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Biomedical subjects

L Ellis

Publications and source records attributed to L Ellis.

At least 109 records · Page 6Linked to original sources
PubMed1989

Expression in heterologous cells of the unusual cytoplasmic domain of rat brain 5B4/NCAM-ld.

Monoclonal antibody 5B4 recognizes a carboxy-terminal epitope common to large (approximately 180 kDa) and short (approximately 140 kDa) forms of neural cell adhesion molecules (NCAM-ld and NCAM-sd, respectively). The deduced primary sequence of rat brain 5B4/NCAM-ld predicts a large cytoplasmic domain (390 amino acids, Mr 39,284) of striking amino acid composition (52% proline, alanine, serine and threonine) and little predicted alpha or beta secondary structure: its function is unknown. To directly test the deduced topology of the protein, and especially the solubility and stability of its unusual cytoplasmic domain, we have constructed a cDNA expression vector designed to express this domain independently as a soluble protein (designated 5B4cyt) in a heterologous cell system (simian Cos cells). 5B4cyt is indeed soluble, but migrates anomalously on SDS-PAGE under denaturing and reducing conditions as two species of approximately 77 and approximately 80 kDa. In pulse chase experiments, the approximately 77 kDa band chases into the approximately 80 kDa band with a t1/2 of approximately 1 h. The difference in mobility is apparently a consequence of the rapid phosphorylation of the approximately 77 kDa species. The approximately 88 kDa phospho-form is reasonably stable with a t1/2 of approximately 6 h. These results are consistent with the deduced topology of 5B4/NCAM-ld, and demonstrate the feasibility of this experimental approach for exploring the biochemistry and structure of its unusual cytoplasmic domain.

Animals↗
PubMed1989

Isolation and sequence of lambda gt11 cDNA clones encoding the 5B4 antigen expressed on sprouting neurons.

Monoclonal antibody (mAb) 5B4 recognizes a developmentally regulated membrane glycoprotein (Mr approximately 185,000-255,000) expressed on sprouting neurons. The amino acid sequence deduced from lambda gt11 cDNA clones encoding the transmembrane and cytoplasmic domains of the 5B4 antigen is co-linear with that of chick NCAM-ld. The significant level of overall sequence identity (75%) demonstrates that the 5B4 antigen is rat brain NCAM. The 5B4 epitope maps to the carboxy-terminus common to both NCAM-ld and NCAM-sd.

Amino Acid Sequence↗
PubMed1989

Delinquency, sidedness, and sex.

One hundred and thirty male and 213 female college students responded to a self-report delinquency and crime measure and to a scale of sidedness preferences. Consistent with most of the literature on official delinquency and crime, left handedness was positively associated with involvement in several forms of delinquency (including some degree of criminality) among males. Other measures of sidedness among males largely failed to correlate with delinquency, however. Among females, sidedness was much less systematically related to delinquency than among males.

Adolescent↗
PubMed1989

Effects of auditory masking on lingual vibrotactile magnitude-estimation scaling responses of young children.

Studies of lingual vibrotactile magnitude-estimation scaling have focused primarily on the responses of normal young adults. Little is known about the scaling responses of young children to suprathreshold vibratory stimulation because of the complexities of the experimental procedure. Binaural auditory masking is one aspect of the procedure that has recently been studied in adults. The purpose of this study was to investigate the effects of binaural auditory masking on lingual vibrotactile magnitude-estimation responses of young children. Four children whose mean age was 4.6 yr. completed magnitude-estimation scaling tasks for eight lingual vibrotactile suprathreshold intensity levels (6, 10, 16, 20, 26, 30, 36, 40 dB SL) under masking and no masking conditions. Results suggest possible effects of auditory masking on the lingual vibrotactile magnitude-estimation scaling responses of these young children that have not been found for such responses of adults.

Auditory Perception↗
PubMed1989

Membrane proteins of the nerve growth cone and their developmental regulation.

