Fasciola hepatica: humoral and cytokine responses to a member of the saposin-like protein family following delivery as a DNA vaccine in mice

The humoral and cellular responses to DNA vaccination of BALB/c mice with a novel antigen from the Fasciola hepatica saposin-like protein family (FhSAP-2) have been investigated. Two constructs were produced containing the FhSAP-2 DNA sequence, one intended for extracellular secretion of FhSAP-2 protein, and one expressing FhSAP-2 in the cytoplasm of a transfected cell. The constructs were tested in HEK 293T cells, with the secretory construct producing less detectable FhSAP-2 relative to cytoplasmic construct when observed by fluorescence. The size of expressed protein was confirmed by Western blot of cell lysate, but FhSAP-2 was undetectable in cell supernatants. Both, secretory and cytoplasmic constructs as well as FhSAP-2 recombinant protein were tested in mice. The antibody response elicited in mice vaccinated with the rFhSAP-2 induced high levels of IgG(1), IgG(2), and IgE as well as high levels of IL-10 and IFNgamma indicating a mixed Th1/Th2 response. Vaccination of mice intramuscularly with the cytoplasmic FhSAP-2 construct resulted in a dominant IgG(2a) isotype antibody as well as a dominant IFNgamma cytokine, with significant IgE, IgG(1), and IL-10 responses also present, suggesting a mixed Th1/Th2 profile. Isotype and cytokine profiles elicited by the FhSAP-2 secretory construct were similar to those obtained with the cytoplasmic construct but at levels that were significantly lower. The results demonstrate that FhSAP-2 can be delivered as a DNA vaccine construct and induces a stronger Th1 response than the recombinant protein alone. This could result in an improvement in the immunoprophylactic potential of this candidate vaccine against F. hepatica.     

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.     

半乳糖配体介导大鼠白介素10基因在大鼠肝脏内的靶向表达

目的 探讨大鼠白介素10（rIL-10）基因是否可通过半乳糖配体介导的脂质体转染法在大鼠肝脏内靶向表达。方法将已构建好的rIL-10基因真核表达质粒与半乳糖配体转染试剂按jetPEI．Gal／DNA（N／P=10）比例混合,通过尾静脉注射转移至大鼠体内。RT—PCR法和ELISA法检测rIL-10基因转移至体内0h、24h、7d和16d后大鼠肝、肾、脾和肺组织及血清中rIL-10的表达情况。结果rIL-10基因转移前大鼠肝、肾、脾和肺组织末扩增出明显rIL-10mRNA表达,转移7d后rIL-10表达主要分布在肝组织。肝组织中rIL-10mRNA表达在基因转移24h和7d时显著升高。血清中的rIL-10浓度在转移后24h和7d浓度分别为（107．92±12．26）pg／ml和（33．2±13．15）pg／ml。结论rIL-10基因通过半乳糖配体介导的脂质体转染法可有效的转移至大鼠体内,并可在肝脏靶向表达一周左右时间。     

Angelman syndrome and severe infections in a patient with de novo 15q11.2–q13.1 deletion and maternally inherited 2q21.3 microdeletion

