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Biomedical subjects

P Moran

Publications and source records attributed to P Moran.

At least 91 records · Page 5Linked to original sources
PubMed1992

Proteins containing an uncleaved signal for glycophosphatidylinositol membrane anchor attachment are retained in a post-ER compartment.

Glycophosphatidylinositol (GPI)-anchored membrane proteins are initially synthesized with a cleavable COOH-terminal extension that signals anchor attachment. Overexpression in COS cells of hGH-DAF fusion proteins containing the GPI signal of decay accelerating factor (DAF) fused to the COOH-terminus of human growth hormone (hGH), produces both GPI-anchored hGH-DAF and uncleaved precursors that retain the GPI signal. Using hGH-DAF fusion proteins containing a mutated, noncleavable GPI signal, we show that uncleaved polypeptides are retained inside the cell and accumulate in a brefeldin A-sensitive, Golgi-like juxtanuclear structure. Retention requires the presence of either a functional or a noncleavable GPI signal; hGH-DAF fusion proteins containing only the COOH-terminal hydrophobic domain (a component of the GPI signal) are secreted. Immunofluorescence analysis shows colocalization of the retained, uncleaved fusion proteins with both a Golgi marker and with p53, a marker of the ER-Golgi intermediate compartment. Since N-linked glycosylation is postulated to facilitate the transport of proteins to the cell surface, we engineered a glycosylation site into hGH-DAF. Glycosylation failed to completely override the transport block, but allowed some uncleaved hGH-DAF to pass through the secretory pathway and acquire endoglycosidase H resistance. The retained molecules remained endoglycosidase H sensitive. We suggest that the uncleaved fusion protein is retained in a sorting compartment between the ER and the medial Golgi complex. We speculate that a mechanism exists to retain proteins containing an uncleaved GPI signal as part of a system for quality control.

Amino Acid Sequence↗
PubMed1991

Glycophospholipid membrane anchor attachment. Molecular analysis of the cleavage/attachment site.

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophosphatidylinositol (GPI) membrane anchor in a process involving proteolytic removal of 17-31 COOH-terminal residues. Previous work suggested that two elements are required for anchor addition, a COOH-terminal hydrophobic domain (the GPI signal) and an element located NH2-terminal to it, postulated to be the cleavage/attachment site. Using [3H]ethanolamine (a component of the anchor) to tag the COOH terminus, we isolated and sequenced a COOH-terminal tryptic peptide, thereby identifying Ser-319 as the COOH-terminal residue attached to the GPI anchor. This indicates that a 28-residue peptide is removed during processing and localizes the cleavage/attachment site precisely to the region previously shown to be required for anchor attachment (between 10 and 20 residues NH2-terminal to the hydrophobic domain). Since DAF contains multiple cryptic cleavage/attachment sites, we used a GPI-linked human growth hormone-DAF fusion to study the structural requirements for cleavage/attachment. Our results show that while sequences immediately NH2-terminal to the attachment site are not required for anchor addition, deletion of Ser-319 abolishes both anchor attachment and transport to the cell surface. Systematic replacement of the attachment site serine with all possible amino acids indicated that alanine, aspartate, asparagine, glycine, or serine efficiently support GPI anchor attachment while valine and glutamate are partially effective. All other substitutions including cysteine (permitted at the attachment site in other GPI-anchored proteins) abolish both GPI anchor attachment and transport to the cell surface, resulting in accumulation of uncleaved fusion protein in internal compartments (endoplasmic reticulum and Golgi). These results support the general rule that the residue at the cleavage/attachment site must be small. Further, addition of a GPI anchor appears to be necessary for transport to the cell surface in transfected COS cells.

Amino Acid Sequence↗
PubMed1991

A nonfunctional sequence converted to a signal for glycophosphatidylinositol membrane anchor attachment.

