Clinical relevance of serum ionized magnesium concentration in dogs with myxomatous mitral valve disease.

BACKGROUND: Hypomagnesemia is associated with a poor prognosis in humans with congestive heart failure (CHF), but studies in veterinary medicine are limited. HYPOTHESIS: Serum ionized magnesium concentration [iMg2+ ] would decrease as CHF progresses compared with the initial diagnostic levels and that lower [iMg2+ ] would be negatively associated with prognosis in dogs with CHF. ANIMALS: A total of 181 client-owned dogs with myxomatous mitral valve disease (MMVD) were included. They were classified into the preclinical stage (NO-CHF, n = 108), stage C (n = 42), and stage D (n = 31) based on the American College of Veterinary Internal Medicine MMVD classification. METHODS: This is a retrospective study from 2 referral centers. The [iMg2+ ] was compared among the NO-CHF, stage C, and stage D groups. Kaplan-Meier curves and the log-rank test were used to compare the incidence of death between groups. Multivariable Cox regression analysis was used to estimate the association of hypomagnesemia with the death. RESULTS: In the stage D group, the [iMg2+ ] was lower than that in the NO-CHF (P < .0001) and stage C groups (P < .003). In the Kaplan-Meier survival analysis, the 1-year cumulative survival rate in hypomagnesemic dogs was 53% compared with 91.5% in normomagnesemic dogs (log-rank test, P < .0001). In the multivariable Cox analysis, lower concentration of [K+ ] and [iMg2+ ], along with higher Evel , were associated with negative prognoses. Specifically, hypomagnesemia was associated with an approximately 4-fold increased risk of death (hazard ratio = 4.015; 95% confidence interval, 1.537-10.488; P = .005). CONCLUSIONS AND CLINICAL IMPORTANCE: Assessing the [iMg2+ ] might serve as a potential marker for estimating the severity and prognosis indirectly in dogs with MMVD. Combining [iMg2+ ] measurement with other diagnostic methods, such as echocardiography, could improve the prognostic evaluation of MMVD in dogs.

SOXE group transcription factors regulates the expression of FoxG1 during inner ear development.

The transcription factor FOXG1 plays an important role in inner ear development; however, the cis-regulatory mechanisms controlling the inner-ear-specific expression of FOXG1 are poorly understood. In this study, we aimed to identify the element that specifically regulates FoxG1 expression in the otic vesicle, which develops into the inner ear, through comparative genome analysis between vertebrate species and chromatin immunoprecipitation. The cis-regulatory element (E2) identified showed high evolutionary conservation among vertebrates in the genomic DNA of FoxG1 spanning approximately 3 Mbp. We identified core sequences important for the activity of the otic-vesicle-specific enhancer through in vitro and in vivo reporter assays for various E2 enhancer mutants and determined the consensus sequence for SOX DNA binding. In addition, SoxE, a subfamily of the Sox family, was simultaneously expressed in the otic vesicles of developing embryos and showed a similar protein expression pattern as that of FoxG1. Furthermore, SOXE transcription factors induced specific transcriptional activity through the FoxG1 Otic enhancer (E2b). These findings suggest that the interaction between the otic enhancer of FoxG1 and SOXE transcription factor, in which the otic expression of FoxG1 is evolutionarily well-conserved, is important during early development of the inner ear, a sensory organ important for survival in nature.

TWIK-1 BAC-GFP Transgenic Mice, an Animal Model for TWIK-1 Expression.

TWIK-1 is the first identified member of the two-pore domain potassium (K2P) channels that are involved in neuronal excitability and astrocytic passive conductance in the brain. Despite the physiological roles of TWIK-1, there is still a lack of information on the basic expression patterns of TWIK-1 proteins in the brain. Here, using a modified bacterial artificial chromosome (BAC), we generated a transgenic mouse (Tg mouse) line expressing green fluorescent protein (GFP) under the control of the TWIK-1 promoter (TWIK-1 BAC-GFP Tg mice). We confirmed that nearly all GFP-producing cells co-expressed endogenous TWIK-1 in the brain of TWIK-1 BAC-GFP Tg mice. GFP signals were highly expressed in various brain areas, including the dentate gyrus (DG), lateral entorhinal cortex (LEC), and cerebellum (Cb). In addition, we found that GFP signals were highly expressed in immature granule cells in the DG. Finally, our TWIK-1 BAC-GFP Tg mice mimic the upregulation of TWIK-1 mRNA expression in the hippocampus following the injection of kainic acid (KA). Our data clearly showed that TWIK-1 BAC-GFP Tg mice are a useful animal model for studying the mechanisms regulating TWIK-1 gene expression and the physiological roles of TWIK-1 channels in the brain.

