Journal: Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology

Article Title: Rho and Rac, but not ROCK, are required for secretion of human and mouse eosinophil-associated RNases

doi: 10.1111/cea.13292

Figure Lengend Snippet: CCL11-mediated EAR secretion from eosinophils requires Rho activity. RNase activity was measured in supernatants of mCCL11-stimulated mouse (A) and hCCL11-stimulated human (B) eosinophils in the presence of Rho GTPase blocker, C3-transferase, as described in Material and Methods. Data are mean ± SD of five independent experiments (A) or mean ± SD from triplicate wells and are representative of four independent experiments (B). Asterisks represent P values < 0.05 (*), or < 0.01 (**).

Article Snippet: In some experiments, cells were pretreated for two hrs at 37°C with the Rho blocker, C3 transferase (5 and 10 μg/ml, Cytoskeleton Inc., Denver, CO, USA), or for 20 min at 37°C with the vesicle formation inhibitor, brefeldin A (10 μM, Enzo Life Sciences LTD., Exeter, UK), the ROCK inhibitor, Y27632 (30, 50 and 100 μM, Calbiochem-EMD), the Rac inhibitor, EHT 1684 (5 and 10 μM, Tocris Bioscience, Bristol, UK) and the microtubule polymerization blocker, nocodazole (10 and 100 μM, Calbiochem-EMD), before and during chemokine stimulation.

Techniques: Activity Assay

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: A novel transgenic mouse model of lysosomal storage disorder

doi: 10.1152/ajpgi.00313.2015

Figure Lengend Snippet: Mice homozygous for transgene (Tg +/+ ) exhibit the bloated phenotype

Article Snippet: To examine whether Tg -positive mice are homozygous or heterozygous, 1 ng of genomic DNA extracted from each mouse was run in a triplicate using a TaqMan assay (assay identification no. Rn00587789_m1; ABI) relative to the endogenous control gene, rps15 (assay identification no. Rn01408642_g1; ABI).

Techniques:

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: A novel transgenic mouse model of lysosomal storage disorder

doi: 10.1152/ajpgi.00313.2015

Figure Lengend Snippet: Sex distribution of the bloated phenotype

Article Snippet: To examine whether Tg -positive mice are homozygous or heterozygous, 1 ng of genomic DNA extracted from each mouse was run in a triplicate using a TaqMan assay (assay identification no. Rn00587789_m1; ABI) relative to the endogenous control gene, rps15 (assay identification no. Rn01408642_g1; ABI).

Techniques:

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: A novel transgenic mouse model of lysosomal storage disorder

doi: 10.1152/ajpgi.00313.2015

Figure Lengend Snippet: Liver histology from mice with different phenotypes. Pathology of bloated mice is independent of Cacnb2 genotype, but correlates with transgene (Tg) genotype; only Tg+/+ mice exhibit enlarged liver sinusoids filled with immune cells. Cacnb2+/+ mice with zero Tg (Tg−/−; A) or heterogeneous for Tg (Tg+/−; B) have healthy livers with long slender sinusoids (black arrows) that travel through the hepatocytes (blue arrow). In contrast, Cacnb2+/− mice (C) or Cacnb2+/+ mice (D) homozygous for Tg (Tg+/+) have distended liver sinusoids (black arrow) filled with large, occasionally multinucleated macrophages containing abundant amphophilic, foamy cytoplasmic material. Hepatocytes also contained foamy material (blue arrow). Cacnb2−/−/Tg+/− mice (E) have normal liver histology, whereas Cacnb2−/−/Tg+/+mice (F) exhibit liver pathology similar to other Tg+/+ mice (C and D).

Article Snippet: To examine whether Tg -positive mice are homozygous or heterozygous, 1 ng of genomic DNA extracted from each mouse was run in a triplicate using a TaqMan assay (assay identification no. Rn00587789_m1; ABI) relative to the endogenous control gene, rps15 (assay identification no. Rn01408642_g1; ABI).

Techniques:

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: A novel transgenic mouse model of lysosomal storage disorder

doi: 10.1152/ajpgi.00313.2015

Figure Lengend Snippet: Mass spectroscopy analysis of 10 mg of total liver lipid from mice with different genotypes. Left: example chromatographs of lipid extracted from a bloated Cacnb2+/+/Tg+/+ (gray bottom traces) mouse (#197L) and a healthy Cacnb2+/+/Tg+/− (black top traces) litter mate (#197R); chromatograms are displayed as 100% of the most abundant peak. NL, normalized intensity level. Right: summary bar graphs of lipid content per milligram liver weight for the different genotypes: total lipid (A), cholesterol (B), cholesterol esters (C), and triglycerides (D). Values are means ± SE; n = 3/group. *P ≤ 0.05. **P ≤ 0.01. ***P ≤ 0.005.

Article Snippet: To examine whether Tg -positive mice are homozygous or heterozygous, 1 ng of genomic DNA extracted from each mouse was run in a triplicate using a TaqMan assay (assay identification no. Rn00587789_m1; ABI) relative to the endogenous control gene, rps15 (assay identification no. Rn01408642_g1; ABI).

