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P Stanley

Publications and source records attributed to P Stanley.

At least 19 recordsLinked to original sources
PubMed1992

E. coli hemolysin interactions with prokaryotic and eukaryotic cell membranes.

The hemolysin toxin (HlyA) is secreted across both the cytoplasmic and outer membranes of pathogenic Escherichia coli and forms membrane pores in cells of the host immune system, causing cell dysfunction and death. The processes underlying the interaction of HlyA with the bacterial and mammalian cell membranes are remarkable. Secretion of HlyA occurs without a periplasmic intermediate and is directed by an uncleaved C-terminal targetting signal and the HlyB and HlyD translocator proteins, the former being a member of a transporter superfamily central to import and export of a wide range of substrates by prokaryotic and eukaryotic cells. The separate process by which HlyA is targetted to mammalian cell membranes is dependent upon fatty acylation of a non-toxic precursor, proHlyA. This is achieved by a novel mechanism directed by the activator protein HlyC, which binds to an internal proHlyA recognition sequence and provides specificity for the transfer of fatty acid from cellular acyl carrier protein.

Acylation↗
PubMed1992

Peripheral congenital arteriovenous fistulae: observe, operate, or obturate?

In 1976, we presented our experience in the surgical management of patients with peripheral congenital arteriovenous fistulae (CAVF). This report updates our experience and specifically describes subsequent experience with intraarterial embolization therapy. Twenty-six infants and children with CAVFs have been treated at Children's Hospital of Los Angeles from 1966 to 1990. The majority of lesions were lower extremity (12), followed by upper extremity (8), shoulder girdle (3), and thorax/neck (3). Seventeen patients had lesions in multiple locations. All patients under 1 year of age had upper extremity lesions, all in the 5- to 10-year age group had lower extremity lesions, and all over 15 years of age at presentation required amputation. Twelve patients had initial embolization therapy and 12 had surgery. Seven of eight patients followed after embolization have improved. Embolization is not effective in large or ulcerated lesions. All patients with embolization alone have residual disease and three of eight have discrepancy in extremity length. Surgery has a 50% complication rate and 7 of 10 followed surgery patients have residual disease. Radiologic catheter techniques and embolization provide a valuable alternative therapy for CAVFs and should be used as the primary form of treatment in most cases.

Adolescent↗
PubMed1992

Communicating bronchopulmonary foregut malformations: classification and embryogenesis.

Communicating bronchopulmonary foregut malformations (CBPFMs) are characterized by a fistula between an isolated portion of respiratory tissue (ie, a lung, a lung lobe, or a segment) and esophagus or stomach. We combine our 30-year (1959 to 1989) experience of 6 cases with 51 reported patients to propose a CBPFM classification supported by a proposed embryogenesis theory. Group I (16%): anomaly is associated with esophageal atresia and tracheoesophageal fistula. Group II (33%): one lung originates from the lower esophagus. Group III (46%): an isolated anatomic lung lobe or segment communicates with the esophagus or stomach. Group IV (5%): A portion of the normal bronchial system communicates with the esophagus. The portion of the lung served by the communicating bronchus receives systemic blood supply. The right and left lung sacs curve dorsally to embrace the lower esophagus during normal lung development. At this stage a part of the lung bud joins the esophagus. This segment then breaks away from the main pulmonary anlage to form a CBPFM. CBPFMs should be considered in the workup of infants with respiratory distress and/or recurrent pneumonias. Patients with suspected pulmonary sequestration should undergo contrast studies to exclude a gastrointestinal communication.

Bronchial Fistula↗
PubMed1992

Cloning and expression of the murine gene and chromosomal location of the human gene encoding N-acetylglucosaminyltransferase I.

