Biomedical subjects
Publications and source records attributed to L Lum.
On the basis of our studies of the fluctuation of the hematopoietic stem cell phenotype with cell cycle trnsit, we hypothesize that the ability of marrow stem cells to convert to nonhematopoietic cells will also vary at different points in the cell cycle. The new biology of stem cells has an impact on many fields including developmental biology and stem cell biology and the clinical potential is enormous.
A number of structurally and functionally diverse membrane proteins are released from the plasma membrane in a process termed protein ectodomain shedding. Ectodomain shedding may activate or inactivate a substrate or change its properties, such as converting a juxtacrine into a paracrine signaling molecule. Here we have characterized the activities involved in protein ectodomain shedding of the tumor necrosis factor family member TRANCE/OPGL in different cell types. The criteria used to evaluate these activities include (a) cleavage site usage, (b) response to activators and inhibitors of intracellular signaling pathways, and (c) sensitivity to tissue inhibitors of metalloproteases (TIMPs). At least two TRANCE shedding activities emerged, both of which are distinct from the tumor necrosis factor alpha convertase. One of the TRANCE sheddases is induced by the tyrosine phosphatase inhibitor pervanadate but not by phorbol esters, whereas the other is refractory to both of these stimuli. Furthermore, the pervanadate-regulated sheddase activity is sensitive to TIMP-2 but not TIMP-1, which is consistent with the properties of a membrane type matrix metalloprotease. This study provides insights into the properties of different activities involved in protein ectodomain shedding and has implications for the functional regulation of TRANCE by potentially more than one protease.
BACKGROUND: The clinical profile of severe upper airway obstruction, a challenging acute pediatric emergency, has not been extensively documented in the developing nations of the tropics. METHODS: The diagnostic categories, severity of illness and outcome from 63 episodes of severe upper airway obstruction in 56 children admitted to the Pediatric Intensive Care Unit between January 1994 and December 1999 were reviewed. Outcome variables studied included requirement for ventilation, mortality and complications. Severity of illness was determined with the Pediatric Risk of Mortality (PRISM) II score. RESULTS: Viral croup (29%) was the most common diagnosis, followed by mediastinal malignancy (13%), bacterial tracheitis (11%) and Pierre Robin syndrome (11%). There were no admissions for acute epiglottitis. Thirty episodes (48%) required ventilation for a median duration of 4.0 days. Bacterial tracheitis (100%) and subglottic stenosis (100%) were the most likely diagnoses requiring ventilation. Difficulty in intubation was encountered in 13 episodes (43%) involving, in particular, patients with bacterial tracheitis (83%; P = 0.006). Only two patients required a tracheostomy. The overall mortality was 11%. The PRISM score for all categories was generally low (mean 10.3 +/- 1.0; median 9.0). Non-survivors had a significantly higher PRISM II score than survivors (27.4 +/- 9.7 vs 8.1 +/- 4.9, respectively; P = 0.002) and were more likely to include children with bacterial tracheitis and mediastinal malignancy. CONCLUSIONS: There is marked heterogeneity in the causes of upper airway obstruction in the tropics with viral croup remaining the most common. A significant proportion required ventilation, but outcome is generally favorable, except in those with bacterial tracheitis and mediastinal malignancy.
Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a member of the TNF family, is a dendritic cell survival factor and is essential for osteoclastogenesis and osteoclast activation. In this report we demonstrate (i) that TRANCE, like TNF-alpha, is made as a membrane-anchored precursor, which is released from the plasma membrane by a metalloprotease; (ii) that soluble TRANCE has potent dendritic cell survival and osteoclastogenic activity; (iii) that the metalloprotease-disintegrin TNF-alpha convertase (TACE) can cleave immunoprecipitated TRANCE in vitro in a fashion that mimics the cleavage observed in tissue culture cells; and (iv) that in vitro cleavage of a TRANCE ectodomain/CD8 fusion protein and of a peptide corresponding to the TRANCE cleavage site by TACE occurs at the same site that is used when TRANCE is shed from cells into the supernatant. We propose that the TRANCE ectodomain is released from cells by TACE or a related metalloprotease-disintegrin, and that this release is an important component of the function of TRANCE in bone and immune homeostasis.
