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Biomedical subjects

G Lam

Publications and source records attributed to G Lam.

17 recordsLinked to original sources
PubMed1992

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.

Bacterial Toxins↗
PubMed1992

The correlation between rises in intracellular calcium and PGI2 production in cultured vascular endothelial cells.

Elevation of intracellular calcium in response to trypsin, bradykinin, thrombin or histamine is associated with a proportional increase in PGI2 production in cultured human umbilical vein endothelial cells (HUVEC), bovine pulmonary artery endothelial cells (CPAE), and bovine aortic endothelial cells (BAEC). The major agonists that induce increases in intracellular calcium and PGI2 production are thrombin and trypsin in HUVEC, bradykinin in CPAE, and bradykinin and trypsin in BAEC. These results suggest that endothelial cells derived from different species or sites require different agonists to induce increases in intracellular calcium and PGI2 production and that only agonists which increase intracellular calcium can stimulate PGI2 production.

Animals↗
PubMed1989

IFN-gamma and IFN-alpha induce the expression and synthesis of Leu 13 antigen by cultured human endothelial cells.

Leu 13 is a 16 kDa human lymphocyte surface Ag and monoclonal anti-Leu 13 induces T cell aggregation and alters T cell proliferation. In previous studies using anti-Leu 13, Leu 13 Ag has been detected on endothelial cells (EC) of arteries, capillaries, and veins in human tissue sections. In contrast, in this study using the same antibody, Leu 13 Ag could not be detected on unstimulated cultured human umbilical vein endothelial cells (HUVEC) by indirect immunofluorescence, immunoperoxidase staining, or immunoisolation of radiolabeled Ag from labeled endothelial cells. However, when HUVEC were cultured with IFN-gamma, Leu 13 Ag was demonstrable using all three detection techniques. Leu 13 Ag was detectable after 24 h of incubation with IFN-gamma (30 U/ml) and was maximally expressed after 72 h. After removal of IFN-gamma, Leu 13 Ag progressively declined back to basal levels by 4 days. Induction of Leu 13 Ag expression by IFN-gamma was suppressed by cycloheximide. Although Leu 13 Ag expression was also induced by IFN-alpha, PMA, and IL-1 were inactive. When HUVEC were incubated with IFN-gamma, surface-labeled with 125I, and then solubilized, anti-Leu 13 immunoisolated a 16-kDa radioactive band that comigrated with Leu 13 immunoisolated from T cells. Similar results were obtained using HUVEC that were metabolically labeled with 35S-methionine. These results demonstrate that HUVEC stimulated with IFN-gamma and IFN-alpha express Leu 13 Ag on their surface and suggest that exposure of EC to IFN may induce EC to develop new functional properties at sites of inflammatory and immune reactions.

Antibodies, Monoclonal↗
PubMed1989

Thrombin and histamine stimulate the phosphorylation of elongation factor 2 in human umbilical vein endothelial cells.

The effects of thrombin and histamine on protein phosphorylation in intact cultured human umbilical vein endothelial cells (HUVEC) prelabeled with 32PO4 were investigated. Incubation of HUVEC with either thrombin or histamine, agonists known to induce rapid transient increases in intracellular calcium levels in HUVEC, caused a rapid reversible increase in the phosphorylation of a protein with a Mr = 100,000 independent of the presence of extracellular calcium. Immunological and biochemical studies demonstrated that this Mr = 100,000 protein is elongation factor 2 (EF-2), a substrate previously shown to be phosphorylated by calcium/calmodulin-dependent protein kinase III (Nairn, A. C., and Palfrey, H. C. (1987) J. Biol. Chem. 262, 17299-17303). EF-2 is crucial for protein synthesis because it catalyzes the translocation of peptidyl-tRNA on the ribosome. Phosphoamino acid analysis of the EF-2 immunoprecipitated from HUVEC revealed that all of the thrombin-stimulated phosphorylation occurred on threonine. EF-2 was also phosphorylated when HUVEC were treated with the calcium ionophore, ionomycin. Phosphorylation of EF-2 was not increased by treatment with D-Phe-Pro-Arg-chloromethyl ketone thrombin, phorbol dibutyrate, forskolin, or 8-bromo-cGMP. The transient nature of the phosphorylation of EF-2 is consistent with it having a role in mediating some of the transient effects of thrombin and histamine on endothelial cell protein synthesis and functional capabilities.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1989

Gene mapping by enzymatic amplification from flow-sorted chromosomes.

