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Biomedical subjects

L Ellis

Publications and source records attributed to L Ellis.

At least 127 records · Page 7Linked to original sources
PubMed1986

Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose.

Insulin stimulates the autophosphorylation of tyrosine residues of the beta subunit of the insulin receptor (IR); this modified insulin-independent kinase has increased activity toward exogenous substrates in vitro. We show here that replacement of one or both of the twin tyrosines (residues 1162 and 1163) with phenylalanine results in a dramatic reduction in or loss of insulin-activated autophosphorylation and kinase activity in vitro. In vivo, these mutations not only result in a substantial decrease in insulin-stimulated IR autophosphorylation but also in a parallel decrease in the insulin-activated uptake of 2-deoxyglucose. Furthermore, a truncated IR protein (lacking the last 112 amino acids) has an unstable beta subunit; this mutant has no kinase activity in vitro or in vivo and does not mediate insulin-stimulated uptake of 2-deoxyglucose. IR autophosphorylation is thus implicated in the regulation of IR activities, with tyrosines 1162 and 1163 as major sites of this regulation.

Amino Acid Sequence↗
PubMed1986

The 5B4 antigen expressed on sprouting neurons contains alpha-2,8-linked polysialic acid.

Monoclonal antibody 5B4 recognizes a large (approximately 185,000-255,000 Da) developmentally regulated membrane glycoprotein, whose expression on fetal rat neurons is coincident with neuronal sprouting. By the use of two prokaryotic-derived probes specific for recognizing alpha-2,8-linked polysialosyl units, we demonstrate the presence of this unusual carbohydrate moiety (a characteristic of neural cell adhesion molecules, NCAMs) on the fetal form of the 5B4 antigen. The 5B4 antigens expressed in adult rat brain (approximately 140,000 and 180,000 Da) do not contain polysialic acid.

Animals↗
PubMed1986

Linking functional domains of the human insulin receptor with the bacterial aspartate receptor.

A hybrid receptor has been constructed that is composed of the extracellular domain of the human insulin receptor fused to the transmembrane and cytoplasmic domains of the bacterial aspartate chemoreceptor. This hybrid protein can be expressed in rodent (CHO) cells and displays several functional features comparable to wild-type insulin receptor. It is localized to the cell surface, binds insulin with high affinity, forms oligomers, and is recognized by conformation-specific monoclonal antibodies. Although most of the expressed protein accumulates as a 180-kDa proreceptor, some processed 135-kDa receptor can be detected on the cell surface by covalent cross-linking. Expression of the hybrid receptor inhibits the insulin-activated uptake of 2-deoxyglucose by CHO cells. Thus, this hybrid is partially functional and can be processed; however, it is incapable of native transmembrane signaling. The results indicate that the intact domains of different types of receptors can retain some of the native features in a hybrid molecule but specific requirements will need to be satisfied for transmembrane signaling.

Amino Acid Sequence↗
PubMed1986

Mechanisms of receptor-mediated transmembrane communication.

Our experiments with the hIR protein have been designed to address a very general question of transmembrane receptor structure and function: What are the roles and interactions of the various deduced structural domains of such molecules in the initiation of the response of cells to extracellular signals? All of the evidence to date supports the previous hypothesis based on biochemical data that the IR requires ligand-activated TPK functions to initiate the insulin response by cells (for review, see Kahn 1985). Thus, mutations that compromise hIR TPK activity (site-directed point mutations or deletions) result in a concomitant decrease in at least one aspect of insulin action (glucose uptake; Ellis et al. 1986a). Other studies utilizing microinjection of antibodies to inhibit the receptor kinase have extended this conclusion to include a critical role for the receptor kinase in insulin's ability to stimulate ribosomal protein S6 phosphorylation in CHO cells, glycogen synthetase in hepatoma cells, glucose uptake in adipocytes (Morgan and Roth 1987), and frog oocyte maturation (Morgan et al. 1986). Second, analyses of cell lines that express experimentally truncated hIR TPKs demonstrate that, when membrane-anchored, this TPK domain is in fact capable of autonomous hormone-independent IR function: Such cells exhibit a constitutively elevated, insulin-independent uptake of 2-deoxyglucose (Ellis et al. 1987). Finally, by substitution of a homologous TPK for that of hIR, we find that although such a hybrid is capable of insulin-dependent transmembrane signaling (phosphorylation of the hybrid beta-subunit on tyrosine residues), the hybrid IR.ros molecule does not function as an IR in such cells: It mediates neither short-term (uptake of 2-deoxyglucose) nor long-term (incorporation of [3H]thymidine) effects of insulin (L. Ellis et al., in prep.). Together, these results suggest that (1) the hIR TPK domain conveys a substrate specificity for the insulin response and (2) that a functional hIR extracellular domain alone is not sufficient for generation of the insulin response (e.g., ligand-induced aggregation, or simple delivery of insulin into the cell). With the linking of the extracellular and cytoplasmic domains of the hIR molecule has evolved a cellular mechanism for the control of hIR TPK activity; the result is that cells which express the IR are now insulin responsive, and the physiological responses associated with the hormone are ligand-activated. Thus, the uncontrolled state of autonomous TPK activity, with the associated constitutive physiological response (e.g., as exhibited by the spBam hIR mutant), is circumvented.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling.

