Fluorescent glucagon derivatives. I. Synthesis and characterisation of fluorescent glucagon derivatives.

The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), 1.7.10(-8), 1.8.10(-8) and 5.4.10(-9) M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. From the IC50 values Kd values of 2.16.10(-9), 4.10(-9), 2.10(-9) and 1.72.10(-9) M were calculated for the binding of NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp25-glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp25-glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp25-glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp25-glucagon and 2-thiolTrp25-glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp25-glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.

Phosphoryl exchange is involved in the mechanism of the insulin receptor kinase.

The cytoplasmic kinase domain of the human insulin receptor (IRKD; M(r) 49 kDa) has been over-expressed in insect cells using the baculovirus expression system. To investigate the kinase mechanism, we have compared the stoichiometry of ADP formation and phosphoryl transfer. After an initial phase of autophosphorylation, ATP is consumed without a stoichiometric increase in incorporated phosphate. During substrate phosphorylation using poly(Glu:Tyr) (4:1) phosphoryl transfer comes close to ATP turnover, which is independent of the presence of the substrate, indicating an increased efficiency (i.e. ATP turnover/phosphate incorporation) of phosphoryl transfer. Autophosphorylation under pulse-chase conditions suggests the existence of a phosphoenzyme intermediate.

Autophosphorylation of the two C-terminal tyrosine residues Tyr1316 and Tyr1322 modulates the activity of the insulin receptor kinase in vitro.

Previously, several studies have demonstrated that autophosphorylation of the C-terminal tyrosine residues (Tyr1316 and Tyr1322) affects the signaling properties of the insulin receptor in vivo. To assess the biochemical consequences of the C-terminal phosphorylation in vitro, we have constructed, purified and characterized 45 kDa soluble insulin receptor kinase domains (IRKD), either with (IRKD) or without (IRKD-Y2F) the two C-terminal tyrosine phosphorylation sites, respectively. According to HPLC phosphopeptide mapping, autophosphorylation of the three tyrosines in the activation loop of the IRKD-Y2F kinase (Tyr1146, Tyr1150, and Tyr1151) was not affected by the mutation. In addition, the Y2F mutation did not significantly change the Km values for exogenous substrates. However, the mutation in IRKD-Y2F resulted in a decrease in the maximum velocities of the phosphotransferase reaction in substrate phosphorylation reactions. Moreover, the exchange of the tyrosines in IRKD-Y2F led to an increase in the apparent Km values for ATP, suggesting a cross-talk of the C-terminus and the catalytic domain of the enzyme. In addition, as judged by size exclusion chromatography, conformational changes of the enzyme following autophosphorylation were abolished by the removal of the two C-terminal tyrosines. These data suggest a regulatory role of the two C-terminal phosphorylation sites in the phosphotransferase activity of the insulin receptor.

Identification of Ser-1275 and Ser-1309 as autophosphorylation sites of the insulin receptor.

We have identified Ser-1275 and Ser-1309 as novel serine autophosphorylation sites by direct sequencing of HPLC-purified tryptic phosphopeptides of the histidine-tagged insulin receptor kinase IRKD-HIS. The corresponding peptides (Ser-1275, amino acids 1272-1292; Ser-1309, amino acids 1305-1313) have been detected in the HPLC profiles of both the soluble kinase IRKD, which contains the entire cytoplasmic domain of the insulin receptor beta-subunit, and the insulin receptor purified from human placenta. In contrast, a kinase negative mutant, IRKD-K1018A, did not undergo phosphorylation at either the tyrosine or serine residues, strongly suggesting that insulin receptor kinase has an intrinsic activity to autophosphorylate serine residues.

Gastric carcinoma: intestinal metaplasia and tumor growth patterns as indicators of prognosis.

We have reviewed 200 cases of gastric carcinoma treated between 1970 and 1980 to assess the value of intestinal metaplasia in the stomach and tumor growth patterns in determining prognosis. Intestinal metaplasia was found to be more frequently associated with early gastric tumors, expanding-type tumors, and tumors located in the antrum. The survival rate was 53% with intestinal metaplasia and 34% without. Sixty-three percent of expanding tumors with metaplasia survived. If the lymph nodes were not involved, the survival rate with metaplasia was 81%. We conclude that intestinal metaplasia and growth patterns are valuable in predicting outcome. Preoperative evaluation of gastric tumors should include multiple endoscopic mucosal biopsy specimens. If intestinal metaplasia is present, the improved possibility of survival should influence the surgeon in the choice of operative treatment.

