Value of a QALY and VSI estimated with the chained approach.

The value of a quality-adjusted life-year (QALY) and the value of a statistical injury (VSI) are important measures within health economics and transport economics. Several studies have, therefore, estimated people's willingness to pay (WTP) for these estimates, but most results show scale insensitivity. The 'original' chained approach (CA) is a method developed to mitigate this problem by combining the contingent valuation (CV) with standard gamble (SG). In contrast to the version of the CA applied by the previous research of the WTP for a QALY, the original version allows the value of major health gains to be estimated without having the respondents express their WTP directly. The objective of this study was to estimate the value of a QALY and VSI in the context of non-fatal road traffic accidents using the original CA to test if the approach, applied to a wide range of health gains, is able to derive valid estimates and a constant value of a QALY which the previous research has not been able to show. Data were collected from a total of 800 individuals in the Swedish adult general population using two web-based questionnaires. The values of a QALY based on trimmed estimates were close to constant at €300,000 irrespective of the size of the QALY gain. The study shows that the original CA method may be a valid method to estimate the value of a QALY and VSI for major health losses. It also supports the use of a higher threshold value for a QALY than that which is currently applied by several health technology assessment agencies in different countries.

Measuring the end-of-life premium in cancer using individual ex ante willingness to pay.

For the assessment of value of new therapies in healthcare, Health Technology Assessment (HTA) agencies often review the cost per quality-adjusted life-year (QALY) gained. Some HTA agencies accept a higher cost per QALY gained when treatment is aimed at prolonging survival for patients with a short expected remaining lifetime, a so-called end-of-life (EoL) premium. The objective of this study is to elicit the existence and size of an EoL premium in cancer. Data was collected from 509 individuals in the Swedish general population 20-80 years old using a web-based questionnaire. Preferences were elicited using subjective risk estimation and the contingent valuation (CV) method. A split-sample design was applied to test for order bias. The mean value of a QALY was MSEK4.8 (€528,000), and there was an EoL premium of 4-10% at 6 months of expected remaining lifetime. Using subjective risk resulted in more robust and valid estimates of the value of a QALY. Order of scenarios did not have a significant impact on the WTP and the result showed scale sensitivity. Our result provides some support for the use of an EoL premium based on individual preferences when expected remaining lifetime is short and below 24 months. Furthermore, we find support for a value of a QALY that is above the current threshold of several HTA agencies.

Ectopic lipid storage and insulin resistance: a harmful relationship.

Obesity increases the risk of metabolic diseases, including insulin resistance and type 2 diabetes, as well as cardiovascular disease. In addition to lipid accumulation in adipose tissue, obesity is associated with increased lipid storage in ectopic tissues, such as skeletal muscle and liver. Furthermore, lipid accumulation in the heart may result in cardiac dysfunction and heart failure. It has recently been demonstrated that intracellular lipid accumulation in ectopic tissues leads to pathological responses and impaired insulin signalling. Here, we will review the current understanding of how lipid storage and lipid droplet physiology affect the risk of developing metabolic diseases.

Rhinovirus infections in western Sweden: a four-year molecular epidemiology study comparing local and globally appearing types.

Human rhinovirus (HRV) is a highly prevalent pathogen and a major cause of acute respiratory tract infection (ARTI). HRV express less seasonality than other viral ARTIs, which typically appear as seasonal epidemics lasting for 1-2 months. The aim of this study was to investigate the seasonal patterns of HRV types over four consecutive years in one geographic region. HRV identified in respiratory samples from 114 patients over a four-year period were analysed by VP4/VP2 sequencing. HRV-A was found in 64, HRV-B in 11 and HRV-C in 37 cases. Overall, 33 different HRV-A types, nine B types and 21 C types were found. As many as 21 of the HRV types appeared during several seasons, with a maximum time-span of four years. Some types appeared during successive seasons and, in some cases, phylogenetic analysis indicated extended periods of circulation locally. Most of the strains were closely related to HRV identified in other parts of the world during the same time period. HRV strains that circulate locally represent many types and seem to reflect that HRV infections are highly globalised. The existence of simultaneous or successive epidemics with different HRV types in combination with the ability of each type to remain in the local population over extended periods of time may contribute to explaining the high rate of HRV infections.

