Monitoring the activation of neuronal nitric oxide synthase in brain tissue and cells with a potentiometric immunosensor

An all solid state potentiometric immunosensor (ASPI) has been developed to study the activation process of neuronal nitric oxide synthase (nNOS), the enzyme involved in the synthesis of nitric oxide generated under physiological conditions. At first, an all solid state H⁺-selective ISE was fabricated with the carboxylated poly(vinyl chloride) (PVC-COOH) film containing H⁺ ionophore, antibody was then immobilized on the polymer layer. The immunocomplex formation was detected by monitoring pH change due to interaction between urease labeled secondary antibody and antigen. Experimental parameters such as the amount of phosphorylated nNOS immobilized on the electrode surface and pH responses due to the antibody–antigen reaction were studied in detail. The calibration plot of the potentiometric potential vs. phosphorylated nNOS concentration exhibited a linear relationship in the range of 3.4–340.0 μg/ml. The calibration sensitivity of the phosphorylated nNOS immunosensor was −0.073 ± 0.002 mV/μg ml⁻¹. The detection limit of nNOS was determined to be 0.2 μg/ml based on five-time measurements (95% confidence level, k = 3, n = 5). The reliability of the immunosensor was examined with rat brain tissues as well as neuronal cells, and the results shown were good, implying a promising approach for a novel electrochemical immunosensor platform with potential applications to clinical diagnosis.     

Atomistic mechanisms of huntingtin N‐terminal fragment insertion on a phospholipid bilayer revealed by molecular dynamics simulations

The huntingtin protein is characterized by a segment of consecutive glutamines (Q_N) that is responsible for its fibrillation. As with other amyloid proteins, misfolding of huntingtin is related to Huntington's disease through pathways that can involve interactions with phospholipid membranes. Experimental results suggest that the N‐terminal 17‐amino‐acid sequence (htt^NT) positioned just before the Q_N region is important for the binding of huntingtin to membranes. Through all‐atom explicit solvent molecular dynamics simulations, we unveil the structure and dynamics of the htt^NTQ_N fragment on a phospholipid membrane at the atomic level. We observe that the insertion dynamics of this peptide can be described by four main steps—approach, reorganization, anchoring, and insertion—that are very diverse at the atomic level. On the membrane, the htt^NT peptide forms a stable α‐helix essentially parallel to the membrane with its nonpolar side‐chains—mainly Leu‐4, Leu‐7, Phe‐11 and Leu‐14—positioned in the hydrophobic core of the membrane. Salt‐bridges involving Glu‐5, Glu‐12, Lys‐6, and Lys‐15, as well as hydrogen bonds involving Thr‐3 and Ser‐13 with the phospholipids also stabilize the structure and orientation of the htt^NT peptide. These observations do not significantly change upon adding the Q_N region whose role is rather to provide, through its hydrogen bonds with the phospholipids' head group, a stable scaffold facilitating the partitioning of the htt^NT region in the membrane. Moreover, by staying accessible to the solvent, the amyloidogenic Q_N region could also play a key role for the oligomerization of htt^NTQ_N on phospholipid membranes. Proteins 2014; 82:1409–1427. © 2014 Wiley Periodicals, Inc.     

The complete mitochondrial genome of the wild silkworm moth, Actias selene

The complete mitochondrial genome (mitogenome) of Actias selene (Lepidoptera: Saturniidae) was determined to be 15,236 bp, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. The arrangement of 13 PCGs was similar to that of other sequenced lepidopterans. The AT skew of the mitogenome of A. selene was slightly negative, indicating a higher number of T compared to A nucleotides. The nucleotide composition of the mitogenome of A. selene was also biased toward A+T nucleotides (78.91%). All PCGs were initiated by ATN codons, except for the gene encoding cytochrome c oxidase subunit 1 (cox1), which may be initiated by the TTAG, as observed in other lepidopterans. Three genes, including cox1, cox2, and nad5, had incomplete stop codons consisting of just a T. With an exception for trnS1(AGN), all the other tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA. The A+T-rich region of the mitogenome of A. selene was 339 bp in length, and contains several features common to the Lepidopteras, including non-repetitive sequences, a conserved structure combining the motif ATAGA and an 18-bp poly-T stretch and a poly-A element upstream of trnM gene. Phylogenetic analysis showed that A. selene was close to Saturniidae.     

Involvement of cell proliferation in the process of follicular atresia in the guinea pig

Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs.     

Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues

Hepatocellular carcinoma (HCC) is a highly malignant tumor, and chronic infection with hepatitis B virus is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we used two-dimensional fluorescence DIGE to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 hepatitis B virus-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio >/= 2, p 相似文献   

Peroxisome-targeted and tandem repeat multimer expressions of human antimicrobial peptide LL37 in Pichia pastoris

Although the human antimicrobial peptide LL37 has a broad spectrum of antimicrobial activities, it easily damages host cells following heterologous expressions. This study attempted two strategies to alleviate its damage to host cells when expressed in Pichia pastoris using the AOX1 promoter. Tandem repeat multimers of LL37 were first designed, and secretion expression strains GS115-9K-(DPLL37DP)_n (n?=?2, 4, 6 and 8) containing different copies of the LL37 gene were constructed. However, LL37 tandems still killed the cells after 96?hr of induction. Subsequently, peroxisome-targeted expression was performed by adding a peroxisomal targeting signal 1 (SKL) at the C-terminus of LL37. The LL37 expression strain GS115-3.5K-LL37-SKL showed no significant inhibition in the cells after induction. Antibacterial activity assays showed that the recombinant LL37 expressed in peroxisomes had good antimicrobial activities. Then, a strain GS115-3.5K-LL37-GFP-SKL producing LL37, green fluorescent protein, and SKL fusion proteins was constructed, and the fusion protein was confirmed to be targeting the peroxisomes. However, protein extraction analysis indicated that most of the fusion proteins were still located in the cell debris after cell disruption, and further studies are required to extract more proteins from the peroxisome membrane.     

Rhizobium qilianshanense sp. nov., a novel species isolated from root nodule of Oxytropis ochrocephala Bunge in China

During a study of the diversity and phylogeny of rhizobia isolated from root nodules of Oxytropis ochrocephala grown in the northwest of China, four strains were classified in the genus Rhizobium on the basis of their 16S rRNA gene sequences. These strains have identical 16S rRNA gene sequences, which showed a mean similarity of 94.4 % with the most closely related species, Rhizobium oryzae. Analysis of recA and glnA sequences showed that these strains have less than 88.1 and 88.7 % similarity with the defined species of Rhizobium, respectively. The genetic diversity revealed by ERIC-PCR fingerprinting indicated that the isolates correspond to different strains. Strain CCNWQLS01^T contains Q-10 as the predominant ubiquinone. The major fatty acids were identified as feature 8 (C18: 1ω7c and/or C18: 1ω6c; 67.2 %). Therefore, a novel species Rhizobium qilianshanense sp. nov. is proposed, and CCNWQLS01^T (= ACCC 05747^T = JCM 18337^T) is designated as the type strain.