A new approach to gene mapping which combines enzymatic amplification with high-resolution flow sorting of human chromosomes has been devised. Reliable amplification from as few as 200 chromosomes has been demonstrated. This method, with particular application to mapping the position of chromosomal translocations, has been used to show that the breakpoint for the constitutional translocation t(11;22)(q23;q11) lies proximal to the genes c-ets-1, Thy-1, and T3 delta and distal to the int-2 gene. The mapping was confirmed by Southern analysis to much larger numbers of chromosomes sorted from the same cell line. Control reactions for the bcl-2 gene on chromosome 18 and the C alpha gene of the IGH locus on chromosome 14 demonstrated the discrimination which can be achieved.

Base Sequence↗
PubMed1989

Assignment of the gene encoding the beta-subunit of the human fibronectin receptor (beta-FNR) to chromosome 10p11.2.

A cDNA corresponding to the beta-subunit of the human fibronectin receptor (beta-FNR) was used as a probe in Southern blot analysis of mouse/human somatic cell hybrid DNAs and in in situ hybridization to metaphase chromosomes. The beta-FNR cDNA detects sequences present on human chromosome 10 as well as recognizing homologous sequences in the genome of the mouse parent of the somatic cell hybrids. In situ hybridization refined the localization of human sequences reacting with the beta-FNR cDNA to 10p11.2. The A-1A5 monoclonal antibody which recognizes the beta-subunit of the fibronectin receptor on the cell surface was used to confirm that the sequences present on chromosome 10 correspond to those required for expression of beta-FNR.

Animals↗
PubMed1988

Rearrangement of T-cell receptor and immunoglobulin heavy chain genes in childhood acute mixed lineage leukaemia.

Rearrangement of the beta and gamma chain genes of the TCR gene complex and of the Ig heavy chain genes were examined in three cases of childhood acute mixed lineage leukaemia. Blast cells, classified morphologically as acute lymphoblastic leukaemia (ALL) in one child and acute non-lymphocytic leukaemia (ANLL) in the other two, all co-expressed markers associated with both T (CD7, TdT) and myeloid (CD33) cells. Cytogenetic analysis detected abnormalities associated with myeloid leukaemia. Immunoglobulin heavy chain genes were not rearranged in two patients but a novel rearrangement was seen in the third. No rearrangement of the beta or gamma chains of the T-cell receptor complex were seen. Acute mixed lineage leukaemia may thus arise from a pluripotent precursor cell capable of both lymphoid and myeloid differentiation.

Antigens, Differentiation↗
PubMed1988

Cellular and molecular studies on infant null acute lymphoblastic leukemia.

