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1.  Simultaneous monitoring of cerebral metal accumulation in an experimental model of Wilson’s disease by laser ablation inductively coupled plasma mass spectrometry 
BMC Neuroscience  2014;15(1):98.
Background
Neuropsychiatric affection involving extrapyramidal symptoms is a frequent component of Wilson’s disease (WD). WD is caused by a genetic defect of the copper (Cu) efflux pump ATPase7B. Mouse strains with natural or engineered transgenic defects of the Atp7b gene have served as model of WD. These show a gradual accumulation and concentration of Cu in liver, kidneys, and brain. However, still little is known about the regional distribution of Cu inside the brain, its influence on other metals and subsequent pathophysiological mechanisms. We have applied laser ablation inductively coupled plasma mass spectrometry and performed comparative metal bio-imaging in brain sections of wild type and Atp7b null mice in the age range of 11–24 months. Messenger RNA and protein expression of a panel of inflammatory markers were assessed using RT-PCR and Western blots of brain homogenates.
Results
We could confirm Cu accumulation in brain parenchyma by a factor of two in WD (5.5 μg g−1 in the cortex) vs. controls (2.7 μg g−1) that was already fully established at 11 months. In the periventricular regions (PVR) known as structures of prominent Cu content, Cu was reduced in turn by a factor of 3. This corroborates the view of the PVR as efflux compartments with active transport of Cu into the cerebrospinal fluid. Furthermore, the gradient of Cu increasing downstream the PVR was relieved. Otherwise the architecture of Cu distribution was essentially maintained. Zinc (Zn) was increased by up to 40% especially in regions of high Cu but not in typical Zn accumulator regions, a side effect due to the fact that Zn is to some degree a substrate of Cu-ATPases. The concentrations of iron (Fe) and manganese (Mn) were constant throughout all regions assessed. Inflammatory markers TNF-α, TIMP-1 and the capillary proliferation marker α-SMA were increased by a factor of 2–3 in WD.
Conclusions
This study confirmed stable cerebral Cu accumulation in parenchyma and discovered reduced Cu in cerebrospinal fluid in Atp7b null mice underlining the diagnostic value of micro-local analytical techniques.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2202-15-98) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2202-15-98
PMCID: PMC4156608  PMID: 25142911
Wilson’s disease; Bio-imaging; LA-ICP-MS; Copper; ATPase7B
2.  Targeted disruption of the mouse Csrp2 gene encoding the cysteine- and glycine-rich LIM domain protein CRP2 result in subtle alteration of cardiac ultrastructure 
Background
The cysteine and glycine rich protein 2 (CRP2) encoded by the Csrp2 gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells. It exhibits a bimodal subcellular distribution, accumulating at actin-based filaments in the cytosol and in the nucleus. In order to analyze the function of CRP2 in vivo, we disrupted the Csrp2 gene in mice and analysed the resulting phenotype.
Results
A ~17.3 kbp fragment of the murine Csrp2 gene containing exon 3 through 6 was isolated. Using this construct we confirmed the recently determined chromosomal localization (Chromosome 10, best fit location between markers D10Mit203 proximal and D10Mit150 central). A gene disruption cassette was cloned into exon 4 and a mouse strain lacking functional Csrp2 was generated. Mice lacking CRP2 are viable and fertile and have no obvious deficits in reproduction and survival. However, detailed histological and electron microscopic studies reveal that CRP2-deficient mice have subtle alterations in their cardiac ultrastructure. In these mice, the cardiomyocytes display a slight increase in their thickness, indicating moderate hypertrophy at the cellular level. Although the expression of several intercalated disc-associated proteins such as β-catenin, N-RAP and connexin-43 were not affected in these mice, the distribution of respective proteins was changed within heart tissue.
Conclusion
We conclude that the lack of CRP2 is associated with alterations in cardiomyocyte thickness and hypertrophy.
doi:10.1186/1471-213X-8-80
PMCID: PMC2529283  PMID: 18713466
3.  Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts 
BMC Cell Biology  2000;1:4.
Background
The hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer.
Results
With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE™6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively.
Conclusions
Our results indicate that FuGENE™6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells.
doi:10.1186/1471-2121-1-4
PMCID: PMC29065  PMID: 11178102

Results 1-3 (3)