Angelman syndrome is a neurodevelopmental disorder characterized by mental retardation, severe speech disorder, facial dysmorphism, secondary microcephaly, ataxia, seizures, and abnormal behaviors such as easily provoked laughter. It is most frequently caused by a de novo maternal deletion of chromosome 15q11–q13 (about 70–90%), but can also be caused by paternal uniparental disomy of chromosome 15q11–q13 (3–7%), an imprinting defect (2–4%) or in mutations in the ubiquitin protein ligase E3A gene UBE3A mostly leading to frame shift mutation. In addition, for patients with overlapping clinical features (Angelman-like syndrome), mutations in methyl-CpG binding protein 2 gene MECP2 and cyclin-dependent kinase-like 5 gene CDKL5 as well as a microdeletion of 2q23.1 including the methyl-CpG binding domain protein 5 gene MBD5 have been described. Here, we describe a patient who carries a de novo 5 Mb-deletion of chromosome 15q11.2–q13.1 known to be associated with Angelman syndrome and a further, maternally inherited deletion 2q21.3 (~ 364 kb) of unknown significance. In addition to classic features of Angelman syndrome, she presented with severe infections in the first year of life, a symptom that has not been described in patients with Angelman syndrome. The 15q11.2–q13.1 deletion contains genes critical for Prader–Willi syndrome, the Angelman syndrome causing genes UBE3A and ATP10A/C, and several non-imprinted genes: GABRB3 and GABRA5 (both encoding subunits of GABA A receptor), GOLGA6L2, HERC2 and OCA2 (associated with oculocutaneous albinism II). The deletion 2q21.3 includes exons of the genes RAB3GAP1 (associated with Warburg Micro syndrome) and ZRANB3 (not disease-associated). Despite the normal phenotype of the mother, the relevance of the 2q21.3 microdeletion for the phenotype of the patient cannot be excluded, and further case reports will need to address this point.     

Catalytic DNA and RNA for Targeting MDR1 mRNA

Abstract

Design, synthesis and properties of catalytic NAs for targeting MDR1 mRNA are reported.     

Cpn20: Siamese twins of the chaperonin world

The chloroplast cpn20 protein is a functional homolog of the cpn10 co-chaperonin, but its gene consists of two cpn10-like units joined head-to-tail by a short chain of amino acids. This double protein is unique to plastids and was shown to exist in plants as well plastid-containing parasites. In vitro assays showed that this cpn20 co-chaperonin is a functional homolog of cpn10. In terms of structure, existing data indicate that the oligomer is tetrameric, yet it interacts with a heptameric cpn60 partner. Thus, the functional oligomeric structure remains a mystery. In this review, we summarize what is known about this distinctive chaperonin and use a bioinformatics approach to examine the expression of cpn20 in Arabidopsis thaliana relative to other chaperonin genes in this species. In addition, we examine the primary structure of the two homologous domains for similarities and differences, in comparison with cpn10 from other species. Lastly, we hypothesize as to the oligomeric structure and raison d’être of this unusual co-chaperonin homolog. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.     

Synthesis, characterization and luminescence property of heterobimetallic trinuclear Mo(W)-Ag-S clusters containing 1,2-bis(diphenylphosphino)-1,2-dicarba-closo-dodecaborane

Reactions of (NH₄)₂MS₄ or (NH₄)₂MOS₃ (M = Mo, W) with AgSCN and closo carborane diphosphine ligand 1,2-(PPh₂)₂-1,2-C₂B₁₀H₁₀ (L) in CH₂Cl₂ yielded four heterobimetallic trinuclear Mo(W)-Ag-S clusters: [Ag₂MoS₄L₂] (1), [Ag₂WS₄L₂] (2), [Ag₂MoOS₃L₂] (3) and [Ag₂WS₄L₂] (4), respectively. All the new clusters have been characterized by elemental analysis, FT-IR, UV-Vis, ¹H and ¹³C NMR spectroscopy and their molecular structures (except for 3) were further confirmed by single-crystal X-ray diffraction. X-ray crystal structure analysis showed that the closo carborane diphosphine ligand was coordinated bidentately to Ag(I) atom through its two phosphorus atoms, resulting in a stable five-member chelating ring between the diphosphine ligand and the metal. The coordination sphere of the central M atom, as well as all the Ag atoms, was tetrahedron. The skeletons of these clusters could be classified into two types: with (NH₄)₂MS₄, the three metal atoms (two Ag atoms and one M atom) are in a linear conformation, while with (NH₄)₂MOS₃, the conformation of the heterobimetallic trinuclear cluster is butterfly shaped. The luminescence properties of the clusters were investigated in CH₂Cl₂ solution at room temperature and for the first time the butterfly-shaped Ag-W-S cluster containing the Ag₂WS₄ core has been proved to show luminescence property.