Biomedical subjects
Publications and source records attributed to J Stock.
The components of the Escherichia coli chemosensory system have been identified and their activities characterized, but how sensory information is processed to give an integrated response remains an open question.
Telomere length, measured by terminal restriction fragments, was examined in tissues from human fetuses of gestational ages estimated as 15-19 weeks. The length of telomeres was similar in most fetal tissues. However, there were significant variations in telomere length among fetuses, with no apparent relationship between gestational age and telomere length. We conclude that synchrony in telomere length exists among tissues of the human fetus. This synchrony is apparently lost during extrauterine life.
In the few years since its gene was first cloned, the Aequorea victoria green fluorescent protein (GFP) has become a powerful tool in cell biology, functioning as a marker for gene expression, protein localization and protein dynamics in living cells. GFP variants with improved fluorescence intensity and altered spectral characteristics have been identified, but additional GFP variants are still desirable for multiple labeling experiments, protein interaction studies and improved visibility in some organisms. In particular, long-wavelength (red) fluorescence has remained elusive. Here we describe a red-emitting, green-absorbing fluorescent state of GFP that is generated by photoactivation with blue light. GFP can be switched to its red-emitting state easily with a laser or fluorescence microscope lamp under conditions of low oxygen concentration. This previously unnoticed ability enables regional, non-invasive marking of proteins in vivo. In particular, we report here the use of GFP photoactivation to make the first direct measurements of protein diffusion in the cytoplasm of living bacteria.
The histidine protein kinase CheA is a multidomain protein that mediates stimulus-response coupling in bacterial chemotaxis. We have previously shown that the purified protein exhibits an equilibrium between inactive monomer and active dimer (Surette, M., Levit, M., Liu, Y., Lukat, G., Ninfa, E., Ninfa, A., and Stock, J. (1996) J. Biol. Chem. 271, 939-945). We report here a study of the kinetics of phosphorylation of the isolated phosphoacceptor domain of CheA catalyzed by the isolated catalytic domain of the protein. The reaction fits Michaelis-Menten kinetics (Km = 0.26 mM for ATP and 0. 10 mM for phosphoacceptor domain; kobs = 17 min-1). The catalytic domain exhibits the same equilibrium between inactive monomers and active dimers as the full-length CheA protein. Thus, CheA dimerization is an intrinsic property of this domain, independent of any other portion of the molecule and is required for its catalytic activity. In equimolar mixtures of full-length CheA and catalytic domain, homodimers and heterodimers are formed in equal concentration, indicating that all of the determinants for the dimerization are localized entirely on the catalytic domain. An analysis of the kinetics of phosphorylation catalyzed by CheA-catalytic domain heterodimers indicates half of the sites reactivity. The rate of CheA phosphorylation within this heterodimer is over 5-fold greater than that observed in CheA homodimers. The dramatic increase in activity within this asymmetric dimer raises the possibility that CheA activation by receptors involves a mechanism that directs catalysis to one active site while preventing interference from the other.
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It is has been proposed that hormone receptors which have only a single transmembrane sequence mediate signaling via hormone-induced monomer-to-dimer transitions. But recent studies of analogous receptors in bacteria indicate that dimerization may be only a prerequisite for signaling.
Phosphoprotein phosphatase 2A (PP2A) is one of the four major protein serine/threonine phosphatases found in all eukaryotic cells. We have shown that the 36-kDa catalytic subunit of PP2A is carboxyl methylated in eukaryotic cells, and we have previously identified and purified a novel methyltransferase (MTase) that is responsible for this modification. Here, we describe a novel protein carboxyl methyl-esterase (MEase) from bovine brain that demethylates PP2A. The enzyme has been purified to homogeneity as a monomeric 46-kDa soluble protein. The MEase is highly specific for PP2A. It does not catalyze the demethylation of other protein or peptide methylesters. Moreover, MEase activity is dramatically inhibited by nanomolar concentrations of okadaic acid, a specific inhibitor of PP2A. From these results, we conclude that PP2A methylation is controlled by two specific enzymes, a MTase and a MEase. Since PP2A methylation is highly conserved in eukaryotes ranging from human to yeast, it is likely that this system plays an important role in phosphatase regulation.
