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Biomedical subjects

S J Lauer

Publications and source records attributed to S J Lauer.

At least 37 records · Page 2Linked to original sources
PubMed1996

Stage I and II Hodgkin's disease in the pediatric population. Long-term follow-up of patients staged predominantly clinically.

Between January 1, 1970, and December 31, 1990, 42 consecutive pediatric patients were treated at the Medical College of Wisconsin Affiliated Hospitals for early-stage Hodgkin's disease. Thirty patients were clinically staged. Twelve underwent staging laparotomy as a part of staging work-up. Thirty-one patients were treated with radiation therapy (RT) alone. Eleven were treated with combined chemotherapy and RT. For the entire group, overall survival at 5, 10, and 15 years was 98, 98, and 92%, respectively. Disease-free survival was 86, 86, and 79, respectively. There was no significant difference in overall survival or disease-free survival comparing clinically versus pathologically staged patients. There was a trend toward improved disease-free survival favoring pathologically staged patients; however, this difference did not reach statistical significance (p = 0.07). The long-term results of this series fail to show statistically significant superior disease-free or overall survival with surgical staging.

Adolescent↗
PubMed1995

Repetitive low dose oral methotrexate and intravenous mercaptopurine treatment for patients with lower risk B-lineage acute lymphoblastic leukemia. A Pediatric Oncology Group pilot study.

BACKGROUND: This trial evaluated the toxicity and preliminary efficacy of a repeated oral low dose (LD) methotrexate schedule with intravenous mercaptopurine infusions as intensification therapy for children with lower risk B-lineage acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS. From December 1986 to January 1991, 96 children with newly diagnosed, lower risk ALL were enrolled. Vincristine, L-asparaginase, and prednisone were used for remission induction. Age-based methotrexate was administered intrathecally (IT) for central nervous system (CNS) prophylaxis. An outpatient-based intensification treatment included LD methotrexate 30 mg/M2 every 6 hours for 5 doses, followed by intravenous mercaptopurine 1000 mg/M2 for 6 hours every 2 weeks for 12 courses. Leucovorin rescue was administered 48 hours after methotrexate treatment was begun. Maintenance therapy included standard daily oral mercaptopurine, weekly intramuscular methotrexate, and IT methotrexate every 12 weeks, for 2 years. RESULTS: All patients had disease remission. Thirty-two patients relapsed; there were 17 isolated bone marrow relapses, 10 isolated CNS relapses, 2 isolated testicular relapses, 1 marrow plus CNS relapse, 1 marrow plus testicular relapse, and 1 CNS plus testicular relapse. Event free survival (EFS) at 4 years was 66% (standard error, 7%) by Kaplan-Meier analysis. Complications associated with LD methotrexate/mercaptopurine courses were few and resulted in hospital readmissions in 2.4% of courses. Two patients were unable to comply with the oral LD methotrexate schedule and received intravenous methotrexate. Three patients were unable to complete scheduled maintenance because of hepatic or hematopoietic dysfunction. CONCLUSIONS: Low dose methotrexate/mercaptopurine can be administered safely on an outpatient basis to children with lower risk B-lineage ALL. However, there was a higher than expected incidence of bone marrow and CNS relapse. The reasons for this outcome were not completely clear but raise the possibility that LD methotrexate therapy may be less effective in preventing relapse than are higher dose, parenteral methotrexate regimens.

Administration, Oral↗
PubMed1994

Overexpression of hepatic lipase in transgenic rabbits leads to a marked reduction of plasma high density lipoproteins and intermediate density lipoproteins.

