Recombinant protein of heptad-repeat HR212, a stable fusion inhibitor with potent anti-HIV action in vitro

HR212, a recombinant protein expressed in Escherichia coli, has been previously reported to inhibit HIV-1 membrane fusion at low nanomolar level. Here we report that HR212 is effective in blocking laboratory strain HIV-1IIIB entry and replication with EC50 values of 3.92±0.62 and 6.59±1.74 nM, respectively, and inhibiting infection by clinic isolate HIV-1KM018 with EC50 values of 44.44±10.20 nM, as well as suppressing HIV-1- induced cytopathic effect with an EC50 value of 3.04±1.20 nM. It also inhibited HIV-2ROD and HIV-2CBL-20 entry and replication in the μM range. Notably, HR212 was highly effective against T20-resistant strains with EC50 values ranging from 5.09 to 7.75 nM. Unlike T20, HR212 showed stability sufficient to inhibit syncytia formation in a time-of-addition assay, and was insensitive to proteinase K digestion. These results suggest that HR212 has great potential to be further developed as novel HIV-1 fusion inhibitor for treatment of HIV/ AIDS patients, particularly for those infected by T20-resistant variants.

Molecular and evolutionary analyses of formyl peptide receptors suggest the absence of VNO-specific FPRs in primates

Formyl peptide receptors (FPRs) were observed to expand in rodents and were recently suggested as candidate vomeronasal chemosensory receptors. Since vomeronasal chemosensory receptors usually underwent positive selection and evolved concordantly with the vomeronasal organ (VNO) morphology, we surveyed FPRs in primates in which VNO morphology is greatly diverse and thus it would provide us a clearer view of VNO-FPRs evolution. By screening available primate genome sequences, we obtained the FPR repertoires in representative primate species. As a result, we did not find FPR family size expansion in primates. Further analyses showed no evolutionary force variance between primates with or without VNO structure, which indicated that there was no functional divergence among primates FPRs. Our results suggest that primates lack the VNO-specific FPRs and the FPR expansion is not a common phenomenon in mammals outside rodent lineage, regardless of VNO complexity.

金环蛇蛇毒丝氨酸蛋白酶抑制剂和三种胡蜂蜂毒的研究

蛇毒和蜂毒是提供药理学活性分子的丰富来源，它们富含肽和蛋白，包括一 些酶类和毒素。 丝氨酸蛋白酶抑制剂广泛存在于动物、植物和微生物体内，参与许多重要的 生理过程，如血液凝集、纤维蛋白溶解、细胞凋亡、发育以及炎症反应和补体活 化等（van Gent D. et al., 2003）。通过凝胶过滤、离子交换和反向高压液相色谱， 我们从金环蛇毒液中纯化得到一种天然的丝氨酸蛋白酶抑制剂，命名为 bungaruskunin。并且从该蛇的毒腺cDNA 文库中克隆到了它的核苷酸序列。 bungaruskunin 预测的前体由83 个氨基酸组成，包括含有24 个氨基酸的信号肽 和含有59 个氨基酸的成熟肽。它与一种由红腹伊澳蛇（Pseudechis porphyriacus） 的cDNA 预测到的丝氨酸蛋白酶抑制剂blackelin 具有最大相似性，达64％。 Bungaruskunin 是一种Kunitz 型的蛋白酶抑制剂，具有一个保守的Kunitz 结构域， 能够抑制胰蛋白酶、胰凝乳蛋白酶和弹性蛋白酶。通过对金环蛇毒腺cDNA 文库 的筛选，我们还得到了另外两条β-bungarotoxin B 链，Bungaruskunin 的整体结 构与β-bungarotoxin B 链相似，特别是它们都具有高度保守的信号肽序列。这些 发现强烈地表明蛇毒Kunitz/BPTI 蛋白酶抑制剂与神经毒性的类似物可能起源于 共同的祖先。 肥大细胞脱粒肽是从膜翅目昆虫的毒液中鉴别出的一个小肽家族，是一种具 有潜在的药物治疗作用的诱导活性分子（Xueqing Xu et al., 2006）。来源于蜂类的 缓激肽样的类似物vespakinin 家族是一种具有调节和激素功能的活性成分，与哺 乳动物和两栖动物的缓激肽类似（Nakajima T., 1984）。本研究对三种胡蜂的 毒液进行了一系列的活性检测，发现黑尾胡蜂的蜂毒对白色念珠菌Candida albicans 和金黄色葡萄球菌 Staphylococcus aureus 有抑制作用。凹纹胡蜂和黑尾 胡蜂的蜂毒具有微弱的磷酯酶A2 活性。通过凝胶过滤和反向高压液相色谱，没 有得到相关的活性组分。通过对三种胡蜂毒腺cDNA 文库的筛选，我们得到了2 条来源于黑尾胡蜂的核苷酸序列，Blast 分析表明，其中一条编码类似肥大细胞 脱粒肽，但未克隆到全长，序列比对结果显示其与来源于大胡蜂（Vespa magnifica） 的Mastoparan-like peptide 12c precursor（GenBank accession A0SPI0）的核苷酸序 列相似性达98％（Xueqing Xu et al., 2006）；另一条编码缓激肽类似物，命名为 Hw-bradykinin，序列比对结果显示其与来源于大胡蜂（Vespa magnifica）的 vespakinin-M precursor（GenBank accessionABG75944）的核苷酸相似率达96% (Zouhong Zhou et al., 2006)。

