Length of metacarpal and metatarsal bones in five Iranian sheep breeds and their associations with ungula measurements

peer-reviewedBackground This study aimed to measure the length of metacarpal and metatarsal bones in five Iranian sheep breeds and to correlate the length of the bones with ungula measurements. Thoracic and pelvic limbs of 2-year-old, previously untrimmed, pastured Afshari, Moghani, Kurdi, Makoui, and Lori–Bakhtiari ewes, (n = 20 ewes per breed) were collected after slaughter. The following lengths were recorded in the metacarpal and metatarsal bones: from the margo proximalis lateralis to the lateral (L1) and medial (D1) cartilago physialis; from the margo proximalis lateralis to the margo abaxialis of the lateral (L2) and medial (D2) caput; from the cartilago physialis lateralis to the margo abaxialis of the lateral caput (X1); from the cartilago physialis medialis to the margo distalis of the caput ridge (X2) and from the margo axialis of cartilago physialis to the margo axialis of the lateral caput (X3). Additionally, measurements of the ungula including pars dorsalis length, pars mobilis lateralis and medialis height, pars dorsalis height to the ground and to the solea cornea, thickness of the solea in the pars dorsalis, pars mobilis lateralis and medialis, solea cornea length and angulus dorsalis were recorded in the medial and lateral digits of the thoracic and pelvic limbs. Data on length of the metatarsal and metacarpal bones were analysed using mixed model equations while Pearson correlations were calculated between metacarpal and metatarsal bones and ungula measurements. Results Lori- Bakhtiari and Moghani ewes had greater L1, L2, and D1 and D2 while X1, X2 and X3 was greater in Kurdi ewes (P  0.05). Low to moderate correlations were observed between bone and ungula measurements (P < 0.05). Conclusion Under the conditions of this study, differences in metacarpal and metatarsal bone measurements were observed between breeds but no asymmetry was observed between lateral and medial bones. Results indicate an association between metacarpal and metatarsal bones ungula measurements. This could provide baseline information for the development and/or improvement of current ungula health protocols in the studied sheep breeds

Knockout or Knock-in? A Truncated D2 Receptor Protein Is Expressed in the Brain of Functional D2 Receptor Knockout Mice

Null mice for the dopamine D2 receptor (D2R) have been instrumental in understanding the function of this protein. For our research, we obtained the functional D2R knockout mouse strain described initially in 1997. Surprisingly, our biochemical characterization showed that this mouse strain is not a true knockout. We determined by sequence analysis of the rapid 3′ amplification of cDNA ends that functional D2R knockout mice express transcripts that lack only the eighth exon. Furthermore, immunofluorescence assays showed a D2R-like protein in the brain of functional D2R knockout mice. We verified by immunofluorescence that the recombinant truncated D2R is expressed in HEK293T cells, showing intracellular localization, colocalizing in the Golgi apparatus and the endoplasmic reticulum, but with less presence in the Golgi apparatus compared to the native D2R. As previously reported, functional D2R knockout mice are hypoactive and insensitive to the D2R agonist quinpirole. Concordantly, microdialysis studies confirmed that functional D2R knockout mice have lower extracellular dopamine levels in the striatum than the native mice. In conclusion, functional D2R knockout mice express transcripts that lead to a truncated D2R protein lacking from the sixth transmembrane domain to the C-terminus. We share these findings to avoid future confusion and the community considers this mouse strain in D2R traffic and protein–protein interaction studies

Characterizing HSF1 Binding and Post-Translational Modifications of hsp70 Promoter in Cultured Cortical Neurons: Implications in the Heat-Shock Response.

Causes of lower induction of Hsp70 in neurons during heat shock are still a matter of debate. To further inquire into the mechanisms regulating Hsp70 expression in neurons, we studied the activity of Heat Shock Factor 1 (HSF1) and histone posttranslational modifications (PTMs) at the hsp70 promoter in rat cortical neurons. Heat shock induced a transient and efficient translocation of HSF1 to neuronal nuclei. However, no binding of HSF1 at the hsp70 promoter was detected while it bound to the hsp25 promoter in cortical neurons during heat shock. Histone PTMs analysis showed that the hsp70 promoter harbors lower levels of histone H3 and H4 acetylation in cortical neurons compared to PC12 cells under basal conditions. Transcriptomic profiling data analysis showed a predominant usage of cryptic transcriptional start sites at hsp70 gene in the rat cerebral cortex, compared with the whole brain. These data support a weaker activation of hsp70 canonical promoter. Heat shock increased H3Ac at the hsp70 promoter in PC12 cells, which correlated with increased Hsp70 expression while no modifications occurred at the hsp70 promoter in cortical neurons. Increased histone H3 acetylation by Trichostatin A led to hsp70 mRNA and protein induction in cortical neurons. In conclusion, we found that two independent mechanisms maintain a lower induction of Hsp70 in cortical neurons. First, HSF1 fails to bind specifically to the hsp70 promoter in cortical neurons during heat shock and, second, the hsp70 promoter is less accessible in neurons compared to non-neuronal cells due to histone deacetylases repression

Effect of heat shock on histone PTMs of the rat <i>hsp70</i> promoter in PC12 cells and cortical neurons.

<p>Comparison of four histone PTM levels: H3Ac (A) and H4Ac (B), H3K4me2 (C) and H3K4me3 (D), at the <i>hsp70</i> gene promoter under control (white bars) and heat shock (black bars) conditions, in cortical neurons and PC12 cells. Cells were heat shocked at 42°C for 1h or kept unstressed. Data is expressed as fold of enrichment over basal condition (unstressed) and correspond to the mean plus SEM of three independent experiments. Statistical analyses were performed by Two-way ANOVA followed by Sidak's multiple comparisons test: *p < 0.05 compared to control.</p

Augmented H3Ac mediated by HDACs inhibition is associated with increased expression of Hsp70 in cortical neurons.

