MiR-15b is a key regulator of proliferation and apoptosis of chondrocytes from patients with condylar hyperplasia by targeting IGF1, IGF1R and BCL2.

OBJECTIVE: This study aimed to explore potential microRNAs (miRNAs), which participate in the pathological process of condylar hyperplasia (CH) through targeting specific proliferation- and apoptosis- related genes of chondrocytes. METHODS: Insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R) and B-cell CLL/lymphoma 2 (BCL2) in CH cartilage were detected by real-time polymerase chain reaction (PCR), Western blot, immunohistochemistry and immunofluorescence. MiRanda and TargetScanS algorithms were used to predict certain miRNAs in CH chondrocytes concurrently modulating the above three genes. MiR-15b was screened and identified using real-time PCR. After transfection of miR-15b mimics or inhibitor into CH chondrocytes, expression of the above three genes was detected by real-time PCR and western blot, meanwhile, cell proliferation and apoptosis was examined by CCK8, cell cycle assays, flow cytometry and Hoechst staining. Dual luciferase activity was performed to identify the direct regulation of miR-15b on IGF1, IGF1R and BCL2. RESULTS: Expression of IGF1, IGF1R and BCL2 increased in CH cartilage. Seven microRNAs concurrently correlated with IGF1, IGF1R and BCL2. Among them, only miR-15b significantly changed in CH chondrocytes. Overexpression of miR-15b in CH chondrocytes suppressed the expression of IGF1, IGF1R and BCL2, while it increased when miR-15b was knockdown. Furthermore, miR-15b suppressed their expression by directly binding to its 3'-UTR in these cells. Besides, miR-15b hampered chondrocytes proliferation through targeting IGF1 and IGF1R and accelerated chondrocytes apoptosis through targeting BCL2. CONCLUSION: Suppressed miR-15b contributed to enhanced proliferation capacity and weakened apoptosis of chondrocytes through augmentation of IGF1, IGF1R and BCL2, thereby resulting in development of CH.

RNA interference-mediated functional characterization of aquaporin genes in Tribolium castaneum.

An obvious challenge faced by most terrestrial insects is maintaining water homeostasis in an arid environment. Current research suggests aquaporins may be evolved to meet the challenge. However, up to now, this suggestion has not been verified in any insect that feeds upon solid food with mandibulate mouthparts. In the present paper, nine putative aquaporin genes [Tribolium castaneum big brain, T. castaneum Drosophila integral protein (TcDrip), T. castaneum Pyrocoelia rufa integral protein (TcPrip), T. castaneum aquaporin 12-like, T. castaneum entomoglyceroporin 1 (TcEglp1), TcEglp2, TcEglp3, TcEglp4 and TcEglp5] were identified in T. castaneum. The transcripts of the nine genes were easily detectable in the foregut, midgut, hindgut-Malpighian tubule complex, fat body and carcass (except gut and fat body). Amongst them TcDrip, TcPrip, TcEglp1, TcEglp3 and TcEglp5 were highly transcribed in the hindgut-Malpighian tubule complex; TcEglp4 was abundantly expressed in both the fat body and hindgut-Malpighian tubule complex. RNA interference (RNAi)-mediated knockdown of TcEglp3 caused a grey larval cuticle, in contrast to a smooth and bright cuticle in control larvae. Approximately 40% of the TcEglp3 RNAi larvae had their hindguts protruding from the anus; their fresh wet faeces were attached to the hindgut. Another 20% of these treated larvae did not defaecate normally; wet brown faeces were adhered to the anal area. As a result, the larval growth was inhibited and about 60% larval lethality occurred. Silencing of TcEglp4 or TcDrip exhibited similar but weaker defective phenotypes as those of the TcEglp3-silenced larvae. Therefore, Eglp3, Eglp4 and Drip may contribute to the conductance of water in the alimentary canal and Malpighian tubules in T. castaneum.

[Clinical effect and safety of pegylated interferon-α-2b injection (Y shape, 40 kD) in treatment of HBeAg-positive chronic hepatitis B patients].

