Lacticaseibacillus casei CNRZ1874 supplementation promotes M1 alveolar macrophage activation and attenuates Mycoplasma pneumoniae pneumonia.

AIMS: To evaluate the protective effect of intestinal supplementation with Lacticaseibacillus casei CNRZ1874 on the inflammatory response induced by Mycoplasma pneumoniae in C57BL/6 J mice, and provide a potential strategy for alleviating M. pneumoniae pneumonia. METHODS AND RESULTS: C57BL/6 J mice were gavaged with L. casei CNRZ1874 or PBS for 7 consecutive days, and then infected with M. pneumoniae on day 8. Treatment with L. casei CNRZ1874 significantly reduced M. pneumoniae loads in the lungs and alleviated the lung inflammation on day 3 and 10 after pathogen infection. Importantly, oral administration with L. casei CNRZ1874 promoted M1 alveolar macrophages activation as evidenced by increased expression of iNOS, TNF-α, and CXCL1, while inhibited M2 alveolar macrophages activation as the expression of Arg1 and Chi3l3 were significantly decreased. In consistent with the M1 alveolar macrophages activation and enhanced mycoplasma clearance, the level of TNF-α was increased while the level of IL-4 was decreased in lung tissue from the L. casei CNRZ1874 group compared with the control group. However, oral administration with L. casei CNRZ1874 may not influence adaptive immunity induced by M. pneumoniae as evaluated by M. pneumoniae specific antibodies and T cells responses in spleen. CONCLUSIONS: Intestinal supplementation with L. casei CNRZ1874 can promote M1 alveolar macrophages activation, which contributes to the clearance of M. pneumoniae and attenuation of M.pneumoniae pneumonia.

Exogenous Hydrogen Sulfide Regulates Mycoplasma pneumoniae Lipid-Associated Membrane Proteins to Induce Expression of Heme Oxygenase-1 and Proinflammatory Cytokines.

This study was designed to investigate the effect of exogenous hydrogen sulfide (H2S) on the secretion of Heme oxygenase (HO-1) and proinflammatory cytokines in human mononuclear cell line THP-1 stimulated by lipid-associated membrane proteins (LAMPs) prepared from Mycoplasma pneumoniae (M. pneumoniae) and explore its regulatory mechanism. Cultured cells were stimulated with M. pneumoniae LAMPs after pretreatment with H2S to analyze the production of proinflammatory cytokines and HO-1 by enzyme-linked immunosorbent assay (ELISA) and Western blot. The results showed that THP-1 cells, which were stimulated by LAMPs after pretreatment with H2S, had decreased production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by inhibiting the mitogen-activated protein kinases (MAPKs)/nuclear factor-kappa B (NF-κB) signaling pathway and increased expression of HO-1 by activating the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. Our results indicate that H2S may play an important role in attenuating inflammation induced by M. pneumoniae LAMPs due to its ability to decrease the production of IL-6 and IL-8 and increase the expression of the HO-1. These findings support further studies for possible clinical applications.

Haplotype analyses of the APOA5 gene in patients with familial combined hyperlipidemia.

BACKGROUND: Familial combined hyperlipidemia (FCH) is the most common genetic lipid disorder with an undefined genetic etiology. Apolipoprotein A5 gene (APOA5) variants were previously shown to contribute to FCH. The aim of the present study was to evaluate the association of APOA5 variants with FCH and its related phenotypes in Dutch FCH patients. Furthermore, the effects of variants in the APOA5 gene on carotid intima-media thickness (IMT) and cardiovascular disease (CVD) were examined. MATERIALS AND METHODS: The study population consisted of 36 Dutch families, including 157 FCH patients. Two polymorphisms in the APOA5 gene (-1131T>C and S19W) were genotyped. RESULTS: Haplotype analysis of APOA5 showed an association with FCH (p=0.029), total cholesterol (p=0.031), triglycerides (p<0.001), apolipoprotein B (p=0.011), HDL-cholesterol (p=0.013), small dense LDL (p=0.010) and remnant-like particle cholesterol (p=0.001). Compared to S19 homozygotes, 19W carriers had an increased risk of FCH (OR=1.6 [1.0-2.6]; p=0.026) and a more atherogenic lipid profile, reflected by higher triglyceride (+22%) and apolipoprotein B levels (+5%), decreased HDL-cholesterol levels (-7%) and an increased prevalence of small dense LDL (16% vs. 26%). In carriers of the -1131C allele, small dense LDL was more prevalent than in -1131T homozygotes (29% vs. 16%). No association of the APOA5 gene with IMT and CVD was evident. CONCLUSION: In Dutch FCH families, variants in the APOA5 gene are associated with FCH and an atherogenic lipid profile.