OBJECTIVES: To determine the association between severity of sepsis with outcome and severity of illness in children with multiple organ dysfunction syndrome (MODS). MATERIALS: Four hundred and ninety-five consecutive paediatric intensive care unit (PICU) admissions were analysed. multiple organ dysfunction syndrome was defined as simultaneous dysfunction of >/= 2 organ system and sepsis by the American College of Chest Physicians and Society of Critical Care Medicine Consensus Conference definition. RESULTS: Eighty-four patients developed MODS. The incidence of sepsis, severe sepsis and septic shock in these patients was 10.7%, 23.8% and 17.9%, respectively. Worsening categories of sepsis were associated with: (1) a higher mean admission Paediatric Risk of Mortality (PRISM II): 36.6 +/- 25.9, 56.8 +/- 32.1 and 73.6 +/- 28.5%, respectively (P = 0. 005), (2) a larger number of organ dysfunctions: mean MODS index of 37%, 46% and 58%, respectively (P = 0.007), and (3) a higher mortality: 22.2%, 65% and 80%, respectively (P = 0.03). CONCLUSION: Presence of sepsis, severe sepsis and septic shock was associated with an increasing severity of illness, increased number of organ dysfunctions and a distinct risk of mortality among critically ill children.
Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that play a role in fertilization, myoblast fusion, neuronal development, and cleavage of the membrane-anchored cytokine tumor necrosis factor-alpha. Here, we report the cloning and cDNA sequencing of the mouse metalloprotease disintegrin MDC15 and an analysis of its processing in the secretory pathway. A notable difference between mMDC15 and its putative human orthologue (hMDC15, metargidin) is the presence of the peptide sequence TDDC instead of the RGDC found in the disintegrin domain of hMDC15. In a Western blot analysis the majority of mMDC15 was found to lack the pro-domain in all mouse tissues examined. Pulse-chase experiments in transiently transfected COS-7 cells suggest that mMDC15 is processed by a pro-protein convertase in a late Golgi compartment, since (i) addition of brefeldin A or monensin blocks pro-domain removal, (ii) all detectable processed mMDC15 is endoglycosidase H -resistant, and (iii) a recombinant soluble form of the trans-Golgi network pro-protein convertase furin can mimic mMDC15 processing in vitro. Cell-surface trypsinization revealed that more than half of mature mMDC15 is intracellular. Immunolocalization provided evidence for a strong perinuclear accumulation in a region resembling the trans-Golgi network and/or endosomal compartments. This study provides the first characterization of the intracellular processing of a metalloprotease disintegrin, and highlights the potential role of pro-protein convertases in removal of the inhibitory pro-domain. These results further suggest possible intracellular functions for mMDC15, such as in protein maturation, in addition to a potential role in cell-surface proteolysis or cell adhesion.
Here we report the cloning and initial biochemical characterization of the mouse metalloprotease/disintegrin/cysteine-rich (MDC) protein meltrin beta and the analysis of the mRNA expression of four MDC genes (meltrin alpha, meltrin beta, mdc9, and mdc15) in bone cells, including osteoclasts and osteoblasts. Like most other MDC proteins, the predicted meltrin beta protein consists of a signal sequence, prodomain, metalloprotease domain with a predicted catalytic site, disintegrin domain, cysteine-rich region, epidermal growth factor repeat, transmembrane domain, and cytoplasmic domain with putative signaling motifs, such as potential SH3 ligand domains. Northern blot analysis indicates that meltrin beta is widely expressed, with the highest expression in bone, heart, and lung. RNase protection studies revealed expression of all four MDC genes analyzed here in osteoblasts, whereas only mdc9 and mdc15 mRNAs were detectable in osteoclast-like cells generated in vitro. Treatment of primary osteoblasts with 10 nM calcitriol increased meltrin beta expression more than 3-fold, and both meltrin alpha and meltrin beta expression is apparently regulated in a differentiation-associated manner in a mouse osteoblastic cell line, MC3T3E1. Collectively, these results suggest that meltrin alpha and meltrin beta may play a role in osteoblast differentiation and/or function but are not likely to be involved in osteoclast fusion.