The COOH terminus of decay-accelerating factor (DAF) contains a signal that directs glycophosphatidylinositol (GPI) membrane anchor attachment in a process involving concerted proteolytic removal of 28 COOH-terminal residues. At least two elements are required for anchor addition: a COOH-terminal hydrophobic domain and a cleavage/attachment site located NH2-terminal to it, requiring a small amino acid as the acceptor for GPI addition. We previously showed that the last 29-37 residues of DAF, making up the COOH-terminal hydrophobic domain plus 20 residues of the adjacent serine/threonine-rich domain (including the anchor addition site), when fused to the COOH terminus of human growth hormone (hGH) will target the fusion protein to the plasma membrane via a GPI anchor. In contrast, a similar fusion protein (hGH-LDLR-DAF17, abbreviated HLD) containing a fragment of the serine/threonine-rich domain of the LDL receptor (LDLR) in place of the DAF-derived serine/threonine-rich sequences, does not become GPI anchored. We now show that this null sequence for GPI attachment can be converted to a strong GPI signal by mutating a pair of residues (valine-glutamate) in the LDLR sequence at a position corresponding to the normal cleavage/attachment site, to serine-glycine, as found in the DAF sequence. A single mutation (converting valine at the anchor addition site to serine, the normal acceptor for GPI addition in DAF) was insufficient to produce GPI anchoring, as was mutation of the valine-glutamate pair to serine-phenylalanine (a bulky residue). These results suggest that a pair of small residues (presumably flanking the cleavage point) is required for GPI attachment. By introducing the sequence serine-glycine (comprising a cleavage-attachment site for GPI addition) at different positions in the LDLR sequence of the fusion protein, HLD, we show that optimal GPI attachment requires a processing site positioned 10-12 residues NH2-terminal to the hydrophobic domain, the efficiency anchor attachment dropping off sharply as the cleavage site is moved beyond these limits. These data suggest that the GPI signal consists solely of a hydrophobic domain combined with a processing site composed of a pair of small residues, positioned 10-12 residues NH2-terminal to the hydrophobic domain. No other structural motifs appear necessary.

Amino Acid Sequence↗
PubMed1991

Fusion of sequence elements from non-anchored proteins to generate a fully functional signal for glycophosphatidylinositol membrane anchor attachment.

Glycophosphatidylinositol (GPI) membrane anchor attachment is directed by a cleavable signal at the COOH terminus of the protein. The complete lack of homology among different GPI-anchored proteins suggests that this signal is of a general nature. Previous analysis of the GPI signal of decay accelerating factor (DAF) suggests that the minimal requirements for GPI attachment are (a) a hydrophobic domain and (b) a cleavage/attachment site consisting of a pair of small residues positioned 10-12 residues NH2-terminal to a hydrophobic domain. As an ultimate test of these rules we constructed four synthetic GPI signals, meeting these requirements but assembled entirely from sequence elements not normally involved in GPI attachment. We show that these synthetic signals are able to direct human growth hormone (hGH), a secreted protein, to the plasma membrane via a GPI anchor. Our results indicate that different hydrophobic sequences, derived from either the prolactin or hGH NH2-terminal signal peptide, can be linked to different cleavage sites via different hydrophilic spacers to produce a functional GPI signal. These data confirm that the only requirements for GPI-anchoring are a pair of small residues positioned 10-12 residues NH2 terminal to a hydrophobic domain, no other structural motifs being necessary.

Amino Acid Sequence↗
PubMed1991

Fluid uptake in laser endometrial ablation.

Fluid uptake was measured from accurate preoperative and postoperative patient weights for 25 consecutive patients undergoing Nd:YAG laser endometrial ablation under general anaesthesia. One patient had a uterine perforation with 5.1 litres of fluid deposited in the peritoneal cavity. In the remaining 24 cases, maximum fluid uptake was 5.1 litres, three patients absorbed more than four litres and eight absorbed more than two litres. In those same 24 patients, the mean fluid uptake was 1.5 litres (SD 1.6) and six patients without tubal ligation had a higher mean uptake (2.2 litres, SD 1.7) than the eighteen with surgically occluded Fallopian tubes (1.3 litres, SD 1.4) but the difference was not statistically significant. Fluid uptake has the potential to cause significant morbidity in this procedure. Both procedure duration and volume of uterine irrigation fluid were poor predictors of fluid gain. Observation of the ongoing volume deficit between infused and collected fluid provided intraoperative monitoring of fluid gain that prevented complications from fluid overload in healthy patients. However, this method of monitoring fluid uptake may be insufficient to prevent either complications from glycine absorption, if glycine is used for irrigation, or fluid overload in patients with cardiac disease.