Objective: To investigate the clinical effect and safety of long-acting pegylated interferon-α-2b (Peg-IFN-α-2b) (Y shape, 40 kD) injection (180 µg/week) in the treatment of HBeAg-positive chronic hepatitis B (CHB) patients, with standard-dose Peg-IFN-α-2a as positive control. Methods: This study was a multicenter, randomized, open-label, and positive-controlled phase III clinical trial. Eligible HBeAg-positive CHB patients were screened out and randomized to Peg-IFN-α-2b (Y shape, 40 kD) trial group and Peg-IFN-α-2a control group at a ratio of 2:1. The course of treatment was 48 weeks and the patients were followed up for 24 weeks after drug withdrawal. Plasma samples were collected at screening, baseline, and 12, 24, 36, 48, 60, and 72 weeks for centralized detection. COBAS® Ampliprep/COBAS® TaqMan® HBV Test was used to measure HBV DNA level by quantitative real-time PCR. Electrochemiluminescence immunoassay with Elecsys kit was used to measure HBV markers (HBsAg, anti-HBs, HBeAg, anti-HBe). Adverse events were recorded in detail. The primary outcome measure was HBeAg seroconversion rate after the 24-week follow-up, and non-inferiority was also tested. The difference in HBeAg seroconversion rate after treatment between the trial group and the control group and two-sided confidence interval (CI) were calculated, and non-inferiority was demonstrated if the lower limit of 95% CI was > -10%. The t-test, chi-square test, or rank sum test was used according to the types and features of data. Results: A total of 855 HBeAg-positive CHB patients were enrolled and 820 of them received treatment (538 in the trial group and 282 in the control group). The data of the full analysis set showed that HBeAg seroconversion rate at week 72 was 27.32% in the trial group and 22.70% in the control group with a rate difference of 4.63% (95% CI -1.54% to 10.80%, P = 0.1493). The data of the per-protocol set showed that HBeAg seroconversion rate at week 72 was 30.75% in the trial group and 27.14% in the control group with a rate difference of 3.61% (95% CI -3.87% to 11.09%, P = 0.3436). 95% CI met the non-inferiority criteria, and the trial group was non-inferior to the control group. The two groups had similar incidence rates of adverse events, serious adverse events, and common adverse events. Conclusion: In Peg-IFN-α regimen for HBeAg-positive CHB patients, the new drug Peg-IFN-α-2b (Y shape, 40 kD) has comparable effect and safety to the control drug Peg-IFN-α-2a.

Expression of bovine Mx1 protein inhibits the replication of foot-and-mouth disease virus in BHK-21 cells.

Mx proteins belonging to the dynamin superfamily of large GTPases inhibit replication of a wide range of RNA viruses. In this study, we examined whether bovine Mx1 protein could interfere with the replication of foot-and-mouth disease virus (FMDV). For this purpose we established cloned BHK-21 cells expressing bovine Mx1 protein (BM1 cells) and infected them with FMDV serotype O. Cloned BHK-21 cells expressing neomycin resistance instead of Mx1 protein (BH1 cells) and original BHK-21 cells served as negative controls. The results showed that the expression of bovine Mx1 protein reduced viral yields by 90% and levels of viral VP1 mRNA by 60%. These findings correlated with a significant reduction of viral antigen detectable in infected cells by immunofluorescent assay. These results demonstrate that bovine Mx1 protein interferes with the replication of FMDV.

Insulin-like growth factor-1 boosts the developing process of condylar hyperplasia by stimulating chondrocytes proliferation.

OBJECTIVE: The etiology of Condylar hyperplasia (CH) of human temporomandibular joint (TMJ) remains largely unknown. Our previous study has demonstrated that enriched insulin-like growth factor-1(IGF-1) was expressed in the proliferation and hypertrophic layers of CH cartilage. Accordingly, this study was aimed to investigate whether IGF-1 regulates CH chondrocytes proliferation in condylar cartilage overgrowth and explore the molecular mechanism of IGF-1 involved in. METHODS: Chondrocytes were isolated from 6 CH and 3 normal cartilage (NC) specimens and cultured in alginate beads or monolayer, treated with IGF-1 or specific inhibitors such as 7-[trans-3-[(azetidin-1-yl)methyl]cyclobutyl]-5-(3-benzyloxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (NVP-AEW541), U0126, and LY294002. Thereafter, cellular proliferation capacity was evaluated by Cell Viability Analyzer (alginate beads culture) or 3-(4,5-dimethylthiazo(-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (monolayer culture). Gene expression levels of IGF-1, IGF-1 receptor (IGF-1R), collagen type II, type X and those genes associated with proliferation were evaluated by realtime PCR. Protein levels of IGF-1 and IGF-1R synthesized by CH chondrocytes were accessed by enzyme-linked immunosorbent assay (ELISA) and western blotting. RESULTS: CH chondrocytes enhanced cellular proliferation capacity and expressed significantly higher levels of messenger RNA (mRNA) and protein expressions of IGF-1 and IGF-1R, as compared with NC chondrocytes. Furthermore, enriched IGF-1 enhanced CH chondrocytes proliferation, up-regulated the expressions of specific genes associated with cellular proliferation and elevated the gene expression of collagen type II A1 (COL2A1). Besides, IGF-1-mediated CH chondrocytes proliferation mainly depended on the mitogen-activated protein kinase (MAPK)-ERK pathway. CONCLUSIONS: IGF-1 promotes human TMJ cartilage overgrowth in the developing process of CH by enhancing chondrocytes proliferation via MAPK-ERK pathway.