Cux2 expression regulated by Lhx2 in the upper layer neurons of the developing cortex.

The laminar structure, a unique feature of the mammalian cerebrum, is formed by a number of genes in a highly complex process. The pyramidal neurons that make up each layer of the cerebrum are functionally characterized by specific gene expressions. In particular, Cux1 and Cux2, which are specifically expressed in layer II-IV neurons, are known to regulate dendritic branching, spine morphology, and synapse formation. However, it is still unknown how their expression is regulated transcriptionally. Here we constructed Cux2-mCherry transgenic mice that reproduce the cortical layer II-IV-specific expression of Cux2, a member of the Cut/Cux/CDP family, using BAC transgenesis and a variety of coordinated cortical layer markers that are known to date. Our immunohistochemistry analysis shows that mCherry was expressed in cortical layer II-IV and the corpus callosum in the same way as endogenous Cux2 without ectopic expression. We also identified a region of 220 bp that is highly conserved in mammals and controls specific cerebral expression of Cux2, using comparative genome analysis and in vivo reporter assays. Furthermore, we confirm that Lhx2, whose expression in cortical layer II-IV is similar to that of the Cux2 enhancer, can act as a transcriptional activator. These results suggest that cortical layer II-IV expression of Cux2 can be regulated by the interaction of Cux2-E1 and Lhx2, and that their failure to co-regulate is associated with neurodevelopmental disorders such as autism and schizophrenia.

Effects of irradiation and leukoreduction on down-regulation of CXCL-8 and storage lesion in stored canine whole blood.

White blood cells (WBCs) and storage period are the main factors of transfusion reactions. In the present study, cytokine/chemokine concentrations after leukoreduction (LR) and irradiation (IR) in stored canine whole blood were measured. Red blood cell storage lesion caused by IR and LR were also compared. Blood samples from 10 healthy Beagles were divided into four groups (no treatment, LR-, IR-, and LR + IR-treated). Leukocytes were removed by filtration in the LR group and gamma radiation (25 Gy) was applied in the IR group. Immunologic factors (WBCs, interleukin-6 [IL-6], C-X-C motif chemokine ligand 8 [CXCL-8], and tumor necrosis factor-alpha) and storage lesion factors (blood pH, potassium, and hemolysis) were evaluated on storage days 0, 7, 14, 21, and 28. Compared to the treated groups, IL-6 and CXCL-8 concentrations during storage were significantly higher in the control (no treatment) group. LR did not show changes in cytokine/chemokine concentrations, and storage lesion presence was relatively mild. IR significantly increased CXCL-8 after 14 days of storage, but IR of leukoreduced blood did not increase CXCL-8 during 28 days of storage. Storage lesions such as hemolysis, increased potassium, and low pH were observed 7 days after IR and storage of blood, regardless of LR. IR of leukoreduced blood is beneficial to avoid immune reactions; however, storage lesions should be considered upon storage.

CCN3 secretion is regulated by palmitoylation via ZDHHC22.

Normal extracellular secretion of nephroblastoma overexpressed (NOV, also known as CCN3) is important for the adhesion, migration, and differentiation of cells. In previous studies, we have shown that the intracellular accumulation of CCN3 inhibits the growth of prominent neurons. Increased intracellular CCN3 can be induced through various processes, such as transcription, detoxification, and posttranslational modification. In general, posttranslational modifications are very important for protein secretion. However, it is unclear whether posttranslational modification is necessary for CCN3 secretion. In this study, we have conducted mutational analysis of CCN3 to demonstrate that its thrombospondin type-1 (TSP1) domain is important for CCN3 secretion and intracellular function. Point mutation analysis confirmed that CCN3 secretion was inhibited by cysteine (C)241 mutation, and overexpression of CCN3-C241A inhibited neuronal axonal growth in vivo. Furthermore, we demonstrated that palmitoylation is important for the extracellular secretion of CCN3 and that zinc finger DHHC-type containing 22 (ZDHHC22), a palmityoltransferase, can interact with CCN3. Taken together, our results suggest that palmitoylation by ZDHHC22 at C241 in the CCN3 TSP1 domain may be required for the secretion of CCN3. Aberrant palmitoylation induces intracellular accumulation of CCN3, inhibiting neuronal axon growth.