Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, becomes upregulated during cell proliferation and transformation. Here we show that intact ODC activity is needed for the acquisition of a transformed phenotype in rat 2R cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Addition of the ODC inhibitor alpha-difluoromethyl ornithine (DFMO) to the cells (in polyamine-free medium) before shift to permissive temperature prevented the depolymerization of filamentous actin and morphological transformation. Polyamine supplementation restored the transforming potential of pp60v-src. DFMO did not interfere with the expression of pp60v-src or its in vitro tyrosine kinase activity. The tyrosine phosphorylation of most cellular proteins, including ras GAP, did not either display clear temperature- or DFMO-sensitive changes. A marked increase was, however, observed in the tyrosine phosphorylation of phosphatidylinositol 3-kinase and proteins of 33 and 36 kD upon the temperature shift, and these hyperphosphorylations were partially inhibited by DFMO. A DFMO-sensitive increase was also found in the total phosphorylation of calpactins I and II. The well-documented association of GAP with the phosphotyrosine-containing proteins p190 and p62 did not correlate with transformation, but a novel 42-kD tyrosine phosphorylated protein was complexed with GAP in a polyamine- and transformation-dependent manner. Further, tyrosine phosphorylated proteins of 130, 80/85, and 36 kD were found to coimmunoprecipitate with pp60v-src in a transformation-related manner. Altogether, this model offers a tool for sorting out the protein phosphorylations and associations critical for the transformed phenotype triggered by pp60v-src, and implicates a pivotal role for polyamines in cell transformation.     

Stroke mortality and its socioeconomic, racial, and behavioral correlates in Florida

Stroke mortality is associated both with being black and with having low socioeconomic status. However, it is uncertain to what extent that increased risk is related to rates of behavior-related risk factors, such as hypertension, cigarette smoking, obesity, or alcohol consumption. The investigators performed an ecologic analysis to estimate the contributions of behavioral risks, socioeconomic status, and black race to regional variations in stroke mortality rates among persons 55-84 years of age in Florida. They used data from the 1980 census and from the Behavioral Risk Factor Surveillance System (BRFSS) for 1986 through 1988. Weighted multiple linear regression models indicated that regions in Florida with high stroke mortality rates were characterized by high prevalences of poverty, obesity, and hypertension. Although limited by its ecologic design, this study suggests that socioeconomic status and prevalence of behavioral risks contribute independently to interregional disparities in stroke mortality rates in Florida. BRFSS data, now available for more than 45 States, can be used to help clarify the relative contributions of behavioral and other risks to population-based mortality rates.     

The single-stranded DNA aptamer-binding site of human thrombin

A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.     

Optical microscopy. 3. Tracking fluorescently labeled neurons in developing brain

For decades, time-lapse microscopy has been used to track dynamic events associated with biological phenomena. Time-lapse studies of the developing nervous system have been restricted to analysis of dissociated cell cultures or of a series of static images from living organisms. The advent of new fluorescent dyes and video imaging technology has produced novel views of the behavior of neurons in the context of the developing nervous tissue, such as migrations within and away from proliferative zones and navigation of axonal processes to synaptic targets. After fixation of the tissue preparation, time-lapse monitoring can be followed by other analytical techniques and forms of microscopy, e.g., immunocytochemistry or electron microscopy, producing information on the interactions of individual cells whose behavioral histories are known. The power of video time-lapse microscopy of living brain tissue lies in the firsthand documentation of developmental patterning, which in turn can serve as an experimental assay.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Creatinine kinetic modelling: a simple and reliable tool for the assessment of protein nutritional status in haemodialysis patients

While the mathematical modelling of urea kinetics is in wide use for evaluating treatment adequacy and protein nutrition in dialysis patients, the kinetics of creatinine generation in dialysis patients has been relatively unexplored. In this study creatinine kinetic modelling as a clinical tool was investigated in a group of 90 patients treated by haemodialysis (n = 20), haemodiafiltration (60), haemofiltration (7), or biofiltration (3) over a 6-36-month period. A single pool model of creatinine kinetics was employed to obtain monthly values of creatinine distribution space and creatinine appearance rate. Extrarenal creatinine degradation rate, estimated using a clearance of 0.038 l/kg/24 h as suggested by Mitch and co-workers, was added to creatinine appearance rate in urine and dialysate to calculate a corrected creatinine index (CI). Extrarenal degradation accounted for 12 +/- 2% of CI. CI was higher in males (22.4 +/- 4.5 mg/kg/24 h) than females (19.8 +/- 4.8) and decreased with age, falling off more sharply for the female group (CI = 29.9-0.185.age, R = 0.72) than the males (CI = 24.1-0.030.age, R = 0.31). CI was found to correlate strongly with protein catabolic rate determined by urea kinetic modelling (CI = 8.84 +/- 10.91.PCR). Low or reduced CI was associated in this study group with severe malnutrition status and high mortality rate. CI is suggested as a strong predictor of patient morbidity and mortality.     

Effects of dehulling, cooking and storage conditions on protein quality and digestibility of soybeans

A monoclonal antibody against tetrodotoxin was produced. Tetrodotoxin coupled with keyhole limpet hemocyanin was used as an immunogen to BALB/c mice. These mice had no clinical signs for the toxicity of tetrodotoxin during the immunization. The reason may be that the guanidyl group of tetrodotoxin which is an important group for the toxicity was hidden by coupling with keyhole limpet hemocyanin. The monoclonal antibody was highly specific for tetrodotoxin and had no cross-reaction to tetrodotoxin derivatives, paralytic shellfish toxins, keyhole limpet hemocyanin and crude proteins from various organs of puffer fish. Also, tetrodotoxin was neutralized in vitro by this antibody. From the fact that the structural difference between tetrodotoxin and anhydro-tetrodotoxin is recognized by this antibody, it was suggested that this antibody reacted with the OH-groups on C-4 and/or C-9 of tetrodotoxin. In addition, the results from immunization and neutralization tests demonstrated that tetrodotoxin became non-toxic even when one of the active groups of tetrodotoxin was coupled by a molecule.