We have studied the cellular and molecular basis of eight cases of infant null acute lymphoblastic leukemia (ALL). All eight patients were under 9 months of age and presented with leukocyte counts in excess of 60 X 10(9)/L, organomegaly, and in two cases CNS infiltration. Although seven cases were morphologically classified as ALL, one patient had both lymphoid and myeloid features. Phenotypic analysis of leukemic blasts from all patients showed a typical null ALL pattern, ie, CD10 (common ALL antigen)-negative, strongly HLA-DR-positive, and CD19 (B4)-positive. The presence of terminal deoxynucleotidyl transferase (TdT) at presentation was positive in six patients' cells and negative in two. Two patients also expressed the myeloid-associated markers CD33 (MY9) and CD15 (TG1), and coexpression of CD19 and CD33 was confirmed in these two by using dual marker flow cytometry (fluorescence-activated cell sorting). Electron microscopic examination of the same two patients' cells showed the presence of monocytoid blasts that labeled with the pan-B cell antibody B4 (CD19). Short-term culture of one of these patients cells in the presence of phorbol ester resulted in the majority of the cells exhibiting myeloid markers, strong nonspecific esterase positivity, and phagocytic properties. Cytogenetic analysis showed the common feature in 7 of 8 cases to be a break in band 11q23. Molecular analysis of DNA from the blast cells of all eight patients showed rearrangement of the immunoglobulin heavy-chain genes in all cases without, however, any evidence of kappa light-chain rearrangement. T cell receptor genes were present in the germline configuration in all cases. Rearrangements of the c-ets 1 oncogene, which maps to band 11q23, were not detected, thus providing no evidence for involvement of this oncogene in the common disease process. Our data indicate that although infant null ALL may present as a heterogeneous disease the similarity of many features between cases suggests a common derivation from a precursor cell sharing phenotypic and genotypic features of both B and myeloid progenitor cells.

Antigens, Neoplasm↗
PubMed1987

Effect of platelet-derived growth factor on enriched populations of haemopoietic progenitors from patients with chronic myeloid leukaemia.

The effect of pure platelet-derived growth factor and fresh serum on the in-vitro growth of purified haemopoietic progenitors from the peripheral blood of 12 patients with CML was studied. Purified haemopoietic progenitors were prepared using Percoll separation followed by cell sorting with the monoclonal antibody BI.3C5. Both pure PDGF at a concentration of 20 ng/ml and fresh serum significantly increased the numbers of BFU-E (p less than 0.01) and CFU-GEMM (p less than 0.014), but not the CFU-GM. That the PDGF effect was not mediated to any significant extent via prostaglandins, was shown by the lack of inhibitory effect of indomethacin on the growth of purified progenitor cells in the presence of fresh serum. Increased amounts of pure PDGF were required to give maximal stimulation of purified CML peripheral blood progenitors compared to normal bone marrow progenitors. These results show that CML progenitors are capable of responding to PDGF. Whether the quantitative difference in response is due to a reduced proportion of mesenchymal cells in CML peripheral blood compared to normal marrow, or whether CML progenitors are most likely already stimulated by autocrime PDGF or other growth factors remains to be elucidated.

Antibodies, Monoclonal↗
PubMed1987

The response of mouse epidermis to fractionated doses of pi mesons.

An extensive series of preclinical experiments are described in which the relative biological effectiveness (RBE) of pions from TRIUMF has been determined for mouse skin. To measure the RBE at both high and low doses per fraction, a range of fractionation schedules was used, with 1, 2, 4, 10, and 20 fractions. Because the pion dose rate is much lower than the dose rate of X or gamma-rays used in radiotherapy, two sets of reference X ray data were obtained: one at the same dose rate as the pions (15 cGy min-1), and another at the more conventional 150 cGy min-1. This allowed a "biophysical" RBE for equivalent dose rates and a "practical" RBE for preclinical evaluation to be calculated. The pion RBE was significantly higher for 20 small fractions than for fewer large doses. The absolute value of RBE depends upon the dose rate of the reference X ray treatments. The "practical" RBE increased from 1.05 at high doses to 1.45 at the smallest dose per fraction tested. The "biophysical" RBE for equivalent dose rates ranged from 1.20 at high doses to 1.5 for 20 fractions.

Animals↗
PubMed1986

Co-ordinate expression of BI.3C5 and HLA-DR antigens on haemopoietic progenitors from chronic myeloid leukaemia.

