Attenuation of LPA-mediated calcium signaling and inositol polyphosphate production in rat-1 fibroblasts transformed by the v-src oncogene

Alterations in cellular signaling underlie the transforming actions of many oncogenes. The vsrc oncogene tyrosine kinase, pp60vsrc, is known to alter multiple signal transduction pathways, including those involving phosphatidylinositol (PI) metabolism. In this study, we investigated the effects of vsrc-transformation on lysophosphatidic acid (LPA) receptor coupling to intracellular free calcium [Ca2+]i and PI turnover in rat-1 fibroblasts. In normal rat-1 cells, LPA rapidly elevated [Ca2+]i (EC50 = 10nM). In contrast, the ability of LPA to mobilize calcium was markedly attenuated in rat-1-vsrc cells. Further study revealed that the LPA-mediated generation of inositol (1,4,5)P3 and other inositol polyphosphates was also markedly attenuated in the vsrc-transformed cells. Although LPA caused a transient reduction in the level of PI(4,5)P2 in normal rat-1 cells, the agonist elevated the level of PI(4,5)P2 in the vsrc-transformed cells. These findings demonstrate that vsrc-transformation alters the coupling of LPA receptors to PI turnover and calcium signaling in rat-1 cells, and point to G protein-coupled receptor systems as targets for modulation by the vsrc kinase.     

Antenatal glucocorticoid therapy suppresses angiotensin-converting enzyme activity in rats with nitrofen-induced congenital diaphragmatic hernia

BACKGROUND: Carcinoma of the true vocal cord represents the earliest clinically recognizable invasive malignancy in the head and neck region and provides a unique model for studying possible prognostic genetic markers. The aim of this study was to determine whether p53 overexpression correlated with tumor recurrence in a homogenous population of patients with early stage glottic carcinoma treated with radiotherapy alone. METHODS: One hundred and fourteen patients with T1N0M0 squamous cell carcinoma of the glottis were treated with curative radiotherapy between 1976 and 1990. With a median follow-up of 6 years, actuarial local control was 80% with 23 local recurrences. Laryngeal biopsy specimens obtained prior to radiation therapy were analyzed retrospectively in 22 patients. Forty-five patients with local control were used as a control group. p53 overexpression indicating a mutated p53 gene was analyzed by immunohistochemistry using the mouse monoclonal antibody D0-7. RESULTS: Approximately 82% of carcinomas that recurred locally expressed p53 compared with only 29% of those with local control (P < 0.001). No significant relation was noted between p53 expression and histologic grade. Intensity of staining did not predict tumor recurrence. CONCLUSIONS: The authors believe that this case-controlled study demonstrated the role of p53 as an independent prognostic factor in patients with early stage glottic carcinoma.     

Partial liquid ventilation reduces pulmonary neutrophil accumulation in an experimental model of systemic endotoxemia and acute lung injury

OBJECTIVE: To determine whether pulmonary neutrophil sequestration and lung injury are affected by partial liquid ventilation with perfluorocarbon in a model of acute lung injury (ALI). DESIGN: A prospective, controlled, in vivo animal laboratory study. SETTING: An animal research facility of a health sciences university. SUBJECTS: Forty-one New Zealand White rabbits. INTERVENTIONS: Mature New Zealand White rabbits were anesthetized and instrumented with a tracheostomy and vascular catheters. Animals were assigned to receive partial liquid ventilation (PLV, n = 15) with perflubron (18 mL/kg via endotracheal tube), conventional mechanical ventilation (CMV, n = 15) or high-frequency oscillatory ventilation (HFOV, n = 5). Animals were ventilated, using an FIO2 of 1.0, and ventilatory settings were required to achieve a normal PaCO2. Animals were then given 0.9 mg/kg of Escherichia coli endotoxin intravenously over 30 mins. Partial liquid ventilation, conventional mechanical ventilation, or high-frequency oscillatory ventilation was continued for an additional 4 hrs before the animals were killed. A group of animals not challenged with endotoxin underwent conventional ventilation for 4.5 hrs, serving as the control group (control, n = 6). Lungs were removed and samples were frozen at -70 degrees C. Representative samples were stained for histology. A visual count of neutrophils per high-power field (hpf) was performed in five randomly selected fields per sample in a blinded fashion by light microscopy. Lung samples were homogenized in triplicate in phosphate buffer, ultrasonified, freeze-thawed, and clarified by centrifugation. Supernatants were analyzed for myeloperoxidase (MPO) activity by spectrophotometry with o-dianisidine dihydrochloride and hydrogen peroxide at 460 nm. MEASUREMENTS AND MAIN RESULTS: Histologic analysis of lung tissue obtained from control animals showed normal lung architecture. Specimens from the PLV and HFOV groups showed a marked decrease in alveolar proteinaceous fluid, pulmonary vascular congestion, edema, necrotic cell debris, and gross inflammatory infiltration when compared with the CMV group. Light microscopy of lung samples of animals supported with PLV and HFOV had significantly lower neutrophil counts when compared with CMV (PLV, 4 +/- 0.3 neutrophils/hpf; HFOV, 4 +/- 0.5 neutrophils/hpf; CMV, 10 +/- 0.9 neutrophils/hpf; p < .01). In addition, MPO activity from lung extracts of PLV and HFOV animals was significantly lower than that of CMV animals (PLV, 61 +/- 13.3 units of MPO activity/lung/kg; HFOV, 43.3 +/- 6.8 units of MPO activity/lung/kg; CMV, 140 +/- 28.5 units of MPO activity/lung/kg; p < .01). MPO activity from lungs of uninjured control animals was significantly lower than that of animals in the PLV, HFOV, and CMV groups (control, 2.2 +/- 2 units of MPO activity/lung/kg; p < .001). CONCLUSIONS: Partial liquid ventilation decreases pulmonary neutrophil accumulation, as shown by decreased neutrophil counts and MPO activity, in an experimental animal model of ALI induced by systemic endotoxemia. The attenuation in pulmonary leukostasis in animals treated with PLV is equivalent to that obtained by a ventilation strategy that targets lung recruitment, such as HFOV.     

