Development and characterization of an inducible Dicer conditional knockout mouse model of Parkinson's disease: validation of the antiparkinsonian effects of a sigma-1 receptor agonist and dihydromyricetin.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by motor impairment and progressive loss of dopamine (DA) neurons. At present, the acute application of neurotoxic drugs such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) are commonly used to simulate the pathology of PD; however, it is difficult to induce the progressive pathogenesis of PD with these models. In this study, we employed DAT promoter-mediated Cre transgenic mice to establish tamoxifen-inducible Dicer conditional knockout (cKO) mice in an effort to mimic the progressive loss of DA neurons and the development of PD-like behavioral phenotypes. The results showed that Dicer cKO mice exhibited progressive loss of DA neurons in the substantia nigra (SN) following tamoxifen administration. Significant DA loss was observed 6 weeks after tamoxifen administration; accordingly, progressive motor function impairment was also observed. We also found that a significant neuroinflammatory response, as evidenced by microglial proliferation, another hallmark of PD pathogenesis, accompanied the loss of DA neurons. The acute application of levo-DOPA (L-DOPA) relieved the PD-like motor impairments in Dicer cKO mice to exert its antiparkinsonian action, indicating that the model can be used to evaluate the antiparkinsonian efficacy of PD drugs. To further elucidate the potential application of this novel PD animal model for PD drug development, we employed the powerful neuroprotective agent dihydromyricetin (DHM) (10 mg/kg) and the selective sigma-1 receptor agonist PRE-084 (1 mg/kg), both of which were previously shown to produce antiparkinsonian effects. The results indicated that the chronic administration of either DHM or PRE-084 attenuated the Dicer cKO-induced loss of DA neurons and motor impairments, although the two drugs acted through different mechanisms. These data indicate that the Dicer cKO mouse model may be a useful model for investigating the pathological development of PD and intervention-mediated changes. In conclusion, this transgenic mouse model appears to simulate the progressive pathogenesis of PD and may be a potentially useful model for PD drug discovery.

Exploring the influence of drug content on DPI powder properties and potential prediction of pulmonary drug deposition.

Based on dry powder inhaler (DPI) inhalation process, powder properties have a key influence on fluidization and deagglomeration behavior during aerosol generation. The aim of this study was to explore the influence of drug content on DPI powder properties and further reveal the correlations between powder properties and pulmonary deposition efficiency. Using salbutamol sulfate as a model drug, Lactohale® 100 as carrier, carrier-based binary mixtures were prepared at drug content from 0.5 to 10% (w/w), characterized with powder rheometer, faraday cage and Next Generation Impactor. It was demonstrated that drug content had a remarkable influence on powder behavior, and good correlations between powder properties and fine particle fraction (FPF) were established in drug content range 0.5-7%. A negative correlation between basic flowability energy and FPF, reflected a good flowability is beneficial for powder fluidization. Further properties characterization, including aeration ratio, permeability, pre-shear stress and aerodynamic specific charge, suggested a strong interaction is beneficial for powder deagglomeration. It's the first time that interaction indicator and flowability indicator were extracted with principal component analysis (PCA). In conclusion, drug content has a significant influence on powder properties. DPI formulations with a stronger interaction and meanwhile a better flowability are desirable for enhanced pulmonary drug delivery.

Phospholipid-modified poly(lactide-co-glycolide) microparticles for tuning the interaction with alveolar macrophages: In vitro and in vivo assessment.

Controlled drug delivery to the lungs is promising with plentiful advantages over current rapid release products. However, alveolar macrophage clearance has severely hindered the application of inhaled controlled release preparations. The objective of our study was to explore the feasibility to decorate poly(lactide-co-glycolide) (PLGA) microparticles with endogenous phospholipids found in the deep lungs, thus, to regulate the interplay with alveolar macrophages. The influence of the phospholipid amount and type on macrophage uptake of PLGA microparticles was investigated systemically under both in vitro (RAW264.7 and NR8383) and in vivo conditions. The uptake rate (k) by macrophages, in vivo elimination rate from the bronchoalveolar lavage fluid (k') and elimination rate from the whole lung (k″) were used as parameters for evaluation. Our data showed that a modification with dipalmitoyl phosphatidylcholine (DPPC) enhanced the macrophage phagocytosis significantly over the unmodified counterparts. Thereafter, using the same modification ratio, remarkable enhancement of macrophage uptake was found in the presence of different types of other phospholipids, especially with distearoyl phosphatidylethanolamine (DSPE). When replaced by a poly(ethylene glycol)-conjugated version of DSPE the uptake of the modified PLGA microparticles was reduced by ~200%. Meanwhile, the drug content in the lung tissue was improved by 3-fold (area under the curve value). Finally, it was possible to establish a correlation between in vitro phagocytosis and in vivo lung elimination rate for the investigated formulations. Overall, our study demonstrated that phospholipids play an important role in modulating the clearance of microparticle-based drug delivery vehicles, which gives a meaningful insight into the development of prolonged drug release system for inhalation.