A mouse cDNA clone previously isolated from an F9 teratocarcinoma cell library and shown to confer N-acetylglucosaminyltransferase I (GlcNAc-TI) activity on Lec1 Chinese hamster ovary (CHO) cell transfectants [Kumar, R., Yang,J., Larsen,R.D. and Stanley,P. (1990) Proc. Natl. Acad Sci. USA, 87, 9948-9952] has been sequenced. The nucleotide and deduced amino acid sequences are highly homologous to previously described human and rabbit GlcNAc-TI cDNAs. A 1250 bp portion of the mouse cDNA encoding all but the first 34 amino acids of the deduced protein sequence was inducibly expressed in Escherichia coli and gave rise to a prominent fusion protein of mol. wt approximately 45 kDa whose presence correlated with high levels of GlcNAc-TI activity in cell lysates. Probes generated from the cDNA were used to show that the GlcNAc-TI gene is present in a single copy in mammals and that a homologous gene was not detectable (under low-stringency hybridization conditions) in DNA from yeast, sea urchin, Drosophila or Chaenorhaditis elegans. Genomic DNA clones that hybridized to probes generated from the GlcNAc-TI cDNA were isolated from a mouse liver library. Restriction analyses, Southern hybridization and DNA sequence analyses of subcloned genomic DNA fragments and a polymerase chain reaction (PCR) product provided evidence that the coding and 3' untranslated regions of the cDNA reside in a single exon. However, the mouse GlcNAc-TI gene (Mgat-1) includes at least one additional exon 5' of the coding region. Southern analyses of DNA from mouse-human somatic cell hybrids and in situ hybridization were used to locate the human GlcNAc-TI gene (MGAT-1) between positions q31.2 and q31.3 on chromosome 5, a region of chromosome 5 that is syntenic with a region of mouse chromosome 11. Northern analyses of adult mouse tissues revealed two GlcNAc-TI gene transcripts that are differentially expressed in different tissues.

Amino Acid Sequence↗
PubMed1992

Activation of Escherichia coli prohemolysin to the membrane-targetted toxin by HlyC-directed ACP-dependent fatty acylation.

Hemolysin (HlyA) and related toxins of Escherichia coli and other Gram-negative pathogenic bacteria form membrane pores in cells of the host immune system, causing cell dysfunction and death. An insight into the mechanism by which HlyA is targetted to mammalian cell membranes was achieved by establishing in vitro activation of the non-toxic precursor proHlyA. By this approach we have discovered that conversion of proHlyA to the post-translational active HlyA toxin is determined by fatty acylation of proHlyA in an apparently novel process directed by the HlyC homodimer activator protein, and dependent upon the cellular acyl carrier protein (ACP). By further exploiting the in vitro activation system it is now possible to obtain direct evidence that HlyC binds to an internal recognition sequence in the proHlyA precursor, in this way providing specificity for the transfer to proHlyA of a fatty acid moiety carried by the ACP. It is possible that the fatty acid modification determines directly the binding of HlyA to mammalian membrane lipids, thus initiating the toxin interaction with the target cells.

Acyl Carrier Protein↗
PubMed1992

Advances in pediatric interventional radiology.

The past year has seen important papers on the risk of acquired immunodeficiency syndrome in the invasive radiology laboratory with emphasis on awareness and prevention. Interventional catheterization techniques in congenital heart disease are reviewed. Valvoplasty of pulmonic stenosis has the greatest success and least complication rate. The majority of complications appear to be at the vascular access site and are more common in neonates and in procedures that are ultimately unsuccessful. Intrauterine valvoplasty of the aortic valve is reviewed as is angioplasty for a recurrent coarctation of the aorta. Closure of a persistent ductus arteriosus with a percutaneously introduced prosthesis and occlusion of congenital coronary artery arteriovenous fistulae with a variety of embolic agents is reviewed. The use of balloon-expandable stents in congenital heart disease is discussed. Traumatic rupture of the aorta, aortic and great vessel involvement in Takayasu's disease, and mucopolysaccharidosis are reviewed along with imaging of congenital anomalies of the aortic arch. The diagnosis of vascular complications of renal transplants is summarized.

Angioplasty, Balloon↗
PubMed1992

Surgery for critical aortic stenosis in newborns is still good therapy after 25 years.

Because of the high operative mortality in newborn infants with critical aortic stenosis, new therapeutic modalities have emerged. The authors reviewed their results of surgery for this condition in newborn infants between January 1964 and December 1990. Thirty-seven infants were operated on for critical aortic stenosis, which was diagnosed at a mean patient age of 14.5 days. The surgical procedure was done at a mean patient age of 37 days. Five infants died intraoperatively of ventricular fibrillation at the time of incision. Transventricular valvotomy was attempted in 4 infants, and the remaining 28 infants underwent transaortic valvuloplasty. Overall survival improved markedly in the last 5 years of the study, from 31% to 75%. All patients who had transventricular valvotomy died, as did the only infant with previous percutaneous aortic valvuloplasty. Of the infants who died, 38% weighed less than 3000 g at the time of operation compared with 13% of the survivors (p < 0.05). The duration of cardiopulmonary bypass was also identified as a risk factor (p = 0.001). Of the surviving infants, 93% were followed up at a mean of 66 months. All but one were in New York Heart functional class I or II. The following risk factors were identified for operative mortality: year of surgery, preoperative hemodynamic condition, associated anomalies of the left ventricle, surgical weight less than 3000 g, transventricular valvotomy, year of surgery and prolonged cardiopulmonary bypass. Because of the much improved survival recently, surgery remains a good therapeutic choice for critical aortic stenosis in the newborn infant.