To elucidate the precise metabolic roles of hepatic lipase (HL), a human HL cDNA in a liver-specific expression vector was used to generate transgenic lines in the rabbit, an animal that normally expresses low levels of this enzyme. HL was detected in the plasma of all rabbits only after the administration of heparin; HL activity in transgenic rabbits was found at levels up to 80-fold greater than that in nontransgenic littermates. This increase in enzyme activity was associated with as much as a 5-fold decrease in total plasma cholesterol levels. Expression of the transgene resulted in a dramatic reduction in the level of large high density lipoproteins (HDL1 and HDL2) as well as dense HDL3. A reduction in the quantity of intermediate density lipoproteins (IDL) was also observed. These results demonstrate that HL functions in the metabolism of HDL and IDL, thereby playing a key role in plasma cholesterol homeostasis.

Animals↗
PubMed1994

Recombinant alpha-2B interferon treatment for childhood T--lymphoblastic disease in relapse. A Pediatric Oncology Group Phase II study.

BACKGROUND: Children with chemotherapy refractory T-cell lymphoblastic leukemia/lymphoma were given alpha-interferon (alpha-IFN) to evaluate the efficacy and toxicity of this biologic response modifier. METHODS: Twenty children with T-cell acute lymphoblastic leukemia (T-cell ALL) in marrow relapse and one patient with mediastinal recurrence of T-cell non-Hodgkin's lymphoma (T-cell NHL) were enrolled. All patients had failed at least two previous multiagent drug trials. Recombinant alpha-IFN was given at 30 million U/M2/dose intravenously or subcutaneously for 10 doses over 14 days, followed by 3 doses per week until disease progression occurred. RESULTS: One child had a complete response (< 5% blasts) and three patients a partial response (5-25% blasts) in their bone marrow. All patients eventually showed signs of progressive disease. Significant toxicities included cardiac hypofunction in two patients and profound lethargy in two patients. CONCLUSIONS: alpha-IFN is tolerated in children with T-cell ALL and T-cell NHL and has activity against chemotherapy resistant disease.

Adolescent↗
PubMed1994

Enhanced binding and uptake of remnant lipoproteins by hepatic lipase-secreting hepatoma cells in culture.

Rat hepatoma McA-RH7777 cells transfected with a human hepatic lipase (HL) cDNA synthesized and secreted 50-80 ng of human HL/mg of cell protein at 4 h, approximately 50% of which was bound to cell-surface heparan sulfate proteoglycans (HSPG). The newly synthesized HL possessed enzymatic activity. When rabbit beta-very low density lipoproteins (beta-VLDL) and canine chylomicrons or chylomicron remnants were incubated with HL-secreting cells, remnant binding and uptake were enhanced 3-fold compared with nontransfected cells. Furthermore, fluorescence microscopy showed enhanced uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled beta-VLDL by the HL-transfected cells. When 125I-beta-VLDL were added to conditioned medium from HL-secreting cells, the HL in the media enhanced the binding and uptake of the remnant lipoproteins by nontransfected cells about 3-fold. Likewise, surface-bound HL (without HL in the medium) also was able to mediate the enhanced binding of the remnants. This HL-enhanced binding was shown to be mediated by an interaction with cell-surface HSPG. Heparinase treatment to remove cell-surface HSPG or chlorate treatment to prevent HSPG sulfation of the HL-secreting cells abolished all the HL-mediated enhanced binding and uptake. Furthermore, heparinase pretreatment of nontransfected cells prevented the enhanced binding and uptake of beta-VLDL incubated with conditioned medium from HL-secreting cells. As binding was not enhanced in the absence of HSPG, an HL-HSPG initial interaction appears essential. Addition of apolipoprotein (apo) E to the beta-VLDL did not facilitate HL-mediated binding and uptake; in fact, beta-VLDL from apoE-null mice demonstrated a similar degree of enhanced binding as did rabbit beta-VLDL with or without added apoE. On the other hand, beta-VLDL from transgenic mice overexpressing binding-defective apoE(Arg142-->Cys) did not display any enhanced binding and uptake by the HL-secreting cells, and it appears that the apoE(Arg142-->Cys) actually inhibited the HL-mediated interaction. This mutant form of apoE is associated with a dominant mode of expression of type III hyperlipoproteinemia in contrast to the more commonly occurring recessive disorder. Impaired HL interaction with the apoE(Arg142-->Cys) beta-VLDL may contribute to remnant lipoprotein accumulation in the plasma of patients with this mutant form of apoE. Thus, HL contributes to the enhanced cell association of specific types of remnant lipoproteins by initiating their binding to cell-surface HSPG.