Haemopoietic cells isolated from the peripheral blood of patients with chronic myeloid leukaemia (CML), have been extensively purified and enriched using either Percoll density gradients or Percoll density gradients combined with elutriation. The quantitative expression of the BI.3C5 associated antigen and the co-expression of BI.3C5 and HLA-DR antigens on these two populations has been studied using either single or simultaneous two colour FACS sorting, following by in-vitro culture for single and multilineage haemopoietic progenitors thus obtained. The data show that the CFU-GEMM are always found in the most strongly BI.3C5 positive fraction, irrespective of the separation procedure and that the bulk of the CFU-GEMM co-express BI.3C5 and HLA-DR. The cell types initiating these CFU-GEMM are morphologically immature blasts. The more mature cells of the myelomonocytic and erythroid lineages forming single lineage colony types show variable BI.3C5 expression, although most are HLA-DR positive. Such enriched populations of malignant progenitors could provide a useful source of material to study both gene expression and the molecular mechanisms underlying malignant transformation.

Antibodies, Monoclonal↗
PubMed1985

The human leucocyte-common antigen: differential expression of framework and restricted antigenic determinants on early haemopoietic progenitors.

Monoclonal antibodies have been raised against human LC determinants. One, F10.89.4, recognizes a 'framework' epitope on all LC molecules; the other, F8.11.13, recognizes a 'restricted' epitope present on only a subset of these molecules which are found mainly on B and a subpopulation of T cells. A previous study of leukaemias showed that some early lymphoid and myeloid leukaemic cells totally lack LC (35% of ALLs and AMLs are F10.89.4-, F8.11.13-). In contrast, a proportion of myeloid leukaemias carried both 'framework' and 'restricted' epitopes (30% AMLs and AMMLs are F10.89.4+, F8.11.13+). To determine whether comparable heterogeneity exists in normal bone marrow we have analysed LC expression during haemopoiesis, using FACS separated populations and in vitro progenitor assays. Our data show that the great majority of haemopoietic progenitors express the LC 'framework' epitope. These can be separated by size into myeloid (large) and lymphoid (small) progenitor populations. However, very few myeloid progenitors (11% CFU-GM, 6% CFU-GEMM) express the additional 'restricted' LC F8.11.13 epitope. Most F8.11.13+ progenitors are CFU-lymphoid; these generate both T and B lymphocytes, but show a preference for the B lineage. Thus there is some molecular heterogeneity of LC during normal haemopoiesis, but this is far less extensive than that found in leukaemias.

Adult↗
PubMedDate not supplied

Phenylpropanolamine pharmacokinetics in dogs after intravenous, oral, and oral controlled-release doses.

An adaption of a published high performance liquid chromatographic (HPLC) assay for phenylpropanolamine (PPA) in plasma was used to examine PPA pharmacokinetics in dogs. Plasma was extracted into ethyl acetate after the addition of 3.5 per cent sodium carbonate, and was then back-extracted into aqueous acetic acid. The acetic acid was injected onto a cyano column using a mobile phase of acetonitrile, dilute hydrochloric acid, and sodium heptane sulfonate. Detection was by UV absorbance at 210 nm. The relative standard deviation of replicate assays averaged 5.2 per cent over a concentration range of 50-1750 ng ml-1 plasma. PPA extraction recovery exceeded 90 per cent. The limit of detection was 30 ng ml-1 using 0.5 ml plasma and injecting 10 microliter. PPA disposition was characterized in three dogs administered PPA i.v. and orally in immediate-release and controlled-release formulations. The terminal elimination half-life averaged 3.5 +/- 0.5 h after the i.v. dose. Oral absorption from the immediate-release capsule was rapid and bioavailability was 98.2 +/- 6.9 per initial rapid cent. PPA absorption from the controlled-release dosage form was biphasic; an rapid phase was followed by a second, slower absorption phase which continued over 16 h. Plasma PPA concentrations then declined with a half-life roughly parallel to the i.v. and oral immediate-release half-lives. Oral bioavailability from the controlled release tablet was 93.7 +/- 5.9 per cent.

Administration, Oral↗