Identification of lipid binding and lipoprotein lipase activation domains of human apoAV.

ApoAV, a newly discovered apolipoprotein, plays a key role in human triglyceride homeostasis; however, the structure-function correlation of apoAV is not clearly understood. To explore the relationship, wild type and six deletion mutants, that is (AV (Delta(1-51)), AV (Delta(51-128)), AV (Delta(132-188)), AV (Delta(192-238)), AV (Delta(246-299)), AV (Delta(301-343))), of human apoAV expressed in Escherichia coli were studied. All the deleted regions together encompass almost the entire 343 amino acid sequence of wild type apoAV. Circular dichroism spectroscopy showed that the alpha helical content of lipid-free wild type apoAV was 46%. In comparison with wild type apoAV, AV (Delta(192-238)) and AV (Delta(301-343)) displayed significantly decreased lipid binding activities, confirming the importance of these two regions in lipid binding function of apoAV. While, the LPL activation function of apoAV remarkably impaired after deletion of residues 192-238. These findings suggested that the domain (192-238) is absolutely necessary for apoAV in lipid binding and lipoprotein lipase activation.

[Structure analysis of apolipoprotein B 3' variable number of tandem repeats].

OBJECTIVE: To study the distribution, frequency and structure of variable number of tandem repeats (VNTR) in 3' region of apoB gene in Chinese population. METHODS: Genomic DNA was extracted from peripheral blood obtained under consent from randomly-chosen 522 individuals who came to the hospital for physical examination, and used to screen for polymorphisms of 3' VNTR of the apoB gene by employing polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), gradient polyacrylamide gel electrophoresis, cloning and sequencing. RESULTS: Sixteen types of alleles of apoB 3oVNTR were identified, among which heterozygotes were more than homozygotes. The biggest allele is HVE58, and the smallest one is HVE22. HVE34 had the highest frequency (40.4%), followed by HVE32 (34.7%). This showed significant difference from the allelic distribution of other populations (Caucasian and Swedish). Through sequencing of 60 alleles, a new isomer (Y-A=ATAATTAAATATTT) and four new types of alleles were found. CONCLUSION: The Chinese population we studied had a higher frequency of small alleles and showed a difference in allelic structure and frequency distribution from European and American in this populations.

Apolipoprotein E epsilon4 and the risk of unfavorable outcome after aneurysmal subarachnoid hemorrhage.

BACKGROUND: The APOE-E4 allele has been identified as a risk factor for Alzheimer's disease and unfavorable outcomes after brain injuries. The purpose of this study was to confirm that APOE allele polymorphism also represents a risk factor for unfavorable outcomes following aneurysmal subarachnoid hemorrhage (SAH). METHODS: A total of 104 patients with aneurysmal SAH were studied. Computed tomography (CT) scan findings of SAH were assessed by Fisher's grade and clinical neurologic assessment was performed using the Hunt and Hess (H&H)grading system. Serum lipids were also analyzed. Outcomes at 3 months after SAH were determined using the Glasgow Outcome Scale. RESULTS: The distributions of APOE genotypes and alleles of patients were matched with those of control subjects. That 5 of 18 patients with APOE-E4 allele (28%) had an unfavorable outcome was significantly different from those without APOE-E4 (8%, chi2, p = 0.032; OR = 4.34, 95% CI 1.20-15.75). However, the presence or absence of E2 or E3 alleles had no significant difference. The relative hazard of APOE-E4 for unfavorable outcome exited after adjustment for clinical assessment (OR = 6.95, 95% CI 1.21-39.75). Total serum cholesterol, low-density lipoprotein and apolipoprotein B were elevated in patients with unfavorable rather than favorable outcomes. CONCLUSION: Our findings confirmed that the patients with APOE-E4 allele were predisposed to unfavorable outcomes after aneurysmal SAH even though an association between APOE and incidence of the SAH may not exist. The effect of APOE on neurobiology and lipoprotein metabolism seems to partially explain the difference in outcomes and deserves further study.

The cDNA Sequence and Its Tissue Expression of Tree Shrew Apolipoprotein CI.