Animals↗
PubMed1994

Overexpression of human apolipoprotein C-III in transgenic mice results in an accumulation of apolipoprotein B48 remnants that is corrected by excess apolipoprotein E.

Overexpression of human apolipoprotein (apo) C-III in the plasma of transgenic mice results in hypertriglyceridemia, with up to a 20-fold elevation in plasma triglyceride. Nearly all of the triglyceride accumulates in the d < 1.006 g/ml lipoprotein fraction, which consists predominantly of apoB48-containing particles having a low apoE:apoB48 ratio in contrast to normal mice. The transgenic and nontransgenic d < 1.006 g/ml lipoproteins are similar in size, and they are equivalent substrates for lipoprotein lipase in vitro. Total apoB100 levels are similar in transgenic and normal plasma, but apoB48 levels are increased in transgenic mice. The transgenic d < 1.006 g/ml particles are poor competitors for the binding of low density lipoproteins to the low density lipoprotein receptor in vitro, which is corrected by the addition of exogenous apoE. The rate of clearance of labeled chylomicron remnants in apoC-III-transgenic mice was about half that in nontransgenic mice. The lipoprotein alterations are accompanied by up to a 5-fold increase in circulating nonesterified fatty acids, which may be the cause of fatty livers and increased liver triglyceride production also observed in the transgenic mice. These observations indicate that the primary defect leading to hypertriglyceridemia in apoC-III overexpressers is an impaired clearance of apoB48 remnants due to apoE insufficiency. Therefore, transgenic mice that overexpressed human apoE were cross-bred with the apoC-III overexpressers. Transgenic progeny that produced both human apoE and human apoC-III had normal levels of plasma triglyceride and normal amounts of apoB48 remnants. Thus, our studies suggest that a function of apoC-III is to modulate the apoE-mediated clearance of lipoproteins, and that the concentration of apoC-III relative to apoE is a key determinant of triglyceride levels in plasma.

Animals↗
PubMed1994

Intensive intravenous methotrexate and mercaptopurine treatment of higher-risk non-T, non-B acute lymphocytic leukemia: A Pediatric Oncology Group study.

PURPOSE: To determine the potential efficacy and toxicity of intravenous (i.v.) methotrexate (MTX) and mercaptopurine (MP) as postremission intensification treatment for children with B-lineage acute lymphoblastic leukemia (ALL) at higher risk to relapse. PATIENTS AND METHODS: Eighty-three patients (age 1 to 20 years) with higher-risk B-lineage ALL were entered onto this protocol. Following standard four-drug remission induction, 80 patients received 12 intensive 2-week cycles of MTX/MP: MTX 200 mg/m2 i.v. push, then 800 mg/m2 i.v. 24-hour infusion on day 1; MP 200 mg/m2 i.v. in 20 minutes, then 800 mg/m2 i.v. 8-hour infusion day 2; MTX 20 mg/m2 intramuscularly day 8; and MP 50 mg/m2 by mouth days 8 to 14. Age-based triple intrathecal therapy (MTX, hydrocortisone, and cytarabine) was administered for CNS prophylaxis. Continuation therapy was weekly MTX/MP (as on days 8 to 14) for 2 years. RESULTS: Eighty-one patients (98%) entered remission. There were 28 relapses (marrow, n = 11; marrow and CNS, n = 2; isolated CNS, n = 9; testes, n = 5; ovaries, n = 1). No overt relapse occurred during the intensive phase of therapy. The event-free survival (EFS) rate at 4 years is 57.4% +/- 9.1% (SE). Hematologic, mucosal, and infectious toxicities were seen in 12%, 9%, and 5% of intensive MTX/MP courses, but were generally mild. CONCLUSION: Combined data from this and our previous trial suggest that intensive MTX/MP may produce long-term disease-free survival in 70 to 75% of children with B-lineage ALL. In comparison to other intensive regimens, intensive MTX/MP is easy to administer, effective, and relatively nontoxic. If patients at risk for failure of MTX/MP can be identified prospectively, more aggressive regimens could be restricted to this smaller (25% to 30%) cohort.

