Incorporation of channel-forming peptides in a Hg-supported lipid bilayer

The channel-forming peptides gramicidin and alamethicin were incorporated in a mercury-supported lipid bilayer composed of a tethered thiolipid monolayer with a self-assembled dioleoylphosphatidylcholine monolayer on top of it. The thiolipid consists of a hexapeptide chain with a high tendency to form a 3₁₀-helical structure, which terminates at the N-terminus end with a sulfydryl group for anchoring to the metal while the C-terminus end is covalently linked to the polar head of dimyristolylphosphatidylethanolamine. The hexapeptide moiety has two triethyleneoxy side chains that impart a satisfactory hydrophilicity and are intended to keep the anchored thiolpeptide chains sufficiently apart, so as to accommodate water molecules and inorganic ions and to create a suitable environment for the incorporation of integral proteins. Changes in the conductance of this biomimetic membrane following the incorporation of gramicidin and alamethicin were detected by impedance spectroscopy. The surface dipole potential of the hexapeptide chain and the transmembrane potential of the lipid bilayer were estimated by using a simple electrostatic model of the mercury|solution interphase.     

精氨酸-天冬氨酸抗涎腺腺样囊性癌实验性肺转移的研究

目的 研究精氨酸－天冬氨酸（Arg-Asp,RD)抗涎腺腺样囊性癌实验性肺转移（salivary adenoid cystic carcinoma lung metastation,SACC-LM)的作用。方法 观察不同剂量、不同时间RD灌胃对SACC－LM的影响。结果30mg/kg和120mg/kgRD灌胃可抑制SACC－LM的形成；7．5mg/kg、30mg/kg、120mg/kgRD灌胃均可延长SACC－LM鼠的生存期。结论 RD毒性小、可经口给药，具有抗SACC－LM的作用。     

HLA-G mediated immune regulation is impaired by a single amino acid exchange in the alpha 2 domain

The trade-off from HLA class I expression to HLA-G expression support the immune evasion of malignant cells. The essential role of the virtually invariant HLA-G in immune tolerance, tumor immunology and its expression frequency in immune privileged tissues is known; however the specific importance of allelic subtypes in immune responses is still not well understood. HLA-G¹01:01, ¹01:03 and ¹01:04 are the most prevalent allelic variants differing at residues 31 and 110, respectively. In cytotoxicity assays applying K562 cells transduced with the HLA-G variants as targets and NK cells as effectors the differential protective potential of HLA-G variants was analyzed. Their peptide profiles were determined utilizing soluble HLA technology. An increased protective potential of HLA-G¹01:04 could be observed. All variants exhibit a unique peptide repertoire with marginal overlap, while G¹01:04 differs in its peptide anchor profile substantially. The functional differences between HLA-G subtypes could be explained by the constraint of the bound peptides, modifying the pHLA-G accessible surface. For the first time a contribution of amino acid alterations within the HLA-G heavy chain for peptide selection and NK cell recognition could be observed. These results will be a step towards understanding immune tolerance and will guide towards personalized immune therapeutic strategies.     

Characterization of HCV Genotype 5a Envelope Proteins: Implications for Vaccine Development and Therapeutic Entry Target

Background:

Hepatitis C virus (HCV) is one of the major causes of cirrhosis and hepatocellular carcinoma with an estimation of 185 million people with infection. The E2 is the main target for neutralizing antibody responses and the variation of this region is related to maintenance of persistent infection by emerging escape variants and subsequent development of chronic infection. While both E1 and E2 are hypervariable in nature, it is difficult to design vaccines or therapeutic drugs against them.

Objectives:

The objective of this study was to characterize genotype 5a E1 and E2 sequences to determine possible glycosylation sites, conserved B-cell epitopes and peptides in HCV that could be useful targets in design of vaccine and entry inhibitors.

Patients and Methods:

This study was conducted through PCR amplification of E1 and E2 regions, sequencing, prediction of B-cell epitopes, analysis of N-linked glycosylation and peptide design in 18 samples of HCV genotype 5a from South African.

Results:

Differences in the probability of glycosylation in E1 and E2 regions were observed in this study. Three conserved antigenic B-cell epitopes were predicted in the E2 regions and also 11 short peptides were designed from the highly conserved residues.

Conclusions:

This study provided conserved B-cell epitopes and peptides that can be useful for designing entry inhibitors and vaccines able to cover a global population, especially where genotype 5a is common.     

