Novel modulators of amyloid-beta precursor protein processing

Proteolytic processing of the amyloid precursor protein (APP) is modulated by the action of enzymes alpha-, beta- and gamma-secretases, with the latter two mediating the amyloidogenic production of amyloid-beta (Abeta). Cellular modulators of APP processing are well known from studies of genetic mutations (such as those found in APP and presenilins) or polymorphisms (such as the apolipoprotein E4 epsilon-allele) that predisposes an individual to early or late-onset Alzheimer's disease. In recent years, several classes of molecule with modulating functions in APP processing and Abeta secretion have emerged. These include the neuronal Munc-18 interacting proteins (Mints)/X11s, members of the reticulon family (RTN-3 and RTN-4/Nogo-B), the Nogo-66 receptor (NgR), the peptidyl-prolyl isomerase Pin1 and the Rho family GTPases and their effectors. Mints and NgR bind to APP directly, while RTN3 and Nogo-B interact with the beta-secretase BACE1. Phosphorylated APP is a Pin1 substrate, which binds to its phosphor-Thr668-Pro motif. These interactions by and large resulted in a reduction of Abeta generation both in vitro and in vivo. Inhibition of Rho and Rho-kinase (ROCK) activity may underlie the ability of non-steroidal anti-inflammatory drugs and statins to reduce Abeta production, a feat which could also be achieved by Rac1 inhibition. Detailed understanding of the underlying mechanisms of action of these novel modulators of APP processing, as well as insights into the molecular neurological basis of how Abeta impairs leaning and memory, will open up multiple avenues for the therapeutic intervention of Alzheimer's disease.     

Pitavastatin prevents NMDA-induced retinal ganglion cell death by suppressing leukocyte recruitment

Excitotoxicity is a major cause of retinal ganglion cell (RGC) death during ischemic diseases such as vessel occlusion and diabetic retinopathy. However, the underlying mechanisms are not well understood. Statins, inhibitors of the HMG-CoA reductase, have neuroprotective effects in addition to their original role in lowering cholesterol. We hypothesize that pitavastatin, a recently introduced potent statin, is protective against N-methyl-d-aspartic acid (NMDA)-induced RGC death. Pitavastatin, administered by gavage, abolished NMDA-induced loss of RGCs. To elucidate the mechanisms underlying the neuroprotective effect of pitavastatin, we investigated its impact on inflammation. NMDA increased the expression of interleukin-1beta and TNF-alpha, and endothelial adhesion molecules, including ICAM-1, and induced leukocyte accumulation in the retinal vessels. Pitavastatin significantly reduced NMDA-induced leukocyte accumulation and up-regulation of endothelial adhesion molecules, whereas cytokine expression was unaffected. Systemic blockade of ICAM-1 in wild-type mice or absence of CD18 in gene-deficient (CD18(-/-)) mice significantly suppressed NMDA-induced leukocyte accumulation and RGC death. These findings suggest a novel and causative role for inflammatory leukocyte recruitment in NMDA-induced excitotoxicity. Furthermore, we show the novel neuroprotective effect of statins against excitotoxicity-induced RGC death. Statins or other anti-inflammatory agents may thus have therapeutic benefits in excitotoxicity-associated neuronal diseases through blockade of leukocyte recruitment.     