Evaluation of macrophage-specific promoters using lentiviral delivery in mice.

In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSPs fused with green fluorescent protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343-bp proximal part and the 150-bp proximal part) and two variants (in forward and reverse orientation) of a previously characterized synthetic promoter derived from elements of transcription factor genes. We transduced a number of cell lines and primary cells in vitro. In addition, hematopoietic stem cells were transduced with MSPs and transferred into lethally irradiated recipient mice. Fluorescence activated cell sorting analysis was performed to determine the GFP expression in different cell populations both in vitro and in vivo. We showed that MSPs can efficiently be used for lentiviral gene delivery and that the 150-bp proximal part of the CD68 promoter provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.

Retracted: 147 reduced syntaxin-5 in skeletal muscle of patients with type 2 diabetes. A link between lipid storage and insulin resistance.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This abstract has been retracted at the request of Jan Borén, co-author, because of conscious fabrication, corruption or suppression of basic material and conscious preparation and presentation of falsified results in the abstract by one of the authors.

High cytokine levels in perforated acute otitis media exudates containing live bacteria.

Acute otitis media (AOM) is an inflammatory response to microbes in the middle ear, sometimes associated with rupture of the tympanic membrane. Human leukocytes produce different patterns of inflammatory mediators in vitro when stimulated with Gram-positive and Gram-negative bacteria, respectively. Here, we investigated the cytokine and prostaglandin E2 (PGE2) responses in middle ear fluids (MEFs) from children with spontaneously perforated AOM, and related the mediator levels to the presence of pathogens detected by culture (live) or PCR (live or dead). Furthermore, the in vivo cytokine pattern was compared with that induced in leukocytes stimulated by dead bacteria in vitro. MEFs with culturable pathogenic bacteria contained more interleukin (IL)-1ß (median: 110 µg/L vs. <7.5 µg/L), tumour necrosis factor (TNF) (6.3 µg/L vs. <2.5 µg/L), IL-8 (410 µg/L vs. 38 µg/L) and IL-10 (0.48 µg/L vs. <0.30 µg/L) than culture-negative fluids, irrespective of PCR findings. IL-6 and PGE2 were equally abundant (69-110 µg/L) in effusions with live, dead or undetectable bacteria. Cytokine levels were unrelated to bacterial species and to the presence or absence of virus. Similar levels of TNF and IL-6 as found in the MEFs were obtained by in vitro stimulation of leukocytes, whereas 11 times more IL-1ß and 3.5 times more IL-8 were produced in vivo, and 22 times more IL-10 was produced in vitro. Vigorous production of proinflammatory cytokines accompanies AOM with membrane rupture, regardless of the causative agent, but the production seems to cease rapidly once the bacteria are killed and fragmented. IL-6 and PGE2, however, remain after bacterial disintegration, and may play a role in the resolution phase.

Comparison of serum and whole blood levels of cytomegalovirus and Epstein-Barr virus DNA.

The monitoring of viral DNA levels after transplantation is crucial for prevention of complications from cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection but there is no consensus as to which matrix is the most adequate. To compare serum and whole blood (WB) as specimens for measuring viral DNA, clinical samples from a 3-year period were studied, with focus on cases where serum and WB were drawn on the same day. In 1896 paired serum and WB samples, CMV DNA was detected in both specimen types in 472 samples with 0.18 log higher levels (P<0.001) in WB than in serum (median level 2.73 vs. 2.56 log copies/mL), and in only either serum or WB in 127 and 108 samples, respectively, generally at levels below 1000 copies/mL. In 664 paired samples, EBV DNA was detected in both serum and WB in 160 samples, with 1.48 log higher levels (P<0.001) in WB (median 4.2 vs. 2.4 log copies/mL), in only WB in 227 cases with a median at 3.0 log copies/mL, and only in serum in 14 samples at low levels. The correlation between serum and WB DNA levels was weaker for EBV than for CMV (R(2) 0.31 vs. 0.74). We conclude that either serum or WB may be used for monitoring CMV and EBV DNA levels, that EBV DNA is detected post transplant in >50% of WB samples and at 30 times higher levels than in serum, and that post-transplantation lymphoproliferative disorder (PTLD) may develop without further increase of EBV DNA in WB. Identification of PTLD may require EBV DNA testing in both specimen types or complementary tests.

