p53 functional loss in a colon cancer cell line with two missense mutations (218leu and 248trp) on separate alleles

We have sequenced p53 in three colon cancer cell lines capable of autonomous proliferation. SNU-C1 and SNU-C4 cells, whose autonomous growth is dependent upon autocrine stimulation of epidermal growth factor receptor (EGFR), had wildtype p53 sequence of exons 4-9. In contrast, an EGFR ligand-independent cell line, SNU-C5, had heterozygous missense mutations affecting codons 218 (valine to leucine) and 248 (arginine to tryptophan) of p53. Bacterial cloning of p53 from SNU-C5 cells showed that the 248trp and 218leu mutants were both expressed and on separate alleles. 248trp is a common 'hot spot' mutant of p53 with variable dominant negative activity depending on the celullar context. Valine 218, in contrast, is rarely affected by mutation in cancers and is located in a region of the hydrophobic core domain away from 'hot spot' DNA contact sights. However, valine 218 is completely conserved across species, prompting us to investigate the function of 218leu in SNU-C5 cells. SNU-C5 cells exhibited complete loss of normal p53 function as evidenced by over-expression of p53 protein and by failure to show induction of p53, waf-1, mdm-2 or G1/S arrest in response to the DNA damaging agent, bleomycin. In a yeast p53 functional assay (FASAY), 50% of the clones were unable to transactivate a p53-specific promoter required for yeast colony expansion at 25, 30 or 37 degrees C. Sequencing of the p53 insert from several randomly selected wild-type and mutant yeast clones revealed that 218leu-bearing clones retained their ability to transactivate the p53-specific promoter. As expected, the 248trp-bearing clones lost this function. These data indicate that although 218leu retains normal transactivation activity on a p53 promoter in yeast at physiological temperatures, it is not capable of normal p53 function in the presence of a 248trp allele in SNU-C5 cells. It remains unclear whether the strong dominant negative activity of 248trp in SNU-C5 cells is related to the cellular context or to an unresolved abnormality of 218leu function.     

Enhancement of intestinal absorptive capacity with omental flaps

The ability of pedicled omental flaps to revascularize isolated jejunal segments was determined in the initial phase of this project. These bowel segments were capable of surviving independent of their mesenteric perfusion, and absorptive function was equal to that of controls as measured by D-xylose assays. In the second phase of this research, we studied in 10 dogs the absorptive capacity of isolated jejunal segments with both an intact mesentery and an omental flap sutured to the antimesenteric border compared with controls that were perfused only by the mesentery. Absorption was measured at 1, 2, 5, and 10 weeks after application of the omental flap. Absorptive function was augmented an average of 25.8% at 2 weeks, reaching 67.6% (p < 0.001) at 5 weeks. This result remained consistent at 10 weeks. Laser Doppler and colored microsphere studies were performed during a secondary laparotomy at 10 weeks and revealed 42.3% and 53.5% increases, respectively, in blood flow to bowel segments receiving both mesenteric and omental perfusion. This finding suggested that the augmentation of absorptive function was a result of increased blood flow.     

Thrombolytic therapy and proteolysis of factor V

OBJECTIVES: We sought to determine the extent of Factor V proteolysis during thrombolytic therapy. BACKGROUND: Thrombin- or Factor Xa-activated Factor V is an essential cofactor in the prothrombinase complex. In purified systems, plasmin, the major product of thrombolytic therapy, is known to first activate then inactivate Factor V. METHODS: We used quantitative gel electrophoresis and Western blotting to analyze the cleavages in plasma Factor V after thrombolytic therapy. RESULTS: The addition of streptokinase to plasma resulted in the activation then inactivation of Factor V, confirming previous results using purified reagents. We also identified the Factor V fragments resulting from the action of thrombin and plasmin. After thrombolytic therapy, there was considerable Factor V cleavage. The cleavage patterns were consistent with the action of plasmin, with little evidence for the action of thrombin. In the Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries trial (n = 17), we observed an average 58% loss of intact Factor V at 6 h (range 1% to 91%). Samples from the Thrombolysis in Myocardial Infarction trial, Phase II (n = 12), collected on a shorter time scale, showed a loss of up to 99% at 50 min, with the loss of intact Factor V associated with the plasma concentration of plasminogen activator. Samples from patients with bleeding (n = 12) showed extensive Factor V cleavage. CONCLUSIONS: Factor V cleavage in thrombolytic therapy is primarily plasmin mediated, rapid and often extensive. It is likely that transient increases, as well as longer term losses, of Factor V cofactor activity play a role in both ischemic and hemorrhagic events subsequent to thrombolytic therapy. The extensive loss of Factor V in some patients may affect the estimation of heparinization.     

