A novel potential effector of M-Ras and p21 Ras negatively regulates p21 Ras-mediated gene induction and cell growth.

Here, we report the identification and characterization of a new member of the RalGDS-family, which is widely expressed and interacts strongly and selectively with the GTP-bound forms of M-Ras and p21 Ras. This Ras pathway modulator (RPM), also termed RGL3, exhibited Ras-binding and catalytic domains typical of the RalGDS-family of guanine nucleotide exchange factors, and was most similar to Rlf (RalGDS-like factor), but was distinguished by a unique proline-rich region with multiple candidate SH3-domain binding sites. RPM/RGL3 resembled AF-6 and Nore1 in interacting strongly with constitutively active M-Ras and p21 Ras. In contrast to Rlf, transiently expressed RPM/RGL3 did not activate an Elk-1-inducible reporter gene alone or in combination with activated p21 Ras, but strongly inhibited induction of this reporter gene by co-expression of activated H-Ras or MEKK-1. This inhibitory effect was independent of the Ras binding domain and required a second signal provided by p21 Ras or MEKK-1, but not Raf-1 or M-Ras. Expression of RPM/RGL3 also strongly inhibited cell growth of fibroblasts transformed by an activated Src Y527F. Thus, RPM/RGL3 is a novel potential effector of both p21 Ras and M-Ras with the novel function of negatively regulating Elk-1-dependent gene induction downstream of p21 Ras or MEKK-1.

M-Ras, a widely expressed 29-kD homologue of p21 Ras: expression of a constitutively active mutant results in factor-independent growth of an interleukin-3-dependent cell line.

M-Ras, a recently identified homologue of p21 Ras, is widely expressed, with levels of the 29-kD protein in spleen, thymus, and NIH 3T3 fibroblasts equaling or exceeding those of p21 Ras. A G22V mutant of M-Ras was constitutively active and its expression in an interleukin-3 (IL-3)-dependent mast cell/megakaryocyte cell line resulted in increased survival in the absence of IL-3, increased growth in IL-4, and, at high expression levels, in factor-independent growth. Expression of M-Ras G22V, however, had a negative effect on growth in the presence of IL-3, suggesting that M-Ras has both positive and negative effects on growth. Expression of M-Ras G22V in NIH-3T3 fibroblasts resulted in morphological transformation and growth to higher cell densities. M-Ras G22V induced activation of the c-fos promoter, and bound weakly to the Ras-binding domains of Raf-1 and RalGDS. Expression of a mutant of M-Ras G22V that was no longer membrane-bound partially inhibited (40%) activation of the c-fos promoter by N-Ras Q61K, suggesting that M-Ras shared some, but not all, of the effectors of N-Ras. An S27N mutant of M-Ras, like the analogous H-Ras S17N mutant, was a dominant inhibitor of activation of the c-fos promoter by constitutively active Src Y527F, suggesting that M-Ras and p21 Ras shared guanine nucleotide exchange factors and are likely to be activated in parallel. Moreover, M-Ras was recognized by the monoclonal anti-Ras antibody Y13-259, commonly used to study the function and activity of p21 Ras. Mammalian M-Ras and a Caenorhabditis elegans orthologue exhibit conserved structural features, and these are likely to mediate activation of distinctive signaling paths that function in parallel to those downstream of p21 Ras.

Human mitogen-activated protein kinase kinase 7 (MKK7) is a highly conserved c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activated by environmental stresses and physiological stimuli.

We report the cloning of a novel human activator of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 7 (MKK7). The mRNA for MKK7 is widely expressed in humans and mice and encodes a 47-kDa protein (419 amino acids), as determined by immunoblotting endogenous MKK7 with an antibody raised against its N terminus. The kinase domain of MKK7 is closely related to a Drosophila JNK kinase dHep (69% identity) and to a newly identified ortholog from Caenorhabditis elegans (54% identity), and was more distantly related to MKK4, MKK3, and MKK6. MKK7 phosphorylated and activated JNK1 but failed to activate p38 MAPK in co-expression studies. In hematopoietic cells, endogenous MKK7 was activated by treatment with the growth factor interleukin-3 (but not interleukin-4), or by ligation of CD40, the B-cell antigen receptor, or the receptor for the Fc fragment of immunoglobulin. MKK7 was also activated when cells were exposed to heat, UV irradiation, anisomycin, hyperosmolarity or the pro-inflammatory cytokine tumor necrosis factor-alpha. Co-expression of constitutively active mutants of RAS, RAC, or CDC42 in HeLa epithelial cells or of RAC or CDC42 in Ba/F3 factor-dependent hematopoietic cells also activated MKK7, suggesting that MKK7 will be involved in many physiological pathways.