Acute suppression of VLDL1 secretion rate by insulin is associated with hepatic fat content and insulin resistance.

AIMS/HYPOTHESIS: Overproduction of VLDL(1) seems to be the central pathophysiological feature of the dyslipidaemia associated with type 2 diabetes. We explored the relationship between liver fat and suppression of VLDL(1) production by insulin in participants with a broad range of liver fat content. METHODS: A multicompartmental model was used to determine the kinetic parameters of apolipoprotein B and TG in VLDL(1) and VLDL(2) after a bolus of [(2)H(3)]leucine and [(2)H(5)]glycerol during a hyperinsulinaemic-euglycaemic clamp in 20 male participants: eight with type 2 diabetes and 12 control volunteers. The participants were divided into two groups with low or high liver fat. All participants with diabetes were in the high liver-fat group. RESULTS: The results showed a rapid drop in VLDL(1)-apolipoprotein B and -triacylglycerol secretion in participants with low liver fat during the insulin infusion. In contrast, participants with high liver fat showed no significant change in VLDL(1) secretion. The VLDL(1) suppression following insulin infusion correlated with the suppression of NEFA, and the ability of insulin to suppress the plasma NEFA was impaired in participants with high liver fat. A novel finding was an inverse response between VLDL(1) and VLDL(2) secretion in participants with low liver fat: VLDL(1) secretion decreased acutely after insulin infusion whereas VLDL(2) secretion increased. CONCLUSIONS/INTERPRETATION: Insulin downregulates VLDL(1) secretion and increases VLDL(2) secretion in participants with low liver fat but fails to suppress VLDL(1) secretion in participants with high liver fat, resulting in overproduction of VLDL(1). Thus, liver fat is associated with lack of VLDL(1) suppression in response to insulin.

Decreased immune reactivity towards a knobless, affibody-targeted adenovirus type 5 vector.

In this study, a prototype Adenovirus type 5 (Ad5) vector deleted of the fiber knob domain and carrying an Affibody molecule as the targeting ligand showed decreased susceptibility to human pre-existing antibodies. This vector, Ad5/R7-Z(taq)Z(taq), has short fibers carrying seven shaft repeats, a non-native trimerization signal and an affibody molecule (Z(taq)) reactive to Taq polymerase. Ad5/R7-Z(taq)Z(taq) could be specifically targeted to 293 cells stably expressing membrane-bound anti-Z(taq) idiotypic affibody called Z(ztaq) (293Z(ztaq)). Sera from 50 blood donors were analyzed for neutralization activity (NA) against the parental Ad5/Fiwt vector and knobless Ad5/R7-Z(taq)Z(taq) on 293Z(ztaq) cells. Twenty-three sera had NA titers (> or =1:64) against Ad5/Fiwt (46%) and only two against Ad5/R7-Z(taq)Z(taq) (4%). Characterization of sera with NA titers showed that the knob domain is one of the targets of the antibodies. Neutralization assays using sera pre-adsorbed on knob and hexon proteins showed that the NA of the sera was carried mainly by anti-knob and anti-hexon antibodies, but in certain sera the anti-hexon antibodies represent the major population of the neutralizing antibodies (NAbs). Our results suggested that a combination of knob deletion and hexon switching could be an effective strategy for Ad vectors to better evade the anti-Ad NAbs.

Overproduction of large VLDL particles is driven by increased liver fat content in man.

