PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma

Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by Heparin-Sepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity. These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma.     

Interplay between hnRNP A1 and a cis-acting element in the 3' UTR of CYP2A5 mRNA is central for high expression of the gene

Our previous evidence suggests that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 plays a part in the regulation of the Cyp2a5 gene by interacting with the 3' untranslated region (UTR) of the CYP2A5 mRNA. However, the exact role of this interaction is not clear. The aim of the present work was to gain further insight into the regulation process of Cyp2a5. For this purpose the 3' UTR of CYP2A5 was fused to the coding region of luciferase mRNA. Luciferase recombinants containing either the full length 3' UTR, or the 3' UTR lacking a previously described 71 nucleotide (nt) region (the hnRNP A1 primary binding site), were transiently expressed in cells expressing or lacking hnRNP A1. The expression of the luciferase recombinants was examined both at mRNA and enzyme activity levels. The results disclosed that the presence of hnRNP A1 was required for the high expression of the recombinant carrying the full length 3' UTR of CYP2A5. Deletion of the hnRNP A1 primary binding site dramatically modified the expression pattern: the mRNA levels and luciferase activities of the deletion mutant were independent from hnRNP A1. These results conclusively demonstrate that the 71 nt region in the 3' UTR of CYP2A5 mRNA can confer hnRNP A1-dependent regulation to a gene. In addition, comparison of RNA levels and luciferase activities suggested that regions flanking the hnRNP A1 binding site could regulate translation of the CYP2A5 mRNA. These results are consistent with a model in which the binding of hnRNP A1 to the 71 nt putative hairpin-loop region in the CYP2A5 mRNA 3' UTR upregulates mRNA levels possibly by protecting the mRNA from degradation.     

Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in prebeta-HDL generation

The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce pre(beta)-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process.     

Prosthetic crowns and other clinical risk indicators of caries among old-old Swedish adults: findings from the KEOHS Project. Kungsholmen Elders Oral Health Study

Objectives: The Kungsholmen Elders Oral Health Study (KEOHS) evaluated the oral health status of generally healthy, community‐dwelling persons over the age of 80 living in Kungsholmen, Sweden. This paper explored possible clinical risk indicators of coronal and root caries among the KEOHS subjects. Design: In this cross‐sectional study, dentate KEOHS subjects received a caries assessment using defined visual, tactile criteria. Setting: Examinations were carried out in two local clinics by standardized examiners. Subjects: One hundred twenty‐nine dentate persons were examined. Main Outcome Measures: The examination identified decayed and filled surfaces, prosthetic crowns, and missing teeth. Results: More root than coronal surfaces had untreated decay, and secondary root caries contributed the greatest number of decayed surfaces. Ninety percent of the examined dentate subjects had at least one prosthetic crown. Root surfaces exposed to crown margins were more likely to have caries than root surfaces not so exposed, particularly among women. The presence of untreated coronal caries (yes/no) was positively associated with having untreated root caries and an intermediate number (14–20) of teeth, but inversely associated with having 4+ prosthetic crowns. Active root caries (yes/no) was positively associated with having untreated coronal caries, 14–20 teeth, and 4+ prosthetic crowns. Nearly 20% of identified root lesions were present at or below the gingival margin, and most (88%) were secondary caries associated with crown margins (65%) or other restorations (23%). Conclusions: Our findings suggest that some dental characteristics, including the presence of prosthetic crowns, are risk indicators for the presence of untreated coronal and root caries.     

Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.     

