The respiratory syncytial virus small hydrophobic protein is phosphorylated via a mitogen-activated protein kinase p38-dependent tyrosine kinase activity during virus infection

The phosphorylation status of the small hydrophobic (SH) protein of respiratory syncytial virus (RSV) was examined in virus-infected Vero cells. The SH protein v.,as isolated from [S-35]methionine- and [P-33]orthophosphate-labelled IRSV-infected cells and analysed by SDS-PAGE. In each case, a protein product of the expected size for the SH protein was observed. Phosphoamino acid analysis and reactivity with the phosphotyrosine specific antibody PY20 showed that the SH protein was modified by tyrosine phosphorylation. The role or tyrosine kinase activity in SH protein phosphorylation was confirmed by the use of genistein, a broad-spectrum tyrosine kinase inhibitor, to inhibit SH protein phosphorylation. Further analysis showed that the different glycosylated forms of the SH protein were phosphorylated, as was the oligomeric form of the protein. Phosphorylation of the SH protein was specifically inhibited by the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580, suggesting that SH protein phosphorylation occurs via a MAPK p38-dependent pathway. Analysis of virus-infected cells using fluorescence microscopy showed that, although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate, at low levels, in the endoplasmic reticulum/Golgi complex, confirming recent observations. However, in the presence of SB203580. an increased accumulation of the SH protein in the Golgi complex was observed, although other virus structures, such as virus filaments and inclusion bodies, remained largely unaffected. These results showed that during RSV infection, the SH protein is modified by an MAPK p38-dependant tyrosine kinase activity and that this modification influences its cellular distribution.

Peptide Tyrosine Arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin secretions of the dusky gopher frog, Rana sevosa

An octadecapeptide was isolated from the skin secretions of the dusky gopher frog (Rana sevosa) on the basis of histamine release from rat peritoneal mast cells. This peptide was purified to homogeneity by HPLC and found to have the following primary structure, YLKGCWTKSYPPKPCFSR, using both Edman degradation chemistry and peptide sequencing using high-resolution mass spectrometry (Q-TOF MS). The peptide, named peptide Tyrosine Arginine (pYR) shares 77.8% homology with peptide Leucine Arginine (pLR). The effects of the natural amidated peptide, non-amidated peptide and C-loop region of pYR on granulopoiesis and neutrophil apoptosis were investigated. All three analogues inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, the mature neutrophil. Thus, pYR is a novel member of an important and emerging new class of amphibian peptides with hemopoietic actions. (c) 2004 Elsevier Inc. All rights reserved.

Glycoprotein VI/Fc receptor gamma chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRgamma (Fc receptor gamma) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRgamma chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induced phosphorylation of the GPVI-regulated proteins Syk and SLP76 (Src homology 2-containing leucocyte protein of 76 kDa). A low level of tyrosine phosphorylation of phospholipase Cgamma2 was observed, which was increased in the presence of U46619, although the degree of phosphorylation remained well below that observed in wild-type platelets (similar to 10%). By contrast, collagen-induced phosphorylation of the adapter ADAP (adhesion- and degranulation-promoting adapter protein) was substantially potentiated by U46619 to levels equivalent to those observed in wild-type platelets. Collagen plus U46619 also induced significant phosphorylation of FAK (focal adhesion kinase). The functional significance of collagen-induced non-GPVI signals was highlighted by the ability of U46619 and collagen to induce the secretion of ATP in FcRgamma chain-deficient platelets, even though neither agonist was effective alone. Protein tyrosine phosphorylation and the release of ATP were abolished by the anti(alpha2 integrin) antibodies Ha1/29 and HMalpha2, but not by blockade of alphaIIbbeta3. These results illustrate a novel mechanism of platelet activation by collagen which is independent of the GPVI-FcRgamma chain complex, and is facilitated by binding of collagen to integrin alpha2beta1.

Chronic neutrophilic leukemia with an associated V617F JAK2 tyrosine kinase mutation

Chronic neutrophilic leukemia (CNL) is a rare disease and can cause considerable diagnostic difficulty. Although the V617F JAK2 mutation has been described by several groups to be associated with classical myeloproliferative disorders (MPD), this same mutation has been detected with a low incidence in atypical MPD, such as CNL. Here we report the presence of the V617F mutation in a CNL patient, who is unusual for having survived for more than 96 months, with little disease progression. It remains to be established what role this mutation, which gives cells a proliferative advantage, might play in the pathogenesis and prognosis of rare atypical MPD.

Effects of Charge Location on the Absorptions and Lifetimes of Protonated Tyrosine Peptides in Vacuo

Nearby charges affect the electronic energy levels of chromophores, with the extent of the effect being determined by the magnitude of the charge and degree of charge-chromophore separation. The molecular configuration dictates the charge chromophore distance. Hence, in this study, we aim to assess how the location of the charge influences the absorption of a set of model protonated and diprotonated peptide ions, and whether spectral differences are large enough to be identified. The studied ions were the dipeptide YK, the tripeptide KYK (Y = tyrosine; K = lysine) and their complexes with 18-crown-6-ether (CE). The CE targets the ammonium group by forming internal ionic hydrogen bonds and limits the folding of the peptide. In the tripeptide, the distance between the chromophore and the backbone ammonium is enlarged relative to that in the dipeptide. Experiments were performed in an electrostatic ion storage ring using a tunable laser system, and action spectra based on lifetime measurements were obtained in the range from 210 to 310 nm. The spectra are all quite similar though there seems to be some changes in the absorption band between 210 and 250 nm, while in the lower energy band all ions had a maximum absorption at similar to 275 nm. Lifetimes after photoexcitation were found to shorten upon protonation and lengthen upon CE complexation, in accordance with the increased number of degrees of freedom and an increase in activation energies for dissociation as the mobile proton model is no longer operative.

Characterisation of the Timing and Prevalence of Receptor Tyrosine Kinase Expression Changes in Esophageal Carcinogenesis

Despite being common in epithelial malignancies, the timing of receptor tyrosine kinase (RTK) up-regulation is poorly understood and therefore hampers the identification of the receptor to target for effective treatment. We aimed to determine if RTK expression changes were early events in carcinogenesis. Esophageal adenocarcinoma and its pre-invasive lesion, Barrett's esophagus, were used for immunohistochemical analysis of the RTK panel, EGFR, ErbB2, ErbB3, Met and FGFR2, by utilising a cohort of patients with invasive disease (n = 367) and two cohorts with pre-invasive disease, one cross-sectional (n = 110) and one longitudinal in time (n = 91). The results demonstrated that 51% of esophageal adenocarcinomas over-expressed at least one of the RTK panel; with 21% of these over-expressing multiple receptors. Up-regulation of RTK expression was an early event corresponding with low grade dysplasia development (25% in areas without dysplasia vs. 63% in low grade dysplasia, p