OBJECTIVE: To evaluate the accuracy of physiologic deadspace (VD/VT) measurement, using a metabolic monitor. DESIGN: Prospective collection of data. SETTING: University-affiliated children's hospital with a 51-bed critical care area. PATIENTS: Infants and children who were sedated and paralyzed and were receiving mechanical ventilation through a cuffed endotracheal tube. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Mixed expired carbon dioxide tension (PECO2) was measured. With the Douglas bag method, mixed expired gas was collected over 15 mins and was analyzed. With the metabolic monitor, FECO2 was measured for 15 mins and the results were averaged. The PECO2 was calculated by multiplying FECO2 by the barometric pressure. The PaCO2 was measured simultaneously. The PECO2 was corrected for the compressible volume in the ventilator circuit. All gas volumes were corrected for body temperature, pressure, and water vapor pressure. The physiologic deadspace/tidal volume ratio (VD/VT) was calculated for both techniques using the Enghoff modification of the Bohr equation. The accuracy of the VD/VT measured, by using the metabolic monitor, was assessed by comparing this measurement against the VD/VT measured by the Douglas bag method. This comparison was done by simple linear regression and correlation and by bias analysis (Bland and Altman method). The magnitude of compressible volume expressed as a fraction of the tidal volume delivered by the ventilator was compared with the error in VD/VT expressed as the difference between the uncorrected and corrected VD/VT. Sixteen paired measurements were made in 12 children. The VD/VT measured by the metabolic monitor correlated well with the VD/VT measured by the Douglas bag method (r2 = .99; p < .0001). There was no correlation between the bias of VD/VT and the average VD/VT. As the magnitude of compressible volume increased, the error in VD/VT increased (r2 =.61; p < .001). CONCLUSIONS: The VD/VT can be measured reliably and accurately in intubated pediatric patients using a metabolic monitor. The metabolic monitor method is a convenient and simple alternative to the standard Douglas bag method.
The guinea pig sperm protein fertilin (previously termed PH-30) plays an important role in sperm-egg fusion, and was the first recognized membrane-anchored metalloprotease/disintegrin protein. Fertilin is a heterodimeric glycoprotein which undergoes at least two distinct proteolytic processing steps. Fertilin alpha is processed first, in the testis, whereas fertilin beta is processed separately during sperm maturation in the epididymis. The final processing of fertilin beta occurs immediately adjacent to its predicted integrin ligand domain, and exposes an epitope recognized by a fusion blocking monoclonal antibody. Here, we demonstrate that one or more serine protease activities associated with testicular sperm can process fertilin beta in vitro in a fashion that closely mimics the processing pattern observed in vivo during epididymal sperm maturation. In contrast, several proteases that were added to testicular sperm did not mimic the pattern observed in vivo. These findings raise the intriguing possibility that a fertilin beta converting protease(s) active in vivo may originate from sperm, instead of from the epididymal epithelium. Further, we show that fertilin alpha is most likely processed intracellularly in the secretory pathway based on three observations: (i) only processed fertilin alpha, but not the precursor pro-alpha can be cell-surface biotinylated; (ii) some processed fertilin alpha is sensitive to endoglycosidase H, suggesting cleavage occurs prior to the medial Golgi apparatus; (iii) a reanalysis of the N-terminus of processed fertilin alpha showed that the proteolytic cleavage site is next to four arginine residues, a consensus sequence for intracellular subtilysin type pro-protein convertases. The N-terminal sequence analysis further showed that processed fertilin alpha contains an intact membrane anchored disintegrin domain, and not a truncated disintegrin domain as reported previously (Blobel, C. P., Wolfsberg, T. G., Turck, C. W., Myles, D. G., Primakoff, P., and White, J. M., Nature 356, 248-252, 1992). Proteolytic processing is thought to play an important role in regulating the function of fertilin, and the present study represents a first step toward a better understanding of protease activities involved in the maturation of fertilin, and potentially other sperm surface proteins.
