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Biomedical subjects

R Gilmore

Publications and source records attributed to R Gilmore.

At least 91 records · Page 5Linked to original sources
PubMed1988

Guanosine triphosphate promotes the post-translational integration of opsin into the endoplasmic reticulum membrane.

Membrane integration of a nascent opsin polypeptide was examined to determine whether insertion of proteins into the endoplasmic reticulum is dependent upon energy provided by ribonucleotide triphosphate hydrolysis. A discrete-sized nascent chain was obtained by in vitro translation of a mRNA which lacked a termination codon yet encoded the first 156 residues of bovine opsin. Ribosomes bearing the newly synthesized opsin chains were post-translationally incubated with canine pancreas microsomal membrane vesicles after addition of exogenous ribonucleotides or ribonucleotide analogues. Post-translational membrane integration and glycosylation of the 156-residue nascent polypeptide was found to require either the presence of guanosine triphosphate or a nonhydrolyzable GTP analogue. ATP did not promote post-translational integration of the nascent polypeptide. Although ribonucleotide hydrolysis was not obligatorily required for integration of opsin, we observed an increase in the proportion of glycosylated opsin chains in post-translational incubations that contained hydrolyzable ribonucleotide triphosphates. We conclude that a GTP-binding protein performs an essential role during integration of opsin into the endoplasmic reticulum.

Animals↗
PubMed1988

Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus.

The M2 protein of influenza A virus is a small integral membrane protein of 97 residues that is expressed on the surface of virus-infected cells. M2 has an unusual structure as it lacks a cleavable signal sequence yet contains an ectoplasmic amino-terminal domain of 23 residues, a 19 residue hydrophobic transmembrane spanning segment, and a cytoplasmic carboxyl-terminal domain of 55 residues. Oligonucleotide-mediated deletion mutagenesis was used to construct a series of M2 mutants lacking portions of the hydrophobic segment. Membrane integration of the M2 protein was examined by in vitro translation of synthetic mRNA transcripts prepared using bacteriophage T7 RNA polymerase. After membrane integration, M2 was resistant to alkaline extraction and was converted to an Mr approximately equal to 7,000 membrane-protected fragment after digestion with trypsin. In vitro integration of M2 requires the cotranslational presence of the signal recognition particle. Deletion of as few as two residues from the hydrophobic segment of M2 markedly decreases the efficiency of membrane integration, whereas deletion of six residues completely eliminates integration. M2 proteins containing deletions that eliminate stable membrane anchoring are apparently not recognized by signal recognition particles, as these polypeptides remain sensitive to protease digestion, indicating that in addition they do not have a functional signal sequence. These data thus indicate that the signal sequence that initiates membrane integration of M2 resides within the transmembrane spanning segment of the polypeptide.

Animals↗
PubMed1988

Integration of membrane proteins into the endoplasmic reticulum requires GTP.

We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.

Cell Membrane↗
PubMed1988

Use of somatosensory evoked potentials in infants and children.

SEPs are a useful, reliable means of assessing somatosensory systems and gaining indirect information about motor systems in children. The factors of complex maturational changes in the CNS and body growth complicate the interpretation of SEPs. Thus, full understanding of factors that affect normative data in infants and children enhance clinical interpretation. SEPs in infancy and childhood in many clinical conditions have been useful in diagnosis and prognosis. The full extent of the clinical applications of SEPs is yet to be explored.

Adolescent↗
PubMed1987

Human milk-derived lactoferrin inhibits mitogen and alloantigen induced human lymphocyte proliferation.

Purified lactoferrin (LF), isolated from human milk, was tested for its effect on human T-lymphocyte proliferative responses to phytohemagglutinin (PHA) and to alloantigens in mixed lymphocyte culture. LF inhibited proliferation in both assays in a dose-dependent manner. The suppressive effect was not due to LF mediated cytotoxicity since washing cells that had been pre-incubated with LF restored their proliferative activity. LF was most effective in suppressing the PHA response when added within 24 h of culture initiation. Iron saturated LF failed to inhibit PHA-induced proliferation, suggesting that the mechanisms of suppression involve the chelating property of LF. The suppressive effect of LF on T-lymphocyte proliferative response in vitro supports the notion that LF has significant immunoregulatory potential in vivo.

Female↗
PubMed1987

Development of lumbar spinal cord and cortical evoked potentials after tibial nerve stimulation in the pre-term newborns: effects of gestational age and other factors.

