百合花粉管中的胞吞/胞吐作用观察分析

应用细胞膜染料FM 4-64结合Zeiss 5 live快速扫描型共聚焦显微镜观察了百合花粉管中的吞排作用.结果显示,培养基中加入FM4-64后,染料迅速进入到花粉管中,并在花粉管顶端形成一个锥形的区域;锥形区域表现出周期性变化,并且与花粉管脉冲生长呈负相关,即锥形区形成期花粉管生长缓慢,而锥形区消退期花粉管生长迅速;在锥形区域的消退期,花粉管顶端,尤其是最顶端快速向前延伸,而在锥形区域的形成期,花粉管最顶端的延伸速度显著下降,但亚顶端区域仍基本维持原有的生长速度.研究发现,花粉管的最顶端既是内吞作用也是胞吐作用的主要场所,但胞吞作用仅局限于花粉管的最顶端,而胞吐作用发生在包括亚顶端在内的整个花粉管顶端;胞吞和胞吐作用在花粉管最顶端交替发生,可能是花粉管脉冲生长的一个非常重要的原因.     

Palmitic Acid Analogs Exhibit Nanomolar Binding Affinity for the HIV-1 CD4 Receptor and Nanomolar Inhibition of gp120-to-CD4 Fusion

Background

We recently reported that palmitic acid (PA) is a novel and efficient CD4 fusion inhibitor to HIV-1 entry and infection. In the present report, based on in silico modeling of the novel CD4 pocket that binds PA, we describe discovery of highly potent PA analogs with increased CD4 receptor binding affinities (K_d) and gp120-to-CD4 inhibition constants (K_i). The PA analogs were selected to satisfy Lipinski''s rule of drug-likeness, increased solubility, and to avoid potential cytotoxicity.

Principal Findings

PA analog 2-bromopalmitate (2-BP) was most efficacious with K_d ∼74 nM and K_i ∼122 nM, ascorbyl palmitate (6-AP) exhibited slightly higher K_d ∼140 nM and K_i ∼354 nM, and sucrose palmitate (SP) was least efficacious binding to CD4 with K_d ∼364 nM and inhibiting gp120-to-CD4 binding with K_i ∼1486 nM. Importantly, PA and its analogs specifically bound to the CD4 receptor with the one to one stoichiometry.

Significance

Considering observed differences between K_i and K_d values indicates clear and rational direction for improving inhibition efficacy to HIV-1 entry and infection. Taken together this report introduces a novel class of natural small molecules fusion inhibitors with nanomolar efficacy of CD4 receptor binding and inhibition of HIV-1 entry.     

Hydrogen peroxide involvement in formation and development of adventitious roots in cucumber

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems. The present study demonstrates that H₂O₂ was generated in seedling explants after the primary roots were removed, and it mediates the auxin response prior to adventitious root formation in cucumber (Cucumis sativus L. Ganfeng 8). When compared with the controls, treatment of cucumber seedling explants after primary roots removal with either 20–40 mM H₂O₂ or 10 μM IAA significantly increased the number of adventitious roots, and treatment with 10–50 mM H₂O₂ significantly increased the fresh weight of adventitious roots. The effects of H₂O₂ on promoting the formation and growth of adventitious roots were eliminated by 2 mM ascorbic acid, 100 U CAT or 1 μM DPI, and the effects of IAA were eliminated by 4 mM ascorbic acid, 100 U CAT or 5 μM DPI. Treatment with either 4 mM ascorbic acid or 1–5 μM DPI inhibited the formation and growth of adventitious roots, and these inhibitory effects were partly reversed by exogenous H₂O₂.Furthermore, a higher concentration of endogenous H₂O₂ was detected in seedling explants 3 h after the primary roots were removed. However, in 10 μM DPI-treated seedling explants, the concentration of endogenous H₂O₂ was markedly reduced by DPI. Results obtained suggest that H₂O₂ may function as a signaling molecule, involved in the formation and development of adventitious roots in cucumber.     