Enzymatic reaction of the immobilized enzyme on porous silicon studied by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

Desorption/ionization on silicon mass spectrometry (DIOS-MS) is a matrix-free technique that allows for the direct desorption/ionization of low-molecular-weight compounds with little or no fragmentation of analytes. This technique has a relatively high tolerance for contaminants commonly found in biological samples. DIOS-MS has been applied to determine the activity of immobilized enzymes on the porous silicon surface. Enzyme activities were also monitored with the addition of a competitive inhibitor in the substrate solution. It is demonstrated that this method can be applied to the screening of enzyme inhibitors. Furthermore, a method for peptide mapping analysis by in situ digestion of proteins on the porous silicon surface modified by trypsin, combined with matrix-assisted laser desorption/ionization-time of flight-MS has been developed.

Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Peptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH4HF2. The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 50degreesC, even 20 fmol cytochrome c could be well digested and detected.

Functional gold nanoparticle-peptide complexes as cell-targeting agents

In this paper, we report a novel approach using peptide CALNN and its derivative CALNNGGRRRRRRRR (CALNNR(8)) to functionalize gold nanoparticles for intracellular component targeting. The translocation is effected by the nanoparticle diameter and CALNNR8 surface coverage. The intracellular distributions of the complexes are change from the cellular nucleus to the endoplasmic reticulum by increasing the density of CALNNR8 at a constant nanoparticle diameter. Additionally, increasing the nanoparticle diameter at a constant density of CALNNR8 leads to less cellular internalization.

Aliphatic Poly(ester-carbonate)s Bearing Amino Groups and Its RGD Peptide Grafting

This article deals with (1) synthesis of novel cyclic carbonate monomer (2-oxo [1,3]dioxan-5-yl)carbamic acid benzyl ester (CAB) containing protected amino groups; (2) ring-opening copolymerization of the cyclic monomer with L-lactide (LA) to provide novel degradable poly(ester-carbonate)s with functional groups; (3) removal of the protective benzyloxycarbonyl (Cbz) groups by catalytic hydrogenation to afford the corresponding poly(ester-co-carbonate)s with free amino groups; (4) grafting of oligopeptide Gly-Arg-Gly-Asp-Ser-Tyr (GRGDSY, abbreviated as RGD) onto the copolymer pendant amino groups in the presence of 1,1'-carbonyldiimidazole (CDI).

Rapid label-free detection of metal-induced Alzheimer's amyloid beta peptide aggregation by electrochemical method

Amyloid beta peptide plays a critical role in the pathogenesis of Alzheimer's disease (AD). Metal ions are highly enriched in cerebral amyloid deposits in AD and are proposed to be able to mediate A beta conformation. Therefore, a rapid, low-cost, and sensitive detection of metal-induced A beta aggregation and their relation to AD is clearly needed for the clinical diagnosis and treatment. In this report, we study metal-induced A beta aggregation by a rapid, label-free electrochemical method and monitor both the aggregation kinetics and the morphology in the absence or presence of Zn (II) and Cu (II).

