Expression and significance of miR-223 in rats with pulmonary fibrosis.

OBJECTIVE: To explore the expression and significance of miR-223 in mice with pulmonary fibrosis. MATERIALS AND METHODS: The rats were separated into a control group (n=15), a sham operation group (n=15), and a model group (n=45) (which was then divided into a 3-day group, a 7-day group, and a 14-day group, with 15 rats in each group). The rat model of pulmonary fibrosis was established. The rats in the model group were injected with bleomycin solution, while those in the control group and sham operation group were given the same operation and injected with the same amount of normal saline. After observing the pulmonary function indexes of the rats on the 3rd, 7th and 14th days after modeling, the rats were sacrificed by cervical dislocation, the pulmonary inflammation and fibrosis of the rats were observed, and the HYP (hydroxyproline) content and miR-223 expression level were determined. Pearson correlation analysis was employed to analyze the correlation between miR-223 and HYP. RESULTS: The pulmonary inflammation score of the model group was significantly higher than that of the sham group and the control group, and the pulmonary inflammation of the model group significantly increased with the increase of time (p<0.05). The pulmonary fibrosis score in the model group was markedly higher than that in the rest two groups, and the pulmonary fibrosis in the model group elevated significantly with the passage of time (p<0.05). The relevant pulmonary function indexes of the model group rats were significantly lower than those of the other two groups, and the pulmonary function of the model group rats gradually decreased with time (p<0.05). As to the HYP, it presented notably higher content in the model group than in the remaining two groups, and its content in the model group rats increased significantly with time (p<0.05). The expression of miR-223 decreased with the increase of fibrosis (p<0.05), and the expression level of miR-223 was negatively correlated with the HYP content (p<0.05). CONCLUSIONS: MiR-572 targeted CDH1 to promote cell metastasis in WT by suppressing EMT.

Thermodynamic and microstructural study of Ti2AlNb oxides at 800 °C.

The high-temperature structural applications of Ti2AlNb-based alloys, such as in jet engines and gas turbines, inevitably require oxidation resistance. The objective of this study is to seek fundamental insight into the oxidation behavior of a Ti2AlNb-based alloy via detailed microstructural characterization of oxide scale and scale/substrate interface after oxidation at 800 °C using X-ray diffraction (XRD), scanning electron microscopy (SEM), electron probe microanalysis (EPMA), and transmission electron microscopy (TEM). The oxide scale exhibits a complex multi-layered structure consisting of (Al,Nb)-rich mixed oxide layer (I)/mixed oxide layer (II)/oxygen-rich layer (III)/substrate from the outside to inside, where the substrate is mainly composed of B2 and O-Ti2AlNb phases. High-resolution TEM examinations along with high-angle annular dark-field (HAADF) imaging reveal: (1) the co-existence of two types (α and δ) of Al2O3 oxides in the outer scale, (2) the presence of metastable oxide products of TiO and Nb2O5, (3) an amorphous region near the scale/substrate interface including the formation of AlNb2, and (4) O-Ti2AlNb phase oxidized to form Nb2O5, TiO2 and Al2O3.

Core-multishell globular oxidation in a new TiAlNbCr alloy at high temperatures.

Oxidation resistance is one of key properties of titanium aluminide (TiAl) based alloys for high-temperature applications such as in advanced aero-engines and gas turbines. A new TiAlNbCr alloy with micro-addition of yttrium has been developed, but its oxidation behavior is unknown. To provide some fundamental insights, high-temperature oxidation characteristics of this alloy are examined via scanning electron microscopy, transmission electron microscopy, electron probe microanalysis, and X-ray diffraction. We show that distinctive core-multishell globular oxidation and "daisy" flower-like oxidation occur exclusively around Y2O3 particles. Globular oxides exhibit multi-layered Y2O3/TiO2/Al2O3-rich/TiO2-rich shell structures from the inside to outside. Flower-like inner oxides consist of core Y2O3 particles surrounded by divergent Al2O3 and oxygen-rich α2-Ti3Al in the near-scale substrate. As the scale-substrate interface moves inward, the inner oxide structures suffer deeper oxidation and transform into the globular oxide structures. Our results demonstrate that the unique oxidation characteristics and the understanding of formation mechanisms pave the way for the exploration and development of advanced oxidation-resistant TiAl-based materials.

[Significance and diagnostic value of synovial fluid anti-cyclic citrullinated peptide antibody and anti-mutated citrullinated vimentin antibodies in patients with serum negative rheumatoid arthritis].