The membrane polypeptides of growth cone fragments ("growth cone particles," GCPs) isolated from fetal rat brain by subcellular fractionation have been analyzed in further detail. The major polypeptides of salt-washed GCP membranes detected by 1-dimensional gel electrophoresis (Ellis et al., 1985b) resolve in 2-dimensional gels as a spot of 52 kDa that comigrates with beta-tubulin and reacts with anti-beta-tubulin; a 46 kDa, pl 4.3, polypeptide (pp46) that has no equivalent in the soluble fraction and is identical to one of the GCP's major phosphoproteins (Katz et al., 1985) and to GAP43 (Willard et al., 1985); a spot of 42 kDa that comigrates with actin; and a species of 34 kDa (p34) without soluble equivalent. The prominent 38 kDa doublet identified in 1-dimensional gels is difficult to resolve in 2-dimensional gels. The major phosphoproteins pp80ac, pp46, and pp40 (Katz et al., 1985), as well as p34 partition into the oil phase of Triton X-114 extracts, suggesting that they are integral membrane proteins, at least in our experimental conditions. The properties of pp46 reported here are in conflict with the highly hydrophilic amino acid sequence predicted for GAP43/B50/F1 (Basi et al., 1987; Karns et al., 1987). Growth-cone and presynaptic membrane proteins are compared as follows. After eye injection of 35S-methionine, GCPs and synaptosomes are isolated from the target areas of optic nerve of fetal and adult rats, respectively. Polypeptides are separated by 1- and 2-dimensional gel electrophoresis and the radiolabeled species identified fluorographically. The comparison of labeled GCP and synaptosome polypeptides shows that all 5 major Coomassie blue-stained polypeptides of GCP membranes (52, 46, 42, 38, 34 kDa) are intensely labeled after eye injection. However, in synaptosomes, these polypeptides are weakly labeled if at all; instead, an intensely labeled polypeptide of 28 kDa, and several additional species not seen in GCPs, have appeared. Therefore, the major growth cone membrane proteins are developmentally regulated, and the rates of synthesis and transport into the axonal ending of neuronal polypeptides change dramatically at the time of synaptogenesis.

Animals↗
PubMed1988

Estrogen and insulin synergism in neurite growth enhancement in vitro: mediation of steroid effects by interactions with growth factors?

Addition of estradiol to organotypic cultures of the fetal murine hypothalamus, preoptic area and cerebral cortex has been shown to elicit a striking enhancement of neurite growth which appears restricted to estrogen receptor-containing explant regions. The mechanisms underlying this response are unknown. An important question is whether the neurite enhancement which follows exposure to estradiol is due directly to the interaction of estrogen with the cell that was stimulated (the receptor-containing cell) or whether intermediate steps involving the possible interaction of estrogen and the endogenous polypeptide neurite-promoting growth factors or their receptors may play an important role. Recent findings in the cultures suggest that the effect of estrogen on neurite growth may involve synergistic interactions between estradiol and insulin-related peptides and may be important in regulating estrogen-responsive neurite growth in the central nervous system. Concurrent addition of estradiol and high levels of insulin (10 micrograms/ml or 50 micrograms/ml) to cultures of the olfactory bulb, hypothalamus, preoptic area and cerebral cortex of the fetal rat and mouse results in a dramatic acceleration and increase of neurite outgrowth which appears localized to estrogen receptor-containing explant regions. The supraphysiological concentrations of insulin required to elicit this response suggest that the factor(s) involved is unlikely to be insulin per se. Insulin may activate the receptor of different but closely related molecules such as the insulin-like growth factors (IGF)-I or -II to which it exhibits a relatively low affinity. Interactions between hormones and endogenous growth factors have been implicated in the modulation or mediation of an increasing number of endocrine-dependent, differentiative processes in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Truncation of the ectodomain of the human insulin receptor results in secretion of a soluble insulin binding protein from transfected CHO cells.