AIMS/HYPOTHESIS: We determined whether hepatic fat content and plasma adiponectin concentration regulate VLDL(1) production. METHODS: A multicompartment model was used to simultaneously determine the kinetic parameters of triglycerides (TGs) and apolipoprotein B (ApoB) in VLDL(1) and VLDL(2) after a bolus of [(2)H(3)]leucine and [(2)H(5)]glycerol in ten men with type 2 diabetes and in 18 non-diabetic men. Liver fat content was determined by proton spectroscopy and intra-abdominal fat content by MRI. RESULTS: Univariate regression analysis showed that liver fat content, intra-abdominal fat volume, plasma glucose, insulin and HOMA-IR (homeostasis model assessment of insulin resistance) correlated with VLDL(1) TG and ApoB production. However, only liver fat and plasma glucose were significant in multiple regression models, emphasising the critical role of substrate fluxes and lipid availability in the liver as the driving force for overproduction of VLDL(1) in subjects with type 2 diabetes. Despite negative correlations with fasting TG levels, liver fat content, and VLDL(1) TG and ApoB pool sizes, adiponectin was not linked to VLDL(1) TG or ApoB production and thus was not a predictor of VLDL(1) production. However, adiponectin correlated negatively with the removal rates of VLDL(1) TG and ApoB. CONCLUSIONS/INTERPRETATION: We propose that the metabolic effect of insulin resistance, partly mediated by depressed plasma adiponectin levels, increases fatty acid flux from adipose tissue to the liver and induces the accumulation of fat in the liver. Elevated plasma glucose can further increase hepatic fat content through multiple pathways, resulting in overproduction of VLDL(1) particles and leading to the characteristic dyslipidaemia associated with type 2 diabetes.

Microbiology profile in women with pelvic inflammatory disease in relation to IUD use.

OBJECTIVE: To study the microbial characteristics of patients with pelvic inflammatory disease (PID) and the possible impact of an intrauterine device (IUD) on the microbial environment in women presenting with PID. METHODS: Case-control study, investigating 51 women with acute PID and 50 healthy women. Endocervical specimens for microbiological investigation were obtained at gynaecological examination. RESULTS: IUD users with PID had significantly more Fusobacteria spp. and Peptostreptococcus spp. than non-IUD users with PID. The finding of combinations of several anaerobic or aerobic microbes was associated with a significantly increased risk of PID and with complicated PID. In IUD users, the combinations of several anaerobic/aerobic microbes were associated with an increased risk of PID, irrespective of duration of IUD use. Long-term IUD use appeared to be associated with an increased risk of a PID being complicated. CONCLUSION: The finding of several anaerobic or aerobic microbes appears to be associated with PID in users of IUD.

Apolipoprotein B: a clinically important apolipoprotein which assembles atherogenic lipoproteins and promotes the development of atherosclerosis.

Apolipoprotein (apo) B exists in two forms apoB100 and apoB48. ApoB100 is present on very low-density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and LDL. ApoB100 assembles VLDL particles in the liver. This process starts by the formation of a pre-VLDL, which is retained in the cell unless converted to the triglyceride-poor VLDL2. VLDL2 is secreted or converted to VLDL1 by a bulk lipidation in the Golgi apparatus. ApoB100 has a central role in the development of atherosclerosis. Two proteoglycan-binding sequences in apoB100 have been identified, which are important for retaining the lipoprotein in the intima of the artery. Retention is essential for the development of the atherosclerotic lesion.

Detection of orientation-specific anti-gp120 antibodies by a new N-glycanase protection assay.

Several functions have been assigned to the extensive glycosylation of HIV-1 envelope glycoprotein gp120, especially immune escape mechanisms, but the intramolecular interactions between gp120 and its carbohydrate complement are not well understood. To analyse this phenomenon we established a new microwell deglycosylation assay for determining N-linked glycan accessibility after binding of gp120-specific agents. Orientation-specific exposition of gp120 in ELISA microplates was achieved by catching with either anti-C5 antibody D7324 or anti-V3 antibody NEA-9205. We found that soluble CD4 inhibited the deglycosylation of gp120 only when gp120 was caught by D7324 and not by NEA9205. In contrast, antibodies from HIV-infected individuals inhibited the deglycosylation best when gp120 was caught by NEA9205. These results demonstrated that both the CD4-binding site and the epitopes recognised by antibodies from HIV-infected individuals have N-glycans in the close vicinity. However, the difference in gp120 orientation indicates that antibodies in HIV-infected individuals, at least partly, bind to epitopes different from the CD4-binding site. Finally, we determined the structural class of the glycan of one V1 glycosylation site of prototype HIV-1 LAI gp120, which remained unsolved from previous studies, and found that it belonged to the complex type of glycans.

Enhanced immunogenicity of a human immunodeficiency virus type 1 env DNA vaccine by manipulating N-glycosylation signals. Effects of elimination of the V3 N306 glycan.