Pre-term neonates are at increased risk for neurological dysfunction. Several investigators have found scalp recorded somatosensory evoked potential studies (SSEPs) after median nerve stimulation useful in the evaluation of newborn infants with asphyxiation and the effects of other adverse prenatal and perinatal factors. In order to evaluate the entire developing neuraxis, we undertook SSEPs after tibial nerve stimulation (PTN-SSEP) in pre-term neonates. Using bilateral simultaneous stimulation, potentials were recorded from the following sites: PF-spT6 (N5), spL1-spT6 (N16), spC7-Fpz (N27), Cz' (1 cm behind the vertex)-Fpz (P55). In all newborns studied, the N5 and N16 were reliably recorded. The N5 appeared relatively independent of the length of the newborns. The N16 correlated inversely with length. The N27 and P55 were recorded in 52% and 65% of the newborns, respectively. N27 inversely correlated modestly with length. The P55 was independent of most factors and probably reflects variable rates of cerebral myelination, neuronogenesis, varying states of alertness, and possibly subclinical encephalopathies. These results demonstrate the feasibility of obtaining such data in pre-term newborns.

Brain↗
PubMed1987

Translation and membrane insertion of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses is likely in the unusual class of glycoproteins with the amino terminus cytoplasmic and the carboxy terminus lumenal or external to the cell. The properties of the membrane insertion of the HN protein of Newcastle disease virus, a prototype paramyxovirus, were explored in wheat germ extracts containing microsomal membranes. HN protein was inserted into membranes cotranslationally, resulting in a glycosylated protein completely resistant to trypsin and proteinase K digestion. No detectable posttranslation insertion occurred. Insertion required signal recognition particle. Signal recognition particle in the absence of membranes inhibited HN protein synthesis. Comparisons of the trypsin digestion products of the HN protein made in the cell-free system with newly synthesized HN protein from infected cells showed that the cell-free product was in a conformation different from that of the pulse-labeled protein in infected cells. First, trypsin digestion of intact membranes from infected cells reduced the size of the 74,000-dalton HN protein by approximately 1,000 daltons, whereas trypsin digestion of HN protein made in the cell-free system had no effect on the size of the protein. Second, trypsin digestion of Triton X-100-permeabilized membranes isolated from infected cells resulted in a 67,000-dalton trypsin resistant HN protein fragment. A trypsin-resistant core of comparable size was not present in the digestion products of in-vitro-synthesized HN protein. Evidence is presented that the newly synthesized HN protein in infected cels contain intramolecular disulfide bonds not present in the cell-free product.

Animals↗
PubMed1986

Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose.

The biosynthesis of insulin in the islets of Langerhans is strongly controlled at the translational level by glucose. We have used a variety of experimental approaches in efforts to dissect the mechanisms underlying the stimulatory effect of glucose. To assess its effects on rates of peptide-chain elongation, isolated rat islets were labelled with [3H]leucine at different glucose concentrations in the presence or absence of low concentrations of cycloheximide. Under these conditions, at glucose concentrations up to 5.6 mM, endogenous insulin mRNA did not become rate-limiting for the synthesis of insulin, whereas stimulation of non-insulin protein synthesis was abolished by cycloheximide at all glucose concentrations, indicating either that insulin synthesis is selectively regulated at the level of elongation at glucose concentrations up to 5.6 mM, or that at these concentrations inactive insulin mRNA is transferred to an actively translating pool. Glucose-induced changes in the intracellular distribution of insulin mRNA in cultured islets were assessed by subcellular fractionation and blot-hybridization using insulin cDNA probes. At glucose concentrations above 3.3 mM, cytoplasmic insulin mRNA was increasingly transferred to fractions co-sedimenting with ribosomes, and relatively more of the ribosome-associated insulin mRNA became membrane-associated, consistent with effects of glucose above 3.3 mM on both the initiation of insulin mRNA and SRP (signal recognition particle)-mediated transfer of cytosolic nascent preproinsulin to the endoplasmic reticulum. When freshly isolated islets were homogenized and incubated with 125I-Tyr-tRNA, run-off incorporation of 125I into preproinsulin was increased by prior incubation of the islets at 16.7 mM-glucose. The addition of purified SRP receptor increased the run-off incorporation of [125I]iodotyrosine into preproinsulin, especially when the islets had been preincubated at 16.7 mM-glucose. These findings taken together suggest that glucose may stimulate elongation rates of nascent preproinsulin at concentrations up to 5.6 mM, stimulates initiation of protein synthesis involving both insulin and non-insulin mRNA at concentrations above 3.3 mM, and increases the transfer of initiated insulin mRNA molecules from the cytoplasm to microsomal membranes by an SRP-mediated mechanism that involves the modification of interactions between SRP and its receptor.

Animals↗
PubMed1986

Evaluation of children and young adults with tethered spinal cord syndrome. Utility of spinal and scalp recorded somatosensory evoked potentials.