Internal diameters of the ventriculo-arterial junctions and great arteries of normal infants and children: a data base for evaluation of congenital cardiac malformations.

In order to obtain reference data for a better evaluation of the operability of infants and children with congenital heart malformations, investigations were made on the growth of the aortic ventriculo-arterial junction along with the aortic arch as well as of the pulmonary root and the pulmonary tree. The internal diameters of the junctions and of various sites of both great arteries were measured in fresh post mortem specimens of 126 children having an age from 21 weeks of gestation up to 10 years after birth and who died from noncardiac diseases. Linear correlations were found between the internal diameters and body length. The post mortem data were in agreement with echocardiographic observations.

A human embryo of 28 mm crown-rump length with cerebral, esophagotracheal and cardiovascular malformations.

The following malformations were observed in a human embryo of 28 mm crown-rump length obtained at operation for tubal rupture in a case of extrauterine pregnancy: 1. Secondary anophthalmia with dysplasia and in part aplasia of the diencephalon. Rudiments of both eyes and eyestalklike proliferations within the diencephalon. No lenses and on the left side only a palpebral fissure. Hypoplasia of the right telencephalic hemisphere and of the right side of diencephalon, mesencephalon and proximal parts of the medulla oblongata. Pseudotumorous proliferations in the diencephalon, in the alar plate of the medulla oblongata (protruding into the fourth ventricle) and in the arachnoid. Hypoplasia of the right internal, middle, and external ear. Dysplasia and in part aplasia of facial osseous elements (cebocephalia). 2. Proximal esophageal atresia with distal tracheoesophageal fistula. 3. A Fallot's tetralogy with right-sided aortic arch and regressive right-sided ductus arteriosus, tricuspid atresia, hypoplasia of the right ventricle with excessive hypertrophy of its wall, and hypoplasia of the pulmonary trunk. Single left superior vena cava and abnormal, semicircular course of the stems of both coronary arteries.

Salvage of both lower extremities using simultaneous bilateral gastrocnemius and soleus muscle flaps: a case report.

We present a case of bilateral lower limb salvage in a young man with extensively exposed tibial bone following thermal injury. The medial head of the respective gastrocnemius muscle was used to cover the proximal third of debrided tibia, and the soleus muscle was used for the middle third coverage of each leg. This provided adequate, stable soft-tissue coverage for full recovery of both extremities and sufficient residual function for unassisted ambulation six months postoperatively.

Use of magnetic resonance spectroscopy in the evaluation of skin flap circulation.

The assessment of events that occur in elevated skin flaps has been largely by indirect methods. A method was sought that gives direct, reproducible, and accurate data about physiological and biochemical changes that occur during flap elevation and during periods of altered blood flow. Because of its ability to monitor changes in the levels of high energy phosphorus metabolites (ATP or adenosine triphosphate, PCr, or phosphocreatin, Pi, or inorganic phosphate), 31p magnetic resonance spectroscopy (MRS) holds promise of providing direct assessment of the metabolic status and biochemical changes that occur during skin flap elevation. MRS monitoring was performed on raised abdominal skin flaps of 12 rats. Abdominal flaps in 4 animals served as controls with and without total vascular occlusion while arterial blood flow was manipulated in 4 flaps and venous flow in 4 flaps. The results have validated the ability of MRS to determine cellular levels of ATP, PCr, and Pi in skin flaps, and to measure intracellular pH through the chemical shift of the Pi resonance.

Reduction mammaplasty with inferiorly based glandular pedicle flap.

We have reduced 60 breasts in 30 women using an inferiorly based glandular pedicle. Our complication rate has been extremely low and the technique offers major time-savings. Our method prevents the serious postoperative breast deformities frequently resulting from traditional methods of breast reduction.

