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The TrkA receptor protein tyrosine kinase is involved in signalling PC12 cell differentiation and cessation of cell division in response to nerve growth factor (NGF). To assess the importance of adaptor proteins and Ras in NGF control of phosphoinositide 3-OH kinase (PI 3-kinase), specific receptor mutations in Trk have been employed. We show that phosphorylation of tyrosine 490, but not 785, of Trk is essential for activation of both Ras and PI 3-kinase in vivo, correlating with tyrosine phosphorylation of Shc and binding of Shc to the adaptor Grb2 and the Ras exchange factor Sos. A mutant receptor that lacks Y490 and Y785, but contains an introduced YxxM motif which binds the regulatory domain of PI 3-kinase, is unable to activate Ras despite causing increased PI 3-kinase activity. This indicates clearly that activation of PI 3-kinase by itself is not sufficient to cause activation of Ras, arguing against a model in which PI 3-kinase acts upstream of Ras. The Shc site of Trk is thus crucial for the activation of Ras and PI 3-kinase.

The incidence of asthma was compared in welders welding in stainless steel (SS) or mild steel (MS). The study was comprised of welders who had been welding for at least 6 months during the preceding 10 year period in four companies, but who had not been welding occupationally prior to this time. The inclusion criteria were met by 42 SS welders with a total welding time of 196 years. Sixteen of these had left work, six because of airway symptoms. Eighty-five MS welders with a total welding time of 403 years were included. Forty-eight of these had left work, 10 citing airway symptoms as a main reason for leaving work, according to responses in a mailed questionnaire. Ex-welders with airway symptoms were in some cases further investigated with spirometry and bronchial provocation tests. In other cases, medical records gave a clear diagnosis. In addition, bronchial responsiveness and lung function were measured and airway symptoms were recorded in presently active welders. Twenty-three of the 26 active SS steel welders and 23 of the 37 active MS steel welders were examined, together with a reference group of 26 (out of 30 invited) vehicle assemblers. There was no difference in the incidence of welding-associated asthma (5% for SS, 7% for MS welders per 1,000 welding-years). Bronchial responsiveness and lung function in active welders was normal and did not differ between MS and SS welders or between welders and a reference group of vehicle fitters. Welders had a significantly higher prevalence of airway symptoms as compared to vehicle fitters.

Background: Activation of the mammalian phosphatidylinositol 3-kinase complex can play a critical role in transducing growth factor responses. The lipid kinase complex, which is made up of p85 alpha and p110 alpha regulatory and catalytic subunits, becomes associated with a number of activated receptor protein tyrosine kinases, but the mechanism of its activation has not yet been defined. Recent evidence indicates that Ras can bind to the p85 alpha/p110 alpha complex. We describe here the functional regulation of the mammalian phosphatidylinositol 3-kinase complex by Ras., Results: Expression of p110 alpha, the catalytic subunit of phosphatidylinositol 3-kinase, in the fission yeast, Schizosaccharomyces pombe, has been used to demonstrate an inhibitory effect of p85 alpha on p110 alpha activity in intact cells; inhibition did not result from a decrease in p110 alpha expression. In this cellular context, we have investigated the effect of a constitutively active mutant of Ras, v-Ras, either on p85 alpha or p110 alpha-alone, or on the p85 alpha/p110 alpha complex. In the presence of the p85 alpha/p110 alpha complex, v-Ras suppressed cell growth, but an effector-domain mutant of v-Ras did not. The growth-suppressive effect of v-Ras was not seen for any other combination of expressed proteins. The phenotype induced by v-Ras was consistent with activation of the p85 alpha/p110 alpha complex: it was sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin, and the cells accumulated 3-phosphorylated polyphosphoinositides. Activation of purified p85 alpha/p110 alpha by purified recombinant Ras in vitro was also demonstrated., Conclusions: The phosphatidylinositol 3-kinase complex, p85 alpha/p110 alpha, shows a suppressed catalytic function in vivo when compared with free p110 alpha. This complex can, however, be activated by Ras. We suggest that the phosphatidylinositol 3-kinase p85 alpha/p110 alpha complex is a downstream effector of Ras.

The ubiquitous Ca(2+)-binding protein calmodulin (CaM) is a key protein in Ca2+ homeostasis and activation of eukaryotic cells. CaM is the molecular link between free Ca2+ in the cell and the inhibition, or activation, of numerous enzymes. Many nuclear functions are under Ca2+/CaM control, and some transcriptional activators are known to be Ca2+ modulated indirectly through Ca2+/CaM-dependent protein kinases. But Ca2+/CaM has not yet been found to directly modulate any transcription factor or other DNA-binding protein. Transcription factors of the basic-helix-loop-helix (bHLH) group are important regulators in numerous systems. Here we report that binding of Ca(2+)-loaded CaM to the bHLH domains of several bHLH proteins directly inhibits their DNA binding. Other bHLH proteins are either less sensitive or resistant. Ca2+ ionophore selectively inhibits transcriptional activation by Ca2+/CaM-sensitive bHLH proteins in vivo, implying that Ca2+ can directly influence transcription through differential CaM inhibition of bHLH domains.

A family of proteins, denoted SL3-3 enhancer factor 1 (SEF1), interacts with DNA sequences in the T cell specific enhancer of SL3-3 murine leukemia virus and in the enhancers of several other viruses. A putative SEF1 binding site was also identified in the T cell specific enhancer of the gene encoding the human T cell antigen receptor (TcR) associated CD3-epsilon polypeptide. In this study we show that the identified sequence is a strong SEF1 binding site, and that purified SEF1 proteins bind specifically to the sequence. We report also that the SEF1 binding site is important for T cell specific activation of transcription by the CD3-epsilon enhancer. We show that SEF1 binding sites are present also in the T cell specific enhancers of other subunits of the TcR-CD3 complex, and that SEF1 proteins appear to play a central role in the T cell specific expression of this set of enhancers.

We have developed computer hardware and software that imports analog waveforms and other measured data from a patient into the PaceBase database system supported by any IBM PC/AT compatible. The programmable A/D converter has the capacity to acquire the pacemaker artifact from the surface ECG leads. Analysis of the pacemaker artifact permits confirmation of pulse width as well as programmability and facilitates discovery of pacemaker hardware failures otherwise undetectable. Continuous recording of real-time surface ECG can be made as other measurements or storing functions are being performed. In this way, sporadic or infrequent intrinsic events are automatically recorded and can be selected to be played back and reviewed or stored on the permanent record. Pacemaker spike detection enhances identification of paced and intrinsic complexes by emphasizing the paced artifact. Even bipolar atrial spikes with pulse widths as short as 0.05 ms are easily identified; myopotential muscle noise is rejected. Enhancement of pacemaker spikes takes the guesswork out of interpretation of ECGs, especially for bipolar systems and when testing or troubleshooting for myopotential tracking or inhibition.

Nuclear factor I (NFI) is shown to be of importance for the activity of the enhancer element of a T-cell leukemogenic murine retrovirus, SL3-3, and for the regulation of this element by glucocorticoid. Each nucleotide of the binding site of the NFI proteins was mutated, and the effects of the mutations were quantitated with an electrophoretic mobility shift assay. Mutations in the inverted repeat of the binding site have symmetric effects which strongly support the notion that NFI proteins preferentially bind to dyad symmetry sites. Such binding sites were shown to be more than 100 fold stronger than the corresponding single binding sites. We find dyad symmetry sequences which are much stronger NFI binding sites than NFI sites identified in different genes and also stronger than previously proposed consensus binding sequences for NFI.