Selective p300 inhibitor C646 inhibited HPV E6-E7 genes, altered glucose metabolism and induced apoptosis in cervical cancer cells.

High risk HPV infection is a causative factor of cervical cancer. The constitutive expression of HPV E6-E7 genes is important for the maintenance of cancer phenotypes. The cellular transcription co-activator p300 plays a crucial role in the regulation of HPV genes thus it was targeted for the inhibition of HPV-associated cervical cancer. In the present study, HPV positive cervical cells were treated with C646, a selective inhibitor of p300, to investigate its influence on HPV E6-E7 expression and cancer cell growth. Results of RT-qPCR, Western-blot and promoter activity assays showed that C646 inhibited the transcription of HPV E6-E7, which was accompanied with the accumulation of p53 protein. Meanwhile, cell proliferation was suppressed, glucose metabolism was disrupted and apoptosis was induced via the intrinsic pathway. Generally, the anti-cervical cancer potential of C646 was demonstrated and a novel mechanism was proposed in this study.

Lack of Cul4b, an E3 ubiquitin ligase component, leads to embryonic lethality and abnormal placental development.

Cullin-RING ligases (CRLs) complexes participate in the regulation of diverse cellular processes, including cell cycle progression, transcription, signal transduction and development. Serving as the scaffold protein, cullins are crucial for the assembly of ligase complexes, which recognize and target various substrates for proteosomal degradation. Mutations in human CUL4B, one of the eight members in cullin family, are one of the major causes of X-linked mental retardation. We here report the generation and characterization of Cul4b knockout mice, in which exons 3 to 5 were deleted. In contrast to the survival to adulthood of human hemizygous males with CUL4B null mutation, Cul4b null mouse embryos show severe developmental arrest and usually die before embryonic day 9.5 (E9.5). Accumulation of cyclin E, a CRL (CUL4B) substrate, was observed in Cul4b null embryos. Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay. The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay. As in human CUL4B heterozygotes, Cul4b null cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues. Together, our results showed that CUL4B is indispensable for embryonic development in the mouse.

Comparative processing and function of human and ferret cystic fibrosis transmembrane conductance regulator.

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTR(ΔF508/ΔF508) animal model may be useful.

Eight-alanine duplication in homeobox D13 in a Chinese family with synpolydactyly.

Human synpolydactyly (SPD), belonging to syndactyly (SD) II, is an inherited autosomal-dominant limb malformation characterized by SD of finger 3 or 4 or toe 4 or 5, usually with digit duplication. Previous studies have demonstrated that homeobox protein D13 (HOXD13) is responsible for this Mendelian disorder. In this paper, we report on a family with SPD - 7 members show typical SPD malformations. We used PCR and Sanger sequencing of DNA from peripheral blood samples and found an 8-Ala expansion in exon 1 of HOXD13 by mutation detection; this variant was absent in unaffected members and in 50 unaffected non-related subjects. This study further confirmed the correlation between SPD and alanine expansion in HOXD13.

Resveratrol modulates angiogenesis through the GSK3ß/ß-catenin/TCF-dependent pathway in human endothelial cells.

Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis due to its potent and specific ability to promote the proliferation and migration of endothelial cells. Resveratrol has been shown to have many health-benefiting effects, including the protection of cardiovascular system. In this study we examined the effect of resveratrol on angiogenesis in human umbilical vein endothelial cells (HUVECs). We observed that resveratrol was able to modulate the expression of VEGF and the formation of vascular network in a biphasic pattern. While resveratrol at low concentrations, from 1 to 10µM, up-regulated the expression of VEGF and promoted angiogenesis, it had opposite effect at high concentrations (20µM and higher). The biphasic effect of resveratrol on angiogenesis was confirmed by chick chorioallantoic membrane assay. Up-regulation of VEGF expression depended on the nuclear accumulation and transcriptional activity of ß-catenin. Correspondingly, GSK3ß, a negative regulator of ß-catenin, turned into a less active state (phosphorylated at Ser9) in cells exposed to 5µM of resveratrol, but became more active at 20µM. We demonstrated that both Akt and ERK signaling pathways, which are known to be critical for angiogenesis, became activated in response to 5µM of resveratrol and functioned to inactivate GSK3ß. Our findings may have implications in the management of cardiovascular diseases and other conditions such as cancer by the use of resveratrol.