Aortic Valve Stenosis↗
PubMed1992

The HlyB/HlyD-dependent secretion of toxins by gram-negative bacteria.

Hemolysin (HlyA) and related toxins are secreted across both the cytoplasmic and outer membranes of Escherichia coli and other pathogenic Gram-negative bacteria in a remarkable process which proceeds without a periplasmic intermediate. It is directed by an uncleaved C-terminal targetting signal and the HlyD and HlyB translocator proteins, the latter of which are members of a transporter superfamily central to import and export of a wide range of substrates by prokaryotic and eukaryotic cells. Our mutational analyses of the HlyA targetting signal and definition for the first time of stages and intermediates in the HlyB/HlyD-dependent translocation allow a discussion of the hemolysin export process in the wider context of protein translocation.

Adenosine Triphosphate↗
PubMed1991

A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in alpha-glucosidase I.

Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.

1-Deoxynojirimycin↗
PubMed1991

Cloning of a human alpha(1,3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on Chinese hamster ovary cell transfectants.

In previous studies, Chinese hamster ovary (CHO) cell genomic DNA transfectants that expressed a human alpha(1,3)-fucosyltransferase (alpha(1,3)Fuc-T) gene were isolated and shown to possess a common approximately 7.5-kilobase (kb) EcoRI fragment that hybridized to an Alu probe (Potvin, B., Kumar, R., Howard, D. R., and Stanley, P. (1990) J. Biol. Chem. 265, 1615-1622). One of these transfectants was used to make a genomic DNA library in lambda ZAP-II from EcoRI-digested, size-selected (6-8 kb) DNA, and plaques that hybridized to an Alu probe were purified. After in vivo excision, two plasmids with DNA inserts greater than or equal to 6 kb were obtained and one of these (D2.1) conferred human alpha(1,3)-Fuc-T activity on CHO transfectants. A partial restriction map of this clone revealed an approximately 3.6-kb PstI fragment that contained an Alu sequence. This fragment was subcloned into pGEM-3Zf(+) and compared by restriction analyses with a previously described approximately 3.6-kb PstI DNA fragment isolated from a human peripheral blood lymphocyte library and shown to encode an alpha(1,3)-Fuc-T gene (Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berhend, T. L., and Marks, R. M. (1990) Cell 63, 475-484). Both approximately 3.6-kb fragments gave identical restriction patterns. In addition, they both caused CHO transfectants to synthesize the Lex determinant Gal beta(1,4)[Fuc alpha(1,3)]GlcNAc beta 1 but not the alpha(2,3)-sialyl-Lex determinant. As expected, these transfectants did not bind to ELAM-1 on activated endothelial cells, since sialyl-Lex is a carbohydrate ligand recognized by ELAM-1. Surprisingly, however, an open reading frame encoded within the approximately 3.6-kb PstI fragment had a sequence identical to that of ELFT, an alpha(1,3)-Fuc-T previously reported to confer ELAM-1 binding on a previously reported to confer ELAM-1 binding on a CHO transfectant (Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R., (1990) Cell 63, 1349-1356). Possible explanations for these apparently disparate results are discussed.

Amino Acid Sequence↗
PubMed1991

Activation of two new alpha(1,3)fucosyltransferase activities in Chinese hamster ovary cells by 5-azacytidine.

Several mammalian alpha(1,3)fucosyltransferases (alpha[1,3]Fuc-T) that synthesize carbohydrates containing alpha(1,3)fucosylated lactosamine units have been identified. Although Chinese hamster ovary (CHO) cells do not express alpha(1,3)Fuc-T activity, the rare mutants LEC11 and LEC12, isolated after mutagenesis or DNA transfection, each express an alpha(1,3)Fuc-T that may be distinguished by several criteria. Two new CHO mutants possessing alpha(1,3)Fuc-T activity (LEC29 and LEC30) have now been isolated after treatment of a CHO cell population with 5-azacytidine (5-AzaC), ethylnitrosourea (ENU), or 5-AzaC followed by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Like LEC12, both mutants possess an N-ethylmaleimide-resistant alpha(1,3)Fuc-T activity that can utilize a variety of acceptors and both express the Lewis X (Lex) determinant (Gal beta[1,4](Fuc alpha[1,3])GlcNAc beta 1)) but not the sialyl alpha(2,3)Lex determinant on cell-surface carbohydrates. However, LEC29 and LEC30 may be distinguished from LEC11 and LEC12, as well as from each other, on the basis of their unique patterns of lectin resistance and their abilities to bind the VIM-2 monoclonal antibody that recognizes carbohydrates terminating in NeuNAc alpha(2,3)Gal beta(1,4)GlcNAc beta(1,3)Gal beta(1,4)(Fuc alpha[1,3])GlcNAc beta and also by the different in vitro substrate specificities and kinetic properties of their respective alpha(1,3)Fuc-T activities. The combined data provide good evidence that the LEC29 and LEC30 alpha(1,3)Fuc-Ts are novel transferases encoded by distinct gene products.