The insulin receptor is an integral transmembrane glycoprotein comprised of two alpha-(approximately 135 kDa) and two beta-(approximately 95 kDa) subunits, which is synthesized as a single polypeptide chain precursor (alpha beta). The primary sequence of the human insulin receptor (hIR) protein, deduced from the nucleotide sequence of cloned human placental mRNAs, predicts two large domains (929 and 403 residues) on either side of a single membrane spanning domain (23 residues); each of these major domains has a distinct function (insulin binding and protein/tyrosine kinase activity, respectively). To experimentally test this deduced topology, and to explore the potential for independent domain function by the hIR extracellular domain, we have constructed an expression plasmid encoding an hIR deletion mutant which is truncated 8 residues from the beginning of the predicted transmembrane domain (i.e., 921 residues). This domain of the hIR is in fact processed into alpha- and truncated beta-subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (alpha beta)2 dimer in the medium. This molecule is recognized by a battery of 13 monoclonal antibodies to epitopes on the IR extracellular domain, four of which block insulin binding and two of which require the native conformation of the IR for recognition. Further, this domain binds insulin with an apparent dissociation constant comparable to that of the wild-type hIR. However, the secreted dimer displays a linear Scatchard plot, while that of the wild-type membrane-associated hIR is curvilinear.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Respiratory syncytial virus.

RSV is now understood to be the most significant viral respiratory pathogen of infants and is capable of causing both bronchiolitis and pneumonia. It is a particular risk to hospitalized infants as the virus is easily spread through close contact. The most vulnerable infants are those who suffer with either congenital heart disease or bronchopulmonary dysplasia who easily fall prey to pulmonary complications of infection. Strict environmental control and the use of protective clothing and eyewear should be implemented to decrease the nosocomial spread of RSV. Available diagnostic studies include viral isolation, fluorescent antibody stains, and enzyme immunoassays. Treatment of the disease is usually supportive but hospitalized patients frequently benefit from aerosolized ribavirin therapy. Hopefully, current vaccine trials will be successful and this pathogen will not only be treatable but will also be preventable.

Child, Preschool↗
PubMed1988

Efficient expression in insect cells of a soluble, active human insulin receptor protein-tyrosine kinase domain by use of a baculovirus vector.

The human insulin receptor (IR) is a transmembrane glycoprotein, whose cytoplasmic domain contains an insulin-activated protein-tyrosine kinase (EC 2.7.1.112). By the use of an appropriately engineered baculovirus expression vector, a soluble cytoplasmic derivative of this domain was expressed in the insect cell line Spodoptera frugiperda (Sf9). At 24 to 48 h after Sf9 cells were infected with recombinant virus, a protein of the size expected for this domain (approximately 48 kilodaltons) constituted a major band when total cell lysates of metabolically labeled cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. This protein (designated AchIRPTK) was immunoprecipitated by three monoclonal antibodies, each of which recognizes a distinct antigenic site of the IR cytoplasmic domain and requires the native structure of the protein for recognition and one of which binds at or near the physiologically relevant site(s) of IR autophosphorylation. In vivo, AchIRPTK was phosphorylated on both tyrosine and serine residues. When affinity purified, the kinase was active in vitro; it autophosphorylated exclusively on tyrosine residues, and phosphorylated the exogenous substrates histone H2b and poly(Glu-Tyr). The expression of an active IR protein-tyrosine kinase molecule in this heterologous cell system provides an efficient experimental method for producing this domain in quantity for enzymatic and structural studies.

Animals↗
PubMed1988

Effect of auditory masking on lingual vibrotactile thresholds and magnitude estimation scaling responses.

Auditory masking has become a frequently employed part of the procedure used in vibrotactile research. Research investigating the effect of auditory masking on lingual vibrotactile thresholds of sensitivity has shown that there is little difference between lingual vibrotactile thresholds under masking and no masking conditions. The purpose of the present study was to extend the investigation of the effect of auditory masking to include lingual vibrotactile suprathreshold scaling responses. 20 young adult subjects of mean age 19 yr. completed lingual vibrotactile-threshold and magnitude-estimation scaling tasks under conditions of bilateral auditory masking and no masking. Similar lingual vibrotactile-threshold values and magnitude-estimation power-function exponents for the conditions of masking and no masking were noted.