Pharmacologic enhancement of functional recovery in free muscle transfers.

To date, most free muscle transfers have been marginally successful as functioning units, producing no more than 50 percent of the tension generated by an identical non-transferred muscle. Functional recovery depends both on revascularization and re-innervation, even after careful microvascular anastomosis, remains limited. Forskolin is a robust stimulator of adenylate cyclase and has been used to stimulate peripheral nerve regeneration in vivo; Forskolin may also directly increase capillary growth. Chronic infusion of Forskolin for the first 21 days following orthotopic transplantation of rabbit rectus femoris, with neurovascular anastomosis, stimulated a two-fold increase in the force-generating capacity of the transplant nine to 10 months after surgery. These results, in a model similar to clinical free muscle transfer, offer promise that Forskolin treatment can improve functional recovery by improving muscle re-innervation.

A single substitution of the insulin receptor kinase inhibits serine autophosphorylation in vitro: evidence for an interaction between the C-terminus and the activation loop.

We examined the effects of mutations of tyrosine and serine autophosphorylation sites on the dual specificity of the insulin receptor kinase (IRKD) in vitro using autophosphorylation and substrate phosphorylation and phosphopeptide mapping. For comparable studies, the recombinant kinases were overexpressed in the baculovirus system, purified, and analyzed. The phosphate incorporation into the enzymes was in the range of 3-4.5 mol/mol, and initial velocities of autophosphorylation were reduced up to 2-fold. However, the mutation Y1151F in the activation loop inhibited phosphate incorporation in the C-terminal serine residues 1275 and 1309, due to a 10-fold decrease of the initial velocity of serine autophosphorylation. Although the K(M) and V(MAX) values of this mutant were only slightly altered in substrate phosphorylation reactions using a recombinant C-terminal insulin receptor peptide (K(M): Y1151F, 9.9 +/- 0.4 microM; IRKD, 6.1 +/- 0.2 microM; V(MAX): Y1151F, 72 +/- 4 nmol min(-)(1) mg(-)(1); IRKD, 117 +/- 6 nmol min(-)(1) mg(-)(1)), diminished phosphate incorporation into serine residues of the peptide was observed. In contrast, the phosphorylation of a recombinant IRS-1 fragment, which was shown to be phosphorylated markedly on serine residues by IRKD, was not affected by any kinase mutation. These results underline that IRKD is a kinase with dual specificity. The substrate specificity toward C-terminal serine phosphorylation sites can be modified by a single amino acid substitution in the activation loop, whereas the specificity toward IRS-1 is not affected, suggesting that the C-terminus and the activation loop interact.

The insulin receptor: a protein kinase with dual specificity?

We have studied serine phosphorylation of the beta-subunit of highly purified human placental insulin receptors. Each purification step was analyzed with respect to phosphotyrosine and phosphoserine content, incorporated in the beta-subunit of the insulin receptor. Independent of the purification state the analysis of the phosphoamino acids of the insulin receptor beta-subunit showed tyrosine and serine phosphorylation in an insulin dependent manner. In the presence of insulin up to seven phosphates per alpha beta-half receptor, indicating a ratio of Tyr(P) and Ser(P) of approximate 3:1 were incorporated, while in the absence of the hormone this ratio did not exceed 1:10. Comparison of the phosphorylation reactions on tyrosine and serine residues makes it highly probable that both phosphoryltransfer reactions obey the same hormone dependence. Half maximal incorporation of total phosphate in the receptor protein was about 5 minutes in contrast to the half maximal serine phosphorylation of about 8 minutes. Our data corroborate that autophosphorylation of serine residues is an intrinsic activity of the receptor kinase itself suggesting a dual-specificity type protein kinase.