Adolescent↗
PubMed1993

Intensive alternating drug pairs for treatment of high-risk childhood acute lymphoblastic leukemia. A Pediatric Oncology Group pilot study.

BACKGROUND: To prevent drug resistance, the authors designed a protocol that featured early intensive rotating drug pairs as part of the therapy for acute lymphoblastic leukemia (ALL). METHODS: After prednisone, vincristine, asparaginase, and daunorubicin induction, 12 intensive treatments (ABACABACABAC) were given in 30 weeks: A--intermediate-dose methotrexate (IDMTX) plus intermediate-dose mercaptopurine (MP); B--cytosine arabinoside (AC) plus daunorubicin (DNR); C--AC plus teniposide (VM-26). Triple intrathecal chemotherapy (AC, MTX, and hydrocortisone) was given for central nervous system (CNS) prophylaxis. Continuation therapy consisted of weekly MTX and daily MP until 2.5 years of continuous complete remission had been achieved. RESULTS: Seventy-four children (age range, 1-19 years) at high risk of relapse were treated. Of 55 with B-lineage (early pre-B, pre-B) ALL, 24 have failed (2 induction failures, 2 deaths from infection, and 20 relapses). The event-free survival (EFS) rate at 4 years was 55.5% (standard error [SE] +/- 7.7%). Of 19 patients with T-cell ALL, 12 have failed (2 induction failures and 10 relapses). The EFS rate at 4 years was 32.6% (SE +/- 26.8%). Toxicities were significantly more common after AC and DNR or AC and VM-26 than IDMTX and MP. There were no toxicity-related deaths during intensive treatments. CONCLUSION: Early intensive rotating therapy is tolerable and warrants consideration for additional trials of patients with high-risk, B-lineage ALL.

Administration, Oral↗
PubMed1993

A far-downstream hepatocyte-specific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice.

The human apolipoprotein (apo) E and apoC-I genes are located 5 kilobases apart in the same transcriptional orientation on chromosome 19, and they are expressed at high levels in the liver with lower levels of expression in selected other tissues. Analysis of a series of overlapping human apoE and apoC-I genomic fragments in transgenic mice revealed that the expression of these transgenes in the liver requires a common cis-acting regulatory domain. This hepatic control region (HCR) was localized to a 764-base pair region that is located about 18 kilobases downstream of the apoE promoter and about 9 kilobases downstream of the apoC-I promoter. All the transgenic animals that had been prepared with a construct that contained this region had relatively high levels of transgene expression in the liver, whereas constructs that lacked this region showed no expression in the liver. In situ hybridization studies showed that the HCR directed apoE and apoC-I transgene expression in hepatocytes. When the HCR from the apoE/C-I gene locus was ligated proximal to a human apoA-IV gene fragment, which is not normally expressed in the liver, the resulting apoA-IV/HCR fusion construct was expressed at high levels in the liver, indicating that the HCR could direct high level liver expression of a heterologous promoter/gene construct. Expression of the apoE transgene in the liver and kidney, and perhaps other tissues, required the presence of a nonspecific proximal enhancer element in the apolipoprotein E gene promoter, located between 161 and 141 bp relative to the transcription initiation site. However, the proximal apoE gene promoter, including this enhancer element, contained no sequences capable of directing hepatocyte expression in the absence of the HCR. Thus, the far-downstream HCR appears to contain all of the sequences necessary for determining high level liver-specific gene expression.