Acoustic Stimulation↗
PubMed1987

Antibody-induced down-regulation of a mutated insulin receptor lacking an intact cytoplasmic domain.

Insulin receptor down-regulation was studied in various Chinese hamster ovary (CHO) cell lines expressing transfected human insulin receptor cDNAs. In addition to a cell line expressing the normal receptor (CHO.T line), three lines expressing mutated receptors were studied: the CHO.T-t line, which expresses a receptor with a degraded cytoplasmic domain due to the removal of the C-terminal 112 amino acids, and the CHO.YF1 and CHO.YF3 lines, in which important autophosphorylation sites of the receptor kinase (tyrosines-1162 and -1163) have been replaced by phenylalanine. A monoclonal anti-receptor antibody, but not insulin itself, was found to down-regulate cell surface receptor levels in all four cell lines by 60-80% after 18-h treatment at 37 degrees C. Down-regulation of the CHO.T and CHO.T-t receptors occurred at similar antibody concentrations and with a similar time course, although the maximum level of CHO.T-t down-regulation (60%) was generally lower than the level of CHO.T down-regulation (80%). Pulse-chase labeling of these two cell types with [35S]methionine revealed that antibody treatment of both CHO.T and CHO.T-t cells resulted in a similar increase in the rate of degradation of mature receptor subunits. These results indicate that antibody-induced down-regulation of the insulin receptor in these cells can occur in the absence of various autophosphorylation sites of the receptor and that the mechanism of antibody-induced down-regulation is different from that for insulin.

Animals↗
PubMed1987

Sexual orientation as a continuous variable: a comparison between the sexes.

Sexual orientation was measured as a continuous variable based upon reported percentage of sexual fantasies and percentage of sexual experiences involving members of the same sex. In the present sample (which probably somewhat overrepresented the homosexual end of the continuum) about one-third of both males and females reported at least occasionally fantasizing about sexually interacting with members of the same sex. The survey indicated about one-third of males also reported having had at least one intimate sexual experience with the same sex, whereas only 10% of females did so. Also, virtually all females who sexually fantasized about the same sex only did so occasionally, whereas most of the males who fantasized about the same sex did so much more exclusively. Although these results cannot be considered representative of the distribution of sexual orientation in any natural population, they suggest that there are advantages in measuring sexual orientation as a continuous variable instead of as one with only a few discrete categories. The results also support other studies that have concluded that when deviations from exclusive heterosexuality are present, they are likely to be much more extreme among males than among females.

Adult↗
PubMed1987

Heterologous transmembrane signaling by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells.

A hybrid receptor molecule composed of the extracellular ligand-binding domain of the human insulin receptor and the transmembrane and cytoplasmic (protein-tyrosine kinase) domains of the chicken sarcoma virus UR2 transforming protein p68gag-ros has been constructed and expressed in Chinese hamster ovary (CHO) cells. The hybrid is processed normally into alpha and hybrid beta subunits, is expressed on the cell surface at high levels, and binds insulin with near-wild-type affinity. Furthermore, insulin stimulates the phosphorylation on tyrosine residues of the hybrid beta subunit in vivo and the phosphorylation of an exogenous substrate [poly(Glu,Tyr)] in vitro. Thus the hybrid is capable of heterologous transmembrane signaling. However, the hybrid mediates neither the insulin-activated uptake of 2-deoxyglucose nor the incorporation of [3H]thymidine into DNA, suggesting that the physiological response(s) mediated by ligand-activated protein-tyrosine kinases may utilize distinct intracellular mechanisms for postreceptor signaling.

Animals↗
PubMed1987

Activation of transforming potential of the human insulin receptor gene.