Adult↗
PubMed1990

Imaging of non-small cell lung cancers with a monoclonal antibody, KC-4G3, which recognizes a human milk fat globule antigen.

To determine the role of lung cancer tumor imaging with monoclonal antibodies directed against high molecular weight human milk fat globule antigens, we administered i.v. 111In-KC-4G3 to 24 patients with advanced non-small cell lung cancer. One mg of 111In-KC-4G3 was mixed with 0, 9, 49, 99, or 499 mg of unlabeled KC-4G3 and infused i.v. over 1 to 5 h. The mean 111In-KC-4G3 radiochemical purity was greater than 97% and the resultant immunoreactivity averaged 62%. Successful imaging of cancer sites was accomplished in 92% of 24 patients, and 57% of 91 total lesions were visualized. Successful localization of tumor sites related to size (P less than 0.001), with 81% of lesions greater than 3.0 cm in diameter, 50% of lesions 1.5 to 3 cm, and 6% of lesions less than 1.5 cm successfully imaging, and to location (P less than 0.05), with 69% of pulmonary lesions, 80% of soft tissue lesions, and only 32% of bone metastases being visualized. Nonspecific reticulo-endothelial uptake of radioactivity was a major problem. Approximately 35% of 111In was chelated to serum transferrin by 24 and 48 h after infusion. The mean t 1/2 beta for plasma radioisotope and immunoreactive KC-4G3 was 29 and 27 h, respectively. There was no correlation between total infused antibody dose and imaging success or between total dose and effect on 111In and KC-4G3 kinetics. Circulating free KC-4 antigen was measurable in all but one patient before study. Tumor biopsy following infusion could demonstrate antibody presence but not saturable antigen binding. We conclude that (a) 111In-KC-4G3 demonstrates successful tumor localization in non-small cell lung cancers bearing generally high expression of its antigen and (b) further investigations to diminish nonspecific radioactivity for imaging and utilization of high dose radiolabeled antibody for therapeutic intent are warranted.

Antibodies, Monoclonal↗
PubMed1990

Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements.

The CaMV 35S and Ti plasmid mannopine synthetase (mas) promoters are commonly used by plant genetic engineers. To combine their useful properties, we constructed hybrid promoters incorporating elements from both. These promoters were spliced to the beta-glucuronidase reporter gene and introduced into tobacco and tomato plants by Agrobacterium cocultivation. T1 and T2 transgenic plant populations transformed with different constructs were assayed for the marker enzyme. Comparisons were made based on the range of expression levels found for each promoter construct. We found that a hybrid promoter incorporating the mas region from +65 to -301 and the 35S enhancer region from -90 to -941 had new and interesting properties. This promoter, called Mac, expressed gus at a level three to five times that expressed by a double 35S promoter in the leaves, and 10 to 15 times in hypocotyls and roots. The Mac promoter, however, showed only marginal wound inducibility. Five- to seven-fold wound induction required the presence of the region from -301 to -613 of mas. Reiteration of the 35S enhancer region, from -90 to -430, behind the 35S TATA box region or the mas +65 to -301 region had a smaller effect on expression, ranging from equal to twice the level of the single enhancer control.

Cloning, Molecular↗
PubMed1990

Human immunodeficiency virus-like, nonreplicating, gag-env particles assemble in a recombinant vaccinia virus expression system.

We report the assembly of human immunodeficiency virus (HIV)-like particles in African green monkey kidney cells coinfected with two recombinant vaccinia viruses, one carrying the HIV-1 gag and protease genes and the other the env gene. Biochemical analysis of particles sedimented from culture supernatants of doubly infected cells revealed that they were composed of gag proteins, primarily p24, as well as the env proteins gp120 and gp41. Thin-section immunoelectron microscopy showed that these particles were 100 to 120 nm in diameter, were characterized by the presence of cylindrical core structures, and displayed the mature gp120-gp41 complexes on their surfaces. Furthermore, thin-section immunoelectron microscopy analysis of infected cells showed that particle assembly and budding occurred at the plasma membrane. Nucleic acid hybridization suggested that the particles packaged only the gag mRNA but not the env mRNA. Therefore, the system we present is well suited for studies of HIV virion maturation. In addition, the HIV-like particles provide a novel and attractive approach for vaccine development.

