Molecular dynamics simulation of human interleukin-4: comparison with NMR data and effect of pH,counterions and force field on tertiary structure stability

The human protein interleukin-4 (IL-4) has been simulated at two different pH values 2 and 6, with different amounts of counterions present in the aqueous solution, and with two different force-field parameter sets using molecular dynamics simulation with the aim of validation of force field and simulation set-up by comparison to experimental nuclear magnetic resonance data, such as proton–proton nuclear Overhauser effect (NOE) distance bounds, ³ J(HN,HC_α) coupling constants and backbone N–H order parameters. Thirteen simulations varying in the length from 3 to 7 ns are compared.

At pH 6 both force-field parameter sets used do largely reproduce the NOE's and order parameters, the GROMOS 45A3 set slightly better than the GROMOS 53A6 set. ³ J values predicted from the simulation agree less well with experimental values. At pH 2 the protein unfolds, unless counterions are explicitly present in the system, but even then the agreement with experiment is worse than at pH 6. When simulating a highly charged protein, such as IL-4 at pH 2, the inclusion of counterions in the simulation seems mandatory.     

Selenium and selenium-antagonistic elements in nutritional cancer prevention

Selenium is an essential trace element with antioxidative, antimutagenic, antiviral and anticarcinogenic properties. There is increasing evidence that the dietary selenium intakes are sub-optimal in the populations of many countries and that human cancer mortalities would significantly decline if additional selenium was made available either through supplementation or the fortification of certain foods. An important property of selenium is its interaction with other elements that may be present in foods, the water, the workplace and the environment, e.g. As, Cu, Ni, Co, Cr, Mn, Zn, Cd, Sn, Pb, Hg, Bi, Mo, Ag, Au, etc. The sequestration of elements by selenium represents an efficient natural detoxification mechanism for some of these elements but also results in the physiological inactivation of selenium. Animal experiments confirm that the chronic exposure to low levels of these elements abolishes the cancer-protective effect of selenium. Human cancer is likewise significantly determined by the interactions of selenium with other elements, as evidenced by epidemiological, ecological and case-control studies. Cadmium, for example, is a key risk-increasing element for prostate cancer; for breast cancer, Cd, Cr, Zn are mainly contributing; for bronchial cancer (in smelter workers), Cd, As, Cr, Sb, Co, La, all these elements are in a reciprocal relationship with Se. While selenium remains the key cancer-protective trace element, the interpretation of its mode of action necessitates consideration of the effects of selenium antagonistic elements.     

Shining light on nanotechnology to help repair and regeneration

Phototherapy can be used in two completely different but complementary therapeutic applications. While low level laser (or light) therapy (LLLT) uses red or near-infrared light alone to reduce inflammation, pain and stimulate tissue repair and regeneration, photodynamic therapy (PDT) uses the combination of light plus non-toxic dyes (called photosensitizers) to produce reactive oxygen species that can kill infectious microorganisms and cancer cells or destroy unwanted tissue (neo-vascularization in the choroid, atherosclerotic plaques in the arteries). The recent development of nanotechnology applied to medicine (nanomedicine) has opened a new front of advancement in the field of phototherapy and has provided hope for the development of nanoscale drug delivery platforms for effective killing of pathological cells and to promote repair and regeneration. Despite the well-known beneficial effects of phototherapy and nanomaterials in producing the killing of unwanted cells and promoting repair and regeneration, there are few reports that combine all three elements i.e. phototherapy, nanotechnology and, tissue repair and regeneration. However, these areas in all possible binary combinations have been addressed by many workers. The present review aims at highlighting the combined multi-model applications of phototherapy, nanotechnology and, reparative and regeneration medicine and outlines current strategies, future applications and limitations of nanoscale-assisted phototherapy for the management of cancers, microbial infections and other diseases, and to promote tissue repair and regeneration.     

