[Hyperlipidemia in children].
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Adolescent↗
Biomedical subjects
T J Jensen
Publications and source records attributed to T J Jensen.
Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR).
Circumstantial evidence has accumulated suggesting that CFTR is a regulated low-conductance Cl- channel. To test this postulate directly, we have purified to homogeneity a recombinant CFTR protein from a high-level baculovirus-infected insect cell line. Evidence of purity included one- and two-dimensional gel electrophoresis, N-terminal peptide sequence, and quantitative amino acid analysis. Reconstitution into proteoliposomes at less than one molecule per vesicle was accomplished by established procedures. Nystatin and ergosterol were included in these vesicles, so that nystatin conductance could serve as a quantitative marker of vesicle fusion with a planar lipid bilayer. Upon incorporation, purified CFTR exhibited regulated chloride channel activity, providing evidence that the protein itself is the channel. This activity exhibited the basic biophysical and regulatory properties of the type of Cl- channel found exclusively in CFTR-expressing cell types and believed to underlie cAMP-evoked secretion in epithelial cells.
Animals↗
Mislocalization of delta F508 CFTR in cystic fibrosis sweat gland.
Misprocessing and mislocalization of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been described for the major CF-causing mutation (delta F508) in heterologous expression systems in vitro. We have generated monoclonal antibodies (mAbs) to CFTR with the aim of localizing the protein and its CF variants in vivo. Of the tissues where CFTR was observed, only the sweat gland is readily available and does not undergo secondary changes due to CF disease pathology. Sweat ducts from CF patients homozygous for delta F508 did not show the typical apical membrane staining seen in control biopsies. This demonstrates that the biosynthetic arrest and intracellular retention of delta F508 CFTR initially observed in vitro does occur in vivo and emphasizes the need to focus efforts on understanding the mislocalization.
Amino Acid Sequence↗
Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance.
The nature of involvement of the cystic fibrosis gene product (CFTR) in epithelial anion transport is not yet understood. We have expressed CFTR in Sf9 insect cells using the baculovirus expression vector system. Reactivity with antibodies against 12 different epitopes spanning the entire sequence suggested that the complete polypeptide chain was synthesized. Immunogold labeling showed localization to both cell-surface and intracellular membranes. Concomitant with CFTR expression, these cells exhibited a new cAMP-stimulated anion permeability. This conductance, monitored both by radioiodide efflux and patch clamping, strongly resembled that present in several CFTR-expressing human epithelial cells. These findings demonstrate that CFTR can function in heterologous nonepithelial cells and lend support to the possibility that CFTR may itself be a regulated anion channel.
Animals↗
[Recommendations for clinical-chemical departments: lipid-lipoprotein analysis].
The section for preventive cardiology within the Danish Society for Cardiology has established a lipid group with representatives from The Danish Society for Clinical Chemistry, The Danish Society for Internal Medicine, The Danish Society for Cardiology, The Danish Society of Hypertension, The Danish College of General Practitioners, and The Danish Paediatric Society. The lipid group has elaborated recommendations for clinical chemical departments regarding lipid and lipoprotein analyses. The group suggests that doctors ordering lipid and lipoprotein analyses are offered the following: S-Cholesterol (total), substance conc., (fPt)S-Triglycerides, substance conc., S-HDL-cholesterol, substance conc., and (fPt)S-LDL-cholesterol, substance conc. (calculated). It is recommended that the biological variation be minimized by sampling in a sitting position after a 15 minutes' rest and by basing the clinical decision on a minimum of 2-3 determinations with an interval of about one month. The analytical variations should be reduced to below 3% (calculated as the variation coefficient), and it is recommended that laboratories participate in external quality control systems at least four times annually by reporting at least two human reference materials with different concentrations. As the use of reference intervals dependent on age and sex, based on random samplings of the background population, are less informative, it is recommended to refer to cutoff values for the clinical decision. The following cutoff values are recommended: S-Cholesterol (total), substance conc.: 6 mmol/l, (fPt)S-Triglycerides, substance conc.; 2.5 mmol/l, S-HDL-cholesterol, substance conc.: 0.9 mmol/l (fPt)-LDL-cholesterol, substance conc. (calculated): 4.5 mmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)
Chemistry, Clinical↗
Transformed sweat gland and nasal epithelial cell lines from control and cystic fibrosis individuals.
We undertook to extend the in vitro lifespan of epithelial cell cultures useful for the study of the cellular defect underlying cystic fibrosis (CF). Primary cultures from sweat glands of four CF and four non-CF and from nasal polyps of one non-CF and two CF individuals were transformed using a chimaeric virus, Ad5/SV40 1613 ori-. The extended lifespans ranged from 20 to more than 250 population doublings beyond that of the primary cultures. Despite some degree of aneuploidy (as assayed by total cellular DNA content) all samples tested retained at least one copy of the region of chromosome 7 containing the CF gene (as assayed by probing with flanking DNA markers). Epithelial characteristics, including an epithelioid morphology, tight junctions and desmosomes, apical microvilli, keratin networks, and dome formation were positive in the majority of cells examined, although variably expressed. All cells tested demonstrated outwardly rectifying chloride channels by patch clamp, with some from non-CF cells responsive to the catalytic subunit of cyclic AMP-dependent protein kinase. The cells were used for DNA transfection assays with selectable marker genes in appropriate vectors, in order to develop methodology for assaying the function of the CF gene product and the effects of mutations.
Cell Division↗
Bicarbonate permeability of the outwardly rectifying anion channel.
Single anion-selective channels have been studied in cultured human epithelial cells using the patch-clamp technique. Three cell types were used as models for different anion transport systems: (i) PANC-1, a cell line derived from the pancreatic duct, (ii) T84, a Cl-secreting colonic cell line, and (iii) primary cultures of sweat duct epithelium. Outwardly rectifying anion-selective channels were observed in all three preparations and were indistinguishable with respect to conductance, selectivity and gating. Striking similarities between HCO3- and Cl-secreting epithelia, and the high density of outward rectifiers in pancreatic cells prompted us to study HCO3 permeation through this channel. HCO3 permeability was significant when channels were bathed in symmetrical 150 mM HCO3 solutions, Cl-HCO3 mixtures, and under bi-ionic conditions with outwardly and inwardly directed HCO3 gradients. Permeability ratios (PHCO3/PCl) estimated from bi-ionic reversal potentials ranged from 0.50 to 0.64, although conductance ratios greater than 1.2 were observed with high extracellular pH. Chloride did not inhibit HCO3 permeation noticeably but rather had a small stimulatory effect when present on the opposite side of the membrane. The prevalence of outward rectifiers in PANC-1 and their permeability to bicarbonate suggests the channel may have a dual role in HCO3 secretion; to allow Cl recycling at the apical membrane and to mediate some of the HCO3 flux. Defective modulation of this channel in cystic fibrosis might provide a common basis for dysfunction in epithelia having very different anion transport properties (e.g., HCO3 secretion, Cl secretion and Cl absorption.
Bicarbonates↗