大肠杆菌F18菌毛操纵子全基因克隆、表达及生物学活性

利用PCR技术以猪产肠毒素大肠杆菌F18标准菌株107/86和2134P基因组DNA为模板成功地扩增出编码F18ab和F18ac完整菌毛操纵子fed基因。将它们分别克隆入表达质粒载体pET-22b( ),结合酶切和核苷酸序列分析证明了PCR预期扩增产物的正确性。然后将克隆的重组载体DNA转化至大肠杆菌BL21(DE3),构建和筛选出分别含F18ab和F18ac完整fed基因的重组菌,经过IPTG诱导表达,在电镜下观察到上述两种重组菌能分别大量表达F18ab和F18ac菌毛。用热抽提法提纯其诱导表达的F18ab和F18ac菌毛,经SDS-PAGE电泳和考马斯亮蓝染色发现提纯后菌毛获单一分子量约为15kDa蛋白条带,免疫家兔后制备出高效价的兔抗血清,玻板凝集试验和Western blot结果表明:体外诱导表达的F18ab和F18ac菌毛具有和野生F18菌毛相同的抗原性。用表达F18ab和F18ac菌毛的上述2株重组菌分别进行小肠上皮细胞体外吸附试验和吸附抑制试验,结果表明:2株重组菌和野生菌株一样具有较强的粘附易感仔猪小肠上皮细胞的能力,而用表达F18ab和F18ac重组菌提纯的菌毛制备出兔抗血清都能有效地抑制上述重组菌或野生菌株对易感仔猪小肠上皮细胞的吸附结合。     

Induction of an effective anti-tumor immune response and tumor regression by combined administration of IL-18 and Apoptin

Immunization strategies using plasmid DNA can potentially improve humoral and cellular immune responses that protect against cancer and infectious diseases. The chicken anemia virus-derived Apoptin protein exhibits remarkable specificity in its ability to induce apoptosis in tumor cells, but not in normal diploid cells. Interleukin-18 (IL-18) is a Th1-type cytokine that has demonstrated potential as a biological adjuvant in murine tumor models. In this study, we analyzed the anti-tumor potential and mechanism of action of simultaneous Apoptin and IL-18 gene transfer in C57BL/6 mice bearing Lewis lung carcinoma (LLC). Here we report that the growth of established tumors in mice immunized with pAPOPTIN in conjunction with pIL-18 was significantly inhibited compared with the growth of tumors in mice immunized with the empty vector (EV) or pAPOPTIN alone. Furthermore, the immunization of mice with pAPOPTIN in conjunction with pIL-18 elicited strong natural killer activity and LLC tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro. In addition, T cells from lymph nodes of mice vaccinated with pIL-18 or pAPOPTIN + pIL-18 secreted high levels of the Th1 cytokine IL-2 and IFN-γ, indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with Apoptin together with IL-18 may be a novel and powerful strategy for cancer immunotherapy.     

Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11β-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11β-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18β-glycyrrhetinic acid (18β-GA) derivatives (2–5) and their inhibitory activities against rat hepatic11β-HSD1 and rat renal 11β-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds’ ability to inhibit human 11β-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18β-GA derivatives 2 and 3 with apparent selectivity for rat 11β-HSD1 showed a high percentage inhibition for human microsomal 11β-HSD1 at 10 μM and exhibited IC₅₀ values of 400 and 1100 nM, respectively. The side chain modified 18β-GA derivatives 4 and 5, although showing selectivity for rat 11β-HSD1 inhibited human microsomal 11β-HSD1 with IC₅₀ values in the low micromolar range.     

Gene therapy legislation in The Netherlands

Several regulatory organisations are involved in the assessment of clinical gene therapy trials involving genetically modified organisms (GMOs) in The Netherlands. Medical, ethical and scientific aspects are, for instance, evaluated by the Central Committee on Research Involving Human Subjects (CCMO). The Ministry of Housing, Spatial Planning and the Environment (VROM) is the competent authority for the environmental risk assessment according to the deliberate release Directive 2001/18/EC. A Gene Therapy Office has been established in order to streamline the different national review processes and to enable the official procedures to be completed as quickly as possible. Although the Gene Therapy Office improved the application process at the national level, there is a difference of opinion between the EU member states with respect to the EU Directive according to which gene therapy trials are assessed, that urges for harmonisation. This review summarises the gene therapy legislation in The Netherlands and in particular The Netherlands rationale to follow Directive 2001/18/EC for the environmental risk assessment.     

Variability of latewood-widths and -stable isotope ratios in a sessile oak tree (Quercus petraea (Matt.) Liebl.)

