A point mutation at the ATP-binding site of the EGF-receptor abolishes signal transduction.

The EGF-receptor (EGF-R) is a transmembrane glycoprotein with intrinsic protein tyrosine kinase (TK) activity. To explore the importance of the receptor TK in the action of EGF, we have used transfected NIH-3T3 cells expressing either the normal human EGF-R or a receptor mutated at Lys721, a key residue in the presumed ATP-binding region. The wild-type receptor responds to EGF by causing inositol phosphate formation, Ca2+ influx, activation of Na+/H+ exchange and DNA synthesis. In contrast, the TK-deficient mutant receptor fails to evoke any of these responses. It is concluded that activation of the receptor TK is a crucial signal that initiates the multiple post-receptor effects of EGF leading to DNA synthesis. Furthermore, the results suggest that tyrosine phosphorylation plays a role in the activation of the phosphoinositide signalling system.     

Aggregation and Sedimentation of Thalassiosira weissflogii (diatom) in a Warmer and More Acidified Future Ocean

Increasing Transparent Exopolymer Particle (TEP) formation during diatom blooms as a result of elevated temperature and pCO₂ have been suggested to result in enhanced aggregation and carbon flux, therewith potentially increasing the sequestration of carbon by the ocean. We present experimental results on TEP and aggregate formation by Thalassiosira weissflogii (diatom) in the presence or absence of bacteria under two temperature and three pCO₂ scenarios. During the aggregation phase of the experiment TEP formation was elevated at the higher temperature (20°C vs. 15°C), as predicted. However, in contrast to expectations based on the established relationship between TEP and aggregation, aggregation rates and sinking velocity of aggregates were depressed in warmer treatments, especially under ocean acidification conditions. If our experimental findings can be extrapolated to natural conditions, they would imply a reduction in carbon flux and potentially reduced carbon sequestration after diatom blooms in the future ocean.     

Cytoplasmic juxtamembrane region of the insulin receptor: a critical role in ATP binding, endogenous substrate phosphorylation, and insulin-stimulated bioeffects in CHO cells.

We have expressed in CHO cells a mutant receptor (IR delta 960) from which 12 amino acids in the juxtamembrane region (A954-D965), including Tyr960, have been deleted. The mutant receptor bound insulin normally but exhibited an increased Km for ATP during autophosphorylation. Upon prolonged incubation in vitro, or at high ATP concentrations such as those observed in vivo, autophosphorylation of IR delta 960 was similar to wild type, and the in vitro phosphotransferase activity of the autophosphorylated IR delta 960 was normal. These results suggest that the deletion did not cause a nonspecific structural disruption of the catalytic domain of IR delta 960. In vivo autophosphorylation of the IR delta 960 receptor was reduced by 30% after 2 min of insulin stimulation and was similar to the wild-type receptor after 30 min of insulin stimulation. However, the mutant receptor was defective in insulin-stimulated tyrosyl phosphorylation of the endogenous substrate pp185. In addition, IR delta 960 was deficient in mediating insulin stimulation of glycogen and DNA synthesis. Thus, autophosphorylation of the insulin receptor is necessary but not sufficient for signal transmission. These data extend the hypothesis that the cytoplasmic juxtamembrane region of the insulin receptor is important for its interactions with ATP, intracellular substrates, and other proteins and is broadly necessary for biological signal transmission.     

Effects of 28Si Ions, 56Fe Ions,and Protons on the Induction of Murine Acute Myeloid Leukemia and Hepatocellular Carcinoma

Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the carcinogenic effects of 300 MeV/n ²⁸Si or 600 MeV/n ⁵⁶Fe ions in a mouse model for radiation-induced acute myeloid leukemia and hepatocellular carcinoma. C3H/HeNCrl mice were irradiated with 0.1, 0.2, 0.4, or 1 Gy of 300 MeV/n ²⁸Si ions, 600 MeV/n ⁵⁶Fe ions or 1 or 2 Gy of protons simulating the 1972 solar particle event (1972SPE) at the NASA Space Radiation Laboratory. Additional mice were irradiated with ¹³⁷Cs gamma rays at doses of 1, 2, or 3 Gy. All groups were followed until they were moribund or reached 800 days of age. We found that ²⁸Si or ⁵⁶Fe ions do not appear to be substantially more effective than gamma rays for the induction of acute myeloid leukemia. However, ²⁸Si or ⁵⁶Fe ion irradiated mice had a much higher incidence of hepatocellular carcinoma than gamma ray irradiated or proton irradiated mice. These data demonstrate a clear difference in the effects of these HZE ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis. Also seen in this study was an increase in metastatic hepatocellular carcinoma in the ²⁸Si and ⁵⁶Fe ion irradiated mice compared with those exposed to gamma rays or 1972SPE protons, a finding with important implications for setting radiation exposure limits for space-flight crew members.     

Regioselective preparation of 5-hydroxypropranolol and 4′-hydroxydiclofenac with a fungal peroxygenase

An extracellular peroxygenase of Agrocybe aegerita catalyzed the H₂O₂-dependent hydroxylation of the multi-function beta-adrenergic blocker propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol) and the non-steroidal anti-inflammatory drug diclofenac (2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid) to give the human drug metabolites 5-hydroxypropranolol (5-OHP) and 4′-hydroxydiclofenac (4′-OHD). The reactions proceeded regioselectively with high isomeric purity and gave the desired 5-OHP and 4′-OHD in yields up to 20% and 65%, respectively. ¹⁸O-labeling experiments showed that the phenolic hydroxyl groups in 5-OHP and 4′-OHD originated from H₂O₂, which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of drug metabolites.     

The omega-hydroxylation of arachidonic acid by human polymorphonuclear leukocytes

Incubations of [1-14C]arachidonic acid with unstimulated human polymorphonuclear leukocytes resulted in the formation of four new metabolites in a previously described reverse-phase HPLC system. Three of these metabolites were largely suppressed in a CO/O2 (80/20, by vol.) atmosphere indicating a cytochrome-P450-dependent monooxygenase reaction. In agreement with this assumption is their NADPH/O2-dependent formation in the microsomal fraction. One metabolite was identified by gas chromatography/mass spectrometry analysis as omega-hydroxy-arachidonic acid and the two others were secondary products identified as omega-carboxy-arachidonic acid and 5,20-dihydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid. Since the affinity for arachidonate of the omega-monooxygenase was quite low and the presence of LTB4 suppressed the omega-hydroxylation of arachidonate, we conclude that the known LTB4 omega-monooxygenase is responsible for the formation of omega-hydroxy-arachidonate. It is unlikely, however, that significant concentrations of these metabolites are formed by activated polymorphonuclear leukocytes in vivo. The fourth metabolite remains tightly associated with the leukocytes but has not been further characterized.     

Einige Beobachtungen über Doppelbrechung am Lebenden Protoplasten,an Verschiedenen Zellorganellen Sowie der Zellwand

Ohne ZusammenfassungMit 5 Textabbildungen.