Cellular disintegrins are a family of membrane-anchored proteins with structural homology to snake venom metalloproteases and disintegrins. We report here the cDNA cloning and initial biochemical characterization of the first cellular disintegrin protein with an RGD sequence in its disintegrin domain, which we propose to name metargidin (for metalloprotease-RGD-disintegrin protein). The domain organization of metargidin is identical with that of previously reported members of the cellular disintegrin family, comprising (i) a pro- and a metalloprotease domain including a zinc-binding consensus motif, (ii) a disintegrin domain containing the RGD motif, (iii) a cysteine-rich domain, (iv) an epidermal growth factor-like domain, (v) a hydrophobic transmembrane domain, and (vi) a cytoplasmic tail with proline-rich sequences that could act as potential SH3 ligands. Antibodies raised against the cytoplasmic tail of metargidin recognize a glycoprotein of 110 kDa in MDA-MB-468 mammary epithelial carcinoma cells, which can be cell surface-biotinylated, indicating its localization in the plasma membrane. A second protein of 56 kDa co-immunoprecipitates with metargidin, suggesting that it is part of a protein complex. These features are consistent with a model in which metargidin is an integrin ligand which, as a transmembrane protein, might function in cell-cell adhesion and/or signaling.
The importance of transcriptional control of gene expression has been documented in relation to several genetic defects in man. Mutations in the promoter region of genes have been shown to be responsible for defects which are characterized by over or under expression of genes. Genetic polymorphisms of the milk protein genes, and their high level of tissue specific expression during lactation, represent a suitable system for studying regulation of gene expression. Quantification of the allele specific protein products has demonstrated unequal allelic contribution in the expression of the beta lactoglobulin (beta-LG) alleles A and B. The DNA sequence analysis of the beta-LG promoter revealed allele specific polymorphisms related to the coding region genotypes.
Twenty-six of fifty-eight patients undergoing autologous bone marrow transplantation (autoBMT) or peripheral stem cell transplantation (PSCT) for Hodgkin's disease had progression of lymphoma (Hodgkin's or non-Hodgkin's) during the course of their follow-up. The majority of progressions, 81% (21/26), occurred within the first year of transplant; 12% (3/26) occurred at three years or more. Three patients developed a non-Hodgkin's lymphoma; all B-cell tumors primarily involving the gastrointestinal tract. The majority of patients (23/26) received at least one therapy after progression and 65% (17/26) of patients received multiple therapies. One patient who received a second BMT is alive without evidence of disease at 49 months following the second autologous BMT. The median survival for the entire group is 11 months. Forty-six percent (12/26) of patients survived more than one year and twenty-three percent (6/26) survived more than two years after disease progression. Post-progression survival is significantly related to time to progression.
Interaction between a 70-amino acid and zinc-binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady-state kinetics, heavy-atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain-C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased "stickiness" of carbamoyl phosphate to the Zn domain-C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain-C trimer complex was about 10(-2) that from C trimer.(ABSTRACT TRUNCATED AT 250 WORDS)
Peripheral blood progenitor cell (PBPC) populations used for transplantation were analyzed for the presence of CD34+ cells, colony-forming cells (initial CFC), and long-term culture initiating cells (LTC-IC) cultured on irradiated stroma for 5 weeks. Thirty-eight leukapheresis products were studied from 11 patients with breast cancer, 2 with non-Hodgkin's lymphoma and 1 with ovarian cancer harvested during recovery from either cyclophosphamide (CY) chemotherapy or cyclophosphamide-VP16 with G-CSF (CY-VP-G). CY-VP-G products had a threefold higher median number of mononuclear cells collected, a fivefold higher median concentration of CD34 and LTC-IC and a threefold higher concentration of initial-CFC when compared with CY products. CY-VP-G products had a significantly higher ratio of CFU-GM to BFU-E than the CY-mobilized products. Significant correlations of r = 0.89 and r = 0.68 were observed when comparing CD34 and CFC in products from CY or CY-VP-G patients, respectively. Analysis of the regression lines indicated that slopes of these regression lines were significantly different with a ratio of CD34 to initial CFC of 15:1 in the CY-VP-G products versus 5.2:1 with the CY products. These data indicate a higher cloning efficiency of the CD34+ population in the products from CY-mobilized patients. Significant correlations of r = 0.9 (CY) and r = 0.53 (CY-VP-G) were observed when the initial CD34 concentration and the LTC-IC were compared. Comparison of initial CFC with LTC-IC also showed significant correlations (r = 0.94, CY; r = 0.58, CY-VP-G) in samples from both patient groups.(ABSTRACT TRUNCATED AT 250 WORDS)
