Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.     

Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles

An in vitro human reconstructed epidermis model (SkinEthic) used for screening acute and chronic skin irritation potential was validated against in vivo data from skin tolerability studies. The irritation potential of sodium lauryl sulfate (SLS), calcipotriol and trans-retinoic acid was investigated. The in vitro epidermis-like model consists of cultures of keratinocytes from human foreskin on a polycarbonate filter. The modulation of cell viability, the release and gene expression of proinflammatory cytokines, interleukins 1α and 8, and morphological changes were evaluated during 3 days as endpoints representative for an inflammatory reaction. The cumulative irritation potential of the topical products was evaluated in a human clinical study by visual scoring and biophysical measurement of inflammatory skin reaction after repeated 24 h applications over 3 weeks under Finn chamber patches. All topical products that were nonirritating in the human study were noncytotoxic and did not induce cytokine expression in the in vitro acute model (day 1 exposure). All irritating controls exhibited specific cell viability and cytokine patterns, which were predictive of the in vivo human data. The ranking of mild to moderate skin irritation potential was based on the lack of cytotoxicity and the presence of cytokine patterns including gene expression specific for each irritant, using the chronic in vitro model (up to 3 days exposure). The human reconstructed epidermis model SkinEthic was shown to be a reliable preclinical tool predicting the irritation potential of topical products. Moreover, it is a useful model in a two-step tiered strategy for screening acute and chronic irritation potential for the selection of vehicles for new topical drugs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stimulation of HeLa cell growth by physiological concentrations of 4-hydroxynonenal

The aim of this study was to analyze the growth response of HeLa cells over a prolonged period of time to a single exposure of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids. Furthermore, the growth modulating effect of serum factors, particularly albumin, on the growth pattern was examined. The effects of HNE on the growth rate and viability of the cells, as well as on the incorporation of labelled amino acids were monitored daily over a period of four days. Fetal calf serum not only had a growth stimualting effect but also modulated the action of HNE. In neither respect was albumin able to substitute for serum indicating that the influence of serum was not exerted via an albumin–HNE conjugate. HNE had a clear dose-dependent effect and a distinction could be made between a supraphysiological concentration (100 μM), which was primarily cytotoxic and a physiological range (below 10 μM) which showed growth modulatory effects. These effects consisted of a transient inhibition in the initial phase of the cell growth, which under optimal conditions (in presence of serum) was followed by a period of increased proliferation, compared to untreated control cultures, until confluence was attained. It is suggested that HNE is not only a toxic product of lipid peroxidation, but a physiological growth regulating factor as well.     

人睫状神经营养因子的原核表达,纯化及其生物效应

人睫状神经营养因子（ｈＣＮＴＦ）克隆入ｐＢＶ２２０中，在ＤＨ５α菌株中表达，重组蛋白以包含体的形式存在，表达量为菌体总蛋白的５０％左右。经比较发现用２ｍｏｌ／Ｌ脲洗涤包含体可溶解大量可溶性细菌蛋白，且包含体损失较小。在高浓度变性剂条件下进行ｓｅｐｈａｒｃｙｌＳ－２００凝胶过滤，解决了纯化中ｈＣＮＴＦ易聚合的问题，在低浓度变性剂条件下进行ＤＥＡＥ离子交换，有利于蛋白活性的保持。经两步纯化后得到均一性ｈＣＮＴＦ，纯度达９５％以上。在自然状态下使ｈＣＮＴＦ复性。纯化复性后的ｈＣＮＴＦ对无血清培养的鸡胚背根节神经元和脊髓腹角运动神经元有明显的维持存活和促进生长发育的生物效应。     

ALBUMIN CAN REVERSE THE RELEASE OF POTASSIUM FROM HUMAN ERYTHROCYTES TREATED WITH THE NON-IONIC DETERGENT,BRIJ 58

Exchange of erythrocyte intracellular (i/c) K⁺for extracellular (e/c) Na⁺in human erythrocytes treated with sub-CMC concentrations of the non-ionic detergent Brij 58 can be stopped by reincubation in serum or albumin containing solutions. The progressive equilibration of the K⁺contents of detergent-treated human erythrocytes with the incubation medium was reversed by an albumin-mediated withdrawal of detergent molecules from the cell. Re-establishment of near normal [K⁺] in terms of K⁺/kg water proceeds in two ways: (i) a metabolism-dependent net accumulation of K⁺ions; and (ii) a metabolism-independent shrinkage of erythrocytes, this being the more significant factor.     

Environmental needs and social justice

Despite the growth in awareness and practical action to maintain biodiversity, environmental degradation and ecosystem destruction has continued at a high rate over the last 20 years. The roots of this lie in the predominant international economic order, underpinned by lifestyle demands for increased material consumption. Net flow of wealth from South (less developed) to North (more developed) nations has exacerbated a spiral of increased poverty and environmental degradation in the former. Global environmental conservation depends upon a radical change of direction with the principle of equity as the starting point. Notwithstanding the importance of continuing to add to local and small-scale conservation achievements, the prospect of radical change happening seems small, despite it being in the long-term self-interest of the North. The concept of equity is, apparently, unacceptable to Northern electorates.     

生态意识及其主要特点

一、生态意识产生的时代背景生态意识作为人类思想的先进观念,产生于20世纪后半叶。产业革命以来二百多年,人类依据先进科学技术武装的强大生产力,无限制地向自然进     

A method to study zhizosphere processes in thin soil layers of different proximity to roots

Frankia is the diverse bacterial genus that fixes nitrogen within root nodules of actinorhizal trees and shrubs. Systematic and ecological studies of Frankia have been hindered by the lack of morphological, biochemical, or other markers to readily distinguish strains. Recently, nucleotide sequence of 16 S RNA from the small ribosomal subunit has been used to classify and identify a variety of microorganisms. We report nucleotide sequences from portions of the 16 S ribosomal RNA from Frankia strains AcnI1 isolated from Alnus viridis ssp. crispa (Ait.) Turrill and PtI1 isolated from Purshia tridentata (Pursh) DC. The number of nucleotide base substitutions and gaps we find more than doubles the previously reported sequence diversity for the same variable regions within other strains of Frankia.     

Polyphasic characterization of Delftia acidovorans ESM-1, a facultative methylotrophic bacterium isolated from rhizosphere of Eruca sativa

In this study, one bacterial strain, ESM-1, was isolated from rhizosphere of Eruca sativa, growing in Al Hofouf, Saudia Arabia, after enrichment with methanol as a sole carbon and energy source in a batch culture. ESM-1 was characterized by a polyphasic approach. The strain was identified as Delftia acidovorans at similarity level of 99.9% of the 16S rRNA gene sequences. Results of the Biolog Gen III MicroPlate test system showed that strain ESM-1 reacted positively to 47 (50%) including the one-carbon compound formic acid, and partially positive to 6 (∼6.4%) out of the 94 different the traits examined. The total cellular fatty acids composition of the strain ESM-1 was (C_16:1ω7c/C_16:1ω6c) and C_16:0) and matched that of Delftia acidovorans at a similarity index of 0.9, providing a robustness to the ESM-1 identification. Furthermore, ESM-1 displayed a complex polar lipid profile consisting of phosphatidylethanolamine, phosphatidylglycerol, glycolipid, aminolipid, in addition to uncharacterized lipids. The DNA G+C content of the strain was 66.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain ESM1-1 was clearly clustered within the Delftia clade and constructed a monophyletic subcluster with Delftia acidovorans NBRC14950. The results addressed that ESM-1 is a facultative methylotrophic bacterium indigenous to Al Hofouf region and opens the door for potential biotechnological applications (e.g., bioremediation) of this strain, in future. Additionally, these findings assure that the total cellular fatty acid analysis and 16S rRNA gene are reliable tool for bacterial characterization and identification.     

Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

β-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of β-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8–7.0 with more than 97% activity. Purified β-galactosidase was optimally active at 50 °C. Kinetic parameters K_m and V_max for purified enzyme were 400 µM and 1.22 × 10⁻¹ U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca²⁺, Mg²⁺ and Mn²⁺ slightly activated the enzyme whereas NH₄⁺, Co²⁺ and Fe³⁺ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that β-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min⁻¹ and t_1/2 of 14.74 min. This is better than other studied β-galactosidases. Both sonicated Pediococcus acidilactici cells and purified β-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of β-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca²⁺ shows that it is suitable for milk processing.