Characterization of nuclear localization signal in the N terminus of CUL4B and its essential role in cyclin E degradation and cell cycle progression.

CUL4A and CUL4B, which are derived from the same ancestor, CUL4, encode scaffold proteins that organize cullin-RING ubiquitin ligase (E3) complexes. Recent genetic studies have shown that germ line mutation in CUL4B can cause mental retardation, short stature, and other abnormalities in humans. CUL4A was observed to be overexpressed in breast and hepatocellular cancers, although no germ line mutation in human CUL4A has been reported. Although CUL4A has been known to be involved in a number of cellular processes, including DNA repair and cell cycle regulation, little is known about whether CUL4B has similar functions. In this report, we tested the functional importance of CUL4B in cell proliferation and characterized the nuclear localization signal (NLS) that is essential for its function. We found that RNA interference silencing of CUL4B led to an inhibition of cell proliferation and a prolonged S phase, due to the overaccumulation of cyclin E, a substrate targeted by CUL4B for ubiquitination. We showed that, unlike CUL4A and other cullins that carry their NLS in their C termini, NLS in CUL4B is located in its N terminus, between amino acid 37 and 40, KKRK. This NLS could bind to importin alpha1, alpha3, and alpha5. NLS-deleted CUL4B was distributed in cytoplasm and failed to promote cell proliferation. Therefore, the nuclear localization of CUL4B mediated by NLS is critical for its normal function in cell proliferation.

RETRACTED: Resveratrol augments the canonical Wnt signaling pathway in promoting osteoblastic differentiation of multipotent mesenchymal cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the authors. The authors were alerted about the loading control in a Western blot analysis of ERK phosphorylation shown in Figure 6B, and they were unable to locate the original scan. Although the authors stand by the conclusion based on this figure, and the conclusion of the entire article, they wish to retract this article.

Analysis of adeno-associated virus progenitor cell transduction in mouse lung.

Although recombinant adeno-associated virus (rAAV) has been widely used in lung gene therapy approaches, it remains unclear to what extent commonly used AAV serotypes transduce adult progenitors in the lung. In this study, we evaluated the life span and proliferative capacity of rAAV1-, 2-, and 5-transduced airway cells in mouse lung, using a LacZ-CRE reporter transgenic model and Cre-expressing rAAV. In this model, the expression of CRE recombinase led to permanent genetic marking of transduced cells and their descendants with LacZ. To investigate whether the rAAV-transduced cells included airway progenitors, we injured the airways of rAAV-infected mice with Naphthalene, while simultaneously labeling with 5-bromodeoxyuridine (BrdU) to identify slow-cycling progenitor/stem cells that entered the cell cycle and retained label. Both rAAV5 and rAAV1 vectors were capable of transducing a subset of long-lived Clara cells and alveolar type II (ATII) cells that retained nucleotide label and proliferated following lung injury. Importantly, rAAV1 and 5 appeared to preferentially transduce conducting airway epithelial progenitors that had the capacity to clonally expand, both in culture and in vivo following lung injury. These studies suggest that rAAV may be a useful vector for gene targeting of airway stem/progenitor cells.

Matrix metalloproteinase-9/tissue inhibitors of metalloproteinase-1 expression and atrial structural remodeling in a dog model of atrial fibrillation: inhibition with angiotensin-converting enzyme.