Animals↗
PubMed1993

ApoA-IV is secreted on discrete HDL particles by the rat hepatoma cell line McA-RH7777 transfected with ApoA-IV cDNA.

In the present study, the synthesis and secretion of transfected apolipoprotein (apo) A-IV was investigated in rat hepatoma McA-RH7777, a cell line that does not express apoA-IV mRNA or protein. An expression plasmid that contained the rat apoA-IV cDNA was transfected into the cells; five stable transformants were selected that harbor different copy numbers of the apoA-IV construct and secrete different amounts of apoA-IV. Gel filtration column chromatography and density gradient ultracentrifugation, combined with gel electrophoresis and electron microscopy techniques, demonstrated that (1) the secreted apoA-IV associated mainly with high-density lipoproteins (HDLs) and only a trace amount of apoA-IV was associated with very-low-density lipoproteins; (2) overexpression of apoA-IV resulted in an increased number of disk-shaped structures (thickness, approximately 8.0 nm and diameter, approximately 22 nm); and (3) the electrophoretic mobilities of the apoA-IV-containing particles differed from those of apoA-I-containing HDL. Expression of apoA-IV exerted no discernible effect on the density distribution or the secretion efficiency of apoB-100. Additionally, secretion of apoB-100 and apoA-IV exhibited opposite responses to serum: apoB-100 secretion was stimulated eightfold after addition of serum, whereas apoA-IV secretion was inhibited by 40%. These results suggest that synthesis of apoA-IV may lead to the formation of a subclass of HDL with a different metabolic fate than that of lipoproteins containing either apoA-I or apoB.

Animals↗
PubMed1992

Accumulation of high levels of methotrexate polyglutamates in lymphoblasts from children with hyperdiploid (greater than 50 chromosomes) B-lineage acute lymphoblastic leukemia: a Pediatric Oncology Group study.

Hyperdiploidy (greater than 50 chromosomes, or a DNA index greater than 1.16) confers a favorable prognosis in B-lineage acute lymphoblastic leukemia of childhood. Children with B-lineage acute lymphoblastic leukemia whose lymphoblasts at diagnosis accumulate high levels of methotrexate (MTX) and MTX polyglutamates (MTXPGs) in vitro experience a better event-free survival than those whose lymphoblasts do not (Blood 76:44, 1990). Lymphoblasts from 13 children with hyperdiploidy (greater than 50 chromosomes) accumulated high levels of MTX-PGs (1,095 and 571 to 2,346 pmol/10(9) cells [median and 25% to 75% intraquartile range]). These levels were higher than those in B-lineage lymphoblasts from 19 children with other aneuploidy (326 and 159 to 775 pmol/10(9) cells) and 15 children with diploidy (393 and 204 to 571 pmol/10(9) cells) (P = .0015). Chromosomal trisomies in hyperdiploid cases were highly nonrandom. Chromosome 9 was not one of the chromosomes involved in trisomies, even though this chromosome contains the gene for folate polyglutamate synthetase, which is the enzyme required for MTXPG synthesis. The correlation between MTXPG level and percentage of S-phase cells was weak, suggesting that increased levels of MTXPGs could not be attributed to elevated proportions of cells in active DNA synthesis. The ability of hyperdiploid lymphoblasts to accumulate high levels of MTXPGs may increase their sensitivity to MTX cytotoxicity, accounting in part for the improved outlook for hyperdiploid patients treated with regimens that emphasize MTX as a primary component of continuation therapy.

Child↗
PubMed1991

Multiple tissue-specific elements control the apolipoprotein E/C-I gene locus in transgenic mice.