A novel peptide-based pan-influenza A vaccine: A double blind,randomised clinical trial of immunogenicity and safety

BackgroundFP-01.1 is a novel synthetic influenza A vaccine consisting of six fluorocarbon-modified 35-mer peptides that encapsulate multiple CD4+ and CD8+ T-cell epitopes and is designed to induce an immune response across a broad population.MethodsFP-01.1 was evaluated for safety and immunogenicity in a randomised, double-blind, placebo-controlled, dose-escalation, phase I clinical study in healthy adult volunteers (n = 49). IFNγ ELISpot assays and multicolour flow cytometry were used to characterise the immune response.ResultsFP-01.1 was safe and well tolerated at all doses tested with a similar adverse event profile in actively vaccinated subjects compared with controls. Maximum immunogenicity was in the 150 μg/peptide dose group where a robust response (243 spots/million PBMC) was demonstrated in 75% subjects compared with 0% in placebo controls. All six peptides were immunogenic. FP-01.1 induced dual CD4+ and CD8+ T cell responses and vaccine-specific T cells cross-recognise divergent influenza strains.ConclusionsThis first-in-human study showed that FP-01.1 has an acceptable safety and tolerability profile and generated robust anti-viral T cell responses in a high proportion of subjects tested. The results support the further clinical testing of FP-01.1 prior to clinical, proof-of-concept, live viral challenge studies.     

Dynorphin-(1–13)-Like Immunoreactivity in Central Nervous System and Pituitary Gland of Spontaneously Hypertensive Rats

We determined by radioimmunoassay concentrations of dynorphin-(1–13)-like immunoreactivity in the central nervous system and pituitary gland of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs). Compared to WKYs, SHRs had significantly lower levels of dynorphin-(1–13)-like immunoreactivity in the hypothalamus and pituitary gland. However, such immunoreactivity in the cerebral cortex, caudate nucleus, diencephalon, brainstem and spinal cord of SHRs and WKYs were similar.     

An Update on the Status of Potent Inhibitors of Metallo-β-Lactamases

The production of metallo-β-lactamases is the most important strategy by which pathogenic bacteria become resistant to currently known β-lactam antibiotics. The emergence of these enzymes is particularly concerning for the future treatment of bacterial infections. There are no clinically available drugs capable of inhibiting any of the metallo-β-lactamases, so there is an urgent need to find such inhibitors. In this review, an up-to-date status of the inhibitors investigated for the inhibition of metallo-β-lactamases has been given so that this rich source of structural information of presently known metallo-β-lactamases could be helpful in generating a broad-spectrum potent inhibitor of metallo-β-lactamases.     

Role of defensins in inflammatory lung disease

The human airways are protected from invading micro-organisms by the highly efficient innate immune system. Antimicrobial peptides that are produced by inflammatory cells and airway epithelial cells are key elements in this innate immune system. A major subgroup of the antimicrobial peptides is the family of defensins - small non-enzymatic and cationic peptides. Besides their extensively studied role in antimicrobial defense, recent studies have demonstrated that defensins are also able to modulate inflammatory responses, to stimulate adaptive immunity and contribute to tissue repair. In line with these observations, increased defensin levels were observed in inflammatory lung diseases, such as cystic fibrosis (CF), diffuse panbroncheolitis (DPB), idiopathic pulmonary fibrosis (IPF) and acute respiratory distress syndrome (ARDS), and in infectious diseases. In the past decade much has been learnt about the activity of defensins and there is abundant evidence for their presence in human inflammatory lung disease. Future studies are required to elucidate their role in the pathogenesis of these diseases.     

Preparation of cell-affinity adsorbents by immobilization of peptides to Sephadex G-10

In this study, affinity adsorbents for the binding of activated blood platelets and endothelial cells were prepared from Sephadex G-10 by immobilization of peptides, derived from the cell-adhesive amino acid sequence RGD (arginine-glycine-aspartic acid). Derivatization of Sephadex G-10 was accomplished by sequential coupling of specific dipeptides (RG and DV) (V = valine) and by coupling of the RGD-containing hexapeptide GRGDSP (S = serine, P = proline). Two types of gel were prepared by sequential coupling, designated as G10 (acetone) and G10 (dimethylformamide) (G10 (DMF)), containing peptides which had been coupled to the outer side of the beads and throughout the porous beads, respectively. The binding capacity of the prepared Sephadex-derivatives amounted up to 2 x 10⁹ human blood platelets per millilitre GRGDV-Sephadex at immobilized peptide concentrations, that were in the nanomole range (per millilitre packed gel) and which differed a factor 10 between G10 (acetone) and G10 (DMF). In a second series of experiments, different amounts of the hexapeptide GRGDSP were coupled to carboxylated Sephadex G-10 and carboxylated Sepharose CL 6B. The binding of human umbilical vein endothelial cells to the resulting materials was studied. Up to 10⁶ endothelial cells attached per ml GRGDSP-derivatized hydrogel at peptide concentrations of 15 nmol GRGDSP/ml Sephadex and at ±300 nmol GRGDSP/ml Sepharose. Substitution of the arginine residue of the RGD-sequence by glutamine abolished the cell-binding activity of the immobilized peptide towards activated blood platelets but not towards endothelial cells. From the results of this study it can be concluded that small peptides can be coupled to the outer side of the porous Sephadex beads, resulting in high effective ligand densities for cell-affinity applications. In this respect, Sephadex G-10, derivatized according to 'the acetone method', is a good alternative for polystyrene and other solid phase materials.