Protein Kinase A-dependent Phosphorylation of Rap1 Regulates Its Membrane Localization and Cell Migration

The small G protein Rap1 can mediate “inside-out signaling” by recruiting effectors to the plasma membrane that signal to pathways involved in cell adhesion and cell migration. This action relies on the membrane association of Rap1, which is dictated by post-translational prenylation as well as by a stretch of basic residues within its carboxyl terminus. One feature of this stretch of acidic residues is that it lies adjacent to a functional phosphorylation site for the cAMP-dependent protein kinase PKA. This phosphorylation has two effects on Rap1 action. One, it decreases the level of Rap1 activity as measured by GTP loading and the coupling of Rap1 to RapL, a Rap1 effector that couples Rap1 GTP loading to integrin activation. Two, it destabilizes the membrane localization of Rap1, promoting its translocation into the cytoplasm. These two actions, decreased GTP loading and decreased membrane localization, are related, as the translocation of Rap1-GTP into the cytoplasm is associated with its increased GTP hydrolysis and inactivation. The consequences of this phosphorylation in Rap1-dependent cell adhesion and cell migration were also examined. Active Rap1 mutants that lack this phosphorylation site had a minimal effect on cell adhesion but strongly reduced cell migration, when compared with an active Rap1 mutant that retained the phosphorylation site. This suggests that optimal cell migration is associated with cycles of Rap1 activation, membrane egress, and inactivation, and requires the regulated phosphorylation of Rap1 by PKA.     

Purification and Characterization of an Extracellular Low Temperature-Active and Alkaline Stable Peptidase from Psychrotrophic Acinetobacter sp. MN 12 MTCC (10786)

An extracellular low temperature-active alkaline stable peptidase from Acinetobacter sp. MN 12 was purified to homogeneity with a purification fold of 9.8. The enzyme exhibited specific activity of 6,540 U/mg protein, with an apparent molecular weight of 35 kDa. The purified enzyme was active over broad range of temperature from 4 to 60 °C with optimum activity at 40 °C. The enzyme retained more than 75 % of activity over a broad range of pH (7.0–11.0) with optimum activity at pH 9.0. The purified peptidase was strongly inhibited by phenylmethylsulfonyl fluoride, giving an indication of serine type. The K _m and V _max for casein and gelatin were 0.3529, 2.03 mg/ml and 294.11, 384.61 μg/ml/min respectively. The peptidase was compatible with surfactants, oxidizing agents and commercial detergents, and effectively removed dried blood stains on cotton fabrics at low temperature ranging from 15 to 35 °C.     

氢化物发生—原子荧光光谱法测定桉树叶、皮、躯干、根中硒含量

以铁氰化钾为掩蔽剂,1.5% KBH4为还原剂,10%的盐酸为载流液,微波消解处理样品,氢化物发生-原子荧光法(HG-AFS)分别测定桉树叶、皮、躯干和根中硒元素含量。加标回收验证了结果的准确性。考察了仪器测定硒的检出限。并对铁氰化钾,聚环氧琥珀酸(PESA),酒石酸,柠檬酸,乙二胺五种掩蔽剂对11种常见干扰元素的掩蔽效果进行了探究,为优良掩蔽剂的选择提供了参考性资料。     

Evaluation of Nutritional Availability and Anti-Tumor Activity of Selenium Contained in Selenium-Enriched Kaiware Radish Sprouts

We estimated the nutritional availability of selenium (Se) in Se-enriched Kaiware radish sprouts (SeRS) by the tissue Se deposition and glutathione peroxidase (GPX) activity of rats administered the sprouts, and examined the effect of SeRS on the formation of aberrant crypt foci (ACF) in the colon of mice administered 1,2-dimethylhydrazine (DMH) to evaluate anti-tumor activity. Male weanling Wistar rats were divided into seven groups and fed a Se-deficient basal diet or the basal diet supplemented with 0.05, 0.10, or 0.15 μg/g of Se as sodium selenite or SeRS for 28 d. Supplementation with Se dose-dependently increased serum and liver Se concentrations and GPX activities, and the selenite-supplemented groups showed a higher increase than the SeRS-supplemented groups. The nutritional availability of Se in SeRS was estimated to be 33 or 64% by slope ratio analysis. Male 4-week-old A/J mice were divided into seven groups and fed a low Se basal diet or the basal diet supplemented with selenite, SeRS, or selenite + non-Se-enriched radish sprouts (NonSeRS) at a level of 0.1 or 2.0 μg Se/g for 9 weeks. After 1 week of feeding, all mice were given six subcutaneous injections of DMH (20 mg/kg) at 1-week intervals. The average number of ACF formed in the colon of mice fed the basal diet was 4.3. At a supplementation level of 0.1 μg Se/g, only SeRS significantly inhibited ACF formation. At a supplementation level of 2.0 μg Se/g, both selenite and SeRS significantly inhibited ACF formation. The addition of NonSeRS to the selenite-supplemented diets tended to inhibit ACF formation, but this was not statistically significant. These results indicate that SeRS shows lower nutritional availability but higher anti-tumor activity than selenite.     

