Role of arginine-292 in the substrate specificity of aspartate aminotransferase as examined by site-directed mutagenesis

X-ray crystallographic data have implicated Arg-292 as the residue responsible for the preferred side-chain substrate specificity of aspartate aminotransferase. It forms a salt bridge with the beta or gamma carboxylate group of the substrate [Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., & Christen, P. (1984) J. Mol. Biol. 174, 497-525]. In order to test this proposal and, in addition, to attempt to reverse the substrate charge specificity of this enzyme, Arg-292 has been converted to Asp-292 by site-directed mutagenesis. The activity (kcat/KM) of the mutant enzyme, R292D, toward the natural anionic substrates L-aspartate, L-glutamate, and alpha-ketoglutarate is depressed by over 5 orders of magnitude, whereas the activity toward the keto acid pyruvate and a number of aromatic and other neutral amino acids is reduced by only 2-9 fold. These results confirm the proposal that Arg-292 is critical for the rapid turnover of substrates bearing anionic side chains and show further that, apart from the desired alteration, no major perturbations of the remainder of the molecule have been made. The activity of R292D toward the cationic amino acids L-arginine, L-lysine, and L-ornithine is increased by 9-16-fold over that of wild type and the ratio (kcat/KM)cationic/(kcat/KM)anionic is in the range 2-40-fold for R292D, whereas this ratio has a range of [(0.3-6) x 10(-6)]-fold for wild type. Thus, the mutation has produced an inversion of the substrate charge specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine releases PGI2 from human pulmonary artery

Histamine caused a triphasic response of human pulmonary artery strips in vitro, consisting of a small initial contraction followed by pronounced relaxation preceding a second contractile response. These characteristics were not seen with other contractile stimuli including 5-hdyroxytryptamine, leukotriene D₄, and KC1. The relaxant component of this response was ablated by removal of endothelium from the vascular strips or by pretreatment of the tissues with 1μM indomethacin. Measurement of the PGI₂ degradation product 6-keto-PGF_1α in supernatants from histamine-challenged tissues confirmed the synthesis of PGI₂. Supernatants from unstimulated or leukotriene-challenged tissues contained no detectable amounts of 6-keto-PGF_1α. The histamine H₁ antagonist diphenhydramine inhibited both the contractile and relaxant responses to histamine whereas the H₂ antagonist cimetidine affected neither component. The released PGI₂ significantly altered the dose-respons curve to histamine without inhibiting the maximal contractile responses. We conclude that histamine induces PGI₂ formation from pulmonary arterial endothelium via an H₁ receptor.     

An innervated cross-finger flap for fingertip reconstruction

An innervated cross-finger flap for treatment of severe fingertip injuries is described. With this method, the dorsal skin over the middle phalanx, together with its sensory nerve, is transferred as a compound skin-nerve flap. A neurorrhaphy is performed between this nerve and the cut end of the digital nerve at the injury site. Seven of eight patients (88 percent) treated with this method (mean follow-up time 14.4 months) achieved measurable two-point discrimination. The average for those who did was 4.8 mm. A group of patients with similar injuries treated with standard cross-finger flaps exhibited slower sensory return that progressed to a lower level. In this group (mean follow-up time 16.3 months), three of six (50 percent) achieved measurable two-point discrimination with a mean value of 9 mm.     

Venous Distensibility in Patients with Varicose Veins

Forearm veins were studied to determine whether patients with primary varicosity of the saphenous veins had a generalized abnormality of the venous system. Distensibility of the superficial forearm and hand veins was measured in 25 patients with varicosity of the saphenous veins, and in 25 control subjects. Patients with saphenous varicosity had a significantly greater distensibility of the undistorted forearm veins than control subjects. Hysteresis of distensibility curves was more prnounced in patients with varicosity than in control subjects; mean hysteresis index was 0.65 ± 0.06 versus 0.28 ± 0.02 in controls. These investigations suggest that an increased distensibility of the venous system is the predisposing factor in the development of varicose veins.     

Behavioural evidence for colour vision in stomatopod crustaceans

If an organism can be taught to respond in a particular way to a wavelength of light, irrespective of that light's intensity, then it must be able to perceive the colour of the stimulus. No marine invertebrate has yet been shown to have colour vision. Stomatopod crustaceans (mantis shrimps) are colourful animals and their eyes have many adaptations which indicate that they are capable of such spectral analysis. We adopted an associative learning paradigm to attempt to demonstrate colour vision. Stomatopods readily learnt to choose some colours from arrays of greys, even when the correct choice colours were darker than the ones they had been trained to. Possible mechanisms underlying colour vision in these animals, and their ecological significance are discussed. A simple model is presented which may help interpret the complex-stomatopod colour vision system and explain some of the learning anomalies.Abbreviations ND neutral density - OD optical density - R8 Retinular cell 8 - R1–7 Retinular cells 1–7 - R1D Distally placed R1–7 retinular cells in mid-band row 1 - e.g. R1P Proximally placed R1–7 retinular cells in mid-band row 1 - D/P Estimate of chromatic signal ratio     

Age-related differences in TGF-α and proto-oncogenes expression in rat liver after a low dose of carbon tetrachloride

The resiliency of rats during early postnatal development to CCl₄ or to an interactive hepatotoxicity of chlordecone (CD) + CCl₄ has been shown to be due to an efficient stimulation of tissue repair. The objective of the current study was to investigate if this is due to efficient expression of transforming growth factor-α (TGF-α) and proto-oncogenes. Postnatally developing (20 day old) and adult (60 day old) male Sprague–Dawley rats were challenged with a single low dose of CCl₄ (100 μL/kg, ip) or corn oil. Liver samples were collected during a time course (0–96 h) after the administration of CCl₄ and used to examine TGF-α and early (c-fos) and late (H-ras and K-ras) proto-oncogenes mRNA expressions. Significant increases in TGF-α, H-ras, and K-ras gene expressions were evident as early as 12 hours after CCl₄ and peaked between 24 and 48 hours in an age-dependent manner as detected by slot-blot analysis. Results of the study revealed three- and twofold increases in TGF-α gene expression in 20 and 60 day old rats, respectively, after CCl₄. There were 3.5- and 2.5-fold increases in H-ras and 4.4- and 3.4-fold increases in K-ras in 20 and 60 day old rats, respectively. In contrast, a 10-fold increase in c-fos mRNA expression was evident in 20 day old rats 1 hour after CCl₄ treatment, returning to the baseline value by 3 hours, whereas in 60 day old rats, this increase was less than twofold. The overall findings of this study indicate that TGF-α and the early and late proto-oncogene mRNA expressions were enhanced in an age- and time-dependent manner in response to a low dose of CCl₄. These results further strengthen the view that the remarkable resiliency of rats to hepatotoxicants during early postnatal development is due to substantial increases in stimulation of hepatocellular regeneration and tissue repair mechanisms, leading to regression of liver injury and recovery. © 1996 John Wiley & Sons, Inc.     

Role of FGFs in skeletal muscle and limb development

Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridgederived factor that participates in limb outgrowth and patterning. © 1994 Wiley-Liss, Inc.