LDL particle size in familial combined hyperlipidemia: effects of serum lipids, lipoprotein-modifying enzymes, and lipid transfer proteins

Small, dense LDL particles are typical for FCHL. Intravascular lipid exchange and net transfer among HDL, LDL, and triglyceride-rich lipoproteins as well as lipolysis in the VLDL-IDL-LDL cascade regulate properties of LDL. We investigated postheparin plasma activities of hepatic lipase (HL) and LPL, and plasma activities of CETP and phospholipid transfer protein (PLTP) in 191 individuals from 37 Finnish FCHL families. LDL peak particle diameter (LDL size) was measured with 2-10% gradient polyacrylamide gel electrophoresis. LDL size was significantly smaller in affected FCHL family members (n = 68) as compared with nonaffected FCHL family members (n = 78) or spouses (n = 45) (25.3 +/- 1.5 nm, 26.8 +/- 1.2 nm, and 26.6 +/- 1.2 nm, respectively, P < 0.001 for both). In affected FCHL family members, serum triglycerides were the strongest correlate for LDL size (r = -0.71, P < 0.001). In univariate correlation analysis LDL size was not associated with HL, LPL, CETP, and PLTP activities. In multivariate stepwise regression analysis, however, serum triglycerides, CETP activity, HL activity, and HDL cholesterol were significant predictors of LDL size in affected FCHL subjects (adjusted r (2) = 0.642).We conclude that serum triglyceride concentration is strongly correlated with LDL size in affected FCHL subjects. After adjustment for serum triglycerides, HL and CETP activities are associated with LDL size in FCHL.     

Role of recycling endosomes and lysosomes in dynein-dependent entry of canine parvovirus

Canine parvovirus (CPV) is a nonenveloped virus with a 5-kb single-stranded DNA genome. Lysosomotropic agents and low temperature are known to prevent CPV infection, indicating that the virus enters its host cells by endocytosis and requires an acidic intracellular compartment for penetration into the cytoplasm. After escape from the endocytotic vesicles, CPV is transported to the nucleus for replication. In the present study the intracellular entry pathway of the canine parvovirus in NLFK (Nordisk Laboratory feline kidney) cells was studied. After clustering in clathrin-coated pits and being taken up in coated vesicles, CPV colocalized with coendocytosed transferrin in endosomes resembling recycling endosomes. Later, CPV was found to enter, via late endosomes, a perinuclear vesicular compartment, where it colocalized with lysosomal markers. There was no indication of CPV entry into the trans-Golgi or the endoplasmic reticulum. Similar results were obtained both with full and with empty capsids. The data thus suggest that CPV or its DNA was released from the lysosomal compartment to the cytoplasm to be then transported to the nucleus. Electron microscopy analysis revealed endosomal vesicles containing CPV to be associated with microtubules. In the presence of nocodazole, a microtubule-disrupting drug, CPV entry was blocked and the virus was found in peripheral vesicles. Thus, some step(s) of the entry process were dependent on microtubules. Microinjection of antibodies to dynein caused CPV to remain in pericellular vesicles. This suggests an important role for the motor protein dynein in transporting vesicles containing CPV along the microtubule network.     

Sperm volume regulation: maturational changes in fertile and infertile transgenic mice and association with kinematics and tail angulation

Laser light scatter analyzed by flow cytometry was used to monitor the volume of viable maturing murine spermatozoa. Upon release, dispersion, and dilution, epididymal sperm from fertile heterozygous c-ros knockout mice were smallest in the cauda region and largest in the corpus region. Cauda sperm from both infertile homozygous c-ros knockout and GPX5-Tag2 transgenic mice were abnormally large. When incubated, corpus and cauda sperm from normal mice became slightly enlarged and later returned to a smaller size. This suggests an immediate swelling due to high intracellular osmolality, which triggers a regulatory volume decrease (RVD) that results in a net volume reduction. Normal caput sperm increased in size continuously and became larger than the more mature sperm, indicating a lack of RVD. The ion-channel blocker quinine induced dose-dependent size increases in normal cauda sperm but not in caput sperm. Dose-dependent quinine action on mature sperm also included induction of tail angulation, and suppression of straight-line velocity and linearity. The kinematic effects were more sensitive, with a quicker onset, but they diminished with time in contrast to tail angulation, which intensified. These results suggest that kinematic changes are an early phenomenon of swelling, which gradually accumulates at the cytoplasmic droplet to cause flagellar angulation. Disruption of the epididymal maturation of sperm volume regulation capacity would hinder the transport of sperm in the female tract, and may thereby explain infertility under certain conditions, but may also provide a novel approach to male contraception.