Transient elastohydrodynamic lubrication analysis of a novel metal-on-metal hip prosthesis with a non-spherical femoral bearing surface.

Effective lubrication performance of metal-on-metal hip implants only requires optimum conformity within the main loaded area, while it is advantageous to increase the clearance in the equatorial region. Such a varying clearance can be achieved by using non-spherical bearing surfaces for either acetabular or femoral components. An elastohydrodynamic lubrication model of a novel metal-on-metal hip prosthesis using a non-spherical femoral bearing surface against a spherical cup was solved under loading and motion conditions specified by ISO standard. A full numerical methodology of considering the geometric variation in the rotating non-spherical head in elastohydrodynamic lubrication solution was presented, which is applicable to all non-spherical head designs. The lubrication performance of a hip prosthesis using a specific non-spherical femoral head, Alpharabola, was analysed and compared with those of spherical bearing surfaces and a non-spherical Alpharabola cup investigated in previous studies. The sensitivity of the lubrication performance to the anteversion angle of the Alpharabola head was also investigated. Results showed that the non-spherical head introduced a large squeeze-film action and also led to a large variation in clearance within the loaded area. With the same equatorial clearance, the lubrication performance of the metal-on-metal hip prosthesis using an Alpharabola head was better than that of the conventional spherical bearings but worse than that of the metal-on-metal hip prosthesis using an Alpharabola cup. The reduction in the lubrication performance caused by the initial anteversion angle of the non-spherical head was small, compared with the improvement resulted from the non-spherical geometry.

The expressions of IGF-1, BMP-2 and TGF-ß1 in cartilage of condylar hyperplasia.

Condylar hyperplasia is a complex post-natal growth abnormality of the mandible and condyle, which leads to facial asymmetry. We investigated the distributions of insulin-like growth factors (IGF-1), bone morphogenetic protein-2 (BMP-2) and transforming growth factor-ß1 (TGF-ß1) in cartilage of condylar hyperplasia and revealed relationships between age and the cartilaginous thickness. Twenty patients with condylar hyperplasia were divided into four histopathological types. The cartilaginous thickness and age in different histological types were analysed, and the localizations of IGF-1, BMP-2 and TGF-ß1 were detected by immunohistochemistry analysis. The cartilaginous thickness of condylar hyperplasia significantly increased. The cartilaginous thickness of type III was significantly thicker than type I and type II, Bivariate correlation revealed a significant correlations between age and the cartilaginous thickness (r = 0·68, P = 0·01). However, the expressions of IGF-1, BMP-2 and TGF-ß1 were the strongest in type I. In almost all types of condylar hyperplasia, the presence of IGF-1 and BMP-2 was found mainly in the proliferative chondrocyte layer and the hypertrophic chondrocyte layer, and only a few in the calcified chondrocyte layer. The presence of TGF-ß1 widely distributed from the fibrous articular surface to the calcified cartilage. These findings suggest that the proliferative activity of cartilage in condylar hyperplasia is strongly associated with age and cartilaginous thickness. Therefore, the four pathological types of condylar hyperplasia seem more likely to be four discontinuous stages.

Histological evaluation of condylar hyperplasia model of rabbit following distraction osteogenesis of the condylar neck.