Animals↗
PubMed1989

Pharmacology of the memory process.

Rather than reviewing once again the published literature on drugs which are capable of modifying the memory process, the authors stress the methodological difficulties which impede the development of such products. At the moment, substances which act on neurotransmission systems, and in particular cholinergic and gabaergic agents, seem to be good candidates. The choice of the molecule to be studied in man depends on the neurochemical physiopathology of the memory disorder which one seeks to correct. For this reason, one tries to select an animal model of memory which is closest to the clinical situation pertaining to man on which the tested molecule might act favorably.

Animals↗
PubMed1988

The histological effects on the guinea pig external ear of several constituents of commonly used aural preparations.

Medicament contact dermatitis can be troublesome in external ear disease. In this work the effects on the guinea pig external ear of instillation twice daily for 3 weeks of benzalkonium chloride, Cerumol, gentamicin, the pharmaceutical base for Gentisone HC, hydrocortisone and propylene glycol were studied using light microscopy, thickness measurements of tympanic membrane and meatal epidermis, and the mitotic index of meatal epidermis. Benzalkonium chloride induced severe inflammatory changes and markedly increased the thickness measurements and meatal mitotic index. Cerumol also increased the thickness measurements and the mitotic index; there was additionally low grade chronic inflammation in the dermis. Prophylene glycol increased the mitotic index and thickness measurements though no signs of inflammation were seen. Other compounds did not produce significant changes except that hydrocortisone reduced the mitotic index. Contact dermatitis to benzalkonium chloride may be important in persistent ear disease. Cerumenolytic agents ought not to be used for prolonged periods or as prophylaxis.

Animals↗
PubMed1987

The Framingham type A scale, vigilant coping, and heart-rate reactivity.

Forty-eight subjects, approximately half of whom were male and half female, took part in an experiment designed to measure coping-strategy choice and heart-rate reactivity in the face of an electric shock threat. All subjects completed, in addition, the Framingham Type A scale and other Framingham psychosocial scales measuring anger expression and emotional lability. Among female subjects, Type A was ex hypothesi associated with choice of a vigilant, predictability-seeking coping style. Type A was also ex hypothesi associated with greater heart-rate reactivity. Among males, the Type A scale did not predict either coping-strategy choice or heart-rate reactivity. Vigilant coping was associated with suppressed anger in both sexes. Results are discussed with reference to a wider body of research on Type A behavior pattern and coronary heart disease.

Adaptation, Psychological↗
PubMed1987

Cardiovascular unwinding, type A behaviour pattern and locus of control.

Three studies are reported, in which subjects were exposed for a period of three minutes to a threat of possible shock. On average subjects showed higher heart rates before the trial than after the trial, and higher rate during the first minute of the trial than the final minute. Results are reported here which show that Type A behaviour pattern and internal locus of control are consistently related to less decline in heart rate.

Adolescent↗
PubMed1987

Autologous blood transfusion for major vascular surgery using the Sorenson Receptal Device.

Twenty-five patients having aortic surgery had blood scavenged using the Sorenson Receptal Device (Group A) and were compared with twenty-five patients having homologous blood transfusion (Group H). Mean intraoperative blood loss was similar in both groups, Group A 3224 (SD 2392) ml, Group H 2999 (SD 1579) ml, but the mean homologous blood replacement was significantly different intraoperatively, Group A 1.2 (SD 1.7) units, Group H 2.7 (SD 1.8) units. Total intra-hospital homologous blood replacement was not significantly different, Group A 4.0 (SD 3.4) units, Group H 5.5 (SD 5.8) units. Mean haemoglobin concentration in the scavenged blood was 8.5 (SD 2.1) g/dl compared to 10.8 (SD 2.4) g/dl in the median aged homologous blood units crossmatched for Group H. Mean red cell half life in the scavenged blood was the same as that for the homologous blood, 24 (SD 5) days, but plasma-free haemoglobin and bacterial contamination was greater in the scavenged blood. There was no difference in the incidence of postoperative renal dysfunction, coagulopathy or mortality between the two groups of patients.

Aged↗