The mRNA was isolated and purified from tree shrew (TS) liver tissue. A cDNA library of the liver tissue was then constructed by using the mRNA as the template by reverse transcription. Two apolipoprotein CI (apoCI) cDNA clones were identified in the library with an anti-serum to TS apoCI. Sequencing and analysizing of the clones showed that both were the apoCI cDNA sequences in which the larger one was determined as 380 nucleotides. It contains 21 bp and 95 bp in 5' and 3' untranslated regions respectively, and 264 bp in an open reading frame, encoding an 88 aa apoCI precursor (a 26 aa signal peptide, and a 62 aa mature protein whose length is the same as those of rat, mouse and dog, but longer than those of human and baboon by 5 aa residues). The function domains in the protein sequence deduced from the cDNA were predicted by comparison of conservative regions in apoCIs from different species. The results of Northern blot indicated in TS the presence of apoCI mRNA not just mainly in the liver but also found in the intestine, suggesting the mRNA distribution different from those in other mammals and primates.

Cloning and characterization of cholesteryl ester transfer transfer protein isolated from the tree shrew.

OBJECTIVE: To obtain the nucleotide sequence and deduced amino acid sequence of cholesteryl ester transfer protein (CETP) cDNA from the tree shrew (Tupaia glis). METHODS: The cDNA sequence of the tree shrew CETP was obtained by utilizing the technique of switching mechanism at 5' end of RNA transcript (SMART) and rapid amplification of cDNA end (RACE) from the first strand of the cDNA. The amino acid sequence of CETP was deduced from the cDNA sequence and its primary and secondary structures were predicted. RESULTS: The sequence of CETP cDNA from tree shrew (GenBank accession number AF334033) covers 1636 bp, including 178 bp at the 3' end of the untranslated region and a 1458 bp fragment in a coding region, which provides the complete sequence of mature tree shrew CETP, although not the initiator methionine. The first 24 bp encodes a partial signal peptide. The mature protein consists of 477 amino acids and is longer than the human version by one amino acid (Gly318). Comparing this amino acid sequence with those of other animals' CETPs, the identity between tree shrew and human and rabbit CETP is 88% and 82%, respectively. The protein is extremely hydrophobic as it contains many hydrophobic residues, especially at the C-terminal, consistent with its function in the transfer of neutral lipids. The amino acid residues concerning with binding and transferring neutral lipids are highly conserved. There is a deletion of an N-linked glycosylation site at Asn342 in the tree shrew CETP protein that may participate in the removal of peripheral cholesterol and cholesteryl ester by increasing its activity of transferring cholesteryl ester. CONCLUSION: The possible glycosylation in the tree shrew CETP may be involved in the molecular mechanism of its insusceptibility to atherosclerosis.

[Differentially expressed genes in vascular endothelial cell line ECV 304 induced by high density lipoprotein].

OBJECTIVE: To isolate and clone the differentially expressed genes in vascular endothelial cell induced by high density lipoprotein (HDL) so as to study the anti- atherogenesis molecular mechanism of HDL. METHODS: Differential display reverse transcription PCR method was used to analyze the differentially expressed cDNA in vascular endothelial cell line ECV 304 induced by HDL. After sequencing and homology research, several differentially expressed cDNA fragments were confirmed by Northern blot analysis. RESULTS: Up-regulated and down-regulated cDNA fragments in ECV 304 induced by HDL were isolated. Nine cDNA fragments were highly homologous to the known human genes and four were fragments of novel genes. The known up-regulated genes included genes of human STE20-like kinase, PBK1 protein, transglutaminase, myosin alkali light chain, apobec-1 binding protein 1, and death- associated protein (DAP). The known down-regulated genes included genes of ribosomal protein L7a (RPL7A), voltage-dependent anion channel isoform 2, and glycinamide ribonucleotide transformylase. Northern blot analysis revealed that the expression levels of transglutaminase and apobec-1 binding protein 1 were upregulated by 50% and 70% respectively. CONCLUSION: HDL upregulates the expression of apobec-1 binding protein 1 and transglutaminase in endothelial cells. The high level expression of transglutaminase and apobec-1 binding protein 1 in endothelial cells induced by HDL may be related to anti-atherogenesis function of HDL.

[Analysis of lecithin-cholesterol acyltransferase cDNA and protein sequence from tree shrew].