A retrovirus containing part of the human insulin receptor (hIR) gene was constructed by replacing ros sequences in the avian sarcoma virus UR2 with hIR cDNA sequences coding for 46 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains of the beta subunit of hIR. The resulting virus, named UIR, contains the hIR sequence fused to the 5' portion of the UR2 gag gene coding for p19. UIR is capable of transforming chicken embryo fibroblasts and promoting formation of colonies in soft agar; however, it does not form tumors in vivo. A variant that arose from the parental UIR is capable of efficiently inducing sarcomas in vivo. UIR-transformed cells exhibit higher rates of glucose uptake and growth than normal cells. The 4-kilobase UIR genome codes for a membrane-associated, glycosylated gag-hIR fusion protein of 75 kDa designated P75gag-hir. P75gag-hir contains a protein tyrosine kinase activity that is capable of undergoing autophosphorylation and of phosphorylating foreign substrates in vitro; it is phosphorylated at both serine and tyrosine residues in vivo.

Animals↗
PubMed1987

A membrane-anchored cytoplasmic domain of the human insulin receptor mediates a constitutively elevated insulin-independent uptake of 2-deoxyglucose.

Insulin stimulates the autophosphorylation of the beta-subunit of the insulin receptor (IR) on tyrosine residues. Mutations which compromise IR autophosphorylation in vivo result in a decrease of the insulin-activated uptake of 2-deoxyglucose. These results are consistent with previous results which implicate IR autophosphorylation in the generation of the insulin response by cells. To further explore the specificity of the IR tyrosine phosphokinase (TPK) domain in IR function, we have altered the human IR (hIR) cDNA to encode truncated insulin-independent TPKs, which are expressed in chinese hamster ovary (CHO) cells as either membrane-anchored or cytosolic proteins. Both mutant hIRs exhibit TPK activity in vitro, although the cytosolic form is approximately 20 times more active. The carbohydrate moiety of the membrane-anchored form is of the high mannose type, consistent with an intracellular localization for this mutant hIR. The two mutant hIRs mediate very different physiological responses in transfected cells: the membrane-anchored, but not the cytosolic, hIR TPK mediates a constitutively elevated (135% the maximum insulin-stimulated response in CHO cells) insulin-independent uptake of 2-deoxyglucose. These results thus suggest that the hIR TPK is in fact specific for this aspect of IR function and, when membrane-associated, can mediate the insulin-independent uptake of 2-deoxyglucose. Neither of these mutant hIRs appears to transform CHO cells.

Animals↗
PubMed1987

The human insulin receptor cDNA: a new tool to study the function of this receptor.

The human insulin receptor (hIR) is an integral transmembrane glycoprotein comprised of two alpha and two beta subunits. An immediate consequence of insulin binding to the extracellular alpha subunit is the autophosphorylation of tyrosine residues on the intracellular domain of the beta subunit. The placental hIR cDNA has been cloned and sequenced, providing the primary structural features of the protein. In order to investigate the functions of the beta subunit and particularly the role of autophosphorylation and tyrosine phosphokinase (TPK) activity (a feature shared by other receptors and oncogene proteins) in transmembrane signalling, we designed an expression system of the hIR cDNA in eucaryotic cells. Superexpressing CHO cell lines that contain about 10(6) functional hIR/cell have been developed. In these cells half maximum stimulation of glucose uptake occurs at 5 X 10(-10)M insulin, whereas normal CHO cells require 5 X 10(-12)M insulin. In this expression system we have carried out site-directed mutagenesis experiments in which domains of the molecule have been deleted or particular amino acids have been replaced by others. The replacement of either or both the tyrosine residues 1162 and 1163 compromise an autophosphorylated site that is important for kinase function and the insulin response. Expression of an isolated membrane-bound form of the beta-subunit produces a 6 fold increase in glucose uptake. This insulin-independent effect disappears if the twin tyrosines are mutated or if the beta subunit is expressed in the cytoplasm. These studies also show that the C terminal 112 amino acid portion of the beta subunit is important for the stability of this protein.

Amino Acid Sequence↗