DNA encoding HIV-1 env is a poorly efficient B-cell immunogen and one probable explanation is that the numerous gp120 N-linked glycans gp120 may interfere with B-cell epitope presentation. The N306 glycan in gp120 shields HIV-1 from neutralizing antibodies. A DNA immunogen lacking the N306 glycosylation signal (T308A) was constructed to determine whether this glycan affected the immune response. Mice were immunized intranasally twice with DNA containing either the wild type or the mutant env. Two additional groups were primed with wild type or mutant env and boosted with rgp160 protein, containing the complete set of N-linked glycans. Immunization with DNA alone resulted in priming of B-cell clones but was not sufficient to induce a complete antibody response. Animals primed with the N306 mutant and subsequently boosted with rgp160 protein displayed higher serum IgG-binding titers to gp120 than animals primed with wild type env DNA. The manipulation of the glycosylation sites of the env DNA strongly primes antibody responses (but non-neutralizing) as well as T-cell responses to the wild type strain gp160. However, priming with mutant plasmid did not result in higher neutralization titers to wild type or T308A-mutated virus than did the wild type plasmid. With the N306 mutant DNA we thus immunized a non-neutralization epitope, but obtained strong env-binding IgG after rgp160 boosting.

Protection of neutralization epitopes in the V3 loop of oligomeric human immunodeficiency virus type 1 glycoprotein 120 by N-linked oligosaccharides in the V1 region.

The V3 region of the human immunodeficiency virus type 1 envelope protein gp120 constitutes a potential neutralization target, but the oligosaccharide of one conserved N-glycosylation site in this region protects it from neutralizing antibodies. Here, we determined whether N-linked glycans of other gp120 domains were also involved in protection of V3 neutralization epitopes. Two molecular clones of HIV-1, one lacking three N-linked glycans of the V1 region (HIV-1(3N/V1)) and another lacking three N-linked glycans of the C2 region (HIV-1(3N/C2)), were created and characterized. gp120 from both mutated viral clones had higher electrophoretic mobilities than gp120 from wild-type virus, confirming loss of N-linked glycans. Wild-type virus and both mutant clones replicated equally well in established T cell lines and all three viruses were able to utilize CXCR4 but not CCR5 as a coreceptor. The induced mutations increased gp120 affinity for CXCR4 but caused no corresponding increase in viral ability to replicate in T cell lines. HIV-1(3N/V1) was neutralized at about 25 times lower concentrations of an antibody to the V3 region than were wild-type virus and HIV-1(3N/C2). Soluble, monomeric gp120 from HIV-1(3N/V1) and wild type virus had identical avidity for the V3 antibody, indicating that the V1 glycans were able to shield V3 only in oligomeric but not monomeric gp120. In conclusion, one or more N-linked glycans of gp120 V1 is engaged in protection of the V3 region from potential neutralizing antibodies, and this effect is dependent on the oligomeric organization of gp120/gp41.

Quantification of cytomegalovirus DNA in BAL fluid: a longitudinal study in lung transplant recipients.

STUDY OBJECTIVES: Cytomegalovirus (CMV) infection is common in patients receiving solid organ transplants, and it is associated with increased morbidity as well as risk for development of chronic rejection. A rapid and sensitive diagnostic method would improve the therapeutic management of CMV infection, including the monitoring of treatment effects. We investigated whether longitudinal determinations of CMV DNA quantities in BAL fluid could be useful for this purpose. DESIGN: CMV DNA levels in 340 BAL samples from 35 consecutive lung transplant recipients were studied during a median of 18 months. Seventeen (49%) of the patients developed CMV disease with pneumonitis. Twenty-seven CMV disease episodes were diagnosed. RESULTS: Patients with CMV disease had a significantly higher mean level of CMV copies per milliliter BAL fluid (1,120 +/- 4,379) compared with those without (180 +/- 1,177, p < 0.01). Viral load as well as acute rejection requiring treatment (>/= A2) were independent risk factors associated with CMV disease. Differences between the groups concerning HLA-DR matching, basic immunosuppressive therapy, and CMV serologic status D/R -/+ vs D/R +/+ were not significant. A diagnostic definition of normality based on the mean level of all episodes without CMV disease +2 SD would discriminate only 9 of the 27 CMV episodes. CONCLUSIONS: Although the viral load is increased during episodes of clinical CMV disease in lung transplant recipients, the quantitative PCR assessment of CMV DNA in BAL fluid is not discriminative enough to be useful as a diagnostic tool for CMV disease.