INTRODUCTION: Matrix metalloproteinases and tissue inhibitors of metalloproteinases regulate extracellular matrix turnover in cardiac tissues. However, alteration of matrix metalloproteinases and tissue inhibitors of metalloproteinases during atrial fibrillation is unclear. This study aims to determine (a) the relationship between altered expressions of matrix metalloproteinases and tissue inhibitors of metalloproteinases and atrial structural remodeling; (b) the role of changes in the atrial angiotensin system and in calcium concentration; and (c) the effect of captopril on the expressions of matrix metalloproteinase-9/tissue inhibitors of metalloproteinase-1 and atrial structural remodeling. METHODS: In left atrial tissue samples, the mRNA expression of angiotensin-converting enzyme, matrix metalloproteinase-9, and tissue inhibitors of metalloproteinase-1; the protein expression of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinase-1; and Ca(2+) concentration and angiotensin II were measured. RESULTS: Compared with controls, dogs under atrial fibrillation showed significantly increased contents of Ca(2+), angiotensin II , and interstitial fibrous tissue (P<.05-.001). The mRNA levels of angiotensin-converting enzyme, matrix metalloproteinase-9, and tissue inhibitors of metalloproteinase-1 were significantly increased as compared with controls (P<.05-.01). The protein level of matrix metalloproteinase-9 was higher, and that of tissue inhibitors of metalloproteinase-1 was lower, in dogs with atrial fibrillation than in controls (P<.01-.001). All findings highlighted above were reversed by treatment with captopril. CONCLUSIONS: Altered expression of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinase-1 contributes to atrial extracellular matrix remodeling and atrial dilatation. Angiotensin-II-mediated intracellular Ca(2+) overload may be the mechanism of altered expression of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinase-1. Angiotensin-converting enzyme inhibitor treatment may attenuate atrial structural remodeling by normalizing the balance between matrix metalloproteinase-9 and tissue inhibitors of metalloproteinase-1.

Mutation in CUL4B, which encodes a member of cullin-RING ubiquitin ligase complex, causes X-linked mental retardation.

We reevaluated a previously reported family with an X-linked mental retardation syndrome and attempted to identify the underlying genetic defect. Screening of candidate genes in a 10-Mb region on Xq25 implicated CUL4B as the causative gene. CUL4B encodes a scaffold protein that organizes a cullin-RING (really interesting new gene) ubiquitin ligase (E3) complex in ubiquitylation. A base substitution, c.1564C-->T, converted a codon for arginine into a premature termination codon, p.R388X, and rendered the truncated peptide completely devoid of the C-terminal catalytic domain. The nonsense mutation also results in nonsense-mediated mRNA decay in patients. In peripheral leukocytes of obligate carriers, a strong selection against cells expressing the mutant allele results in an extremely skewed X-chromosome inactivation pattern. Our findings point to the functional significance of CUL4B in cognition and in other aspects of human development.

[Effects of matrix metalloproteinase-9 and tissue inhibitor-1 of metalloproteinase expression on atrial structural remodeling during chronic atrial fibrillation].

OBJECTIVE: To investigate the matrix metalloproteinase-9 (MMP-9) and tissue inhibitor-1 of metalloproteinase (TIMP-1) mRNA and protein expression in chronic fibrillating human atria and to evaluate the influence of MMP-9 and TIMP-1 expression on the progress of atrial structural remodeling. METHODS: Twenty-four patients with chronic atrial fibrillation (AF) and 12 patients with sinus rhythm as control group underwent transthoracic echocardiography and left atrial appendage (LAA) tissue samples were obtained from these patients during mitral/aortic valve replacement operation. MMP-9 and TIMP-1 protein expressions were detected by immunohistochemistry and their mRNA expressions were determined by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The left atrial and right atrial diameters increased significantly in the fibrillation group in comparison with the control group (57 +/- 6 vs 45 +/- 7, 62 +/- 10 vs 51 +/- 17, P < 0.05 approximately 0.001) The expressions of MMP-9 mRNA and protein in the LAA tissue of the AF group is upregulated (0.70 +/- 0.12 vs 0.53 +/- 0.22, and 2.25 +/- 0.73 vs 1.12 +/- 0.58, P < 0.05 approximately 0.001) and the expressions of TIMP-1 mRNA and protein were downregulated significantly (0.20 +/- 0.07 vs 0.31 +/- 0.15, and 1.12 +/- 0.48 vs 1.75 +/- 0.46, P < 0.05 approximately 0.01). The MMP-9 mRNA level was positively correlated with AF duration and the left atrial diameter (P < 0.05 approximately 0.001). CONCLUSION: There is a selective downregulation of TIMP-1 expression along with the upregulation of MMP-9 in AF, which indicates that the disturbance expression of MMP/TIMP system may promote the process of atrial structural remodeling. Enhanced MMP-9 activity may be a molecular mechanism contributing to the dilation of fibrillating human atria.

[Genetic polymorphism of 5 short tandem repeat loci in Hui population in Ningxia area].

