Remodeling of the human papillomavirus type 11 replication origin into discrete nucleoprotein particles and looped structures by the E2 protein

The human papillomavirus (HPV) DNA replication origin (ori) shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundreds of base pairs. The HPV type 11 ori spans 103 bp and contains three palindromic E2 binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 ori binding protein. These sites are separated by 64 and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 DNA ori bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound, indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies on the dependence of looping on salt, ATP, and DTT using full-length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N-terminal domain and factors that may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.     

Leishmania infantum: prime boost vaccination with C-terminal extension of cysteine proteinase type I displays both type 1 and 2 immune signatures in BALB/c mice

The aims of current study are to describe the immunogenicity and protective efficacy of prime boost vaccine using C-terminal extension (CTE) of cysteine proteinase type I of Leishmania infantum in BALB/c mice. Group I as vaccinated group primed with 100 microg of pcDNA-CTE and 3 weeks later boosted with combination of 30 microg rCTE, 50 microg of CpG and Montanide 720. Groups II and III were served as control groups. Although, this vaccination regimen did not protect mice against the infectious challenge but it was highly immunogenic. IgG2a has been raised strongly against rCTE in contrast to IgG1 and remains high at every time point under study. By analysis of CTE synthetic peptides (CTE100) before challenge, both IgG1 and IgG2a were produced and for all overlapping synthetic peptides (CTE 1-8) IgG1 raised significantly. This statue is changed at 7 weeks after challenge and only CTE2 and CTE3 have shown to induce considerable amount of IgG1. In all groups, the level of IL-5 started to increase with high concentration shortly passing only 3 weeks after infectious challenge. In compare with two control groups, the vaccinated group produced significantly higher level of IL-5 at 7 weeks post-infection. The parasite burden of all groups is similar at 4 weeks post-challenge in both liver and spleen. In contrast, at 8 weeks post challenge, the spleen of the vaccinated group showed significantly higher level of parasite load in compare with two control groups. This study demonstrated that immunization with CTE display both type 1 and 2 immune signatures in experimental murine model of L. infantum infection.     

Mitochondrial DNA heteroplasmy in calves cloned by using adult somatic cell

Adult somatic cell cloned calves were produced by somatic cell nuclear transfer prepared by fusion of cultured ear fibroblast from a Holstein cow into enucleated oocytes of Luxi Yellow cow. In order to determinate the source of mitochondrial DNA of cloned calves, we designed the breed-specific PCR primers by aligning the known D-loop sequences of Bos taurus and analyzed the displacement loop sequences of five live cloned calves by breed-specific primers PCR. The results demonstrated that mtDNA originated from Holstein breed and that from Luxi breed co-exist in all five live calves.     

The UL2 Open Reading Frame of Bovine Herpesvirus 1 Encodes a Uracil-DNA Glycosylase

Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence. To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence downstream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl β-D -thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV-1 UL2 gene does encode a UDGase.     

The role of tropomyosin domains in cooperative activation of the actin-myosin interaction

To establish α-tropomyosin (Tm)'s structure–function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, and wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm–actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasi-equivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca²⁺. Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm–actin interaction cooperatively affects actin to result in generation of greater force and velocity.     

Glutamate antagonism fails to reverse mitochondrial dysfunction in late phase of experimental neonatal asphyxia in rats

Because estrogen plays important neurotrophic and neuroprotective roles in the brain by activating estrogen receptors (ERs), disruption of normal estrogen signaling can leave neurons vulnerable to a variety of insults, including β-amyloid peptide (Aβ). Aroclor1254 (A1254) belongs to the endocrine-disrupting chemical (EDC) polychlorinated biphenyls and has anti-estrogenic properties. In the present study, we evaluated the effect of A1254 on the protective activity of estrogen against Aβ toxicity in differentiated cholinergic SN56 cells. Aged Aβ25-35 causes apoptotic cell death in differentiated SN56 cells, and the cytotoxic evidences are effectively rescued by estrogen. We found that A1254 abolishes the neuroprotective activity of estrogen against Aβ toxicity, and attenuates the suppressive effect of estrogen on Aβ-induced tau phosphorylation and JNK activation. The effects of A1254 on the neuroprotective effects of estrogen in Aβ toxicity are very similar to the effects of the estrogen receptor antagonist ICI182,780. Thus, exposure to EDCs that have anti-estrogenic activity might interfere with normal estrogen-activated neuroprotective signaling events and leave neurons more vulnerable to dangerous stimuli. Our present results provide new understanding of the mechanisms contributing to the harmful effects of EDCs on the function and viability of neurons, and the possible relevance of EDCs in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease.     

利用单链构象多态性检测牛白细胞黏附缺陷症方法的建立

牛白细胞黏附缺陷症(BLAD)是由常染色体上CD18基因单碱基突变(A→G)引起的隐性遗传疾病,隐性基因纯合时导致白细胞表面的β2整合素表达明显减少或缺乏而引起临床发病,患病牛的主要特征是机体免疫力降低、易患病从而影响其生产性能的表现,严重影响了奶牛场的经济效益。该工作利用自行设计的特异性PCR引物,通过对PCR、PCR-SSCP条件的筛选和优化,并结合DNA序列测定,建立了PCR-SSCP检测BLAD 有害基因的新方法,该方法简便快捷、准确率高,使用样品宽泛,适合大样本筛选,为奶牛BLAD有害基因的分型和筛选提供了新的方法和思路,为我国奶牛的分子选育提供了可靠的理论依据。     

Sphingosine 1-phosphate receptors modulate intracellular Ca2+ homeostasis

Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca2+ homeostasis in HeLa cells. Overexpression of S1P2, or S1P3, but not S1P1, leads to a significant increase in cytosolic and mitochondrial [Ca2+] in response to S1P challenge. Moreover, cells ectopically expressing S1P2, or S1P3 exhibited an appreciable decrease of the free Ca2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompanied by a reduced susceptibility to C2-ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca2+ homeostasis and its possible implication in the regulation of cell survival.     

RhoA-ROCK-Myosin pathway regulates morphological plasticity of cultured olfactory ensheathing cells

Olfactory ensheathing cells (OECs) are glial cells in the olfactory system with morphological and functional plasticity. Cultured OECs have the flattened and process-bearing shape. Reversible changes have been found between these two morphological phenotypes. However, the molecular mechanism underlying the regulation of their morphological plasticity remains elusive. Using RhoA FRET biosensor, we found that the active RhoA signal mainly distributed in the lamellipodia and/or filopodia of OECs. Local disruption of these active RhoA distributions led to the morphological change from the flattened into process-bearing shape and promoted process outgrowth. Furthermore, RhoA pathway inhibitors, Toxin-B, C3, Y-27632 or over-expression of DN-RhoA blocked serum-induced morphological change of OECs from the process-bearing into flattened shape, whereas the activation of RhoA pathway by lysophosphatidic acid (LPA) promoted the morphological change from the process-bearing into flattened shape. Finally, ROCK–Myosin–F-actin as a downstream of RhoA pathway was involved in morphological plasticity of OECs. Taken together, these results suggest that RhoA–ROCK–Myosin pathway mediates the morphological plasticity of cultured OECs in response to extracellular cues.