Mechanism of the phosphorylase reaction. Utilization of D-gluco-hept-1-enitol in the absence of primer.

alpha-Glucan phosphorylases from rabbit skeletal muscle, potato tubers and Escherichia coli catalyze the utilization of 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) in the presence of arsenate or phosphate. 1H-NMR analysis in the presence of 2H2O and arsenate indicated formation of 1-[1-2H]deoxy-alpha-D-glucoheptulose with rates comparable to the arsenolysis of poly- or oligosaccharides. The reaction depends on the presence of a dianionic 5'-phosphate group of pyridoxal in the active conformation of the phosphorylases. Heptenitol is the first known substrate of alpha-glucan phosphorylases which does not require a primer. This is explained by the finding that heptenitol is exclusively used as substrate for the degradative pathway of the phosphorylase reaction where it competes with polysaccharide substrates. In the presence of phosphate the reaction product is 1-deoxy-alpha-D-gluco-heptulose 2-phosphate (heptulose-2-P), which subsequently inhibits the reaction. This characterizes heptulose-2-P as an enzyme-derived inhibitor. The Ki = 1.9 X 10(-6) M with potato phosphorylase suggests the formation of a transition-state-like enzyme-ligand complex. These findings, together with the fact that the phosphates of heptulose-2-P and pyridoxal 5'-phosphate are linked by hydrogen bridges [Klein, H. W., Im, M. J., Palm, D. & Helmreich, E. J. M. (1984) Biochemistry 23, 5853-5861], make it likely that both phosphates are involved in phosphorylase catalysis. A catalytic mechanism of phosphorylase action is proposed in which a 'mobile' phosphate anion plays a versatile role. It serves as proton carrier for the substrate activation, it stabilizes the intermediate and acts as a nucleophile which can accept a glycosyl residue reversibly.

Structural requirements for signal transduction of the insulin receptor.

Structural requirements for signal processing by human placental insulin receptors have been examined. Insulin binding has been found to change the physico-chemical properties of (alpha beta)2 receptors solubilized with Triton X-100, indicating a marked alteration of the form, i.e. size and shape, of the molecular complex. (a) The Stokes radius decreases from about 9.5 nm to 7.9 nm, as determined by PAGE with Triton X-100 in the buffer (Triton X-100/PAGE), and from 9.1 nm to 8.7 nm, as assessed by gel filtration. (b) The sedimentation coefficient s20,w rises from 10.1 S to 11.4 S. Upon dissociation of the receptor-hormone complex, the alterations are reversed. After autophosphorylation of hormone-bound (alpha beta)2-insulin receptors, phosphate incorporation was found for 7.9-nm receptor forms when receptor-insulin complexes were crosslinked with disuccinimide suberate prior to Triton X-100/PAGE. However, phosphate incorporation was demonstrated for the 9.5-nm receptor forms when receptor-insulin complexes were not prevented from dissociation. This strongly indicates that the (alpha beta)2 receptor is autophosphorylated after assuming its 7.9-nm form upon insulin binding. Moreover, the insulin-dependent structural alterations are not affected by autophosphorylation. In contrast to (alpha beta)2 receptors, the diffusion and the sedimentation behaviour of alpha beta receptors, which carry a dormant tyrosine kinase even in the hormone-laden state, has been found to be insensitive to insulin binding. Different molecular properties of alpha beta and (alpha beta)2 receptors have also been detected by hormone binding studies. Insulin binding to (alpha beta)2 and alpha beta receptors differs markedly with respect to pH, ionic strength, and temperature. This might indicate that the structure of the hormone binding domain of alpha beta receptor changes on association into the (alpha beta)2 species. Alternatively, distinct hormone-induced conformational alterations at the molecular level of alpha beta and (alpha beta)2 receptor species may lead to the different binding properties. Our data demonstrate that the (alpha beta)2-insulin receptor undergoes extended conformational alterations upon insulin binding. This capacity for structural changes coincides with the hormone-inducable enhancement of tyrosine autophosphorylation of the 7.9-nm insulin-bound receptor form. In contrast, alpha beta receptors appear to be locked in an inactive nonconvertable state. Thus, interaction between two alpha beta receptor units is required to allow extended conformational alterations, which are assumed to be the triggering event for augmented auto-phosphorylation.

Enhancement of peripheral nerve regeneration by pharmacological activation of the cyclic AMP second messenger system.

This paper reviews the history of attempts to study peripheral nerve regeneration, focusing upon pharmacologic agents that may prove to be useful clinically. In particular, forskolin is described as a model for such an agent, and its mechanism of action, as a stimulator of the cyclic AMP second messenger system, is described.