Animals↗
PubMed1991

A subclass of cell surface carbohydrates revealed by a CHO mutant with two glycosylation mutations.

A novel lectin-resistance phenotype was displayed by a LEC10 Chinese hamster ovary (CHO) cell mutant that was selected for resistance to the erythroagglutinin, E-PHA. Biochemical and genetic analyses revealed that the phenotype results from the expression of two glycosylation mutations, LEC10 and lec8. The LEC10 mutation causes the appearance of N-acetylglucosaminyltransferase III (GlcNAc-TIII) activity and the production of N-linked carbohydrates with a bisecting GlcNAc residue. The lec8 mutation inhibits translocation of UDP-Gal into the Golgi lumen and thereby dramatically reduces galactosylation of all glycoconjugates. This reduction in galactose addition does not, however, cause Lec8 mutants to be very resistant to the galactose-binding lectin, ricin. By contrast, the double mutant LEC10.Lec8 behaved like a LEC10 mutant and was highly resistant to ricin. Based on structural studies of cellular glycopeptides as well as glycopeptides of the G glycoprotein of vesicular stomatitis virus grown in mutant cells, it appears that the ricin resistance of LEC10.Lec8 cells is due to the presence of a small number of Gal residues on branched, N-linked carbohydrates that also carry the bisecting GlcNAc residue. Labelling of N-linked cellular carbohydrates with [3H]galactose was found to occur at a low level for a wide spectrum of cellular glycoproteins in independent Lec8 mutants. Studies of the LEC10.Lec8 mutant have, therefore, led to the identification of a subset of structures that are acceptors for Gal when intra-Golgi UDP-Gal levels are limiting. This mutant also illustrates the potential for regulating cell surface recognition by carbohydrate-binding proteins by altering the expression of a single glycosyltransferase such as GlcNAc-TIII.

Animals↗
PubMed1991

Mutational analysis supports a role for multiple structural features in the C-terminal secretion signal of Escherichia coli haemolysin.

We have carried out an extensive mutational analysis of the C-terminal signal which targets the export of the 1024-residue haemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium. Over 60 variants of the HlyA C-terminal 53-amino-acid sequence were created by oligonucleotide-directed mutagenesis and fused to the HlyA N-terminal 830 residues. Transport of the HlyA derivatives by the HlyB/HlyD system was compared with the wild-type level and the data indicate that the HlyA C-terminal export signal lies within the last 48 amino acids and comprises three functional domains: an amphipathic, charged helix between residues 1,977 and R,996; a 13-amino-acid uncharged region from residue T,997 to S,1009; and an 8-amino-acid hydroxylated tail at the extreme C-terminus. Analogous features were found in the C-terminal sequences of an extended family of haemolysins, leukotoxins and proteases which are secreted by HlyB/HlyD-type translocators. In particular, all nine proteins which are secreted into the extracellular medium possess potential extended amphipathic helices. These results suggest a possible role for multiple regions of the HlyA C-terminal export signal in which the first two domains span the membranes and the third domain remains in the cytoplasm.

Amino Acid Sequence↗
PubMed1991

Effects of compaction variables on porosity and material tensile strength of convex-faced aspirin tablets.

The porosity and tensile strength of convex-faced aspirin tablets formed under a compaction pressure in the range 40-320 MPa and at punch velocities in the range 0.008 to 500 mm s-1 have been determined. The material tensile strength, sigma f, was calculated from the observed fracture load, Ps, using the equation of Pitt et al (1988): sigma f = 10 Ps/pi D2(2.84 t/D - 0.126 t/W + 3.15 W/D + 0.01)-1 where D is the tablet diameter, t is the overall tablet thickness and W is the central cylinder thickness. Tablets formed at lower compaction pressures had a higher porosity and lower tensile strength than those formed at higher compaction pressures. Tablets of face curvature ratio (D/R) in the range 0.25-0.67 and a normalized cylinder length (W/D) of 0.2 had the optimum tensile strength. (R is the radius of curvature of the tablet face.) Tablets formed at high compaction rates were significantly weaker than those formed at lower compaction rates.