The growth and differentiation of selected bone marrow CD34+ cells stimulated with hematopoietic growth factors in lipid cultures were evaluated to determine whether cell types that may be useful for reducing the neutropenia associated with high-dose chemotherapy (HDC) can be produced and quantitated in vitro. CD34+ cells enriched from bone marrow were cultured for up to 5 weeks in interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) with or without stem cell factor (SCF) (also termed c-kit ligand). The mixture of IL-3, GM-CSF and G-CSF resulted in an 18-fold increase in cells after 10 to 12 days of culture and a 94-fold increase after 21 days. A 3-fold increase in colony-forming unit granulocyte-macrophage (CFU-GM) was observed after 10 days of culture. The addition of SCF during the first 10 days of culture further augmented the proliferation of cell numbers to 24-fold and colony-forming cells (CFC) to 8-fold after 10 days while cell numbers increased 130-fold after 21 days. Two-color flow cytometry defined phenotypes expressing CD11b and CD15 that represented maturation stages of neutrophils. Maturation of neutrophils in these cultures could be followed by the initial appearance after 3 to 7 days of a CD15+CD11b- phenotype representing promyelocytes, which gave rise after 2 to 3 weeks to a CD15+CD11b+ phenotype representing more mature neutrophil forms (metamyelocytes to segmented neutrophils). In contrast to normal neutrophil development, only a small fraction (10 to 15%) of the culture-derived neutrophils expressed CD16. These data define the kinetics and differentiation of neutrophils and neutrophil precursors from selected CD34+ cells in liquid cultures.
Bacillus subtilis contains a 30 kDa protein which was phosphorylated during late vegetative growth and sporulation. The sequence for the N-terminal 16 amino acids was found to be identical to the predicted sequence for the N-terminus of a small open reading frame, orfY, but diverged from the predicted sequence thereafter. The orfY region was resequenced and contained one less adenine residue than previously reported, resulting in an open reading frame from within orfY through the entire coding region for tsr which follows orfY. The predicted orfY-tsr amino acid sequence showed 24% identity to Escherichia coli fructose-1,6-bisphosphate aldolase. Two mutants in the tsr region had 2-5% of wild-type aldolase and the nucleotide sequences showed missense mutations. These results indicate that orfY-tsr encodes aldolase and should be renamed fba1.
OBJECTIVE: To determine the changes in the endocrine mechanisms of fluid balance after cardiopulmonary bypass in children. DESIGN: Prospective study; analysis of numbered plasma samples performed blind with respect to clinical data. SETTING: Regional paediatric cardiothoracic unit. PATIENTS: Nine patients, median age 4, range 2 to 9 years, five males. Patients under the age of 1 year were excluded because of the frequent blood sampling involved. MAIN OUTCOME MEASURES: Plasma concentrations of atrial natriuretic peptide (ANP), arginine vasopressin, plasma renin activity, aldosterone, noradrenaline and adrenaline, and urinary concentrations of cyclic guanosine monophosphate (cGMP) as measured by radioimmunoassay. RESULTS: After 30 minutes of cardiopulmonary bypass plasma atrial natriuretic peptide (ANP) decreased from (mean (SEM)) 151 (71) pg/ml to 52 (44) pg/ml (NS), and urinary production of its second messenger cyclic guanosine monophosphate (cGMP) decreased from 1286 (600) pmol/ml to 151 (414) pmol (p < 0.05). Other plasma concentrations of hormones studied did not change significantly although arginine vasopressin, adrenaline, and noradrenaline increased whereas aldosterone and plasma renin activity decreased. After cardiopulmonary bypass stopped there was an immediate and significant rise in plasma ANP, but within the next 24 hours plasma ANP declined significantly (p < 0.05), decreasing from 294 (49) pg/ml to 64 (29) pg/ml at 22 hours. In the postoperative period there was a significant correlation between plasma ANP and both mean fluid balance (r = 0.96, p < 0.001) and mean urine output (r = 0.97, p < 0.001). Plasma aldosterone peaked (p < 0.05) at 22 hours after operation, and argine vasopressin peaked (p < 0.05) at two hours and then declined (p < 0.05) to a trough at 24 hours. Plasma renin activity, adrenaline, noradrenaline, and urinary cGMP concentrations, and mean central venous pressure did not change significantly in the postoperative period. CONCLUSION: The changes documented show the differing pattern of release of water balance hormones invoked by cardiopulmonary bypass. The central role of ANP is shown by its strong correlation with urinary output and its similarly strong relation to fluid balance.
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