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Biomedical subjects

L Chan

Publications and source records attributed to L Chan.

At least 415 records · Page 23Linked to original sources
PubMed1986

Human liver fatty acid binding protein gene is located on chromosome 2.

The human gene for liver fatty acid binding protein was assigned to chromosome 2 using human-rodent somatic cell hybrids and Southern filter hybridization of cell hybrid DNA. The filters were hybridized to a radiolabeled cloned cDNA which covers 485 bases. Hybridization of the DNA to a human-specific 7.5-kb DNA fragment in HindIII-digested DNA was used to identify the human liver fatty acid binding protein gene. Results from two cell hybrids with chromosomal rearrangements suggest that the gene is on the short arm of chromosome 2.

Carrier Proteins↗
PubMed1986

Quantification and immunolocalization of apolipoprotein E in experimental atherosclerosis.

We developed a radioimmunoassay for rabbit apolipoprotein E (apo E) for studying their plasma apo E levels and its accumulation in the aorta of rabbits fed a cholesterol diet. Delipidation of plasma did not increase the apo E immunoreactivity and this immunoreactivity was indistinguishable from that in an apo E-phospholipid complex. The antigenic determinants of apo E in lipoprotein particles were therefore fully reacted with our goat anti-apo E antibodies. In our assay system, the non-ionic detergent Tween-20 was found to be necessary to significantly reduce the non-specific binding of 125I-labeled apo E to polystyrene tubes, and yet not interfere with the assay. In rabbits (n = 6) fed a high cholesterol (1%) diet, plasma apo E increased at least 10-fold above baseline levels and reached maximal levels within 17-20 days after the onset of cho-diet feeding. These levels were sharply reduced only 10 days after resuming a normal diet. Plasma total cholesterol levels went through a similar pattern. Thus, the plasma cholesterol concentration can simply be used to monitor the increase of apo E in cholesterol-fed rabbits. All the cholesterol-fed rabbits developed atherosclerotic fatty streak lesions and apo E located mostly in the thoracic region and was significantly correlated with the accumulation of lipids in the areas of lesion. In addition, the apo E deposition was limited to the aortic areas where lipids were present. On the other hand, apo A-I was not detectable in any lesion area. Our data suggest that apo E or apo E-containing lipoproteins, may be involved in the development of atherosclerosis in cholesterol-fed rabbits.

Animals↗
PubMed1986

Magnetic resonance spectroscopy for the determination of renal metabolic rate in vivo.

The method of magnetic resonance spectroscopy has been validated and applied to the determination of renal metabolic rate in vivo. Using an indwelling detector coil, 31P NMR spectra from one kidney of anesthetized rats were quantified. The concentration of ATP was the same as that determined enzymatically, but both ADP and Pi were substantially lower. Only 25% of renal Pi and virtually none of the ADP were detected by NMR. The remainder is assumed to be bound to proteins. These concentrations of metabolites contributed to a significantly increased phosphorylation potential, which in turn should increase the free energy of hydrolysis of ATP. Saturation transfer, a non-destructive magnetic technique for the measurement of chemical exchange, was readily able to detect synthesis of ATP from ADP and inorganic phosphate. The rate of ATP synthesis determined was comparable to that determined in parallel studies of renal oxygen consumption. An ATP:O ratio of approximately 2 was found, indicating that fatty acid is the preferred fuel of respiration of the rat kidney in vivo.

Adenosine Diphosphate↗
PubMed1986

Contributions of nuclear magnetic resonance to renal biochemistry.

31P NMR as a descriptive technique is of interest to nephrologists. Particular contributions of 31P NMR to our understanding of renal function may be enumerated.: "Free" metabolite levels are different from those classically accepted; in particular, ADP and Pi are low with implications for the control of renal metabolism and Pi transport, and, via the phosphorylation potential, for Na+ transport. Renal pH is heterogeneous; between cortex, outer medulla, and papilla, and between cell and lumen, a large pH gradient exists. Also, quantitation between cytosol and mitochondrion of the pH gradient is now feasible. In acute renal failure of either ischemic or nonischemic origin, both ATP depletion and acidification of the renal cell result in damage, with increasing evidence for the importance of the latter. Measurements of renal metabolic rate in vivo suggest the existence of a prodromal phase of acute renal failure, which could lead to its detection at an earlier and possibly reversible stage. Human renal cancers show a unique 31P NMR spectrum and a very acidic environment. Cancer chemotherapy may alter this and detection of such changes with NMR offers a method of therapeutic monitoring with significance beyond nephrology. Renal cortex and medulla have a different T1 relaxation time, possibly due to differences in lipid composition. It seems that NMR spectroscopy has much to offer to the future understanding of the relationship between renal biochemistry and function.