Techniques: Mass Spectrometry

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: A novel transgenic mouse model of lysosomal storage disorder

doi: 10.1152/ajpgi.00313.2015

Figure Lengend Snippet: H&E staining illustrates dramatic changes in adipocytes associated with the pancreas from Tg+/+ mice. A–D: healthy phenotypes. A: adipocytes along with lymph nodes (asterisks), associate with the pancreas of Cacnb2+/+/Tg−/− mice. B: immune cells are found at low density among the adipocytes and in small clusters (arrow) along borders of the adipose tissue. C and D: adipocytes and immune cell clusters from mice heterozygous for Tg (Tg+/−) appear similar to Tg−/− mice whether Cacnb2+/+/Tg+/− (C) or Cacnb2−/−/Tg+/− (D). E–G: mice of varying Cacnb2 genotypes homozygous for Tg (Tg+/+) exhibit extensive changes in adipose tissue. Adipocyte numbers are greatly reduced with thickened extracellular material between cells. Immune cells infiltrated the adipose tissue to such an extent that discriminating lymph vessels from adipose tissue is difficult. E and F: arrows highlight arteries, which remain associated with the adipocytes. H: three arteries and a lymph vessel (arrow) coursing through healthy adipose tissue.

Article Snippet: To examine whether Tg -positive mice are homozygous or heterozygous, 1 ng of genomic DNA extracted from each mouse was run in a triplicate using a TaqMan assay (assay identification no. Rn00587789_m1; ABI) relative to the endogenous control gene, rps15 (assay identification no. Rn01408642_g1; ABI).

Techniques: Staining

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: A novel transgenic mouse model of lysosomal storage disorder

doi: 10.1152/ajpgi.00313.2015

Figure Lengend Snippet: Electron micrographs reveal immune cell infiltration of the exocrine pancreas in Tg+/+ mice. A: healthy acinar tissue from a Cacnb2+/+/Tg−/− mouse. Note the immune cells (asterisks) in interstitial spaces. B: acinar tissue from a Cacnb2+/+/Tg+/+ sibling mouse. Note the increased number of immune cells (asterisk) with extended, thin processes, and the wider interstitial spaces. One immune cell by a capillary (c) contained large lipid droplets (arrow). Magnification, ×1,950.

Article Snippet: To examine whether Tg -positive mice are homozygous or heterozygous, 1 ng of genomic DNA extracted from each mouse was run in a triplicate using a TaqMan assay (assay identification no. Rn00587789_m1; ABI) relative to the endogenous control gene, rps15 (assay identification no. Rn01408642_g1; ABI).

Techniques:

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: A novel transgenic mouse model of lysosomal storage disorder

doi: 10.1152/ajpgi.00313.2015

Figure Lengend Snippet: Tg+/+ mice exhibit changes in protein levels. A: immunohistochemistry of α-amylase. Left: pancreas from Cacnb2+/+/Tg−/− mouse. Right: pancreas from Cacnb2+/+/Tg+/+mouse. B: quantitation of α-amylase immunostaining in pancreas of different genotypes (n = 2–3 mice/group). **P < 0.0016. C: Western blot analysis of exocrine pancreas from Cacnb2+/+/Tg−/− and Cacnb2+/+/Tg+/+ mice. Amylase, α-amylase; TRP, trypsinogen. Lanes 1–3, male Cacnb2+/+/Tg−/−; lanes 4–6, male Cacnb2+/+/Tg+/+; lanes 7–9, female Cacnb2+/+/Tg−/−; lanes 10–12, female Cacnb2+/+/Tg+/+; lanes 13–15, male Cacnb2−/−/Tg+/−; lanes 16–18, male Cacnb2−/−/Tg++. D: quantification of Western blot band densities (n = 3 mice/group). **P = 0.025. E: H&E image of bloated pancreas where islets are plentiful and appear normal. F: insulin/pancreas is significantly lower in bloated Cacnb2−/−/Tg+/+ mice compared with other healthy genotypes. *P < 0.05. **P < 0.01. G: enlargement of area within the square shown in E and stained with hemoxylin and an HRP-conjugated insulin antibody. H: this difference was also observed when insulin content per 25 islets from Cacnb2−/−/Tg+/+ mice was compared with other healthy genotypes. *P < 0.05. In healthy mice, no significant differences were observed in total insulin/pancreas or insulin content/islet, measured from 25 isolated islets. Values are means ± SE.

Article Snippet: To examine whether Tg -positive mice are homozygous or heterozygous, 1 ng of genomic DNA extracted from each mouse was run in a triplicate using a TaqMan assay (assay identification no. Rn00587789_m1; ABI) relative to the endogenous control gene, rps15 (assay identification no. Rn01408642_g1; ABI).

Techniques: Immunohistochemistry, Quantitation Assay, Immunostaining, Western Blot, Staining, Isolation