Visualization of the Activity of Rac1 Small GTPase in a Cell

Rho family G proteins including Rac regulate a variety of cellular functions, such as morphology, motility, and gene expression. Here we developed a fluorescence resonance energy transfer-based analysis in which we could monitor the activity of Rac1. To detect fluorescence resonance energy transfer, yellow fluorescent protein fused Rac1 and cyan fluorescent protein fused Cdc42-Rac1-interaction-binding domain of Pak1 protein were used as intermolecular probes of FRET. The fluorophores were separated with linear unmixing method. The fluorescence resonance energy transfer efficiency was measured by acceptor photobleaching assisted assay. With these methods, the Rac1 activity was visualized in a cell. The present findings indicate that this approach is sensitive enough to achieve results similar to those from ratiometric fluorescence resonance energy transfer analysis

Human Mena Associates with Rac1 Small GTPase in Glioblastoma Cell Lines

Mammarian enabled (Mena), a member of the Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) family of proteins, has been implicated in cell motility through regulation of the actin cytoskeleton assembly, including lamellipodial protrusion. Rac1, a member of the Rho family GTPases, also plays a pivotal role in the formation of lamellipodia. Here we report that human Mena (hMena) colocalizes with Rac1 in lamellipodia, and using an unmixing assisted acceptor depletion fluorescence resonance energy transfer (u-adFRET) analysis that hMena associates with Rac1 in vivo in the glioblastoma cell line U251MG. Depletion of hMena by siRNA causes cells to be highly spread with the formation of lamellipodia. This cellular phenotype is canceled by introduction of a dominant negative form of Rac1. A Rac activity assay and FRET analysis showed that hMena knock-down cells increased the activation of Rac1 at the lamellipodia. These results suggest that hMena possesses properties which help to regulate the formation of lamellipodia through the modulation of the activity of Rac1

Introduction of Autopsy Imaging Redefines the Concept of Autopsy: 37 Cases of Clinical Experience

H Ezawa, R Yoneyama,S Kandatsu, K Yoshikawa,H Tsujii, K Harigaya. Introduction of Autopsy Imaging (AI) Redefines the Concept of Autopsy: 37 Cases of Clinical Experience. Pathology International.53. 865-873. 2003

Risk factors in chronic subdural hematoma: comparison of irrigation with artificial cerebrospinal fluid and normal saline in a cohort analysis.

BACKGROUND: Chronic subdural hematoma (CSDH) is known to have a substantial recurrence rate. Artificial cerebrospinal fluid (ACF) is an effective irrigation solution in general open craniotomy and endoneurosurgery, but no evidence of its use in burr-hole surgery exists. OBJECTIVE: To identify the potential of ACF irrigation to prevent CSDH recurrence. More specifically, to investigate the perioperative and intraoperative prognostic factors, and to identify controllable ones. METHODS: To examine various prognostic factors, 120 consecutive patients with unilateral CSDH treated with burr-hole drainage between September 2007 and March 2013 were analyzed. Intraoperative irrigation was performed with one of two irrigation solutions: normal saline (NS; n = 60) or ACF (n = 60). All patients were followed-up for at least 6 months postoperatively. We also examined the morphological alternations of the hematoma outer membranes after incubation with different solutions. RESULTS: Eleven patients (9.2%) had recurrence. Nine patients (15%) required additional surgery in the NS group, whereas only 2 patients (3.3%) in the ACF group required additional surgery. Among preoperative and intraoperative data, age (<80 years old, P = .044), thrombocyte (>22.0, P = .037), laterality (right, P = .03), and irrigation solution (ACF, P = .027) were related to smaller recurrence rates by log-rank tests. Only the type of irrigation solution used significantly correlated with recurrence in favor of ACF in both Cox proportional hazards (relative hazard: 0.20, 95% confidence interval (CI): 0.04-0.99; P = .049) and logistic regression models (odds ratio, 0.17, 95% CI: 0.03-0.92; P = .04) using these factors. Histological examinations of the hematoma membranes showed that the membranes incubated with NS were loose and infiltrated by inflammatory cells compared with those incubated with ACF. CONCLUSION: Irrigation with ACF decreased the rate of CSDH recurrence

Differentiation of human hematopoietic cells increases expression of a gene transferred by a retroviral vector

To study expression of a retroviral vector in human hematopoietic lineages, two established human hematopoietic cell lines (HL60 and K562) and a human adherent stromal cell line (KM101) were infected with the vector pZIP-SV(X). Expression of the transferred neomycin resistance gene (neor) of pZIP-SV(X) was evaluated as the ability of the cells to form colonies (greater than 50 cells) in an agar assay in the presence of the neomycin analogue, G418. After infection, all three cell lines produced colonies resistant to G418. The level of neor mRNA in separate colonies was analyzed by Northern blot analysis. The neor gene transferred by the vector pZIP-SV(X) was expressed in both human hematopoietic and stromal cell lines. In addition, primary adherent human stromal cells infected with pZIP-SV(X) grew in the presence of G418. To determine if differentiation of hematopoietic cells affects expression of the retroviral vector, HL60 cells infected with pZIP-SV(X) were induced to differentiate, and the level of neor mRNA measured. The amount of neor mRNA increased when HL60 cells were induced to differentiate along the granulocytic pathway. Conversely, when HL60 cells were induced toward monocytoid differentiation (TPA), the level of neor mRNA did not significantly increase. We conclude that the neor gene transferred by a retroviral vector, pZIP-SV(X), is functionally expressed. In addition, expression of the transferred neor gene is regulated during myeloid differentiation of HL60 cells

Collision of advanced gastric adenocarcinoma and gastrointestinal stromal tumour: a case report

We present a case of an 83-year-old female patient with a collision tumour of an advanced Borrmann type 4 gastric cancer and a large gastric gastrointestinal stromal tumour (GIST). According to the deformity of the gastric wall caused by the GIST, type 4 cancer was difficult to identify by oesophagogastroduodenoscopy (OGD). The patient died of progressive gastric cancer related disease. While the mechanism of histogenesis of the simultaneous adenocarcinoma and GIST remains to be determined, the present case suggests that gastric adenocarcinoma has a more adverse effect on prognosis than does GIST. Additionally, this case suggests that thorough inspection of GIST patients is required at the OGD and at the pathology facility, in order to avoid overlooking the underlying cancer