Condylar hyperplasia is the excessive unilateral growth of mandibular leading to facial asymmetry, occlusal disturbance, joint pain and dysfunction. The aim of this study is to evaluate the histological presence of temporomandibular joint in model of condylar hyperplasia by lengthening unilateral condylar neck of distraction osteogenesis. An extra oral distractor was employed to achieve unilateral condylar neck distraction (1·0 mm daily for 7 days). The experimental condylar necks were elongated by 7 mm compared to the contralateral. Eleven adult white rabbits were used. Eight rabbits were, respectively, sacrificed after the post-distraction period (4 or 8 weeks). All animals were evaluated clinically and histomorphometrically. The condyles radiologically showed remodelling, flattening and sclerosis. In 4-week group, thinning of the cartilage was evident, and the trabeculae were long, not multiply connected. A thin, dense fibrous layer covered all over the surface of cartilage. In 8-week group, the cartilaginous layer was similar to thickness of the normal cartilage, but still thinner than control. However, the fibrous layers covering condyle manifested slight degenerative changes, and even depressions and erosions were seen in the cartilage and subchondral bone. The trabeculae showed denser and multiply connected. In 8-week group, the cartilaginous thickness of surgical condyles was significantly thinner than the contralateral. This study indicates that unilateral distraction of condylar neck loads the condyles asymmetrically. Asymmetrical loads affect more on the surgical condyles than the contralateral, and after 8 weeks of the post-distraction, condyle could recover from asymmetrical loads in some degree.

Outcomes comparison of 5 coated cardiopulmonary bypass circuits versus an uncoated control group of patients undergoing cardiac surgery.

UNLABELLED: Attenuated inflammatory response and decreased platelet activation have been claimed repeatedly when biocompatible circuits are used for cardiopulmonary bypass. We evaluated five Health Canada approved biocompatible circuit coatings (BCC) against an un-coated control group to determine their effectiveness in improving post-operative outcomes. Patients were assigned to the Control group or one of the 5 coated circuit groups: 40 Control; 33 Trillium; 32 Phisio; 34 Bioline; 33 X; and 11 GBS. Measured outcomes included: ventilator time; ICU time; post-operative chest tube drainage and transfusion volume; high sensitivity C-reactive protein (hsCRP); tau protein; and pre- and 72-hour post-operative anti-saccadic eye movement test comparisons. RESULTS: 183 patients were enlisted into the study. One arm of the study (GBS) was abandoned after 11 patients on account of inconsistent pressure excursions within the oxygenator and the excessive consumption of platelets necessitating transfusion. Patients in the X-coated group had significantly longer ventilator and intensive care unit (ICU) time compared to the three remaining coated circuit study groups. Though not significant, patients in the X group also demonstrated the highest post-operative chest tube losses, the most platelet transfusions, the highest tau protein levels and the lowest post-operative anti-saccadic eye movement test (ASEMT) results compared to the three remaining coated groups. The patients in the Trillium, Bioline and Phisio groups showed an improvement in ventilator and ICU time relative to the Control group. The diabetic patients in the Trillium, Bioline and Phisio groups showed an improvement in bleeding relative to the diabetic patients in the Control group. CONCLUSION: We compared all 5 coated circuits approved for clinical use in Canada against an uncoated control circuit. Three of the 5 coated circuits (Trillium, Phisio and Bioline BCC) were found to improve ventilator and ICU time compared to Control. Further studies are indicated to validate these results and their impact upon approval criteria, purchasing choices and safe clinical practice, especially as applied to higher risk diabetic patients.

Effect of fluoride iontophoresis on the microtensile bond strength between dentin and two adhesive systems.

AIM: To evaluate the effect of fluoride iontophoresis on the microtensile bond strength (MTBS) between coronal dentin and two resin-based adhesive systems, and to measure quantitatively dentinal tubule occlusion. METHODS: Twelve freshly extracted intact human mandibular third molars were divided randomly into four groups. The superficial occlusal dentin of each tooth was exposed and treated. Group A1: One-Step Plus total-etch adhesive system; group A2: One-Step Plus total-etch adhesive system after fluoride iontophoresis; group B1: ACE BOND SE self-etching adhesive system; group B2: ACE BOND SE self-etching adhesive system after fluoride iontophoresis. A resin composite buildup was made for each tooth, which was then sectioned along its long axis to produce 10 beams (1.0 mm x 1.0 mm) for the microtensile bond strength (MTBS) test. Five dentin disks were cut in half and their occlusal surfaces etched with 6% citric acid. The test halves were treated with fluoride iontophoresis. Four SEM photomicrographs were taken from corresponding sites on each test and each non-treated control half-disk. Image-Pro Plus 4 software quantified the percentage of tubule occlusion. Data were analyzed using one-way ANOVA, chi(2)- and t-tests, with the probability level set at alpha=0.05. RESULTS: The mean MTBS (MPa) for each group was, A1: 30.86 (S.D. 6.84); A2: 25.04 (8.49); B1: 19.22 (6.88); B2: 19.40 (6.92). There were significant differences among all groups (P < or = 0.02), except between groups B1 and B2 (P=0.92). Fluoride iontophoresis resulted in significantly increased dentinal tubule occlusion (P<0.001). CONCLUSIONS: Fluoride iontophoresis treatment reduced significantly the dentin bond strength of One-Step Plus total-etch adhesive, but not that of ACE BOND SE self-etching adhesive. However, the bond strength of the former remained significantly higher than that of the latter adhesive system.

