Divergent Activity Profiles of Type 1 Ryanodine Receptor Channels Carrying Malignant Hyperthermia and Central Core Disease Mutations in the Amino-Terminal Region

The type 1 ryanodine receptor (RyR1) is a Ca²⁺ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in several diseases, including malignant hyperthermia (MH) and central core disease (CCD). Most MH and CCD mutations cause accelerated Ca²⁺ release, resulting in abnormal Ca²⁺ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca²⁺ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca²⁺ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca²⁺ in the ER and an increase in resting cytoplasmic Ca²⁺. RyR1 channel activity was quantitatively evaluated by [³H]ryanodine binding and three parameters (sensitivity to activating Ca²⁺, sensitivity to inactivating Ca²⁺ and attainable maximum activity, i.e., gain) were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca²⁺ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca²⁺ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca²⁺ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.     

Oral Behaviors of Beef Steers in Pen and Pasture Environments

This study observed the behavioral characteristics of 122 steers in eight pens and 1,136 steers at six pastures. Nonhuman animals kept in pens performed less nutritive oral behaviors and more nonnutritive oral behaviors than animals kept at pasture. Although these could not be described as stereotypies, they did represent a replacement of nutritive oral behaviors by nonnutritive oral behaviors, rather than simply an increase in resting time. This could be indicative of a level of oral frustration. At pasture, there was a greater proportion of oral behaviors in animals with low pasture availability as compared to high availability, but this was an increase in nutritive oral behaviors rather than nonnutritive oral behaviors. Factors other than oral frustration—for example, rumen fill—probably drove this increase.     

Gene expression in the vascular wall of the aortic arch in spontaneously hypertensive hyperlipidemic model rats using DNA microarray analysis

Aims

In recent years, there has been an increase in patients with arteriosclerosis and the risk of lifestyle-related diseases. However, the pathogenesis and medication of atherosclerosis have not been elucidated. We developed a rat model of lifestyle-related diseases by feeding a high-fat diet and 30% sucrose solution (HFDS) to spontaneously hypertensive hyperlipidemic rats (SHHR) and reported that this model is a useful model of early atherosclerosis. In order to elucidate the pathogenesis of early atherosclerosis, we searched for atherosclerosis-related genes by microarray analysis using the aortic arch rat model of lifestyle-related diseases.

Main methods

Four-month-old male Sprague–Dawley rats and SHHR were each divided into two normal diet (ND) groups and two HFDS groups. After a four-month treatment, the expression of mRNA in the aortic arch was detected using the oligo DNA microarray one-color method and quantified using real-time PCR.

Key findings

In this study, we detected 39 genes in microarray analysis. Esm1, Retnlb Mkks, and Grem2 showed particularly marked changes in gene expression in the SHHR-HFDS group. Compared with the SD-ND group, the SHHR-HFDS group had an increase in Mkks gene expression of about 26-fold and an approximately 22-fold increase in the expression of Grem2. Similarly, the expression of Esm1 increased by about 12-fold and that of Retnlg by about 10-fold as shown by quantitative real-time PCR.

Significance

This study suggested that these four genes might be important in early atherosclerosis development.     

Qualitative and Quantitative Assessment of Adenosine Triphosphate Stress Whole-Heart Dynamic Myocardial Perfusion Imaging Using 256-Slice Computed Tomography

Background

The aim of this study was to investigate the correlation of the qualitative transmural extent of hypoperfusion areas (HPA) using stress dynamic whole-heart computed tomography perfusion (CTP) imaging by 256-slice CT with CTP-derived myocardial blood flow (MBF) for the estimation of the severity of coronary artery stenosis.

Methods and Results

Eleven patients underwent adenosine triphosphate (0.16 mg/kg/min, 5 min) stress dynamic CTP by 256-slice CT (coverage: 8 cm, 0.27 s/rotation), and 9 of the 11 patients underwent coronary angiography (CAG). Stress dynamic CTP (whole–heart datasets over 30 consecutive heart beats in systole without spatial and temporal gaps) was acquired with prospective ECG gating (effective radiation dose: 10.4 mSv). The extent of HPAs was visually graded using a 3-point score (normal, subendocardial, transmural). MBF (ml/100g/min) was measured by deconvolution. Differences in MBF (mean ± standard error) according to HPA and CAG results were evaluated. In 27 regions (3 major coronary territories in 9 patients), 11 coronary stenoses (> 50% reduction in diameter) were observed. In 353 myocardial segments, HPA was significantly related to MBF (P < 0.05; normal 295 ± 94; subendocardial 186 ± 67; and transmural 80 ± 53). Coronary territory analysis revealed a significant relationship between coronary stenosis severity and MBF (P < 0.05; non-significant stenosis [< 50%], 284 ± 97; moderate stenosis [50–70%], 184 ± 74; and severe stenosis [> 70%], 119 ± 69).

Conclusion

The qualitative transmural extent of HPA using stress whole-heart dynamic CTP imaging by 256-slice CT exhibits a good correlation with quantitative CTP-derived MBF and may aid in assessing the hemodynamic significance of coronary artery disease.     