E-selectin, an endothelial cell adhesion molecule, mediates the initial step of leucocyte adhesion to activated vascular endothelium. The soluble isoform of E-selectin promotes angiogenesis in rat cornea. In the present study, we investigated whether leucocyte adhesion and angiogenesis are also involved in tumour progression and metastasis of colorectal cancer. Therefore, we determined the level of circulating soluble E-selectin in serum samples of 38 patients with colorectal cancer; 20 patients with non-metastatic and 18 patients with metastatic disease. Median levels of soluble E-selectin were found to be significantly higher in metastatic tumour disease (88.7 ng/ml, range 25-203 ng/ml) than in healthy controls (34.9 ng/ml, range 15-59 ng/ml, P = 0.01), in patients with primary tumours or with local recurrences (39.5 ng/ml, range 22-100 ng/ml). Furthermore, there was no correlation with the serum level of C-reactive protein, fibrinogen or tumour necrosis factor alpha suggesting that the elevation of E-selectin is independent of inflammation in tumour patients. Therefore, we propose that elevated soluble E-selectin may reflect increased neovascularisation in metastatic tumour tissue.
BACKGROUND: Fungal sinusitis typically occurs in immunocompromised patients. The authors report four cases of fungal sinusitis in immunocompetent young adults, all of whom had proptosis. METHODS: The diagnosis in all four patients was determined after orbital imaging and sinus biopsies. RESULTS: All four patients required surgical removal of the fungal source and anti-fungal chemotherapy postoperatively. CONCLUSION: Patients with proptosis, ocular pain, or other symptoms suggestive of orbital cellulitis unresponsive to antibiotic treatment should undergo radiographic imaging. If sinus disease is present, biopsy and culture may lead to the diagnosis of fungal disease. Surgical debridement and the appropriate systemic antifungal therapy usually lead to cure.
In bacterial chemotaxis, membrane receptors control the phosphorylation of the response regulator CheY; phospho-CheY has been found to control motility by binding to a switching apparatus attached to the flagellar motor.
Protein phosphatase 2A is the principal carboxyl methylated protein in cytoplasmic extracts derived from Xenopus eggs arrested in interphase. Levels of methylation approach stoichiometric values after 2 hr of incubation at 22 degrees C. Rates of PP2A methylation increase by over 50% upon addition of cAMP. Methylation is inhibited by okadaic acid at concentrations that inhibit PP2A phosphatase activity.
Major advances have been made over the past year in understanding the molecular mechanisms involved in membrane receptor function, and in resolving the global organization of intracellular signaling pathways. Crystallographic and biochemical studies are revealing details of transmembrane signaling mechanisms and the phosphorylation reactions of the two-component regulatory systems. In addition, the discovery of new signal transduction pathways and new inputs into known pathways are providing a clearer view of the basic architecture of the signal transduction networks within the bacterial cell.
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In eukaryotic cells a number of different proteins with important regulatory functions are reversibly methyl-esterified at carboxyl-terminal prenylcysteine residues. These proteins include the low molecular weight GTP-binding proteins, the gamma-subunit of the heterotrimeric G-proteins, and the nuclear lamins. The methylating enzymes that catalyze this type of carboxyl methylation reaction are integral membrane proteins, and the methylated protein products tend to be membrane-associated. Analyses of protein carboxyl methylation in a wide range of vertebrate tissues revealed a major carboxyl-methylated protein that was clearly distinct from those that are modified at prenylcysteine groups (Volker, C., Miller, R.A., McCleary, W.R., Rao, A., Poenie, M., Backer, J.M., and Stock, J.B. (1991) J. Biol. Chem. 266, 21515-21522). This M(r) = 36,000 protein is localized to the cytosol. Unlike the prenylcysteine methyltransferases, the enzyme that catalyzes the methylation of the 36-kDa protein is found in the cytosol. The 36-kDa methylated protein has been purified from bovine brain. Sequence analysis of several peptides clearly shows that the protein is the catalytic subunit of protein phosphatase 2A. A soluble 40-kDa methyltransferase that catalyzes the reaction has also been purified.
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