Aspirin↗
PubMed1991

[Results of the surgical treatment of critical aortic valve stenosis in the newborn infant].

Retrospective studies have been conducted in Lyon (33 patients) and Montreal (24 patients) in order to compare the results of transventricular valvotomy (TVV, 20 cases) and aortic valvotomy with cardiopulmonary bypass (CPB, 37 cases) in neonatal critical aortic stenosis. Clinical, echocardiographic, catheterization and operative data were analyzed in order to determine prognostic factors. Mortality rate was 59%: 30/34 perioperative deaths in the first month, and 4 late deaths after a reintervention for severe residual obstruction. Long term follow up was available for 23 patients (41%) for a 2 to 16 year period (mean 7.5). Five patients (7%) required a reintervention six years after the initial operation. Two of them required valve replacement. Eighteen patients (31%) surviving the initial operation, displayed a satisfactory result, being free of symptoms, endocarditis, reoperation and sudden death. Factors that influenced the outcome included severe heart failure, a left ventricular end-diastolic diameter below 14 mm, an aortic valve annulus below 8 mm, and a poor shortening fraction. Factors that did not influence the prognosis were age, pulmonary hypertension, and the anatomic type of the valve. Trans-aortic valvotomy with CPB was associated with a smaller operative mortality and a better long term result than TVV.

Aortic Valve↗
PubMed1991

[Critical aortic stenosis in newborn infants. Twenty-five years' experience].

Operative mortality for critical aortic stenosis in newborn having been reported as high as 20-80%, the percutaneous aortic valvuloplasty is looked upon as an alternative therapy. We elected to review our surgical experience in the last 25 years before changing our therapeutic approach. Among 37 infants included in the study, 28 were boys and 9 girls. Mean age at diagnosis was 14.5 days (1 to 113 days) and decreased to 4.1 days in the last 5 years. Surgery was done at a mean of 37.2 days; 22 infants were operated on before one month of age and 9 in their first week of life. Five died from ventricular fibrillation at incision. Trans-ventricular valvulotomy was attempted in 4 and remaining 28 had trans aortic valvuloplasty. The overall survival of 40% had improved in last five years to 75%. All patients with trans-ventricular approach died and also did the only child with percutaneous angioplasty. Only 13% patients weighing less than 3 kg survived the operation. Follow-up was of 93.3% with a mean time of 66 months. All but one are in NYHA class 1 or 2. Our study identified the following risk factors: preoperative hemodynamic state, surgical weight, associated anomalies of the left ventricle, transventricular valvulotomy and the year of surgery. In view of improved survival, surgery remains a good therapeutic choice.

Aortic Valve Stenosis↗
PubMed1990

Transfection of a human alpha-(1,3)fucosyltransferase gene into Chinese hamster ovary cells. Complications arise from activation of endogenous alpha-(1,3)fucosyltransferases.

In order to isolate a human gene encoding an alpha-(1,3)fucosyltransferase (alpha-(1,3)Fuc-T), genomic DNA from HL-60 cells was transfected by several methods into Chinese hamster ovary (CHO) cells. Colonies expressing alpha-(1,3)Fuc-T activity were identified by their ability to bind a monoclonal antibody (anti-SSEA-1) that recognizes the carbohydrate product of alpha-(1,3)Fuc-T action. CHO cells do not express alpha-(1,3)Fuc-T activity but contain at least two, silent alpha-(1,3)Fuc-T genes previously identified by their activation in the rare, dominant mutants LEC11 and LEC12. These CHO enzymes were shown to be distinguishable from the alpha-(1,3)Fuc-T activity of HL-60 cells by the latter's comparative inability to transfer fucose to paragloboside and fetuin. Based on these criteria, only 11 isolates from more than 70 putative transfectants examined were found to stably express an alpha-(1,3)Fuc-T activity typical of HL-60 cells. Genomic DNA from two of these isolates was used to generate five independent secondary transfectants with HL-60-like alpha-(1,3)Fuc-T activity. Southern analysis revealed a common DNA fragment that hybridized to an Alu probe in each secondary, providing evidence that a human alpha-(1,3)Fuc-T gene had been transfected. However, in all transfection experiments, isolates that expressed alpha-(1,3)Fuc-T activities similar to CHO-encoded enzymes were also obtained. Several lines of evidence indicated that these cells arose from activation of endogenous CHO alpha-(1,3)Fuc-T genes as a consequence of DNA transfection. These false positives complicated the identification of transfectants expressing a human alpha-(1,3)Fuc-T gene and represent an important consideration in experiments to transfect other glycosyltransferase genes.

Animals↗