A Three-Dimensional Skeletal Reconstruction of the Stem Amniote Orobates pabsti (Diadectidae): Analyses of Body Mass,Centre of Mass Position,and Joint Mobility

Orobates pabsti, a basal diadectid from the lower Permian, is a key fossil for the understanding of early amniote evolution. Quantitative analysis of anatomical information suffers from fragmentation of fossil bones, plastic deformation due to diagenetic processes and fragile preservation within surrounding rock matrix, preventing further biomechanical investigation. Here we describe the steps taken to digitally reconstruct MNG 10181, the holotype specimen of Orobates pabsti, and subsequently use the digital reconstruction to assess body mass, position of the centre of mass in individual segments as well as the whole animal, and study joint mobility in the shoulder and hip joints. The shape of most fossil bone fragments could be recovered from micro-focus computed tomography scans. This also revealed structures that were hitherto hidden within the rock matrix. However, parts of the axial skeleton had to be modelled using relevant isolated bones from the same locality as templates. Based on the digital fossil, mass of MNG 10181 was estimated using a model of body shape that was varied within a plausible range to account for uncertainties of the dimension. In the mean estimate model the specimen had an estimated mass of circa 4 kg. Varying of the mass distribution amongst body segments further revealed that Orobates carried most of its weight on the hind limbs. Mostly unrestricted joint morphology further suggested that MNG 10181 was able to effectively generate propulsion with the pelvic limbs. The digital reconstruction is made available for future biomechanical studies.     

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.     

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.     

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.     

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.     

Priming of human polymorphonuclear leukocytes with granulocyte-macrophage colony-stimulating factor involves protein kinase C rather than enhanced calcium mobilisation.

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.     

Augmented mitogenesis and impaired metabolic signaling mediated by a truncated insulin receptor

Recently, we have described a COOH-terminal deletion mutation of the human insulin receptor (HIR delta CT) that exhibits normal insulin-mediated kinase activity and endocytosis, but is inefficient in stimulating glucose transport and glycogen synthase (McClain, D. A., Maegawa, H., Levy, J., Huecksteadt, T., Dull, T. J., Lee, J., Ullrich, A., and Olefsky, J.M. (1988) J. Biol. Chem. 263, 8904-8911; Maegawa, H., McClain, D. A., Freidenberg, G., Olefsky, J. M., Napier, M., Lipari, T., Dull, T. J., Lee, J., and Ullrich, A. (1988) J. Biol. Chem. 263, 8912-8917). In this paper, we report that despite this defect in metabolic signaling, the truncated receptor exhibits augmented mitogenic activity compared to normal receptors. These results were verified in three independently isolated clones of Rat 1 fibroblasts transfected with the HIR delta CT cDNA. The increase in insulin sensitivity of mitogenic stimulation was proportional to the number of HIR delta CT receptors expressed on the cells. By contrast, only the cells with normal receptors and none of the HIR delta CT clones exhibit increased sensitivity for a metabolic action of insulin, the stimulation of glucose uptake. Stimulation of cells by other mitogens and autoradiographic analysis confirm that the enhanced mitogenic effects seen in HIR delta CT cells are attributable only to the presence of the truncated insulin receptors. These receptors mediate the tyrosine phosphorylation of a number of cellular proteins, and the pattern of these phosphorylations differs quantitatively from that seen in cells with normal receptors. We conclude: 1) The COOH terminus plays a role in signaling metabolic actions of insulin, perhaps through its recognition of substrates for the receptor kinase. 2) By contrast, the COOH terminus is an inhibitory regulator of mitogenesis, and removal of the terminal 43 amino acids converts the receptor from a moderately active growth signaler to a very active one. 3) The changes seen in biologic activities of the HIR delta CT receptor are associated with quantitative changes in substrate phosphorylation by the receptor kinase.