Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G- CSF-mediated activation of signaling complexes of the p21ras route

Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G- CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G- CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R.     

The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.     

Remodeling of myocyte gap junctions in arrhythmogenic right ventricular cardiomyopathy due to a deletion in plakoglobin (Naxos disease).

OBJECTIVES: We tested the hypothesis that defective interactions between adhesion junctions and the cytoskeleton caused by the plakoglobin mutation in Naxos disease lead to remodeling of gap junctions and altered expression of the major gap junction protein, connexin43. BACKGROUND: Naxos disease, a recessive form of arrhythmogenic right ventricular cardiomyopathy, is associated with a high incidence of arrhythmias and sudden cardiac death. Naxos disease is caused by a mutation in plakoglobin, a protein that links cell-cell adhesion molecules to the cytoskeleton. METHODS: Myocardial expression of connexin43 and other intercellular junction proteins was characterized in 4 patients with Naxos disease. Immunohistochemistry was performed in all 4 patients, and immunoblotting and electron microscopy were performed in 1 patient who died in childhood before overt arrhythmogenic right ventricular cardiomyopathy had developed. RESULTS: Connexin43 expression at intercellular junctions was reduced significantly in both right and left ventricles in all patients with Naxos disease. Electron microscopy revealed smaller and fewer gap junctions interconnecting ventricular myocytes. Mutant plakoglobin was expressed but failed to localize normally at intercellular junctions. Localization of N-cadherin, alpha- and beta-catenins, plakophilin-2, desmoplakin-1, and desmocollin-2 at intercalated disks appeared normal. CONCLUSIONS: Remodeling of gap junctions occurs early in Naxos disease, presumably because of abnormal linkage between mechanical junctions and the cytoskeleton. Gap junction remodeling may produce a coupling defect which, combined with the subsequent development of pathologic changes in myocardium, could contribute to a highly arrhythmogenic substrate and enhance the risk of sudden death in Naxos disease.     

Natural bioburden levels detected on rigid lumened medical devices before and after cleaning

Controversy exists concerning the degree of microbial contamination associated with the us of rigid lumened medical devices, the efficacy of standard cleaning techniques used to remove pathogenic microorganisms from lumen channels, and whether patients are placed at risk of cross infection because of microbial contamination. In this study the level and types of microorganisms found on rigid lumened medical devices before and after cleaning in a hospital environment were investigated. The bioburden level after clinical use was found to be relatively low, ranging from 10¹ to 10⁴ colony forming units (CFU) per device. After the instruments were cleaned, none of the devices studied contained bioburden levels greater than 10⁴ CFU and 83% had bioburden levels less than or equal to 10² CFU. The bioburden present before cleaning was comprised of organisms derived from the handling of the device, from the hospital environment, and from the patient. The bioburden present after cleaning was comprised of organisms typically derived from the handling of the device and from the hospital environment. The level of bioburden per device was also related to the anatomic site where the device was used, with lower numbers of organisms found on devices exposed to sterile body sites and the respiratory tract.