To investigate the mechanisms controlling tissue-specific expression of the human apolipoprotein (apo) E/C-I gene locus, human apoE and apoC-I gene constructs containing various lengths of the 5'-flanking or 3'-flanking region were used to create transgenic mice. Several essential tissue-specific regulatory elements were identified in the region between the apoE and the apoC-I genes, as well as in a distal domain found downstream of the apoC-I gene. Most notably, transcription of both the apoE and apoC-I genes in the liver, their major site of expression, required downstream regulatory elements, possibly located within a common regulatory domain more than 2 kilobases 3' of the apoC-I gene (about 14 kilobases downstream of the apoE gene promoter). In the region between the apoE and apoC-I genes, a single strong positive element directed apoE and apoC-I gene expression in the skin. The intergenic region also contained elements that stimulated apoE gene expression in the brain and silenced apoE gene expression only in the kidney. These results demonstrate that multiple independent regulatory elements control expression of the human apoE/C-I gene locus in various tissues. Transgenic mice expressing human apoC-I in the liver exhibited plasma triglyceride levels that were 2-3-fold higher than those in control mice, an effect not found when transgenic human apoE was produced. This result suggests that apoC-I may modulate the metabolism of triglyceride-rich lipoproteins.

Animals↗
PubMed1991

Lipoproteins in pinnipeds: analysis of a high molecular weight form of apolipoprotein E.

We have examined the apolipoprotein content of the lipoproteins of several marine mammals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their apolipoprotein (apo) B-100, apoB-48, and apoA-I migrated to virtually the same position as the comparable human apolipoproteins. In cetaceans (bottlenose dolphins and killer whales), the molecular mass of the apoE was identical to that of human apoE (35 kDa). In contrast, in the lipoproteins of pinnipeds (harbor seals, sea lions, and walrus) there was no protein comparable in size to human apoE; however, there were two proteins in the 40- to 44-kDa range. The protein with the higher apparent molecular weight (44 kDa) was apoA-IV, as determined by NH2-terminal amino acid sequencing. Sequencing of the NH2-terminal 15 amino acids of the lower molecular weight protein (40-42 kDa) revealed no obvious homology with human apoE. However, a human apoE-specific monoclonal antibody, 1D7, bound to the 40- to 42-kDa protein, allowing us to identify that protein as apoE. Sequencing of sea lion apoE cDNA clones demonstrated that sea lion apoE is 311 amino acids in length, 12 residues longer than human apoE. All 12 additional residues are located in the NH2-terminal 31 amino acids, a region that has extremely low homology with the NH2-terminal portion of human apoE. The remainder of the sea lion apoE sequence is 74% homologous to human apoE. The sea lion apoE cDNA was expressed in Chinese hamster ovary (CHO) cells as well as CHO ldlD cells, a cell line that is deficient in O-glycosylation of proteins. The size of the sea lion apoE secreted by these two cell lines, compared with the apoE in sea lion plasma, indicated that the predominant form of apoE in sea lion plasma must be posttranslationally modified. Thus, our studies have demonstrated that the higher apparent molecular weight of pinniped (sea lion) apoE is due to a longer polypeptide chain as well as posttranslational modification of the protein.

Amino Acid Sequence↗
PubMed1990

In the absence of a downstream element, the apolipoprotein E gene is expressed at high levels in kidneys of transgenic mice.

Human apolipoprotein (apo) E gene constructs with 30 or 5 kilobases of 5'-flanking and 1.5 kilobases of 3'-flanking regions were used to create transgenic mice. High levels of human apoE mRNA were present in the transgenic kidney, but none was detected in the liver, which is normally the major source of apoE. When a construct with 5 kilobases of 5'- and 23 kilobases of 3'-flanking regions was used, only trace levels of human apoE mRNA were detected in the kidney, whereas high levels were found in the liver. These results indicated that regulatory elements downstream of the human apoE gene interacted with the transcription initiation complex to stimulate gene expression in the liver while suppressing expression in the kidney. In each case, human apoE was secreted into the plasma. The source of human apoE in the transgenic kidney was the epithelial cells lining the proximal tubule and Bowman's capsule.

Animals↗
PubMed1989

Apolipoprotein B48 RNA editing in chimeric apolipoprotein EB mRNA.

