Constitutive phosphorylation of a Rac GAP MgcRacGAP is implicated in v-Src-induced transformation of NIH3T3 cells

MgcRacGAP plays critical roles in cell division through regulating Rho family small GTPases. As we previously reported, phosphorylation of MgcRacGAP on serine 387 (S387) is induced by Aurora B kinase at the midbody during cytokinesis, which is a critical step of cytokinesis. Phosphorylation of S387-MgcRacGAP converts it from RacGAP to RhoGAP, leading to completion of cytokinesis. Here we show that MgcRacGAP is prominently phosphorylated on S387 even in the interphase of v-Src-transformed NIH3T3 cells in the cytoplasm, but not in the interphase of parental NIH3T3 or H-RasV12-transformed NIH3T3 cells. Interestingly, levels of phosphorylation on S387 (pS387) correlated with soft agar colony-forming abilities of v-Src-transformed NIH3T3 cells. Expression of a phosphorylation-mimic mutant MgcRacGAP-S387D enhanced colony formation of v-Src-transformed NIH3T3 cells. Surprisingly, a Rac1 inhibitor but not kinase inhibitors including Aurora B kinase inhibitor specifically inhibited phosphorylation of S387-MgcRacGAP in v-Src-transformed NIH3T3 cells, suggesting the v-Src-induced pathological positive feedback mechanisms towards Rac1 activation using pS387-MgcRacGAP. These results indicated the difference in the mechanisms between v-Src- and H-RasV12-induced transformation, and should shed some light on pathological roles of disordered phosphorylation of MgcRacGAP at S387 in v-Src-induced cell transformation. ( Cancer Sci 2009; 100: 1675–1679)     

A phase I trial of CPT-11 and S-1 combination chemotherapy in patients with metastatic colorectal cancer

BACKGROUND/AIMS: To establish a safe and practical chemotherapeutic regimen using CPT-11 in combination with an oral 5FU derivative S-1 for patients with metastatic colorectal cancer, a phase I clinical trial was conducted in an outpatient setting. METHODOLOGY: Nine patients with metastatic colorectal cancer were enrolled. S-1 was administered at approved doses of 80 mg/body/day to eligible patients with a body surface area (BSA) of less than 1.25m2, 100 mg/body/day to those with a BSA of 1.25-1.5m2, and 120 mg/body/day to those with a BSA of more than 1.5m2, for 2 weeks followed by 1 week rest, comprising one treatment cycle of 3 weeks. CPT-11 was administered on day 8 of the S-1 cycle. The dose of CPT-11 was escalated from 60-120 mg/m2 by every 20 mg/m2 for every cohort consisting of at least 3 patients in order to define dose-limiting toxicity (DLT), maximal tolerated dose (MTD), and recommended dose (RD) in preparation for a phase II trial. RESULTS: In regard to the hematologic toxicity, a decrease of WBC to less than grade 2 was observed in 2 patients until the dose was escalated to 100 mg/m2 of CPT-11, which delayed the treatment for 1 week in 1 patient. Regarding non-hematologic toxicity, fatigue and gastrointestinal toxicity, including anorexia and nausea/vomiting, at grades 1 and 2 were commonly observed throughout the dose levels. Diarrhea at grade 3 was observed at the 4th cycle of 100 mg/m2 CPT-11 in 2 of 3 patients, both of whom required hospitalization. All patients were able to complete more than 3 treatment cycles, and 1 patient at 80 mg/m2 of CPT-11 was able to receive 31 treatment cycles. Observed tumor responses included 1 partial response (PR), 2 moderate responses, 4 stable diseases, and 2 progressive diseases. Serum CEA level decreased in 7 of the 9 patients enrolled. CONCLUSIONS: These results suggest that this treatment regimen using CPT-11 in combination with oral S-1 therapy is a safe regimen in an outpatient setting and effective for patients with metastatic colorectal cancer. The DLT is diarrhea at a MTD of 100 mg/m2 of CPT-11, and 80 mg/m2 CPT-11 is recommended for the next phase II trial.     

cis9, trans11-Conjugated Linoleic Acid Differentiates Mouse 3T3-L1 Preadipocytes into Mature Small Adipocytes through Induction of Peroxisome Proliferator-activated Receptor γ

