Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.     

The Time Course of Dynamic Computed Tomographic Appearance of Radiation Injury to the Cirrhotic Liver Following Stereotactic Body Radiation Therapy for Hepatocellular Carcinoma

This study aimed to evaluate the dynamic computed tomographic (CT) appearance of focal radiation injury to cirrhotic liver tissue around the tumor following stereotactic body radiation therapy (SBRT) for hepatocellular carcinoma (HCC). Seventy-seven patients with 92 HCCs were observed for >6 months. Sixty-four and 13 patients belonged to Child–Pugh class A and B, respectively. The median SBRT dose was 48 Gy/4fr. Dynamic CT scans were performed in non–enhanced, arterial, portal, and venous phases. The median follow-up period was 18 months. Dynamic CT appearances were classified into 3 types: type 1, hyperdensity in all enhanced phases; type 2, hypodensity in arterial and portal phases; type 3, isodensity in all enhanced phases. Half of the type 2 or 3 appearances significantly changed to type 1, particularly in patients belonging to Child–Pugh class A. After 3–6 months, Child–Pugh class B was a significant factor in type 3 patients. Thus, dynamic CT appearances were classified into 3 patterns and significantly changed over time into the enhancement group (type 1) in most patients belonging to Child–Pugh class A. Child–Pugh class B was a significant factor in the non–enhancement group (type 3).     

Gender Differences in Patients with Takotsubo Cardiomyopathy: Multi-Center Registry from Tokyo CCU Network

Background

The clinical features of gender differences in takotsubo cardiomyopathy (TC) remain to be determined. The aim of this study was to evaluate the differences in clinical characteristics of male and female patients with TC.

Methods

We obtained the clinical information of 368 patients diagnosed with TC (84 male, 284 female) from the Tokyo CCU Network database collected from 1 January 2010 to 31 December 2012; the Network is comprised of 71 cardiovascular centers in the Tokyo (Japan) metropolitan area. We attempted to characterize clinical differences during hospitalization, comparing male and female patients with TC.

Results

There were no significant differences in apical ballooning type, median echocardiography ejection fraction, serious ventricular arrhythmias (such as ventricular tachycardia or fibrillation), or cardiovascular death between male and female patients. Male patients were younger than female patients (median age at hospitalization for male patients was 72 years vs. 76 years for female patients; p = 0.040). Prior physical stress was more common in male than female patients (50.0% vs.31.3%; p = 0.002), while emotional stress was more common in female patients (19.0% vs. 31.0%; p = 0.039). Severe pump failure (defined as Killip Class > III) (20.2% vs. 10.6%; p = 0.020) and cardiopulmonary supportive therapies (28.6% vs. 12.7%, p < 0.001) were more common in male than female patients. Multivariate analysis revealed that male gender (odds ratio = 4.32, 95% CI = 1.41–13.6, p = 0.011) was an independent predictor of adverse composite cardiac events, including cardiovascular death, severe pump failure, and serious ventricular arrhythmia.

Conclusions

Cardiac complications in our dataset appeared to be more common in male than female patients with TC during their hospitalization. Further investigation is required to clarify the underlying mechanisms responsible for the observed gender differences.     

Spatial Change of Cruciate Ligaments in Rat Embryo Knee Joint by Three-Dimensional Reconstruction

This study aimed to analyze the spatial developmental changes of rat cruciate ligaments by three-dimensional (3D) reconstruction using episcopic fluorescence image capture (EFIC). Cruciate ligaments of Wister rat embryos between embryonic day (E) 16 and E20 were analyzed. Samples were sectioned and visualized using EFIC. 3D reconstructions were generated using Amira software. The length of the cruciate ligaments, distances between attachment points to femur and tibia, angles of the cruciate ligaments and the cross angle of the cruciate ligaments were measured. The shape of cruciate ligaments was clearly visible at E17. The lengths of the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) increased gradually from E17 to E19 and drastically at E20. Distances between attachment points to the femur and tibia gradually increased. The ACL angle and PCL angle gradually decreased. The cross angle of the cruciate ligaments changed in three planes. The primordium of the 3D structure of rat cruciate ligaments was constructed from the early stage, with the completion of the development of the structures occurring just before birth.     

High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity

Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 10⁴/cm² showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 10⁴/cm² largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density.     

Excessive ingestion of long-chain polyunsaturated fatty acids during developmental stage causes strain- and sex-dependent eye abnormalities in mice

The eyes are riched in long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic acid [ARA; 20:4 (n−6)] and docosahexaenoic acid [DHA; 22:6 (n−3)]. Despite their abundance in the eyes, ARA and DHA cannot be sufficiently synthesized de novo in mammals. During gestation, eye development is exceptionally rapid, and substantial amounts of LC-PUFAs are needed to ensure proper eye development. Here, we studied the influences of dietary LC-PUFAs in dams (C57BL/6 and C3H/He) on the eye morphogenesis and organogenesis of their pups. Intriguingly, fetuses and newborn mice from C57BL/6 dams fed an LC-PUFA (particularly ARA)-enriched diet displayed a much higher incidence of eye abnormalities such as microphthalmia (small eye) and corneal opacity than those from dams fed an LC-PUFA-poor diet. The effects of LC-PUFAs on eye anomalies were evident only in the female pups of C57BL/6 inbred mice, not in those of C3H/He mice or male C57BL/6 mice. These results demonstrate a gene-by-environment (GxE) interaction in eye development in mice. Furthermore, our molecular analysis suggested the potential roles of Pitx3 and Pax6 in the above interaction involving ARA.     

Age-dependent and cell-population-restricted LRRK2 expression in normal mouse spleen

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinson’s disease (PD), but its true physiological function remains unknown. In the normal mouse, LRRK2 is expressed in kidney, spleen, and lung at much higher levels than in brain, suggesting that LRRK2 may play an important role in these organs. Analysis of age-related changes in LRRK2 expression demonstrated that expression in kidney, lung, and various brain regions was constant throughout adult life. On the other hand, expression of both LRRK2 mRNA and protein decreased markedly in spleen in an age-dependent manner. Analysis of purified spleen cells indicated that B lymphocytes were the major population expressing LRRK2, and that T lymphocytes showed no expression. Consistently, the B lymphocyte surface marker CD19 exhibited an age-dependent decrease of mRNA expression in spleen. These results suggest a possibly novel function of LRRK2 in the immune system, especially in B lymphocytes.     

Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences

The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.