Axial variation of latewood-widths and -stable isotopes (δ¹³C and δ¹⁸O) of a single sessile oak tree (Quercus petraea (Matt.) Liebl.) was analyzed. The influence of sampling height was found to be weak for both, latewood-widths and -stable isotopes in the latewood. The observed trends with stem-height show clearly that possible effects due to cambial aging are overrated by annual effects. In addition, relations between samples from different stem-heights are strong for identical calendar years while they are poor for identical cambial age. This indicates that samples from both, living trees and historical wood can be taken from any height position of a trunk or beam as long they are free from anatomical abnormities. Simple correlation and Gleichläufigkeit were used for validation.     

Molecular phylogeny, scale evolution and taxonomy of centrohelid heliozoa

Heliozoa are ubiquitous, unicellular phagotrophs with slender radiating axopodia for trapping prey. We sequenced 18S rRNA genes from 35 cultured centrohelid heliozoa (18 studied by electron microscopy) and 28 environmental libraries (18 freshwater, 10 marine), yielding 97 new sequences, this exceeding described species. Phylogenetic analyses show two major groups and that ancestral centrohelids probably had inner plate-like tangential and distinct outer radial silica scales, the latter diverging early into contrasting scale types seen in extant Pterocystis/Choanocystis and Acanthocystis/Raphidiophryidae. Scales were lost at least thrice. Pterocystis is paraphyletic, as was the classical family Acanthocystidae; Heterophrys was polyphyletic. Using scale morphology and rRNA sequences, we establish new families Pterocystidae (Pterocystis, Raineriophrys, Chlamydaster), Marophryidae (type Marophrys (Heterophrys) marina gen. et comb. nov.) and Choanocystidae, new suborders Pterocystina (Pterocystidae, Choanocystidae, Heterophryidae) and Acanthocystina (Acanthocystidae, Raphidiophryidae, Marophryidae), and ten new Pterocystis, Acanthocystis and Choanocystis species. Most clades are exclusively freshwater or exclusively marine; evolutionary transitions between these habitats have been rare.     

The purification and characterization of alpha interferons and related cytokine receptors--a personal account

In 1976–1977, I adapted reversed-phase HPLC (RP-HPLC) to peptide and protein purification, starting with pituitary proteins and continuing with the first successful purification to homogeneity of human leukocyte interferon (IFN-). Using this technology, I isolated and characterized 6–8 different leukocyte interferon subtypes, which were later identified as products of the IFN- gene family. Since then, RP-HPLC became a standard procedure for isolation and analysis of proteins. The successful purification of IFN- led to the development of Roferon-A™, a drug used for the treatment of hairy cell leukemia, hepatitis C and a variety of other diseases. Later studies with my colleagues in Israel and abroad led to isolation and discovery of several cytokine receptors and binding proteins, including those of Type I IFNs, TNF and IL-18. The use of HPLC was indispensable in most of these studies.     

Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinases

AIMS: Isolation and characterization of chitinases from a halotolerant Bacillus pumilus. METHODS AND RESULTS: Bacillus pumilus strain SG2 was isolated from saline conditions. It is able to produce chitinase activity at high salt concentration. SDS-PAGE analysis of the B. pumilus SG2 culture supernatant showed two major bands that were induced by chitin. The amino acid sequence of the two proteins, designated ChiS and ChiL, showed a high homology with the chitinase of B. subtilis CHU26, and chitinase A of B. licheniformis, respectively. N-terminal signal peptide of both proteins was also determined. The molecular weight and isoelectric point of the chitinases were determined to be 63 and 74 kDa, and 4.5 and 5.1, for ChiS and ChiL respectively. The genes encoding for both chitinases were isolated and their sequence determined. The regulation of the chitinase genes is under the control of the catabolite repression system. CONCLUSIONS: Secreted chitinase genes and their flanking region on the genome of B. pumilus SG2 have been identified and sequenced. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a multiple chitinases-producing B. pumilus halotolerant strain. We have identified two chitinases by using a reverse genetics approach. The chitinases show resistance to salt.