Colostrum and milk selenium,antioxidative capacity and immune status of dairy cows fed sodium selenite or selenium yeast

Dietary selenium (Se) can be supplemented from organic or inorganic sources and this may affect Se metabolism and functional outcome such as antioxidative status and immune functions in dairy cows. A feeding trial was performed with 16 Holstein-Friesian dairy cows fed with a total mixed ration (0.18 mg Se/kg dry matter (DM)) either without Se supplement (Control, n = 5), or with Se from sodium selenite (Group SeS, n = 5) or Se yeast (Group SeY, n = 6). In Groups SeS and SeY, the Se supplementation amounted to an additional intake of 4 mg Se and 6 mg Se/d during gestation and lactation, respectively. The effect of both Se sources was characterised by milk Se and antioxidant levels, and the phenotyping and functional assessment of phagocytic activity of milk immune cells. Se yeast has been found to increase (p ≤ 0.001) the milk Se and antioxidant levels markedly compared to the control group. The experimental treatment did not affect the immune parameters of the cows. Lymphocyte subpopulations and phagocytosis activity of neutrophilic granulocytes were affected neither by the Se intake nor by the two different dietary supplements. It can be concluded that sodium selenite and Se yeast differ considerably in their effects on antioxidant status in dairy cows. However, the basal dietary Se concentration of 0.18 mg/kg DM seemed to be high enough for the measured immune variables.     

The central role of selenium in the biochemistry and ecology of the harmful pelagophyte,Aureococcus anophagefferens

The trace element selenium (Se) is required for the biosynthesis of selenocysteine (Sec), the 21st amino acid in the genetic code, but its role in the ecology of harmful algal blooms (HABs) is unknown. Here, we examined the role of Se in the biology and ecology of the harmful pelagophyte, Aureococcus anophagefferens, through cell culture, genomic analyses, and ecosystem studies. This organism has the largest and the most diverse selenoproteome identified to date that consists of at least 59 selenoproteins, including known eukaryotic selenoproteins, selenoproteins previously only detected in bacteria, and novel selenoproteins. The A. anophagefferens selenoproteome was dominated by the thioredoxin fold proteins and oxidoreductase functions were assigned to the majority of detected selenoproteins. Insertion of Sec in these proteins was supported by a unique Sec insertion sequence. Se was required for the growth of A. anophagefferens as cultures grew maximally at nanomolar Se concentrations. In a coastal ecosystem, dissolved Se concentrations were elevated before and after A. anophagefferens blooms, but were reduced by >95% during the peak of blooms to 0.05 nℳ. Consistent with this pattern, enrichment of seawater with selenite before and after a bloom did not affect the growth of A. anophagefferens, but enrichment during the peak of the bloom significantly increased population growth rates. These findings demonstrate that Se inventories, which can be anthropogenically enriched, can support proliferation of HABs, such as A. anophagefferens through its synthesis of a large arsenal of Se-dependent oxidoreductases that fine-tune cellular redox homeostasis.     

Tailor-made approach for selective isolation and elution of low-density lipoproteins by immunoaffinity sorbent on silica

Immunoaffinity procedure was developed for isolation of low density lipoprotein (LDL) from biological samples by using silica-derived immunoaffinity sorbent. Sorbent was prepared by immobilization of monoclonal anti-apoB-100 antibody onto macroporous silica particles, using carefully optimized binding chemistry. Binding capacity of the sorbent towards LDL was determined by batch extraction experiments with solutions of isolated LDL in phosphate-buffered saline, and found to be 8 mg LDL/g. The bound LDL fraction was readily released from the sorbent by elution with ammonia at pH 11.2. The total time needed for isolation procedure was less than 1 h, with LDL recoveries being essentially quantitative for samples containing less than 0.3 mg LDL/mL. With higher concentrations, recoveries were less favorable, most probably due to irreversible adsorption caused by LDL aggreggation. However, reusability studies with isolated LDL at concentration 0.2 mg/mL indicate that the developed immunoaffinity material may be used for multiple binding-release cycles, with minor losses in binding capacity. Finally, the sorbent was successfully applied to isolation of LDL from diluted plasma. Apart from its practical implications for LDL isolation, this study provides crucial insights into issues associated with LDL-sorbent interactions, and may be useful in future efforts directed to development of lipoprotein isolation approaches.