Expression of TGF-beta isoforms, TGF-beta receptors, and SMAD molecules at different stages of human glioma.

Human gliomas express TGF-beta but, so far the expression of downstream mediators has been investigated in only a few cell lines. We have examined tissue specimens of 23 gliomas: 3 astrocytomas grade II (AST), 8 anaplastic astrocytomas grade III (AAST), and 12 glioblastoma multiforme grade IV (GBM). We analyzed the mRNA expression of TGF-beta1, TGF-beta2, TGF-beta3, the TGF-beta receptors type I (TbetaR-I) and type II (TbetaR-II), Smad2, Smad3, and Smad4. mRNA expression of IL-10 and CD95 (FAS/APO-1) were also studied. We detected increased mRNA levels of the 3 TGF-beta isoforms, correlating with the degree of malignancy. TGF-beta3 mRNA was increased, particularly in AST and AAST, while TGF-beta1 and TGF-beta2 mRNAs were strongly expressed in GBM. TGF-beta normally up-regulates the TGF-beta receptors, and TbetaR-I and TbetaR-II showed stronger expression in all gliomas when compared to normal tissues. However, the mRNA expression of Smad2, Smad3, and Smad4 was decreased in GBM. IL-10 mRNA expression was detected in glioma tissues but not in glioma cell lines. No marked increase in the expression of soluble CD95 splicing variants was found in the gliomas compared with normal tissue. However, total CD95 mRNA was elevated among GBM tissues.

ADP-ribosylation factor 1 and its activation of phospholipase D are important for the assembly of very low density lipoproteins.

The role of ADP-ribosylation factor 1 (ARF-1) in the assembly of very low density lipoproteins (VLDL) was investigated by expressing dominant-negative mutants in McA-RH7777 cells. Transient expression of ARF-1(T31N), a GDP-restrictive mutant, significantly inhibited apolipoprotein B-100 (apoB-100) VLDL production without influencing the biosynthesis of apoB-100 low density lipoproteins or total apoB production (indicating that it inhibited the second step of VLDL assembly) and without altering total protein production or biosynthesis of transferrin, phosphatidylcholine, or triglycerides. These effects were confirmed in stable inducible transfectants. In contrast, expression of an ARF-1 mutant lacking the N-terminal 17 amino acids, which has no myristoylation site and cannot interact with the microsomal membrane, did not affect VLDL assembly. Thus, active ARF-1 is needed for the second step of the process. To further explore these observations, we developed a cell-free system based on the postnuclear supernatant isolated from McA-RH7777 cells. In this system, 10-15% of the apoB-100 pool was converted to VLDL in a time- and temperature-dependent way. The assembly process was highly dependent on a heat-stable factor in the d > 1.21 g/ml infranatant of fetal calf serum; this factor was not present in low density lipoproteins or VLDL. Brefeldin A inhibited VLDL assembly in this system, as did a synthetic peptide (corresponding to N-terminal amino acids 2-17 of ARF-1) that displaces ARF-1 from the membrane. Thus, active ARF-1 is also needed for cell-free assembly of VLDL. Guanosine 5'-3-O-(thio)triphosphate also inhibited VLDL assembly in this system, indicating that the process requires ongoing hydrolysis of GTP. 1-Butanol, which inhibits the formation of phosphatidic acid (PA) and instead gives rise to phosphatidylbutanol, inhibited VLDL assembly, whereas 2-butanol, which does not inhibit PA formation, failed to do so. Thus, phospholipase D (PLD)-catalyzed formation of PA from phosphatidylcholine is essential for VLDL assembly. In support of this conclusion, exogenous PLD prevented brefeldin A from inhibiting the assembly process. Our results indicate that ARF-1 participates in the second step of VLDL assembly through a process that involves activation of PLD and production of PA.

The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells.

We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.