Investigation of Calmodulin-Peptide Interactions Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

In this report, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to study the binding interactions between calmodulin and two target peptides (melittin and substance P). Various matrix conditions were tested and the less acidic matrix DHAP and THAP were found to favor the survival of the intact calcium-calmodulin as well as the calmodulin-peptide complexes. However, the application of direct MALDI-MS to detect the intact complexes turned out to be very difficult due to the dissociation of the complexes and the formation of nonspecific aggregates. In contrast, the specific binding of the target peptides to calmodulin could be easily deduced using intensity-fading (IF) MALDI-MS. Compared with the nonbinding control, clear reduction in the ion abundances of the target peptides was observed with the addition of calmodulin.

Microarray-based Raman spectroscopic assay for kinase inhibition by gold nanoparticle probes

In this paper, a microarray-based surface-enhanced Raman spectroscopic (SERS) assay for detection of kinase functionality and inhibition has been reported. Biotinylated anti-phosphoserinen antibodies mark the phosphorylation and inhibition events and gold nanoparticles are attached to the antibodies by standard avidin-biotin chemistry, followed by silver deposition for SERS signal enhancement. The avidin conjugated fluorescein is used as SERS probe. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA), its well known substrate, kemptide, and three inhibitors, H89, HA1077, and KN62 have been chosen here to establish the SERS assay. As expected, highly selective inhibition of PKA is demonstrated with the inhibitor H89 and the inhibition assay enable to detect kinase inhibition as well as derive IC50 (half maximal inhibitory concentration) plots.

Prostate cancer cell-specific VEGF siRNA delivery system using cell targeting peptide conjugated polyplexes

A polymeric gene carrier was developed to deliver vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) for prostate cancer cells in a target-specific manner. Prostate cancer-binding peptide (PCP) was conjugated with polyethylenimine (PEI) via a poly(ethylene glycol) (PEG) linker (PEI-PEG-PCP). The PEI-PEG-PCP conjugate could effectively condense siRNA to form stable polyelectrolyte complexes (polyplexes) with an average diameter of approximately 150 nm in an aqueous solution. VEGF siRNA/PEI-PEG-PCP polyplexes exhibited significantly higher VEGF inhibition efficiency than PCP-unmodified polycationic carriers (PEI-PEG or PEI) in human prostate carcinoma cells (PC-3 cells). The enhanced gene silencing activity of VEGF siRNA/PEI-PEG-PCP was maintained even under serum conditions, owing to the steric stabilization of the polyplexes with hydrophilic PEG grafts. Confocal microscopic studies revealed that the siRNA/PEI-PEG-PCP polyplexes were delivered into PC-3 cells in a PCP ligand-specific manner.

Time-dependent DNA condensation induced by amyloid beta-peptide

The major protein component of the amyloid deposition in Alzheimer's disease is a 39-43 residue peptide, amyloid beta (A beta). A beta is toxic to neurons, although the mechanism of neurodegeneration is uncertain. Evidence exists for non-B DNA conformation in the hippocampus of Alzheimer's disease brains, and A beta was reportedly able to transform DNA conformation in vitro. In this study, we found that DNA conformation was altered in the presence of A beta, and A beta induced DNA condensation in a time-dependent manner. Furthermore, A beta sheets, serving as condensation nuclei, were crucial for DNA condensation, and Cu2+ and Zn2+ ions inhibited A beta sheet-induced DNA condensation. Our results suggest DNA condensation as a mechanism of A beta toxicity.

RGD peptide grafted biodegradable amphiphilic triblock copolymer poly(glutamic acid)-b-poly(L-lactide)-b-poly(glutamic acid): Synthesis and self-assembly

A novel amphiphilic biodegradable triblock copolymer (PGL-PLA-PGL) with polylactide (PLA) as hydrophobic middle block and poly(glutamic acid) (PGL) as hydrophilic lateral blocks was successfully synthesized by ring-opening polymerization (ROP) Of L-lactide (LA) and N-carboxy anhydride (NCA) consecutively and by subsequent catalytic hydrogenation. The results of cell experiment of PGL-PLA-PGL suggested that PGL could improve biocompatibility of polyester obviously. The copolymer could form micelles of spindly shape easily in aqueous solution. The pendant carboxyl groups of the triblock copolymer were further activated with N-hydroxysuccinimide and combined with a cell-adhesive peptide GRGI)SY Incorporation of the oligopeptide further enhanced the hydrophilicity and led to formation of spherical micelles. PGL-PLAPGL showed better cell adhesion and spreading ability than pure PLA and the GRGDSY-containing copolymer exhibited even further improvement in cell adhesion and spreading ability, indicating that the copolymer could find a promising application in drug delivery or tissue engineering.