Nonlinear superposition principles: A new numerical method for solving matrix Riccati equations

Previously derived superposition formulae, expressing the general solution of the matrix Riccati equation $\text{W}\text{?}\text{W(t) = A + WB + CW + WDW (A(t), B(t), C(t), D(t), W(t) ?}\text{R}{}^{\mathrm{n\times n}}\text{, n ≥ 2, t ?}\text{R}\text{)}$ in terms of 5 particular solutions, are applied to obtain numerical solutions of this coupled system of nonlinear equations.     

Sequential or simultaneous, same-day anterior decompression and posterior stabilization in the management of vertebral osteomyelitis of the lumbar spine

STUDY DESIGN: A retrospective clinical study of patients with vertebral osteomyelitis of the lumbar spine necessitating surgical treatment. All patients underwent sequential (same-day) or simultaneous anterior decompression and posterior stabilization of the involved vertebrae. OBJECTIVE: To evaluate the efficacy and clinical out-come of sequential or simultaneous anterior and posterior surgical approaches in the management of vertebral osteomyelitis of the lumbar spine. SUMMARY OF BACKGROUND DATA: Anterior approach alone and staged anterior decompression and posterior stabilization have been advocated as the surgical treatment methods of choice for patients with vertebral osteomyelitis of the lumbar spine. The drawbacks of the latter management plan are the necessity to use external support or the delayed patient mobilization and the need for additional anesthesia and surgical trauma. Sequential (same-day) anterior and posterior approaches are used regularly in the surgical management of scoliosis and other spinal deformities. It would appear advantageous to also use the same strategy (i.e., combined same-day double approaches) in the management of vertebral osteomyelitis of the lumbar spine. METHODS: Ten consecutive patients who had a diagnosis of vertebral osteomyelitis of the lumbar spine underwent combined (same-day) anterior and posterior approaches either in a sequential or simultaneous manner. Indications for surgery included neurologic deficit, abscess formation, instability with localized kyphosis formation, and failure of nonoperative treatment. Patients were evaluated clinically and radiographically after surgery. RESULTS: All 10 patients had uneventful surgery. Only one patient required a second surgical procedure because of expulsion of the anterior bone graft and pull-out of instrumentation. All patients were mobilized within the 2 days immediately after surgery. At the mean follow-up examination 30 months after surgery, all patients had regained their motor function and prior ambulatory status. CONCLUSIONS: Patients with lumbar osteomyelitis necessitating surgery can undergo combined, same-day surgery either in a sequential or simultaneous manner. This is a safe and efficient way to control the infection and stabilize the affected segments, allowing for early mobilization of these sick elderly patients.     

How many discoveries have been lost by ignoring modern statistical methods?

Hundreds of articles in statistical journals have pointed out that standard analysis of variance, Pearson product-moment correlations, and least squares regression can be highly misleading and can have relatively low power even under very small departures from normality. In practical terms, psychology journals are littered with nonsignificant results that would have been significant if a more modern method had been used. Modern robust techniques, developed during the past 30 years, provide very effective methods for dealing with nonnormality, and they compete very well with conventional procedures when standard assumptions are met. In addition, modern methods provide accurate confidence intervals for a much broader range of situations, they provide more effective methods for detecting and studying outliers, and they can be used to get a deeper understanding of how variables are related. This article outlines and illustrates these results. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A genetic selection for Caenorhabditis elegans synaptic transmission mutants

We have isolated 165 Caenorhabditis elegans mutants, representing 21 genes, that are resistant to inhibitors of cholinesterase (Ric mutants). Since mutations in 20 of the genes appear not to affect acetylcholine reception, we suggest that reduced acetylcholine release contributes to the Ric phenotype of most Ric mutants. Mutations in 15 of the genes lead to defects in a gamma-aminobutyric acid-dependent behavior; these genes are likely to encode proteins with general, rather than cholinergic-specific, roles in synaptic transmission. Ten of the genes have been cloned. Seven encode homologs of proteins that function in the synaptic vesicle cycle: two encode cholinergic-specific proteins, while five encode general presynaptic proteins. Two other Ric genes encode homologs of G-protein signaling molecules. Our assessment of synaptic function in Ric mutants, combined with the homologies of some Ric mutants to presynaptic proteins, suggests that the analysis of Ric genes will continue to yield insights into the regulation and functioning of synapses.