A cloned approximately 5 kb cDNA (human placenta) contains the coding sequences for the insulin receptor. The nucleotide sequence predicts a 1382 amino acid precursor. The alpha subunit comprises the N-terminal portion of the precursor and contains a striking cysteine-rich "cross-linking" domain. The beta-subunit (the C-terminal portion of the precursor) contains a transmembrane domain and, in the intracellular region, the elements of a tyrosine phosphokinase: an ATP-binding site and a possible tyrosine autophosphorylation site or sites. The overall structure is reminiscent of the EGF receptor; the cross-linking domain of the alpha subunit and several regions of the beta subunit exhibit sequence homology with the EGF receptor. The phosphokinase domain also exhibits homology with some oncogenic proteins that have tyrosine phosphokinase activity, in particular, a striking homology with v-ros. Southern blotting experiments suggest that the coding region spans more than 45 kb. The insulin receptor gene is located on chromosome 19.

Amino Acid Sequence↗
PubMed1985

Elevated serum immunoreactive pancreatic cationic trypsinogen in acute malnutrition: evidence of pancreatic damage.

We used a sensitive probe of pancreatic dysfunction, serum immunoreactive cationic trypsinogen, to study 50 infants and children with varying degrees of malnutrition. Patients were classified into subgroups according to the severity of malnutrition. Mean serum trypsinogen concentration was significantly elevated in 25 patients with "severe" malnutrition (77.4 +/- 42.0 ng/ml, P less than 0.001) and in 23 with "moderate" malnutrition (55.2 +/- 16.1 ng/ml, P less than 0.02) compared with the mean value (32.5 +/- 10.4 ng/ml) for well-nourished controls. The level of circulating trypsinogen tended to rise with increasing severity of malnutrition. There was no relationship between serum trypsinogen and other variables such as age, specific diagnosis, or mode of feeling. Elevated serum trypsinogen levels could not be attributed to renal disease or cystic fibrosis. In patients who showed an improvement in nutritional status, serum trypsinogen tended to revert toward normal. Elevated serum trypsinogen values in acutely malnourished infants and children may result from pancreatic acinar cell damage or regurgitation of enzymes from obstructed pancreatic ducts.

Acute Disease↗
PubMed1985

Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells.

We have placed human insulin receptor cDNA into a vector under the control of the simian virus 40 (SV40) early promoter and tested its function by transient expression in microinjected Xenopus oocytes and by expression in stably transformed CHO cells. The precursor and the alpha and beta subunits of the receptor were detected by immunoprecipitation from extracts of these cells. The human insulin receptor expressed in CHO cells specifically binds 125I-labeled insulin but not insulin-like growth factor I, displays insulin-stimulated autophosphorylation of the beta subunit, and mediates insulin-stimulated 2-deoxyglucose uptake. We conclude that the human insulin receptor is synthesized, processed normally, and functional in this heterologous cell system.

Animals↗
PubMed1985

Nerve growth cones isolated from fetal rat brain. IV. Preparation of a membrane subfraction and identification of a membrane glycoprotein expressed on sprouting neurons.

This study describes the preparation of a membrane subfraction from isolated nerve growth cone particles (GCPs) (see Pfenninger, K. H., L. Ellis, M. P. Johnson, L. B. Friedman, and S. Somlo, 1983, Cell, 35:573-584) and the identification in this fraction of a glycoprotein expressed during neurite growth. While approximately 40 major polypeptides are visible in Coomassie Blue-stained SDS polyacrylamide gels of pelleted (partially disrupted) GCPs, a salt-washed membrane fraction prepared from lysed, detergent-permeabilized GCPs contains only 14% of this protein and has an unusually simple polypeptide pattern of seven major bands. Monoclonal antibodies have been generated to GCP membranes isolated from fetal rat brain. These antibodies have been screened differentially with synaptosomes from adult rat brain in order to identify those which recognize antigens expressed selectively during neurite growth. One such antibody (termed 5B4) recognizes a developmentally regulated membrane glycoprotein that is enriched in GCP membranes and expressed in fetal neurons sprouting in vitro. The 5B4 antigen in fetal brain migrates in SDS polyacrylamide gels as a diffuse band of approximately 185-255 kD, is rich in sialic acid, and consists of a small family of isoelectric variants. Freezing-thawing and neuraminidase digestion result in the cleavage of the native antigen into two new species migrating diffusely around 200 and 160 kD. Prolonged neuraminidase digestion sharpens these bands at about 180 and 135 kD, respectively. In the mature brain, antibody 5B4 recognizes a sparse polypeptide migrating at approximately 140 kD. As shown in the following paper (Wallis, I., L. Ellis, K. Suh, and K. H. Pfenninger, 1985, J. Cell Biol., 101:1990-1998), the fetal antigen is specifically associated with regions of neuronal sprouting and, therefore, can be used as a molecular marker of neurite growth.