Dietary conjugated linoleic acid (CLA) has been reported to exhibit a number of therapeutic effects in animal models and patients, such as anti-hypertensive, anti-hyperlipidemic, anti-arteriosclerotic, anti-carcinogenic, and anti-diabetic effects. However, the underlying mechanism is not well-characterized. In the present study, the effects of cis(c)9, trans(t)11-CLA on the differentiation of mouse 3T3-L1 preadipocytes into mature adipocytes were examined. Treatment with c9, t11-CLA in the presence of insulin, dexamethasone, and 3-isobutyl-1-methyl-xanthine (differentiation cocktail) significantly stimulated the accumulation of triacylglycerol. The microscopic observation of cells stained by Oil Red O demonstrated that c9, t11-CLA increases the amount and proportion of small mature adipocytes secreting adiponectin, a benign adipocytokine, when compared to the differentiation cocktail alone. Furthermore, c9, t11-CLA increased bioactive peroxisome proliferator-activated receptor γ (PPARγ) levels in a nuclear extract of 3T3-L1 cells, suggesting the enhancing effect of this fatty acid on the nuclear transmission of PPARγ, a master regulator of adipocyte differentiation, in 3T3-L1 cells. These results suggest that the therapeutic effects of c9, t11-CLA on lifestyle-related diseases are partially due to the enhanced formation of small adipocytes from preadipocytes via PPARγ stimulation.     

Flexible, tapered-tip tube facilitates conventional orotracheal intubation by novice intubators

Orotracheal intubation is the standard technique for airway management, but several untoward airway complications are possible with this method. To avoid airway trauma caused by the tube tip during intubation, the Parker Flex-Tip tube (PFT), which has a flexible, tapered tip, was developed. It has been reported that the PFT facilitates fiberoptic orotracheal intubation and introducer-guided tracheal intubation. In this study, we compared the PFT to a standard endotracheal tube (SET), regarding the time of intubation during conventional orotracheal intubation and the incidence of postoperative sore throat and hoarseness. One hundred and thirty-four patients scheduled for elective anesthesia using orotracheal intubation were randomized to either the PFT or SET and 132 completed the study. The intubators were classified into three groups: staff anesthesiologists, inexperienced anesthesiologists, and anesthesia trainees. The tube was selected by another anesthesiologist and the time required for intubation was measured. PFT did not shorten the time required for intubation and did not reduce the incidence of sore throat and hoarseness. However, a detailed analysis revealed that the PFT decreased the time required for intubation in the anesthesia trainee group. The PFT may help novice intubators to conduct a smooth intubation.     

Anesthetic and airway management of general anesthesia in a patient with Meckel-Gruber syndrome

Meckel-Gruber syndrome, characterized by occipital encephalocele, microcephaly, polydactyly, cleft lip or palate, mandibular micrognathism, and anatomical abnormality of the larynx and tongue, along with other associated malformations, is in the list of diseases associated with difficult airway. However, there has been no report on the management of general anesthesia and airway management for such patients. A 2-year-old girl with Meckel-Gruber syndrome was scheduled for cardioplasty and gastrostomy for gastroesophageal reflux under general anesthesia. Preoperative examination revealed obesity, microgenia, dysspondylism, proteinuria, hypoplastic kidneys, and stenosis of the anal canal. Although we anticipated some difficulty with the intubation and prepared several alternative methods for intubation, such as a bronchofiberscope and a laryngeal mask airway, tracheal intubation was completed without difficulty using conventional laryngoscopy after inhalational induction with sevoflurane. Because most patients with this syndrome die before and shortly after delivery, those who survive to some age might have less severe deformities.     

Mechanisms underlying the maintenance of muscle hypercontractility in a model of postinfective gut dysfunction

BACKGROUND & AIMS: Acute gastroenteritis is a strong risk factor for the development of irritable bowel syndrome (IBS). We have developed an animal model in which transient acute infection leads to persistent muscle hypercontractility. Here, we investigate the mechanisms underlying the maintenance of this hypercontractility in the postinfective (PI) state. METHODS: Muscle contraction and messenger RNA (mRNA) or protein expression of cytokines were examined from jejunal longitudinal muscle cells of NIH Swiss mice infected with Trichinella spiralis or incubated with or without cytokines. RESULTS: During acute infection, interleukin (IL)-4 or IL-13, transforming growth factor (TGF)-beta1, and cyclooxygenase (COX)-2 were increased in the muscle layer ( P < .05). In the PI phase of the model, T helper (Th)2 cytokines returned to normal, but TGF-beta1 remained in the muscle ( P < .05). Exposure of muscle cells to IL-4 or IL-13 increased TGF-beta1 ( P < .01), COX-2 protein, and prostaglandin (PG)E 2 . Exposure of muscle cells to TGF-beta1 increased PGE 2 ( P < .05) and COX-2 protein. Incubation of tissue with IL-4, IL-13, TGF-beta1, or PGE 2 enhanced carbachol-induced muscle cell contractility ( P < .05). COX-2 inhibitor attenuated TGF-beta1-induced muscle hypercontractility ( P < .05). CONCLUSIONS: These results support the hypothesis that Th2 cytokines induce muscle hypercontractility during infection by a direct action on smooth muscle. The maintenance of hypercontractility results from Th2 cytokine-induced expression of TGF-beta1 and the subsequent up-regulation of COX-2 and PGE 2 at the level of the smooth muscle cell. We propose that PI gut dysfunction reflects mediator production in the neuromuscular tissues and that this may occur in PI-IBS.