Conversion of human interferon-β from a secreted to a phosphatidylinositol anchored protein by fusion of a 17 amino acid sequence to its carboxyl terminus

A number of cell-surface proteins are anchored in plasma membranes by a glycosylated phosphatidylinositol (PI) moiety that is covalently attached to the carboxyl-terminal amino acid of the mature protein. We have previously reported the construction of a cDNA clone of a truncated Platelet-derived growth factor (PDGF) receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. In the construction of the vector, a sequence of 51 base pairs (bp) from the 3′-untranslated region of the receptor cDNA was linked in frame with the external domain coding sequence. The truncated receptor protein with the peptide VTSGHCHEERVDRHDGE fused to its carboxyl terminus was covalently attached to the membrane by a PI linkage and it was released by phosphatidylinositol specific-phospholipase C (PI-PLC). When the 51 bp sequence was deleted, the external domain receptor protein was secreted into the media. To determine whether the PI linkage of the protein was due to the 17 amino acids added, the peptide was fused to the carboxyl terminus of the secreted protein human Interferon-β (hu-IFN-β). Chinese hamster ovary (CHO) cells transfected with the hu-IFN-β cDNA secreted the protein to theconditioned media, whereas CHO cells transfected with the carboxyl terminus modified-hu-IFN-β cDNA did not secrete detectable levels of protein. CHO cells expressing the carboxyl terminus modified-hu-IFN-β were treated with PI-PLC, the media and cell lysates were analyzed by SDS-PAGE after immunoprecipitation with antibodies against hu-IFN-β. The modified protein is anchored to the plasma membrane by a PI linkage and it is specifically released by PI-PLC, whereas a control preparation of CHO cells expressing wild type hu-IFN-β does not show the same pattern. The 17 amino acid peptide fused to the carboxyl terminus of IFN-β directs attachment of a PI anchor and targets the fusion protein to the plasma membrane.     

Geochemical baseline determination and pollution assessment of heavy metals in urban soils of Karachi,Pakistan

Karachi is one of the most populated urban agglomerations in the world. No categorical study has yet discussed the geochemical baseline concentrations of metals in the urban soil of Karachi. The main objectives of this study were to establish geochemical baseline values and to assess the pollution status of different heavy metals. Geochemical baseline concentrations of heavy metals were estimated using the cumulative frequency distribution (CDF) curves. The estimated baseline concentrations of Pb, Cr, Cu, Zn and Fe were 56.23, 12.9, 36.31, 123.03 and 11,776 mg kg⁻¹, respectively. The pollution status of heavy metals in urban soils was evaluated using different quantitative indices (enrichment factor–EF, Geo-accumulation Index–I_geo, and pollution index–PI). Enrichments factors of the selected heavy metals determined by using Fe as a normalizer showed that metal contamination was the product of anthropogenic activities. The urban soils of Karachi were found to have a moderate to moderately severe enrichment with Pb, whereas Cr and Cu has moderate and Zn has minor enrichment. I_geo results indicated moderate soil contamination by Pb at some of the sampling locations. PI for Pb, Cr, Cu and Zn was found in the range of 0.04–3.42, 0.19–1.55, 0.27–2.45 and 0.32–1.57, respectively. Large variations in PI values of Pb revealed that soil in those areas of the city which are influenced by intensive anthropogenic activities have exceptionally high concentrations of Pb. The findings of this study would contribute to the environmental database of the soil of the region and would also facilitate both at the local and the international scales, in a more accurate global environmental monitoring, which will eventually facilitate the development of management and remediation strategies for heavy metal contaminated urban soil.     

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。     

Denaturing gradient gel electrophoresis detection of polymorphism in a PCR fragment of sheep epidermal growth factor gene

The ovine map is not yet well-developed, which represents a problem when looking for markers of a region of interest in sheep. A means of circumventing this is to use comparative mapping. In this study primers were determined using consensus sequences for the epidermal growth factor gene of humans, rats and mice, and an ovine epidermal growth factor gene fragment was amplified by polymerase chain reaction (PCR). A new set of specific ovine primers was chosen to study the polymorphism of this DNA fragment by denaturing gradient gel electrophoresis. Eighty-four individuals belonging to seven sheep breeds were studied with this technique and four alleles were detected. The heterozygosity rate was 0.57. Family analysis showed mendelian inheritance of the alleles. Usually, genetic analysis of type-I loci used in the comparative mapping is based on the detection of restriction fragment length polymorphisms in sheep DNA using cDNA probes from other species. Our work shows that another method, based on PCR and denaturing gradient gel electrophoresis techniques, can be efficiently used.     

Biological activity of Burkholderia (Pseudomonas) cepacia lipopolysaccharide

Abstract Burkholderia cepacia has emerged as an important multiresistant pathogen in cystic fibrosis (CF), associated in 20% of colonised patients with a rapid and fatal decline in lung function. Although knowledge of B. cepacia epidemiology has improved, the mechanisms involved in pathogenesis remain obscure. In this study, B. cepacia lipopolysaccharide (LPS) was assessed for endotoxic potential and the capacity to induce tumour necrosis factor (TNF). LPS preparations from clinical and environmental isolates of B. cepacia and from the closely related species Burkholderia gladioli exhibited a higher endotoxic activity and more pronounced cytokine response in vitro compared to preparations from the major CF pathogen Pseudomonas aeruginosa . This study may help to explain the vicious host immune response observed during pulmonary exacerbations in CF patients colonised by B. cepacia and lead to therapeutic advances in clinical management.     

Thioredoxin-dependent regulation of AIF-mediated DNA damage

The thioredoxin (Trx) system is one major redox system in mammalian cells. One of its component, Trx, is involved in redox homeostasis and many cellular biological processes through participating in disulfide reduction, S-nitrosylation/S-denitrosylation reactions and protein-protein interactions. In this study, we report the identification of a novel interaction between cytosolic/nuclear Trx1 and apoptosis inducing factor (AIF), and the redox sensitivity and biological significance of the Trx-AIF interaction was characterized. Cytosolic Trx1 but not mitochondrial Trx2 was observed to interact with AIF under physiological conditions and Trx1's active site cysteines were crucial for the interaction. Under oxidative stress conditions, Trx-AIF interaction was disrupted. When the treated cells were allowed to recover from oxidative stress by means of removal of the oxidants, interaction between Trx1 and AIF was re-established time-dependently, which underpins the biological relevance of a Trx-dependent redox regulation of AIF-mediated cell death. Indeed, in times of oxidative stress, nuclear translocation of AIF was found to occur concurrently with perturbations to the Trx-AIF interaction. Once localized in the nucleus, reduced Trx1 hindered the interaction between AIF and DNA, thereby bringing about an attenuation of AIF-mediated DNA damage. In conclusion, characterization of the Trx-AIF interaction has led to an understanding of the effect of reduced Trx1 on possibly regulating AIF-dependent cell death through impeding AIF-mediated DNA damage. Importantly, identification of the novel interaction between Trx1 and AIF has provided opportunities to design and develop therapeutically relevant strategies that either promote or prevent this protein-protein interaction for the treatment of different disease states.     

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H₂O₂), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H₂O₂-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury.