Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2

The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y(119) in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y(119) in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y(119)E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y(119)F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y(119) in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-gamma.     

Immunoreactivity of proteolytic degradation products of human prostatic acid phosphatase

Prostatic acid phosphatase (EC 3.1.3.2) was fragmented by trypsin and papain in the presence of sodium dodecyl sulphate. Trypsin-catalysed cleavage gave a peptide of 33 kDa which was subsequently trimmed to 18 kDa, 15 kDa and 13 kDa peptides. Even the small tryptic fragments reacted with antiphosphatase antibodies from rabbit serum and with monoclonal antibody mAb-14. Papain treatment under these conditions resulted in the release of a 40 kDa peptide which was gradually reduced to a 18 kDa peptide. The monoclonal antibody mAb-14 to the prostatic phosphatase was bound exclusively to the 50 kDa subunit of the phosphatase and to the 40 kDa peptide. The results suggest that the monoclonal antibody mAb-14 binding site represents a "local" sequence rather than a "conformational" one and does not require an extensive tertiary folding of the antigen molecule.     

Crystallinity of mineral deposited in arterial walls in the course of arteriosclerosis in diabetics and in patients with normal carbohydrate metabolism

A comparison was made of the amount and crystallinity of mineral deposited in the course of the arteriosclerosis in arterial walls in diabetic patients and in individuals with normal carbohydrate metabolism. The macroscopically unchanged tunica intima, fibro-lipidic plaques and bone-like lamellae were taken from aorta thoracalis , aorta abdominalis, arteriae femorales and arteriae coronariae in the course of the autopsy of 17 insulin dependent diabetic individuals and of 9 persons with arteriosclerosis, but with normal carbohydrate metabolism, called control group. The total inorganic constituents in the three kinds of samples were determined by ashing of dried samples at 600 degrees C for 6 h. Crystallinity of mineral was defined as the ratio of the spins connected with the radiation-induced stable paramagnetic centers present in the crystalline lattice of hydroxyapatite crystal, to the total ash content of the sample. An increase in the amount and in the crystallinity of deposited mineral was observed when consecutive stadia of development of arteriosclerotic lesions were compared. This phenomenon resembles the maturation of mineral observed in the course of bone development.     

Stabilization of human prostate acid phosphatase by cross-linking with diimidoesters

1. Modification of dimeric human prostate acid phosphatase (EC 3.1.3.2) by diimidoesters leads to the formation of water-soluble preparations of high enzymatic activity, resistant to denaturing agents. 2. Monomeric, dimeric, trimeric and tetrameric species were found in SDS-polyacrylamide gel electrophoresis of the phosphatase cross-linked with dimethyl-suberimidate, and dimeric, trimeric and tetrameric enzymatically active species on thin-layer Sephadex 200 gel filtration. This molecular pattern evidenced formation of the inter-subunit covalent linkages. All molecular forms are immunoreactive against the polyclonal rabbit anti-phosphatase antibodies. 3. The catalytic properties of the modified phosphatase are almost the same as those of the native enzyme. Differences in the optical properties between the modified and the native enzymes point to slight conformational transitions in the modified enzyme.     

Constitutive mutation of cysJIH operon in a cysB deletion strain of Salmonella typhimurium.

Summary In a cysB deletion strain a new mutation, denoted cys-2332 was isolated, which causes the constitutive expression of the cysJIH operon. cys-2332 is closely linked to cysJIH and presumably is located in the initiator region of this operon, rendering its expression independent of the cysB gene product and the internal inducer O-acetyl-L-serine. The presence of salfite reductase (encoded by cysI and cysJ) activity in a cysB ^- cys-2332 double mutant indicates that cysG, which is not linked to cysJIH but is required for the synthesis of the sulfite reductase co-factor siroheme, is not controlled by cysB.     

Spin label electron paramagnetic resonance (EPR) studies of Huntington disease erythrocyte membranes.

Several spin-label electron paramagnetic resonance (EPR) studies of red cell membranes appear to show abnormalities in some Huntington disease (HD) patients, but not in others. Both studies measure the W/S ratios, presumably under similar conditions, but have different results. We have studied the W/S ratio in some detail to gain a better understanding of this experimental parameter and to determine its potential application in detecting HD abnormalities. Our results offer little encouragement for continued use of W/S as an indication of membrane defect in HD.