Acute Kidney Injury↗
PubMed1986

Mast cell leukaemia: evidence for bone marrow origin of the pathological clone.

A 57-year-old female patient, admitted for an acute abdominal syndrome, was found to have an extensive proliferation of mast cells both in the peripheral blood and the bone marrow. Cytochemical studies confirmed the mast cell characteristics of the pathological cell population, while the immunophenotype strongly suggested a bone marrow origin of this malignancy. The course of the disease was not affected by antiproliferative treatment and the patient, after progressive general deterioration, died of intractable haemorrhage. On both clinical and haematological criteria it seems possible to distinguish this rare case of primary mast leukaemia from the more common form of tissue mastocytosis with secondary leukaemia.

Bone Marrow↗
PubMed1986

Immunoreactive apolipoprotein E is a widely distributed cellular protein. Immunohistochemical localization of apolipoprotein E in baboon tissues.

Apolipoprotein (apo)E is an important protein determinant in cholesterol homeostasis in man. The protein is synthesized by the liver as well as by a number of extrahepatic tissues. In the present study, immunohistochemical techniques were used to identify apoE in specific cells in various baboon organs. In the 11 tissues studied, the following cell types have been found to harbor apoE immunoreactivity: cerebral astrocytes; thyroid follicular cells; alveolar type II pneumocytes; hepatocytes, and Kupffer cells; adrenocortical cells in zona fasciculata and zona reticularis; adrenal medullary cells; some renal tubular epithelia; some pancreatic islet cells; histiocytic macrophages in lymph nodes and the spleen; some gastric mucosal epithelia; and ovarian oocytes. These observations indicate the wide distribution of apoE in many organs and suggest that the protein might perform other important functions such as regulation of local hormonal homeostasis in addition to its role in cholesterol metabolism.

Adrenal Glands↗
PubMed1986

Estrogen induction of very low density apolipoprotein II synthesis, a major avian liver yolk protein, involves the recruitment of hepatocytes.

The mechanism of steroid hormone action was studied in the cockerel liver. Very low density apolipoprotein II (apo-VLDL-II), a yolk protein, is a low molecular weight apolipoprotein that is inducible by estrogen. The intracellular apo-VLDL-II messenger RNA (mRNA) concentration under various hormonal conditions was examined by a dot-blot assay. The concentration was very low in untreated cockerels (approximately 0.5 molecule per cell). It increased to 8,000 molecules per cell within 24 h of estrogen treatment and reached a maximum level of approximately 70,000 molecules per cell after 14 daily doses of estrogen. The distribution of hepatocytes harboring apo-VLDL-II and its mRNA was studied by immunohistochemistry and by in situ nucleic acid hybridization to cloned [3H]apo-VLDL-II complementary DNA (cDNA). The number of cells containing the immunoreactive protein and the hybridizable mRNA increased from extremely low (0.3% and 0.27%, respectively) to substantial (11% and 10%, respectively) at 24 h after estrogen treatment and to extremely high (94% and 92%, respectively) in maximally treated animals. Our studies indicate that, in addition to enhanced transcription and stabilization of mRNA, the recruitment of liver cells previously not engaged in the synthesis of apo-VLDL-II is an important mechanism by which the hormone induces the hepatic production of this protein. The phenomenon of recruitment and the heterogeneity of the functional capacity of individual hepatocytes to respond to estrogen may be important to our understanding of estrogen action in the liver.

Animals↗
PubMed1986

Somatostatin production by a human medullary thyroid carcinoma cell line.

Somatostatin (SRIF, SRIF-14) is a known product of the normal and malignant parafollicular cell of the thyroid. In this report we characterize SRIF production by the TT cells, a line of transformed calcitonin-producing cells derived from a human medullary thyroid carcinoma. The cells were found to contain (5-12 ng/10(6) cells) and secrete (3-10 ng/10(6) cells X 48 h) immunoreactive SRIF. Molecular sieve chromatography of cell extracts under denaturing conditions showed a major peak with a mol wt slightly larger than 12,700, probably representing pro-SRIF and a second peak which coeluted with SRIF; in one gel chromatogram a very small peak was also noted which coeluted with SRIF-28, but represented less than 0.4% of the total immunoreactive SRIF. Short term secretion of calcitonin and SRIF was stimulated by calcium in vitro (0.5-4 mM) in a dose-related manner. mRNA isolated from the TT cells hybridized to a specific bovine fetal pancreatic SRIF DNA (BFPS-2); there was no hybridization to identical amounts of mRNA from the atT-20/D16, 3T3, or RINC5F cell lines. In vitro translation of the TT cell mRNA followed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the product revealed a single protein band of approximately 13,000 daltons. It was completely abolished when the immunoprecipitation was performed in the presence of excess unlabeled SRIF. Northern transfer of TT cell cytoplasmic RNA and hybridization with FBPS-2 cDNA showed a single hybridizing band with an apparent size of approximately 750 nucleotides. Our observations demonstrate the production of SRIF by a continuous line of human medullary thyroid carcinoma cells and provide a model for studying the biosynthesis and secretion of SRIF in the parafollicular cell.