Cone-beam computed tomographic analysis of maxillary and mandibular changes after high condylectomy combined with orthodontic treatment for active unilateral condylar hyperplasia.

Our aim was to evaluate the efficacy of high condylectomy combined with orthodontic treatment for active unilateral condylar hyperplasia in 25 affected patients, by an analysis of the maxillary and mandibular changes on cone-beam computed tomography (CT). High condylectomy was the sole operative treatment. Variables that reflected the canting of the occlusal plane, the height of the maxillary complex, the buccolingual angulation of the maxillary first molar, the height of the ramus, the total length of the mandible, and the deviation of the chin were measured and compared between the two sides and between time intervals: preoperatively (T1) and the end of treatment (T2). The differences between time intervals in the deviation of the chin (p<0.001) and the canting of the occlusal plane (p<0.001) were significant, but there were no significant differences in the height of the ramus (p=0.476) and the total length of the mandible (p=0.838) between the affected and unaffected sides at T2. There were significant differences between time intervals in the buccolingual angulation on the unaffected side and the height of the maxillary complex on the affected side (p<0.001). Facial asymmetry was corrected and the occlusal plane was improved. In conclusion, high condylectomy as the sole operative treatment combined with orthodontic treatment can provide an alternative method for correction of facial asymmetry associated with active unilateral condylar hyperplasia.

Role of NF-kappaB in TNF-alpha-induced COX-2 expression in synovial fibroblasts from human TMJ.

In the temporomandibular joint (TMJ) synovium, cyclo-oxygenase-2 (COX-2) expression has been believed to be directly related to joint pain and synovitis. Here we investigated the role of Nuclear Factor kappaB (NF-kappaB) in the regulation of COX-2 expression in synovial fibroblasts from human TMJ induced by tumor necrosis factor-alpha (TNF-alpha). By reverse-transcriptase/polymerase chain-reaction (RT-PCR) and Western blotting analysis, TNF-alpha induced a dose- and time-dependent increase in COX-2 expression. Electrophoretic mobility shift assay (EMSA) revealed that transient NF-kappaB activation in the COX-2 promoter was triggered by TNF-alpha. In parallel with transient NF-kappaB activation, the rapid translocation of NF-kappaB, particularly the p65 subunit, from the cytoplasm into the nucleus was demonstrated. Pre-treatment with pyrolidine dithiocarbamate (PDTC), one of the NF-kappaB inhibitors, prevented binding to the COX-2 promoter and expression of COX-2 protein in response to TNF-alpha. These findings indicate that activation of NF-kappaB is responsible for TNF-alpha-induced COX-2 expression in synovial fibroblasts from the TMJ.

Technical note: A special apparatus for facilitating the in situ nylon bag measurement of the ruminal degradation of feedstuffs in cattle.

A new apparatus, named Bang-Bang (BB), which can facilitate the in situ nylon bag measurement of ruminal degradation in beef cattle, is described. The BB is an apparatus specially designed as a replacement of the traditional steel chain or flexible plastic tubes for binding the bags used in the in situ nylon bag (ISNB) method. The BB apparatus consists of cylindrical pedestals, sealing clips, nylon string, and nylon bags. A series of in situ rumen incubations using 3 ruminally cannulated beef steers were conducted to compare the BB with the ISNB method by measuring rumen DM degradation kinetics of 12 different feedstuffs commonly offered to beef cattle in China. Greater ( < 0.01) DM disappearance and less ( < 0.01) relative SD (%) were observed with the BB than with the ISNB method. In addition, effective degradability of DM was highly correlated between the 2 methods (ISNB = (1.01 ± 0.04 × BB) - 4.03 ± 2.12; = 0.99, < 0.001, = 12). These results indicate that the BB apparatus can be effectively used to determine the rumen degradation properties of feedstuffs with advantages over the traditional ISNB technique.

Origin of ultralow Young׳s modulus in a metastable ß-type Ti-33Nb-4Sn alloy.