OBJECTIVE: To explore the significance of synovial fluid (SF) anti-cyclic citrullinated peptide (CCP) antibodies and anti-mutated citrullinated vimentin (MCV) antibodies in the diagnosis of serum negative rheumatoid arthritis (SNRA). METHODS: Enzyme linked immunosorbent assay (ELISA) method was apllied in the detection of two groups of patients with knee joint fluid resistance against CCP antibody and antibody of MCV, the experimental group to SNRA patients, a total of 29 cases, and the control for patients with osteoarthritis (OA), a total of 28 cases, and clinical manifestations and laboratory parameters of the two groups were collected. RESULTS: The positive rate of synovial fluid anti-CCP was 34.5% in the SNRA patients, which was significantly higher than 10.7% in the control patients(χ2=4.571, P<0.05). The positive rate of synovial fluid anti-MCV was 20.7% in the SNRA patients, which was significantly higher than 7.1% in the control patients(χ2=2.167, P>0.05). The SNRA patients of SF anti-CCP and anti-MCV positive had no significant difference from the SNRA patients of SF anti-CCP and anti-MCV negative in age, course and morning stiffness. The levels of erythrocyte sedimentation rate (ESR), C-reactive protein(CRP) and DAS28 scores in the SF anti-CCP positive patients were higher than those of the SF anti-CCP negative patients. The levels of ESR, CRP and DAS28 scores in the SF anti-MCV positive patients were higher than those of the SF anti-MCV negative patients, (all P<0.01). SF anti-CCP had correlation with ESR, CRP(r=0.567, P<0.01; r=0.664, P<0.01). SF anti-MCV had correlation with ESR, CRP (r=0.344, P<0.01; r=0.749, P<0.01). CONCLUSION: SF anti-CCP and anti-MCV are helpful for the diagnosis of SNRA and judgement of SNRA activity.

Role of cysteine residues in human plasma phospholipid transfer protein.

Phospholipid transfer protein (PLTP) belongs to a family of human plasma lipid transfer proteins that bind to small amphophilic molecules. PLTP contains cysteines at residues 5, 129, 168, and 318. Bactericidal/permeability-increasing protein, which is a member of the same gene family, contains an essential disulfide bond between Cys135 and Cys175; these residues, which correspond to Cys129 and Cys168 in PLTP, are conserved among all known members of the gene family. To identify the importance of these and the remaining cysteine residues to PLTP secretion and activity, each was replaced by a glycine by site-directed mutagenesis. The mutant as well as wild-type PLTP cDNAs were cloned into the mammalian expression vector pSV.SPORT1, and the PLTP cDNAs were transfected to COS-6 cells for expression. PLTP Cys129 --> Gly and PLTP Cys168 --> Gly were secretion incompetent. Neither PLTP mass nor activity was detectable in cell lysates and culture medium. Relative to wild-type PLTP, PLTP Cys5 --> Gly and PLTP Cys318 --> Gly exhibited similar specific activities but partially impaired PLTP synthesis and secretion. Intracellular PLTP appeared as two bands of 75 and 51 kDa corresponding to reported molecular masses for the glycosylated and nonglycosylated forms. The specific activities of PLTP Cys5 --> Gly and PLTP Cys318 --> Gly were similar in the cell lysates and medium, suggesting that glycosylation does not affect transfer activity.

Molecular basis of fish-eye disease in a patient from Spain. Characterization of a novel mutation in the LCAT gene and lipid analysis of the cornea.

The genetic and biochemical basis of fish-eye disease (FED) was investigated in a 63-year-old female proband with low plasma HDL cholesterol. Analyses of corneal and plasma lipids of the proband were consistent with impaired lecithin:cholesterol acyltransferase (LCAT) activity. Free cholesterol and phospholipid levels were elevated relative to control values, whereas cholesteryl ester levels were greatly reduced. Fatty acid compositions of corneal lipids from the proband and control subjects differ from the respective fatty acid compositions of their plasma lipids. This suggests that the metabolic pathways and acyl chain specificities for phospholipid, cholesteryl ester, and triglyceride metabolism within the cornea are distinct from those of plasma. Sequencing of the LCAT gene from the proband revealed a novel mutation at nucleotide 399, corresponding to an Arg99-->Cys substitution. Secretion of LCAT (Arg99-->Cys) by transfected COS-6 cells was approximately 50% of that of the wild type, but its specific activity against reassembled HDL was 93% lower than that of wild-type LCAT. The specific activities of wild-type and LCAT (Arg99-->Cys) against LDL were reduced similarly, suggesting that the appearance of the FED phenotype does not require enhanced activity against LDL. Our data support the hypothesis that FED is a partial LCAT deficiency in which poor esterification in specific types of HDL particles may contribute to the appearance of the corneal opacities.

In vitro expression of natural mutants of human lecithin:cholesterol acyltransferase.

Fish-eye disease (FED) and familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) are rare disorders of lipid metabolism linked to mutations in the LCAT gene. Eleven LCAT cDNA constructs associated with FED and FLD were prepared by site-directed mutagenesis and expressed in COS-6 cells. Analysis of total RNA from wild-type, FED, and FLD transfectants revealed that all contained LCAT-specific mRNA. Western blot analysis demonstrated that all LCAT transfectants synthesized LCAT. Mean LCAT secretion by FED transfectants was slightly lower than secretion by wild-type transfectants, whereas secretion by FLD transfectants was much lower. The specific activities of FED and FLD LCAT against model high density lipoproteins were 6% and 11%, respectively, of wild-type activity. The ratios of the LCAT activities against low density lipoproteins to those against model high density lipoproteins decreased in the order FED mutants > FLD mutants approximately wild type. FED and FLD LCAT mutants are different: the former are more active against low density lipoproteins, and the latter are less secretion-competent. The greater reactivity of FED LCAT against low density lipoproteins may explain the relative mildness of the clinical manifestations of FED compared to those of FLD.