Animals↗
PubMed1986

bcr rearrangement and translocation of the c-abl oncogene in Philadelphia positive acute lymphoblastic leukemia.

The Philadelphia (Ph1) chromosome, the cytogenetic hallmark of chronic myeloid leukemia (CML), has also been detected in a significant number of acute lymphoblastic leukemias (ALL). Using in situ hybridization, we demonstrate that in accordance with observations in CML the Ph1 chromosome in ALL patients is the result of a consistent translocation of the c-abl oncogene to the Ph1 chromosome. Southern blot analysis using bcr probes, however, suggests that Ph1-positive ALL includes heterogeneous leukemic subtypes: six ALL patients showed bcr rearrangements as observed in CML; in three other patients recombination involving 5' bcr sequences could be demonstrated, but the corresponding translocated 3' bcr sequences were not detectable. A third group of five patients did not show any bcr rearrangements at all. Northern blot analysis using RNA from three Ph1-positive ALL patients revealed that in the leukemic cells of two patients larger c-abl mRNA transcripts were present, as in CML. In the RNA of one patient without a detectable bcr rearrangement, only the normal c-abl mRNA transcripts are present. The observed heterogeneity in bcr rearrangements of this group of Ph1-positive ALL patients is in contrast with the consistent results obtained in more than 50 Ph1-positive CML patients investigated in chronic and acute states.

Chromosome Deletion↗
PubMed1985

The structure of the human apolipoprotein C-II gene. Electron microscopic analysis of RNA:DNA hybrids, complete nucleotide sequence, and identification of 5' homologous sequences among apolipoprotein genes.

Cloned human apo-C-II cDNA was used as a hybridization probe to identify the human apo-C-II gene in a genomic library constructed in our laboratory. The isolated apo-C-II DNA was studied both by electron microscopy and by direct sequence analysis. Ultrastructural morphological analysis of RNA-DNA hybrids revealed that the apo-C-II gene had complex structures because of regions of inverted complementary sequences in and around the gene forming stem-and-loop structures which interfere with the formation of stable RNA:DNA hybrids. Extensive morphological analysis revealed a minimum of 3 intervening sequences (IVS), and their lengths were measured. Direct sequence analysis of the cloned gene confirmed the presence of 3 IVS. There are 4 Alu type sequences in IVS-I. We sequenced 4340 nucleotides which include 545 nucleotides in the 5' flanking region, the entire gene which spans 3320 nucleotides, and 475 nucleotides in the 3' flanking region which also encompasses an additional Alu sequence. The 5' end of the gene was identified by primer extension and sequencing of the primer extended cDNA. Apo-C-II mRNA structure was deduced from the cDNA sequence, the primer extension experiments, and the genomic sequence. It is 494 nucleotides in length. Its sequence differs from previously published sequences in that there are 7 additional nucleotides before the polyadenylate tail. In the 5' flanking region, nucleotides -234 to -213 encompass a GC-rich region which exhibits high homology (greater than 70%) to the 5' flanking regions of the genes of all the apolipoproteins published to date, namely, apo-A-II (-497 to -471), apo-A-I (approximately -196 to -179), apo-E (-409 to -391), and apo-C-III (approximately -116 to -103). This highly conserved region might represent some evolutionarily conserved sequences from these related genes and/or might represent a region with regulatory function.

Apolipoprotein C-II↗
PubMed1985

Isolation and characterization of the human apolipoprotein A-II gene. Electron microscopic analysis of RNA:DNA hybrids, nucleotide sequence, identification of a polymorphic MspI site, and general structural organization of apolipoprotein genes.