Although there is difficulty in growing a Ti-33Nb-4Sn single crystal due to its ultralow ß-phase stability, the single-crystal elastic constants of metastable ß-type Ti-33Nb-4Sn (wt%) alloy were extracted successfully from its polycrystal by in-situ synchrotron X-ray diffraction technique, to clarify the origin of the ultralow Young's modulus in its polycrystal. It is indicated that compared to binary TiCr, TiV and TiNb alloys, the Ti-33Nb-4Sn alloy possesses slightly lower ß-phase stability with respect to {110}<110>(-)shear (i.e., C׳) but much lower ß-phase stability regarding to {001}〈100〉 shear (i.e., C44). An analysis by the Hill approximation suggests that the ultralow isotropic polycrystalline Young׳s modulus (EH) of Ti-33Nb-4Sn alloy originates from the extremely low shear modulus C44 as well as the relatively low C׳. This indicates that in addition to C׳, C44 has a significant contribution to the Young's modulus of polycrystal, which challenges a conventional understanding that the Young's modulus of ß-type Ti alloys is predominantly determined by C׳.

Antiproliferative efficacy of lipophilic soy isoflavone phytoestrogens delivered by low density lipoprotein particles into cultured U937 cells.

Some fat-soluble bioactive substances incorporated into low density lipoprotein (LDL) may be delivered into cells via LDL receptor pathway influencing cellular functions. In this study, we synthesized a number of fat-soluble isoflavone esters and investigated their incorporation into LDL as well as their delivery into U937 cells. Using an artificial transfer system (Celite dispersion), genistein and daidzein oleates and daidzein dilinoleate were efficiently incorporated into LDL with concentrations ranging between 2.7 to 16.9 isoflavone molecules/LDL particle, while much smaller amounts of unesterified isoflavones and genistein stearates were transferred into LDL. LDL containing 7-oleates or 4',7-dioleates of genistein and daidzein significantly reduced U937 cell proliferation by 36-43%. The strongest inhibitory effect was shown by daidzein 4',7-dilinoleate with 93% reduction of cell proliferation. Neither of the 4'-oleates of genistein and daidzein contained in LDLs exhibited antiproliferative effects although they were incorporated into LDL. In summary, we demonstrated that isoflavones made fat-soluble by esterification can be incorporated into LDL in vitro and delivered into cultured U937 cells via the LDL-receptor pathway, reducing the cell proliferation.

Deformation behavior of metastable ß-type Ti-25Nb-2Mo-4Sn alloy for biomedical applications.

The deformation behavior of metastable ß-type Ti-25Nb-2Mo-4Sn (wt%) alloy subjected to different thermo-mechanical treatments was discussed by the combining results from transmission electron microscope, tensile test and in-situ synchrotron X-ray diffraction. Visible "double yielding" behavior, which is characterized by the presence of stress-plateau, was observed in the solution treated specimen. Upon a cold rolling treatment, the Ti-25Nb-2Mo-4Sn alloy performs nonlinear deformation because of the combined effects of elastic deformation and stress-induced α″ martensitic transformation. After the subsequent annealing, the ß phase is completely stabilized and no stress-induced martensitic transformation takes place on loading due to the inhibitory effect of grain boundaries and dislocations on martensitic transformation. As a result, the annealed specimen exhibits linear elastic deformation.

Apolipoprotein A-I conformation in discoidal particles: evidence for alternate structures.

To define the conformation of apolipoprotein A-I in discoidal particles, the immunoreactivity of a series of epitopes distributed along the apoA-I sequence has been evaluated in lipid-free apoA-I and in lipid-bound form. To this end, reconstituted discoidal lipoproteins, here called LpA-I, and defined by number of apoA-I per particle (e.g., Lp2A-I), have been prepared with palmitoyloleoylphosphatidylcholine, cholesterol, and apoA-I. Four LpA-I have been obtained and studied: two in the Lp2A-I class, 7.8 and 9.6 nm in diameter, and two in the Lp3A-I class, 10.8 and 13.4 nm. The immunoreactivity of all the epitopes tested was significantly different in LpA-I particles compared to lipid-free apoA-I, demonstrating that binding to lipids produces a drastic change in apoA-I conformation. Specific domains in the primary sequence become highly exposed while others are masked. Although the variation in immunoreactivity of the epitopes between various LpA-I was not drastic, significant differences in the calculated ED50 values were observed for a number of antibodies in small versus large particles within each class (Lp2A-I or Lp3A-I), indicating that particle size can modulate apoA-I conformation. In addition, when the competition between pairs of mAbs was analyzed in order to understand the relative position of epitopes, highly significant differences were observed as a function of particle size within each class. In particular, the competition between mAbs recognizing epitopes in the central region of apoA-I was greater in the larger particles than in their small counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)