Costs of poison-related hospitalizations at an urban teaching hospital for children.

BACKGROUND: Childhood poisonings continue to exact a notable toll in injury-related morbidity and economic cost. Because a substantial portion of this morbidity and economic cost is associated with hospitalization, an analysis of the cost of poisoning hospitalizations might help to identify areas in which medical care could be improved and costs could be reduced. OBJECTIVE: To assess the aggregate cost of poison-related hospitalizations and to analyze the trends in categorical poisoning costs during a 4-year period. DESIGN: Cost-benefit analysis of charge data and length of stay (LOS) for poison-related hospitalizations. SETTING: Admissions to one urban children's hospital. PATIENTS: Case mix data were reviewed to identify those children whose hospitalizations had a primary discharge diagnosis related to poisoning in fiscal years 1992 to 1995. MAIN OUTCOME MEASURES: Mean aggregate hospital charges and LOS for poisonings were compared for each of the 4 years of the study. Comparisons were also made with charges and LOS for hospitalizations for all other diagnoses during the same period. Trends in hospital charges and LOS for the most common specific types of poisoning were analyzed individually. Linear regression statistics were used to compare the costs and LOS of specific types of poisoning. RESULTS: There were 638 poison-related hospitalizations during the 4-year study (0.9% of all pediatric hospital admissions). Charges per case decreased from $7934 in fiscal year 1992 to $4968 in fiscal year 1995 (z = -2.74, P = .006); mean LOS decreased from 5.85 days in 1992 to 3.45 days in 1995 (z = 2.84, P = .005). These trends exceeded smaller trends toward decreasing charges and LOS seen also for non-poison-related hospitalizations. Acetaminophen, lead, and antidepressant medications were the most common and most costly specific agents implicated in poison-related hospitalizations during the study period. Linear regression analysis showed a significant decrease in charges (F = 6.35, R2 = 0.09, P = .014) and LOS for acetaminophen (F = 4.30, R2 = 0.063, P < .04) but not for lead or antidepressant poison-related hospitalizations. CONCLUSIONS: Despite an increasing number of children hospitalized for poisoning at one institution during a 4-year period, per case hospital charges decreased substantially. The cost savings were associated with a marked decrease in LOS. Still, poison-related admissions to a single pediatric facility accounted for almost $1 million in hospital charges in fiscal year 1995. A few agents are overrepresented; new poisoning prevention measures aimed at these toxic agents are warranted. We conclude that further outcomes studies are needed to delineate cost-effective improvements in patient care targeted toward poisonings owing to those agents.

Reflux after esophagectomy and interposition of right colon.

OBJECTIVE: To determine the nature of reflux after esophagectomy and interposition of right colon. DESIGN: A case series. SETTING: A university hospital. PATIENTS: Five patients (mean age 43 years) in whom right colon had been interposed after total esophagectomy. INTERVENTIONS: Endoscopy and biopsy, manometry, 24-hour ambulatory pH testing. MAIN OUTCOME MEASURES: Endoscopic and histologic appearances of the colon, type of motor activity in the grafted colon and the type of reflux. RESULTS: In all patients the grafted colon was macroscopically and microscopically normal. There was nonperistaltic motor activity in the interposed colon in two patients. All patients had an abnormal pattern of alkaline reflux, and one patient had abnormal acid reflux. CONCLUSIONS: The clinical effect of alkaline reflux in patients with colonic interposition after total esophagectomy may be slight. Histologic and gross changes are likely to be minimal.

The extent and pattern of gastro-oesophageal reflux in patients with scleroderma oesophagus: the effect of low-dose omeprazole.