Three cloned apolipoprotein A-II genes were isolated from a human genomic cosmid library constructed in our laboratory. An approximately 3-kilobase HindIII insert containing the entire gene was analyzed by RNA:DNA hybridization and electron microscopy. The apo-A-II gene was found to consist of 4 exons and 3 intervening sequences (IVS), and the lengths of each exon and IVS were estimated by direct observation of the hybrids. The entire approximately 3-kilobase HindIII insert was sequenced. The 5' end of the gene was determined by primer extension. The DNA sequence confirms the presence of 4 exons and 3 IVS: exon 1, 34 nucleotides; exon 2, 76 nucleotides; exon 3, 133 nucleotides; exon 4, 230 nucleotides; IVS-I, 169 nucleotides; IVS-II, 299 nucleotides; and IVS-III, 396 nucleotides. A "TATA box" is located at position -29 from the CAP site. A "CAT box" is present at position -78. A "TG" element consisting of (TG)19 is identified at the 3' end of IVS-III. Furthermore, an enhancer core sequence, CTTTCCA, is identified at position -355 in the 5' flanking sequence. At positions -497 to -471 upstream from the CAP site is a stretch of 27 nucleotides that show high homology to stretches of 5' flanking sequences in the apo-C-II, apo-A-I, apo-E, and apo-C-III genes. An Alu dimer sequence is located approximately 300 nucleotides from the 3' end of the gene. Within this Alu sequence, we have identified a polymorphic MspI site. Restriction fragment length polymorphism involving this site has been previously shown to correlate with apo-A-II levels and high density lipoprotein structure. Analysis of conformation by Chou-Fasman analysis and by the helical hydrophobic moment of Eisenberg et al. (Eisenberg, D., Weiss, R. M., and Tergwillager, T. C. (1982). Nature (Lond.) 299, 371-374) indicates that in all of the 5 apolipoproteins characterized at the nucleotide level to date, i.e. apo-C-II, apo-A-II, apo-E, apo-A-I, and apo-C-III, the 2 IVS within the peptide coding regions of the gene tend to occur at regions corresponding to the surface of the polypeptide chain and divide the protein into distinct functional domains.

Apolipoprotein A-II↗
PubMed1985

The human apolipoprotein B-100 gene: a highly polymorphic gene that maps to the short arm of chromosome 2.

Using a cloned cDNA of apolipoprotein B-100 as hydridization probe, we have found high frequence polymorphisms in the apoB-100 gene involving sites for the restriction enzymes EcoRI, BamHI, and HindIII. The major EcoRI polymorphisms involved a 17 kb vs 15 kb variant. The incidence of the various phenotypes was estimated. In addition, other complex polymorphisms involving MspI and TaqI sites were also noted. [32P]-labeled apoB-100 cDNA was used as a probe in chromosome mapping studies to detect the human apoB-100 structural gene sequence in human-Chinese hamster and human-mouse cell hybrids. Southern blot analysis of 14 hybrids localized the gene to the short arm of human chromosome 2.

Apolipoprotein B-100↗
PubMed1985

Apolipoprotein E gene mapping and expression: localization of the structural gene to human chromosome 19 and expression of ApoE mRNA in lipoprotein- and non-lipoprotein-producing tissues.

Apolipoprotein E (apoE) binds to specific cell-surface receptors and appears to be an important determinant in lipoprotein metabolism in man. Cloned human apoE cDNA (pAE155) was used as a probe in chromosome mapping studies to detect the structural gene sequences in human--Chinese hamster cell hybrids. Southern blot analysis of HincII-digested DNAs from 13 hybrids localized the gene to human chromosome 19. This observation indicates that apoE is syntenic to at least two other genes related to lipid metabolism, those for the low-density lipoprotein (LDL) receptor (the LDLR) and apoC-II. The cloned apoE cDNA was further used to detect the presence of apoE mRNA in RNA extracts of various human and baboon tissues. Northern gel analysis using the 32P-labeled pAE155 as a probe demonstrated the presence of hybridizable apoE mRNAs in human liver and in baboon liver, intestine, spleen, kidney, adrenal gland, and brain but not in baboon skeletal muscle. The apoE mRNAs appear to be intact and migrate on an agarose gel under denaturing conditions at approximately 18 S. To assay for the biological activity of the apoE mRNAs in these tissues, they were translated in a reticulocyte lysate system in vitro. Immunoprecipitation with an apoE-specific antiserum followed by sodium dodecyl sulfate gel electrophoresis and fluorography demonstrated that immunoreactive apoE with the expected apparent size was a product of translation of mRNAs from baboon liver, intestine, kidney, spleen, and brain but not that from baboon skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