Apolipoprotein (apo) B occurs in two forms, apoB100 (512 kDa) and apoB48 (240 kDa); both are derived from the same gene. A novel mechanism involving editing of the apoB mRNA causes the formation of apoB48; the first base of codon 2153 is changed from cytosine to uracil, converting a glutamine codon to a premature stop codon. To identify the apoB mRNA sequence elements recognized by the apoB mRNA editing mechanism, two apoB cDNA fragments (354 and 63 base pairs) with codon 2153 near their centers were inserted into a high expression vector of another secreted apolipoprotein, apoE. The resulting vectors, pHEB-354 and -63, were transfected into Chinese hamster ovary cells, HepG2 cells, and apoB48-producing CaCo-2 cells. The secreted chimeric apolipoproteins (apoEB354 and apoEB63) were analyzed for premature truncation, and the mRNA was analyzed for the presence of an edited base. The pHEB-354 construct produced a truncated protein only in CaCo-2 cells, whereas pHEB-63 produced no truncated protein in any of the three cell types. The mRNA was converted to cDNA and amplified by the polymerase chain reaction technique. Differential hybridization of the polymerase chain reaction products with CAA (Gln) and TAA (Stop) specific probes detected an edited base only in cDNA from CaCo-2 cells transfected with pHEB-354, in agreement with the protein analysis. We conclude that the nucleotide sequence of the apoB cDNA insert in pHEB-354 contains sufficient information to be edited in CaCo-2 cells. In these cells, a cryptic polyadenylation site was activated in the edited pHEB-354 mRNA. As a result, CaCo-2 cells transfected with pHEB-354 produced a short, edited pHEB-354 mRNA and a long, unedited pHEB-354 mRNA. Chinese hamster ovary cells transfected with pHEB-354 or CaCo-2 cells transfected with pHEB-63 produced only a full length transcript. Amplification of the pHEB-354 cDNA using 3'-primers upstream and downstream of the poly(A) addition site and hybridization with the TAA probe confirmed these results. This unusual mRNA editing apparently occurs before polyadenylation, probably in the nucleus.

Adenocarcinoma↗
PubMed1989

Glycosylation of human apolipoprotein E. The carbohydrate attachment site is threonine 194.

The glycosylation of human apolipoprotein (apo) E was examined with purified plasma apoE and apoE produced by transfected cell lines. The carbohydrate attachment site of plasma apoE was localized to a single tryptic peptide (residues 192-206). Sequence analysis and amino sugar analysis of this peptide derived from asialo-, monosialo-, or disialo-apoE indicated that the carbohydrate moiety is attached only to Thr194 in monosialo- and disialo-apoE and that asialo-apoE is not glycosylated. Mammalian cells that normally do not express apoE were transfected with human apoE plasmid expression vectors to test the utilization of potential carbohydrate attachment sites and the role of apoE glycosylation in secretion. Site-specific mutants of apoE, designed to eliminate or alter glycosylation sites, were expressed in HeLa cells by acute transfection. Apolipoprotein E(Thr194----Ala) was secreted exclusively as the asialo isoform, confirming that Thr194 is the site of carbohydrate attachment in these cells and indicating that glycosylation of apoE is not essential for secretion. Apolipoprotein E(Thr194----Asn,Gly196----Ser), which introduces a potential site for N-glycosylation at position 194, was secreted with a higher apparent molecular weight than native, O-glycosylated apoE. Studies with tunicamycin indicated that this apoE was N-glycosylated at Asn194. Stably transfected cell lines expressing human apoE were prepared from wild-type Chinese hamster ovary (CHO) cells and from CHO ldlD cells, which are defective in glycosylation. The transfected wild-type cells secreted multiply sialylated apoE. The transfected ldlD cells also secreted high levels of apoE even in the absence of glycosylation, which confirms that glycosylation is not essential for secretion of apoE.

Animals↗