The diagnosis of tethered spinal cord syndrome should be considered in young patients with progressive orthopedic deformities, lower extremity weakness, urinary and fecal incontinence, low back pain, or combinations of these symptoms. Myelographic, computed tomographic, and urodynamic studies are useful for establishing a diagnosis, but contribute little to the evaluation of lower extremity sensory function or to the assessment of electrophysiologic impairment of the spinal cord itself. To determine the diagnostic usefulness of the somatosensory evoked potential after posterior tibial nerve stimulation (posterior tibial nerve somatosensory evoked potential) in tethered spinal cord syndrome, 22 consecutive patients with symptoms of tethered spinal cord syndrome (aged 18 months to 22 years) underwent recording of posterior tibial nerve somatosensory evoked potential; results were correlated with clinical, myelographic, and operative findings. In patients with clinical symptoms but no myelographically demonstrable lesions, posterior tibial nerve somatosensory evoked potentials were within normal limits, suggesting normal physiologic function. In patients with myelographically and operatively confirmed tethering dysraphic lesions, posterior tibial nerve somatosensory evoked potential was predictive of the level and laterality of the lesion. Similarly, ranking the severity of neurological impairment and extent of dysraphism at operation, as well as the extent of abnormality of posterior tibial nerve somatosensory evoked potential, revealed a significant (r = 0.81, p less than 0.001) correlation between clinical severity and posterior tibial nerve somatosensory evoked potential abnormalities. Postoperatively, in 8 patients, posterior tibial nerve somatosensory evoked potential also reflected improved function in relation to the level and type of dysraphic lesion present. These findings indicate that posterior tibial nerve somatosensory evoked potential is a sensitive indicator of neurophysiologic status in patients with tethered spinal cord, and is useful for determining the level of the conus medullaris, degree of spinal cord displacement, and severity of neurological impairment associated with this congenital disturbance of neuraxis formation. Recording of posterior tibial nerve somatosensory evoked potential is noninvasive and offers a more sensitive diagnostic tool than the clinical testing of sensation for detection of the development of neurologic deficits in patients with tethered cord syndrome.

Adolescent↗
PubMed1986

Purification of microsomal signal peptidase as a complex.

We report here the purification to near homogeneity of signal peptidase from canine pancreatic microsomes. Purification was monitored using an improved post-translational assay. A 42-fold enrichment over starting membranes was achieved by selective solubilization in nonionic detergent/high-salt buffer followed by gradient sievorptive anion and cation exchange chromatography, hydroxylapatite chromatography, gel filtration, and sucrose gradient velocity sedimentation. When examined by NaDodSO4/PAGE, the purified enzyme consisted of a complex of six polypeptides with apparent molecular masses of 25, 23, 22, 21, 18, and 12 kDa. The 22- and 23-kDa subunits were shown to be glycoproteins based on their sensitivity to endoglycosidase H and their ability to bind concanavalin A. We suggest that only one subunit of this complex carries out signal peptide cleavage. The structural association of the other subunits in stoichiometric amounts may reflect their requirement in chain translocation across the microsomal membrane.

Animals↗
PubMed1986

Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein.

The requirement for ribonucleotides and ribonucleotide hydrolysis was examined at several distinct points during translocation of a secretory protein across the endoplasmic reticulum. We monitored binding of in vitro-assembled polysomes to microsomal membranes after removal of ATP and GTP. Ribonucleotides were not required for the initial low salt-insensitive attachment of the ribosome to the membrane. However, without ribonucleotides the nascent secretory chains were sensitive to protease digestion and were readily extracted from the membrane with either EDTA or 0.5 M KOAc. In contrast, nascent chains resisted extraction with either EDTA or 0.5 M KOAc and were insensitive to protease digestion after addition of GTP or nonhydrolyzable GTP analogues. Translocation of the nascent secretory polypeptide was detected only when ribosome binding was conducted in the presence of GTP. Thus, translocation-competent binding of the ribosome to the membrane requires the participation of a novel GTP-binding protein in addition to the signal recognition particle and the signal recognition particle receptor. The second event we examined was translocation and processing of a truncated secretory polypeptide. Membrane-bound polysomes bearing an 86-residue nascent chain were generated by translation of a truncated preprolactin mRNA. Ribonucleotide-independent translocation of the polypeptide was detected by cleavage of the 30-residue signal sequence after puromycin termination. Nascent chain transport, per se, is apparently dependent upon neither ribonucleotide hydrolysis nor continued elongation of the polypeptide once a functional ribosome-membrane junction has been established.

Adenosine Triphosphate↗