Ambulatory 24-hour oesophageal pH studies were obtained from 11 patients with scleroderma who expressed either dysphagia (n = 6) or heartburn (n = 5) as their predominant oesophageal symptom. No significant differences were found in the extent of pattern of reflux between these two groups. The pH data of both scleroderma groups were combined and compared to an age- and sex-matched group of control subjects (n = 11). The reflux demonstrated by scleroderma patients was significantly greater than the control group in every category (P < 0.01). The percentage of time the pH was < 4.0 was not significantly different (P > 0.05) upright (29.9 +/- 19.8%) vs. supine (44.2 +/- 28.5%) in patients with scleroderma. Eight scleroderma patients underwent repeat pH studies while taking low-dose omeprazole (20 mg daily) and reflux was reduced significantly (P < 0.01) in all patients. The authors believe that 24-hour ambulatory oesophageal pH-monitoring should be routinely conducted in scleroderma patients to provide quantitative reflux data, even when heartburn is not expressed as a symptom. Omeprazole, 20 mg daily, provides adequate protection from the H+ component of the refluxate.

Tyrosine phosphorylation of receptor beta subunits and common substrates in response to interleukin-3 and granulocyte-macrophage colony-stimulating factor.

The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) consist of two polypeptides each belonging to a new class of molecules referred to as the hemopoietin receptor family. When expressed alone, receptor polypeptides of this family often bind their respective factors with lower affinity than the receptors identified in whole cells. Despite the lack of structural evidence for any enzymatic activity of the receptor polypeptides, both IL-3 and GM-CSF stimulate tyrosine phosphorylation of multiple intracellular substrates. We investigated IL-3 and GM-CSF receptor structure and signaling in a myeloid cell line, FDC-P1, which is dependent on either IL-3 or GM-CSF for growth. Antiphosphotyrosine antibodies were used to immunoprecipitate tyrosine-phosphorylated proteins from 32P-labeled cells or to probe immunoblots. Both IL-3 and GM-CSF stimulated the phosphorylation of a similar pattern of polypeptides on tyrosine. One tyrosine phosphorylated polypeptide migrated with M(r) = 135,000 and increased to 150,000 over a period of 10 min following stimulation of cells with IL-3 or GM-CSF. The M(r) = 135,000-150,000 polypeptide phosphorylated in response to IL-3 was shown to be primarily the Aic-2A polypeptide, the low affinity IL-3 receptor. Phosphatase treatment showed that the dramatic IL-3-induced shift in apparent molecular weight from M(r) = 125,000 in unstimulated cells was entirely due to phosphorylation. The closely related receptor, Aic-2B, was also tyrosine phosphorylated in response to IL-3, although to a lesser extent than Aic-2A. Treatment with GM-CSF resulted in tyrosine phosphorylation of the Aic-2B polypeptide exclusively. It was intriguing that GM-CSF treatment did affect the mobility of the Aic-2A polypeptide on polyacrylamide gels. Together, these results suggest that the Aic-2A polypeptide is part of the IL-3 receptor complex, but not the GM-CSF receptor. In contrast, the Aic-2B polypeptide is a component of the GM-CSF receptor, but it can also be utilized in an IL-3 receptor.

Esophageal reflux before and after isolated myotomy for achalasia.

Four patients with achalasia underwent 24-hour esophageal pH measurements as ambulatory patients before and after limited myotomy without fundoplication. Resting lower esophageal sphincter pressure was reduced from 24.3 +/- 1.3 mm Hg to 7.5 +/- 4.3 mm Hg. No significant differences (p greater than 0.05) were found before and after operation in the total 24-hour pH data distribution (pH 6.24 +/- 0.84 vs 5.75 +/- 1.03), the fraction of time below pH 4.0 (4.8% +/- 5.3% vs 8.0% +/- 6.9%), or the mean duration of reflux episodes greater than 5 minutes (22.8 +/- 18.8 minutes vs 23.0 +/- 10 minutes), all +/- SD. Effective relief of esophageal obstruction in achalasia is feasible by isolated limited myotomy without producing gastroesophageal reflux.