OBJECTIVE: To analyze the genetic polymorphism of 5 short tandem repeat(STR) loci in Hui population in Ningxia area. METHODS: The genetic polymorphisms of five selected STR loci(D11S1984, D14S306, D14S617, D17S1290 and D19S433) in chromosomes 11, 14, 17 and 19 in 144 unrelated individuals in Hui population in Ningxia area were analyzed by PCR amplification, denaturing polyacrylamide gel electrophoresis(PAGE) and silver staining. RESULTS: 10, 8, 11, 13 and 8 alleles, 30, 25, 33, 40 and 23 genotypes of the 5 STR loci in Hui population in Ningxia were detected. The measured values of the heterozygosity of the 5 STR loci were 0.8413, 0.8033, 0.8331, 0.8369 and 0.7703; of the polymorphism information content were 0.8217, 0.7746, 0.8121, 0.8174 and 0.7332; of the discrimination power (DP) were 0.9516, 0.9257, 0.9611, 0.9660 and 0.9135. The calculated discrimination power was 0.9999995. The measured values of paternity exclusion were 0.7046, 0.6367, 0.6911, 0.7012 and 0.5801; the calculated paternity exclusion was 0.9958. The genotype distributions were in accordance with Hardy-Weinberg equilibrium. CONCLUSION: The 5 STR loci have better polymorphism in Hui population in the Ningxia area, and thus could serve as useful markers for population genetics research and for individual identification and paternity test in forensic medicine.

A locus for nonspecific X-linked mental retardation mapped to a 22.3 cM region of Xp11.3-q22.3.

By using several microsatellite markers scattered along the X chromosome, we studied a Chinese family with nonspecific X-linked mental retardation (MRX84) to search for a region including the MRX84 locus that was linked to the markers. Two-point linkage analysis demonstrated linkage between the disorder and several markers located at Xq22.2, with maximum LOD score Z(max) = 2.41 at recombination fraction theta = 0 for DXS1191 and DXS1230, respectively. Recombination events were observed with flanking markers DXS8080 and DXS456, located at Xp11.3 and Xq22.3, respectively, and a region of approximately 22.3 cM was defined. Accordingly, markers distal to Xp11.3 and Xq22.3 segregated independently of the disease. The localized region observed in this Chinese family overlaps with 29 other MRX loci previously reported in Xp11.3-q22.3. These results should contribute to the identification of the disease gene for the MRX84 disorder.

A novel mutation of keratin 9 in epidermolytic palmoplantar keratoderma combined with knuckle pads.

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominantly inherited disease. We studied a family from Shandong, China, having patients suffering from EPPK with a unique symptom-knuckle pads. We noticed that both the hyperkeratosis and knuckle pads in the Chinese family were friction-related. Candidate gene analysis was carried out using linkage analysis and direct sequencing. A novel L160F mutation in keratin 9 was found, and its effects on the secondary structure of keratin 9 were studied. We predict that the L160F mutation is also responsible for the knuckle pads in the family. Our study provides a new clue for the study of the function of keratin 9.

[Gene mapping of a nonsyndromic hearing impairmint family].

OBJECTIVE: To map the gene responsible for nonsyndromic hearing impairment in a consanguineous family. METHODS: Firstly, X chromosome scanning was used to exclude X chromosome. Secondly, candidate gene analyzing and genome scanning were performed by homozygosity mapping. Then, additional markers flanking the tightly linked marker were tested to confirm linkage and decide the candidate region. RESULTS: The nonsyndromic hearing impairment of this family was autosomal recessive. Twenty-five known genes were excluded. Autosomal genome scanning indicated that D17S1293 was tightly linked with disease gene. And further study mapped the disease gene to a 5.07 cM interval bounded by D17S1850 and D17S1818. CONCLUSION: The disease gene of the family is mapped to a 5.07 cM interval between D17S1850 and D17S1818, which is a new locus of autosomal recessive nonsyndromic hearing impairment.

[Application of homozygosity mapping to the fine mapping of the osteoporosis-pseudoglioma syndrome locus].

OBJECTIVE: To evaluate the role of homozygosity mapping in the fine mapping of the genes responsible for the rare autosomal recessive diseases. METHODS: Polymerase chain reaction-single sequence length polymorphism was used to genotype the family members from 8 families with osteoporosis-pseudoglioma syndrome(OPS) for 14 polymorphic loci within candidate region. The OPS candidate region was narrowed by searching for homozygous region in affected. RESULTS: The OPS candidate region was narrowed to a 1 cM interval between D11S1296 and D11S4136. CONCLUSION: Homozygosity mapping is a powerful method for mapping and narrowing the candidate region of the genes responsible for the rare autosomal recessive diseases.