<p>(A) Fold Induction of hsp70 (black circles) and hsp25 (grey squares) transcripts in cortical neurons by different doses of TSA. Data are expressed as mean plus SEM of at least three independent experiments. Statistical analyses were performed by best fit Linear Regression and Spearman’s r correlation analysis where *p < 0.05 for hsp70 mRNA. (B) Comparison of histone acetylation of H3 and H4 at the <i>hsp70</i> gene promoter in cortical neurons under control, heat shock (42°C, 1h) or TSA (50 nM, 15h) treatment. Data is expressed as the percentage of the immunoprecipitated DNA in relation to the input and correspond to the mean plus SEM of at least three independent experiments. Statistical analyses were performed by Two-way ANOVA followed by Tukey's multiple comparisons test: *p < 0.05 compared to control.</p

Neuronal cells show weaker induction of Hsp70 in response to heat shock.

<p>(A) Upper panels: Representative immunoblots of Hsp70 protein in undifferentiated (-NGF) and differentiated (+NGF for 7d) PC12 cells, and cortical neurons (E18.5, 7div) subjected to heat shock (42°C, 2h) or kept under control conditions (C, 37°C). Lower panel: Quantification of the relative Hsp70 protein levels showed in the upper panels. Tubulin and GAPDH were used as loading controls. Statistical analyses were performed by Two-way ANOVA followed by Tukey’s multiple comparisons test. ****p < 0.0001, *p < 0.05 compared with control; ###p <0.001, compared to Hsp70 fold induction in undifferentiated PC12 cells during heat shock. (B, C) Relative abundance of hsp70 and hsp25 transcripts in PC12 cells and cortical neurons. hsp70 and hsp25 mRNA levels were calculated comparing the abundance of each cDNA in cells under control and heat shock conditions. For each sample, cyclophilin mRNA was used as a reference gene. Data are expressed as mean plus SEM of at least three independent experiments. Statistical analyses were performed by Two-way ANOVA followed by Tukey's multiple comparisons test. ****p < 0.0001, *p < 0.05 compared with control; ##p < 0.01, ###p <0.001, compared to Hsp70 mRNA abundance in undifferentiated PC12 cells during heat shock.</p

Analysis of the chromatin environment of rat promoter regions of <i>hsp70</i> and <i>hsp25</i> genes.

<p>H3Ac, H4Ac, H3K4me2 and H3K4me3 were analyzed in the proximal promoter region of the <i>hsp70</i> and <i>hsp25</i> rat genes in undifferentiated PC12 cells (A) and cortical neurons (B), under basal conditions. The graphs show the data from the ChIP assays and correspond to the mean plus SEM of the percentage of DNA immunoprecipitated by each antibody (% of the Input), normalized by the percentage of DNA immunoprecipated by anti-H3 antibody (Relative to Nucleosome Density). Statistical analyses were performed by Two-way ANOVA followed by Bonferroni post hoc multiple comparison test. **p < 0.01. (C) Comparative analysis of the <i>hsp70</i> gene promoter between PC12 and cortical neurons. The graph shows the data as described above for (A) and (B). Statistical analyses were performed by Two-way ANOVA followed by Sidak's multiple comparison test. ****p < 0.0001; *p < 0.05.</p

HSF1 does not bind to the <i>hsp70</i> promoter in neurons during heat shock.

<p>(A) Representative immunoblots of HSF1 protein expression in undifferentiated (-NGF) and differentiated (+NGF for 7d) PC12 cells, and in cortical neurons (E18.5, 7div) subjected to heat shock (42°C, 2h) or kept at control conditions (C, 37°C). (B) Nuclear fractions from cortical neurons at control or, after 1h or 2h of heat shock were analyzed for HSF1 protein detection. (C) HSF1 ability to activate <i>hsp70</i> promoter in PC12 cells was assayed after 24 h of NGF (50 ng/ml) treatment. Data correspond to the mean plus SEM of three independent experiments. Statistical analyses were performed by Two-way ANOVA followed by Tukey's multiple comparisons test: ***p <0.001, compared to pCDNA3.1; ###p< 0.001 (D) PC12 cells and cortical neurons were heat shocked at 42°C for 1h or kept unstressed. Chromatin was immunoprecipitated with an anti-HSF1 antibody and amplified by quantitative real-time PCR using primers flanking the promoter area of both <i>hsp70</i> (upper panel) and <i>hsp25</i> (lower panel) genes. Data are expressed as the percentage of the immunoprecipitated DNA in relation to the Input and correspond to the mean plus SEM of at least three independent experiments. Statistical analyses were performed by Two-way ANOVA followed by Tukey's multiple comparisons test: *p < 0.05 compared with control; #p < 0.05 compared to heat shocked PC12 cells.</p

The <i>hsp70</i> gene shows predominant cryptic transcription initiation sites usage in rat cerebral cortex.

<p>(A) Model of rat <i>hsp70</i> gene based on our transcriptome profiling data analysis. (B) Whole brain transcriptome profiling data shows a predominant transcription start site at the beginning of the gene. (C) Cerebral cortex transcriptome profiling data shows predominant transcriptional start sites inside the codifying sequence of <i>hsp70 gene</i>. The coverage for reads aligned in the positive and negative strand are shown in red and blue, respectively.</p