Animals↗
PubMed1985

Immunolocalization of a neuronal growth-dependent membrane glycoprotein.

Monoclonal antibody (mAb) 5B4 recognizes in the rat a large, developmentally regulated membrane glycoprotein. The larger form of this antigen (185-255 kD) occurs in the developing nervous system and is present in membranes of nerve growth cones, as determined by analysis of a growth cone particle fraction. An immunochemical characterization of this antigen and of a smaller form (140 kD), sparsely present in the mature nervous system, has been described (Ellis, L., I. Wallis, E. Abreu, and K. H. Pfenninger, 1985, J. Cell. Biol., 101:1977-1989). The present paper reports on the localization by immunofluorescence of 5B4 antigen in cultured cortical neurons, developing spinal cord, and the mature olfactory system. In culture, mAb 5B4 stains only neurons; it is sparsely present in neurons at the onset of sprouting while, during sprouting, it appears to be concentrated at the growth cone and in regions of the perikaryon. In the developing spinal cord, 5B4 labeling is faintly detectable on embryonic day 11 but is intense on fetal day 13. At this stage, the fluorescence is observed in regions of the cord where axonal growth is occurring, while areas composed of dividing or migrating neural cells are nonfluorescent. With maturation of the spinal cord, this basic pattern of fluorescence persists initially, but the staining intensity decreases dramatically. In the adult, faint fluorescence is detectable only in gray matter, presumably indicating the presence of the 140 kD rather than the fetal antigen. The only known structure of the adult mammalian nervous system where axonal growth normally occurs is the olfactory nerve. mAb 5B4 intensely stains a variable proportion of olfactory axons in the mucosa as well as in the olfactory bulb. Based on both immunochemical and immunofluorescence data, the 5B4 antigen of 185-255 kD is associated specifically with growing neurons, i.e., neurons that are generating neurites.

Animals↗
PubMed1985

Nerve growth cones isolated from fetal rat brain. II. Cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins and cAMP-dependent protein phosphorylation.

Cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins and cAMP-dependent protein phosphorylation were examined in growth cone particles (GCPs) prepared from fetal rat brain. Several major proteins which specifically bind a photoactivatable analogue of cAMP are observed in GCPs and correspond to isoelectric variants of the regulatory subunits of the cAMP-dependent protein kinase described in adult brain. We found no evidence for differential compartmentalization of specific cAMP-binding proteins in subcellular fractions of fetal brain or within GCPs. cAMP-stimulated phosphoproteins of GCPs are similar to cAMP-dependent protein kinase substrates characterized in nerve terminals (synaptosomes) of adult brain and include the nerve terminal-specific protein, synapsin I. However, as shown in the companion paper (Katz, F., L. Ellis, and K. H. Pfenninger (1985) J. Neurosci. 5: 1402-1411), this synaptic phosphoprotein is not the major kinase substrate in the GCP fraction. The finding of synapsin I in a subcellular fraction prepared from fetal brain suggests that components of the mature nerve terminal are already present in fetal brain during neuronal sprouting and prior to synaptogenesis.

Animals↗
PubMed1985

Nerve growth cones isolated from fetal rat brain. III. Calcium-dependent protein phosphorylation.

Calcium-dependent protein kinase activities have been studied in nerve growth cone particles (GCPs) and compared with those of synaptosomes. GCPs contain a set of phosphoproteins qualitatively similar to that of synaptic nerve terminals. However, major quantitative differences appear to exist: whereas synapsin I phosphorylation is relatively weak, the major kinase substrates of GCPs are a 46,000-dalton membrane protein (calcium/calmodulin dependent) and two acidic proteins of 80,000 and 40,000 daltons, phosphorylated by a calcium/phospholipid-dependent protein kinase. The presence of synaptic kinase activities in GCPs is consistent with their neuronal origin. The role of these kinases in GCPs is not understood at present. They may be involved in growth-related functions and/or may prepare the sprouting neuron for synaptic function.