N-cadherin regulates p38 MAPK signaling via association with JNK-associated leucine zipper protein: implications for neurodegeneration in Alzheimer disease

Synaptic loss, which strongly correlates with the decline of cognitive function, is one of the pathological hallmarks of Alzheimer disease. N-cadherin is a cell adhesion molecule essential for synaptic contact and is involved in the intracellular signaling pathway at the synapse. Here we report that the functional disruption of N-cadherin-mediated cell contact activated p38 MAPK in murine primary neurons, followed by neuronal death. We further observed that treatment with Aβ(42) decreased cellular N-cadherin expression through NMDA receptors accompanied by increased phosphorylation of both p38 MAPK and Tau in murine primary neurons. Moreover, expression levels of phosphorylated p38 MAPK were negatively correlated with that of N-cadherin in human brains. Proteomic analysis of human brains identified a novel interaction between N-cadherin and JNK-associated leucine zipper protein (JLP), a scaffolding protein involved in the p38 MAPK signaling pathway. We demonstrated that N-cadherin expression had an inhibitory effect on JLP-mediated p38 MAPK signal activation by decreasing the interaction between JLP and p38 MAPK in COS7 cells. Also, this study demonstrated a novel physical and functional association between N-cadherin and p38 MAPK and suggested neuroprotective roles of cadherin-based synaptic contact. The dissociation of N-cadherin-mediated synaptic contact by Aβ may underlie the pathological basis of neurodegeneration such as neuronal death, synaptic loss, and Tau phosphorylation in Alzheimer disease brain.     

High-frequency phage-mediated gene transfer among Escherichia coli cells, determined at the single-cell level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3x10(-8) to 2x10(-6), 1x10(-8) to 4x10(-8), and <4x10(-9) to 4x10(-8) per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7x10(-4) to 1x10(-3), 9x10(-4) to 3x10(-3), and 5x10(-4) to 4x10(-3) for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Expression of steroidogenic enzymes and synthesis of sex steroid hormones from DHEA in skeletal muscle of rats

The functional importance of sex steroid hormones (testosterone and estrogens), derived from extragonadal tissues, has recently gained significant appreciation. Circulating dehydroepiandrosterone (DHEA) is peripherally taken up and converted to testosterone by 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD, and testosterone in turn is irreversibly converted to estrogens by aromatase cytochrome P-450 (P450arom). Although sex steroid hormones have been implicated in skeletal muscle regulation and adaptation, it is unclear whether skeletal muscles have a local steroidogenic enzymatic machinery capable of metabolizing circulating DHEA. Thus, here, we investigate whether the three key steroidogenic enzymes (3beta-HSD, 17beta-HSD, and P450arom) are present in the skeletal muscle and are capable of generating sex steroid hormones. Consistent with our hypothesis, the present study demonstrates mRNA and protein expression of these enzymes in the skeletal muscle cells of rats both in vivo and in culture (in vitro). Importantly, we also show an intracellular formation of testosterone and estradiol from DHEA or testosterone in cultured muscle cells in a dose-dependent manner. These findings are novel and important in that they provide the first evidence showing that skeletal muscles are capable of locally synthesizing sex steroid hormones from circulating DHEA or testosterone.     

Regulation of N-cadherin-based cell-cell interaction by JSAP1 scaffold in PC12h cells

We previously reported that the level of c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, increases dramatically during nerve growth factor (NGF)-induced differentiation of PC12h cells. In the present study, we investigated the function of JSAP1 during PC12h cell differentiation by knocking down the level of JSAP1. The depletion of JSAP1 caused NGF-treated PC12h cells to form aggregates and impaired their differentiation. The aggregation was not observed in JSAP1-depleted cells that were untreated or treated with epidermal growth factor. Immunocytochemical studies indicated that N-cadherin, but not E-cadherin, was localized to sites of cell-cell contact in the aggregated cells. Furthermore, an inhibitory anti-N-cadherin antibody completely blocked the aggregation. Taken together, these results suggest that JSAP1 regulates cell-cell interactions in PC12h cells specifically in the NGF-induced signaling pathway, and does so by modulating N-cadherin.     

Porcine germinal angiotensin I-converting enzyme: isolation, characterization and molecular cloning

Germinal angiotensin I-converting enzyme (gACE) was purified to homogeneity from porcine seminal plasma. The molecular weight of the purified enzyme was calculated to be 182,000 on non-denaturing PAGE and 94,000 and 93,000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of two identical subunits in seminal plasma. The K(m), V(max), K(cat) and K(cat)/K(m) values of gACE at optimal pH (pH 7.2) were 680 microM, 1.0 micromol/mg/min, 33.1 s(-1) and 4.87 x 10(4) s(-1) M(-1) for Z-Val-Lys-Met-MCA, respectively. gACE was potently inhibited by EDTA, 1,10-phenanthroline, captopril and lisinopril, and it promptly released the dipeptides His-Leu and Phe-Arg from angiotensin I and bradykinin. Met- and Leu-enkephalins, neuromedine B and beta-neo-endorphin were also good natural substrates for gACE. We determined the structure of gACE cDNA from the porcine testis, and deduced the amino acid sequence of gACE. The cDNA is composed of 2508 bp of nucleotides in length and encodes 745 amino acids in the coding region. The overall homology of amino acid sequences between porcine, human, sheep and rat gACEs is 72.6 to 84.7%. Zinc-binding motif, chloride-binding site and positions of cysteine residues were well conserved.     

A 43-kDa protein of Treponema denticola is essential for dentilisin activity

A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43- and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3' region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.