Effects of site-directed mutagenesis on the serine residues of human lecithin:cholesterol acyltransferase.

Lecithin:cholesterol acyltransferase (LCAT) is a serine protease-type enzyme that esterifies cholesterol in human plasma and is activated by apolipoprotein A-I in high-density lipoproteins. LCAT contains 22 serine residues, including Ser181, which is thought to be part of the catalytic site. In order to determine the importance of these serine residues in LCAT, we prepared six LCAT mutants: LCAT (Ser19-->Ala), LCAT (Ser181-->Gly), LCAT (Ser208-->Ala), LCAT (SEr216-->Ala), LCAT (Ser225-->Ala) and LCAT (Ser383-->Ala). We also replaced the adjacent asparagine residues in two additional mutants, LCAT (Ser19-->Ala, Asn20-->Thr) and LCAT (Ser383-->Ala, Asn384-->Thr), in order to ascertain the effect of the serines on N-glycosylation. The mutant complementary DNA (cDNA) were subcloned into a eukaryotic expression vector (pSG5) and expressed in COS-6 cells. By polymerase chain reaction analysis, LCAT-specific messenger RNA (mRNA) was found in all mutant and wild-type transfectants. Western blot analysis revealed LCAT-specific bands in media and lysates of the transfected cells. With two exceptions, the amounts of LCAT mass secreted by the transfectants were similar to that of the wild type (mean, 90% mass of wild type; range, 34-138%). Except for LCAT (Ser181-->Gly), which was inactive, the specific activities of the remainder of the mutant enzymes were also similar (mean 95% activity of wild type; range, 65-169%). These results indicate that Ser181 is part of the catalytic site and that stereoconservative substitutions for serines have minor effects on the synthesis, secretion and specific activities of human LCAT.

Effects of site-directed mutagenesis on the N-glycosylation sites of human lecithin:cholesterol acyltransferase.

There are four potential N-glycosylation site (Asn-X-Ser/Thr) in human lecithin:cholesterol acyltransferase (LCAT, residues 20, 84, 272, and 384). To study the role of the N-linked sugars, the codon for Asn at these positions was replaced with one for Thr (AAC to ACC). The wild-type and mutant LCAT cDNAs were used to transfect COS-6 cells from which RNA was isolated; cDNAs were synthesized by reverse transcription and subjected to the polymerase chain reaction, which showed that all transfectants synthesized LCAT-specific mRNA. No intracellular or secreted LCAT was detected with the Asn272-->Thr transfectants, indicating that this residue is essential for intracellular processing. All other single-point transfectants were secretion-competent. Although there was detectable LCAT protein inside the cells and in the media of the transfectant, Asn84-->Thr, its specific activity and secreted amount were only 26% and 58% of the wild type, respectively. This implies that Asn84 is critical for full activity but not for intracellular processing. The amount secreted, specific activity, and Vmax of LCAT (Asn20-->Thr) were similar to those of the wild-type LCAT. LCAT (Asn384-->Thr) differed from the wild-type LCAT only by a lower Km. These results suggest that glycosylation at residues 20 and 384 is not essential for intracellular processing, secretion, or activity.

Roles of cysteines in human lecithin:cholesterol acyltransferase.

Human lecithin:cholesterol acyltransferase (LCAT, E.C.2.3.1.43) is a serine-type esterase that contains six cysteines, two of which, Cys31 and Cys184, are free. The remaining cysteines form disulfide links. One of these is between Cys50 and Cys74 and the other is between Cys313 and Cys356. The cDNA of LCAT and mutants in which one or two of the six cysteines were replaced by glycine was expressed in COS-6 cells. Polymerase chain reactions and Northern blot analysis indicated that LCAT mRNA was produced by all transfectants. Western blots of all transfected cells probed with a polyclonal antibody revealed intracellular LCAT. Substitution of glycine for either Cys50, Cys74, Cys313, or Cys356 was associated with a nearly total absence of activity in the medium. No protein was secreted when glycine replaced either of the amino acid residues that link Cys313 and Cys356. The small amounts of the Cys50-->Gly and Cys74-->Gly mutants found in the medium had specific activities that were much lower than that of the wild-type LCAT. All other transfectants secreted immunologically measurable amounts of active enzyme. Mutants in which one or both free cysteines, Cys31 and Cys184, were replaced with glycine were less active than the wild type and only partially inhibited by a sulfhydryl blocking reagent. The substrate specificities of the Cys31-->Gly and Cys184-->Gly mutants differed from that of the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)