Animals↗
PubMed1984

Postoperative recurrence in Crohn's disease. The effect of the initial length of bowel resection and operative procedure.

We reviewed the surgical experience of 61 patients with Crohn's disease who have received surgical treatment over a 32-year period. Sex, age at onset of symptoms, associated systemic abnormalities, presenting symptoms, indication for previous surgery, and site of disease were not significant predictors of postoperative recurrence. Certain extensive resections of the small bowel are associated with a decreased probability of rehospitalization and reoperation. Resection of more than 25 cm of the small bowel and more than 50 cm of the "total" (small plus large) bowel was associated with a decreased likelihood of recurrence. Interestingly, analysis of larger resections (50, 75, 100 cm) failed to document a decreased likelihood of recurrence. The amount of large bowel resected did not predict postoperative recurrence. Bypass and diversion procedures offer a significantly enhanced risk for recurrent disease, whereas procedures employing resection are associated with lower probabilities of recurrent disease. We conclude that technically adequate resections of 25 to 50 cm of the small bowel or the combined small and large bowel are associated with a decreased probability of reoperation or rehospitalization after the initial surgery for Crohn's disease.

Adolescent↗
PubMed1984

Studies on equine prematurity 3: Insulin secretion in the foal during the perinatal period.

The factors influencing beta cell function in the foetal and neonatal foal have been investigated in chronically catheterised foetal foals and in newborn foals delivered either spontaneously at term or by induction at different gestational ages. Insulin was detected in the foetal plasma from as early as 150 days of gestation (term = 340 days) and during the last third of gestation the foetal beta cells responded to exogenous administration of glucose and arginine and to endogenous variations in the glucose level. Insulin secretion by the foetal beta cells was depressed by anaesthesia and surgery. At birth, there was a significant positive correlation between the plasma concentrations of insulin and glucose irrespective of the maturity at birth or type of delivery (r = 0.86, n = 39, P less than 0.01). The slope of this relationship was significantly less than that relating the postoperative foetal concentration but only when delivery was difficult or prolonged was the beta cell sensitivity to glucose completely abolished. At birth, there were no significant differences in the plasma concentrations of insulin or glucose between full term foals delivered spontaneously or by induction. However, the spontaneously delivered foals showed a transient increase in the insulin concentration 15 mins after birth which was not observed in the full term foals delivered by induction. Plasma glucose concentrations were maintained during the 2 h after birth in the absence of sucking in both the induced and the spontaneously delivered full term foals. Premature foals had significantly lower plasma glucose concentrations at birth than full term foals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Nerve growth cones isolated from fetal rat brain: subcellular fractionation and characterization.

The biochemical and functional characterization of the nerve growth cone is of major interest for studies on mechanisms involved in nervous system development. We describe the isolation from fetal brain of membrane-bound fragments of nerve growth cones by density gradient fractionation. These so-called growth cone particles are highly uniform and identifiable on the basis of their organelle complement. Furthermore, they co-purify in mixing experiments with fragments of radiolabeled and light microscopically identified nerve growth cones from primary cultures. The possibility of isolating growth cone fragments in quantity renders feasible the analysis of molecular mechanisms involved in growth cone function.

Animals↗
PubMed1982

Response of the adrenal cortex to tetracosactrin (ACTH1-24) in the premature and full-term foal.

The changes in plasma cortisol concentration in the immediate postnatal period were examined in 3 groups of newborn foals and the response of the adrenal cortex to exogenous ACTH1-24 (tetracosactrin) was tested in 2 of these groups. In full-term Thoroughbred and Pony foals a rise in plasma cortisol occurred between 0 and 30 min after birth, whereas no significant cortisol changes could be detected within 2 h of birth in the group of prematurely delivered foals. These differences in plasma cortisol between term and premature foals were accompanied by differences in blood pH and lymphocyte and neutrophil counts. The high cortisol concentrations in the term animals declined to low levels by 4-12 h after birth. There was also a clear distinction between the responsiveness of the adrenal cortex to ACTH in the term and premature animals. When short-acting tetracosactrin was given as a single i.m. injection (0.125 mg), a rapid rise in plasma cortisol occurred in all term foals whereas no response was detected in the premature group during the same 2-h test period. The adrenal cortical response was also accompanied by a significant fall in the neutrophil/lymphocyte ratio at 2 h after the injection; no such change was seen in the premature animals. After a 24-h period of treatment with depot tetracosactrin (3 doses; 0.4, 0.2, 0.2 mg i.m.), a 2nd test with short-acting tetracosactrin led to a rise in plasma cortisol in term and pre-term foals during the 2-h test period. The depot tetracosactrin also appeared to cause a 3-fold increase in basal plasma cortisol levels in both groups.

Adrenal Cortex↗