system

A membrane bioreactor (MBR) with gravity drain was tested for virus rejection with two coliphages, T4 and f2, which were used as surrogates for human enteric viruses. Virus rejection was investigated by PVDF and PP membrane modules, with the pore sizes of 0.22 and 0.1 μm, respectively. In tap water system, 2.1 lg rejection of coliphage T4 could be achieved by PVDF membrane compared with complete rejection by PP membrane, while for coliphage f2 with smaller diameter, 0.3―0.5 lg rejection of the influent virus was removed by the two membranes. In domestic wastewater system, cake layer and gel layer on the membrane surface changed the cut-off size of the membrane so that there was no significant difference between PP and PVDF for each coliphage. The removal ratios of coliphage T4 and f2 in the MBR were more than 5.5 and 3.0 lg, respectively. Compared with 5.5 lg removal for virus T4 in the MBR system, only 2.1 lg (96.8%―99.9%) removal rate was observed in the conventional activated sludge system with the influent virus concentration fluctuating from 1830 to 57000 PFU/mL. Only 0.8%―22% virus removal was the effect of adsorption to activated sludge, which showed a decreasing tendency with the retention time, while 75%―98% was the effect of virus inactivation by microbial activity. It indicated that the major mechanism of virus removal was not the transfer of viruses from the water phase to the sludge phase but inactivation in the biological treatment process.     

Modulation of animal and human hematopoiesis by β-glucans: a review

β-glucans are cell wall constituents of bacteria, yeast, fungi, and plants. They are not expressed in mammalian cells, but they are recognized by mammalian cells as pathogen-associated molecular patterns by pattern recognition receptors and thus act as biological response modifiers. This review summarizes data on the hematopoiesis-stimulating effects of β-glucans, as well as on their ability to enhance bone marrow recovery after an injury. β-glucans have been shown to support murine hematopoiesis suppressed by ionizing radiation or cytotoxic anti-cancer therapy. They also enhance stem cell homing and engraftment. Basically, two forms of β-glucan preparations have been investigated, namely particulate and soluble ones. β-glucans are generally well tolerated, the particulate forms showing a higher incidence of undesirable side effects. Taken together, the hematopoiesis-stimulating properties of β-glucans predetermine these biological response modifiers to ever increasing use in human medicinal practice.     

Can human prolidase enzyme use different metals for full catalytic activity?

The catalytic hydrolysis of the Gly-Pro substrate by the bimetallic prolidase active site model cluster has been investigated at the DF/B3LYP level of theory, in order to provide fundamental insights into the still poorly understood mechanism of prolidase catalysis. To date, the majority of prolidases exhibits metal-dependent activity, requiring two divalent cations such as Zn(2+), Mn(2+), or Co(2+) for maximal activity. In addition, it has been shown recently that two different metal ions in the active site of human prolidase (Zn and Mn) can coexist, with the protein remaining partially active. With the purpose of identifying which is the most efficient dimetallic center for the prolidase catalyzed reaction, Zn(II), Co(II), and Mn(II) have been examined as potential catalytic metals for this enzyme. Furthermore, to better elucidate the exact roles played by the metals occupying the site 1 and site 2 positions, the hetero-bimetallic active site having Zn and Mn cations has been also investigated, considering the two derivatives Mn1-Zn2 and Zn1-Mn2. The rate-determining step of the hydrolysis reaction is always found to be the nucleophilic addition of the hydroxide ion on the carbonyl carbon of the scissile peptide bond, followed by the less energetically demanding proline-peptide C-N bond scission. The analysis of the involved energy barriers does not indicate clearly a preference for a particular metal by the prolidase enzyme. Instead, we may point out a slightly better behavior of the cobalt-containing cluster as far as both tetrahedral formation and its decomposition are concerned, due to a greater degree of ligands-to-metals charge transfer. The mixed Mn-Zn hetero-dimetallic clusters appear to be also able to perform the hydrolysis of the Pro-Gly substrate, with a slight preference for the Mn1-Zn2 configuration.