FasL induces Fas/Apo1-mediated apoptosis in human embryonic kidney 293 cells routinely used to generate E1-deleted adenoviral vectors

Phosphatidylinositol-4-phosphate 5-kinase (PIP5K) phosphorylates phosphatidylinositol-4-phosphate to produce phosphatidylinositol-4,5-bisphosphate as a precursor of two second messengers, inositol-1,4,5-triphosphate and diacylglycerol, and as a regulator of many cellular proteins involved in signal transduction and cytoskeletal organization. Despite PIP5K playing such an essential role in a number of physiological processes, much still remains to be made clear about its association with plants. Searching the Arabidopsis expression sequence tag database against already known yeast and mammalian PIP5K cDNAs, we identified two clones which partly encode the same Arabidopsis PIP5K and isolated a corresponding full-length cDNA encoding a protein that we designated AtPIP5K1. Recombinant AtPIP5K1 expressed in Escherichia coli possessed a PIP5K activity in vitro. Due to some structural and biochemical differences, AtPIP5K1 was not categorized as either a type I or type II PIP5K. The expression of the AtPIP5K1 mRNA was induced rapidly by treating Arabidopsis plants with drought, salt and abscisic acid, which suggests that AtPIP5K11 is involved in water-stress signal transduction. These data give evidence for a close link between phosphoinositide signaling cascades and water-stress responses in plants.     

Enterogastric bile reflux during technetium-99m-sestamibi cardiac imaging

Twenty-six patients with squamous cell cancer of the cervix were treated with i.v. paclitaxel, 250 mg/m2 over 3 h every 21 days. They received steroid, H1 and H2 blocker premedications, and granulocyte-colony-stimulating factor (G-CSF) support (5 microgram/kg/day). No prior chemotherapy, except as a radiation sensitizer, was allowed. The median age was 50 (range, 36-81) years, and performance status Zubrod was 1 (range, 0-2). Eight (33%) patients had prior surgery, and 22 (92%) had prior radiation therapy. Twenty-four patients were evaluable for response; 2 were later found to be ineligible. Five patients had partial responses (21%; 95% confidence interval, 6-40%), and 14 (58%; 95% confidence interval, 35-78%) had stable disease. The median duration of response was 10 (range, 3-27+) weeks. The responses were within the radiation port (four responses) and outside of it (one response). The median interval from the start of irradiation to the start of paclitaxel in responding patients was 94 weeks, whereas in patients with stable disease it was 68 weeks, and in patients whose disease progressed it was 46 weeks. Eighty-eight percent of the 105 cycles of paclitaxel were administered at a dose of 250 mg/m2 or higher. Granulocytopenia was brief and noncumulative, with grades 3 and 4 experienced by 5 and 3 patients, respectively. G-CSF was used for a median of 7 (range, 2-14) days/cycle. Anemia was mild, with G3 noted in 3 patients, and thrombocytopenia was not significant. Infections and musculoskeletal pain were mild and infrequent. Sensory (14 patients G1 or G2 and 2 patients G3) and motor (4 patients G1 or G2 and 1 patient G3) neurotoxicity was noted. There was no significant cardiovascular toxicity. Paclitaxel is active in patients with squamous cell cancer of the cervix and is well tolerated at this dose schedule with G-CSF support.