Circumventing the heterogeneity and instability of human serum albumin-interferon-alpha2b fusion protein by altering its orientation

Albuferon is a novel long-acting interferon resulted from the direct genetic fusion of human albumin and interferon-alpha2b (HSA-IFN-alpha2b). Albuferon, co-developed by Human Genome Sciences Inc. and Novartis, is currently in late stage development for the treatment of hepatitis C. It was unexpected that HSA-IFN-alpha2b secreted from Pichia pastoris migrated as doublets on non-reducing SDS-PAGE and was prone to form covalent aggregates in aqueous solution. The heterogeneity and instability of HSA-IFN-alpha2b lowered its recovery rate to about 10% and necessitated lyophilized formulation. Site-directed mutagenesis revealed that the heterogeneity and instability of HSA-IFN-alpha2b was caused by the incomplete disulfide bridge formation between Cys1 and Cys98 of IFN-alpha2b. To alleviate the structural perturbation of IFN-alpha2b by HSA, IFN-alpha2b-HSA fusion protein, in which IFN-alpha2b was located at the N-terminus, was created. IFN-alpha2b-HSA was shown to be homogeneous and stable at 37 degrees C for at least 10 days. The improved homogeneity and stability of IFN-alpha2b-HSA increased the recovery rate by 2.5-fold and made the development of stable solution formulation possible. In vitro antiviral assays showed that both fusion proteins retained the activity of IFN-alpha2b, and the EC(50) of HSA-IFN-alpha2b, and IFN-alpha2b-HSA was calculated to be 120+/-12.5, and 160+/-1 1.3ng/ml, respectively. The increased recovery rate and the possibility of solution formulation of IFN-alpha2b-HSA may compensate for its slightly decreased in vitro activity, and makes it to be a promising therapeutic agent that deserves further evaluation.     

Study of the restitution of action potential duration using the artificial neural network

It is widely accepted that the APD (action potential duration) restitution plays a key role in the initializing and maintaining of the reentry arrhythmias. The Luo-Rudy II models paced with different protocols showed that the current APD had a complex relation with the previous APDs and diastole intervals (DIs). This relation could not be accurately described by a single exponential function. We used an artificial neural network to formularize this relation. The results suggested that back-propagation (BP) network could predict the current APD from the information of the first three previous beats. This would help provide a target for potential anti-arrhythmic therapies.     

A high-fat, ketogenic diet induces a unique metabolic state in mice

Ketogenic diets have been used as an approach to weight loss on the basis of the theoretical advantage of a low-carbohydrate, high-fat diet. To evaluate the physiological and metabolic effects of such diets on weight we studied mice consuming a very-low-carbohydrate, ketogenic diet (KD). This diet had profound effects on energy balance and gene expression. C57BL/6 mice animals were fed one of four diets: KD; a commonly used obesogenic high-fat, high-sucrose diet (HF); 66% caloric restriction (CR); and control chow (C). Mice on KD ate the same calories as mice on C and HF, but weight dropped and stabilized at 85% initial weight, similar to CR. This was consistent with increased energy expenditure seen in animals fed KD vs. those on C and CR. Microarray analysis of liver showed a unique pattern of gene expression in KD, with increased expression of genes in fatty acid oxidation pathways and reduction in lipid synthesis pathways. Animals made obese on HF and transitioned to KD lost all excess body weight, improved glucose tolerance, and increased energy expenditure. Analysis of key genes showed similar changes as those seen in lean animals placed directly on KD. Additionally, AMP kinase activity was increased, with a corresponding decrease in ACC activity. These data indicate that KD induces a unique metabolic state congruous with weight loss.     

B cell lymphoma 10 is essential for FcepsilonR-mediated degranulation and IL-6 production in mast cells

The adaptor protein B cell lymphoma 10 (Bcl10) plays an essential role in the functions of the AgRs in T and B cells. In this study, we report that Bcl10 also plays an important role in mast cells. Bcl10 is expressed in mast cells. Although Bcl10-deficient mast cells undergo normal development, we demonstrate that Bcl10 is essential for specific functions of FcepsilonR. Although Bcl10-deficient mast cells have normal de novo synthesis and release of the lipid mediator arachidonic acid, the mutant cells possess impaired FcepsilonR-mediated degranulation, indicated by decreased serotonin release, and impaired cytokine production, measured by release of IL-6. In addition, Bcl10-deficient mice display impaired IgE-mediated passive cutaneous anaphylaxis. Moreover, although Bcl10-deficient mast cells have normal FcepsilonR-mediated Ca(2+) flux, activation of PI3K, and activation of the three types of MAPKs (ERKs, JNK, and p38), the mutant cells have markedly diminished FcepsilonR-mediated activation of NF-kappaB and decreased activation of AP-1. Thus, Bcl10 is essential for FcepsilonR-induced activation of AP-1, NF-kappaB, degranulation, and cytokine production in mast cells.