OBJECTIVE: To acquire cDNA sequence of lecithin-cholesterol acyltransferase (LCAT) from tree shrew and analyze the sequence structure. METHODS: The first strand cDNA was acquired by reverse transcription using mRNA from tree shrew liver as template. By the method of SMART RACE PCR, tree shrew LCAT cDNA was acquired and deduced its amino acids sequence. The sequence and structure of tree shrew LCAT cDNA and amino acid were analyzed and predicted by the molecular software. RESULTS: Tree shrew LCAT cDNA is composed of 1,340 bp, including 2 bp 5' untranslated region (5' UTR), 1,320 bp open reading frame (ORF) which encodes protein precursor of 440 amino acids (24 amino acids signal peptide and 416 amino acids mature peptide), and 18 bp 3' untranslated region (3'UTR). The stop codon is TAA and there is a poly (A) signal sequence AATAAA and a 25 bp poly (A) tail. Tree shrew LCAT cDNA sequence has been accepted by GenBank as a new gene, accession number AF272861 and its homology with human and baboon was 90% and 89%, respectively. CONCLUSION: The sequence of LCAT cDNA in tree shrew has high identity with that of human and other experimental animal species.

Identification of the tree shrew ATP-binding cassette transporter A1 (ABCA1) and its expression in tissues : cDNA sequence and expression of tree shrew ABCA1.

ATP-binding cassette transporter A1 (ABCA1) modulates plasma levels of high density lipoprotein (HDL), a cardiovascular protecting factor. Tree shrew was considered to be an animal protected from atherosclerosis characterized by high proportion of HDL in plasma. The cDNA clones and expression of tree shrew ABCA1 was identified using SMART-RACE and Real-Time PCR techniques respectively. The nucleotide sequence of tree shrew ABCA1 covered 7,762 bp, including a 6,786 bp coding region which encoded a 2,261 amino acids protein with the high identity to human ABCA1 (95%). Tree shrew ABCA1 was expressed in various tissues, the highest in lung, followed by liver, kidney, spleen and cardiac muscle in turn from high to medium expression levels. This pattern was partially different from that of human ABCA1 which was low in kidney and cardiac muscle. This work could shed new light on its role of ABCA1 in the distinctive HDL metabolism in tree shrew.

[cDNA cloning and prokaryotic expression of human cholesteryl ester transfer protein and preparation of its antiserum].

AIM: To clone human cholesteryl ester transfer protein (CETP) cDNA, express and purify human CETP in E.coli and prepare CETP-specific rabbit antiserum. METHODS: by RT-PCR method, the encoding sequence of human cholesteryl ester transfer protein (CETP) was cloned into pET-30b(+) vector. Then BL21 (DE3) of E.coli transformed with recombinant vector pET-CETP was induced to express CETP in high level by IPTG. The expressed protein was purified from SDS-polyacrylamide gel, and the antiserum against CETP was raised in rabbit. The titer and specificity of rabbit antiserum were evaluated by ELISA, Western blot and immunofluorescence assay. RESULTS: The results of SDS-PAGE showed that CETP was expressed in the form of inclusion bodies in BL21(DE3) and the best expression time was about 4 hours. The titer of the rabbit antiserum prepared with CETP purified from SDS-PAGE was 1:5. 12 x 10(5) and the antiserum reacted specifically with CETP expressed in BL21 (DE3) and COS7 cells. CONCLUSION: The preparation of the specific rabbit antiserum against CETP will be valuable for the study on the structure and function of human CETP.

Secreted human apolipoprotein(a) kringle IV-10 and kringle V inhibit angiogenesis and xenografted tumor growth.

Abstract Angiogenesis plays an important role in normal physiology of blood vessel growth, but can contribute to the pathogenesis of diseases, such as cancer. A new anti-angiogenic recombinant kringle protein, composed of the fused domains of human apolipoprotein(a) carboxyl-terminal kringle IV-10 and kringle V, was expressed in Pichia pastoris and human colorectal carcinoma (HCT 116) cells to investigate its influence on angiogenesis and tumor growth. The mature recombinant protein exhibited the characteristic features of kringle-containing proteins (glycosylation and disulfide bond formation) and, when added to cultures of human umbilical vein endothelial cell, resulted in a 31% decrease in proliferation relative to untreated controls (p<0.05). The neo-angiogenesis was diminished by 63% in chick embryos treated with 10 mug recombinant protein compared with 7% for phosphate buffer solution-treated embryos (p<0.01). Transfection of a kringle IV-10-kringle V fusion protein construct into HCT 116 cells decreased tumorigenesis and inhibited tumor growth in vivo without affecting tumor cell proliferation. HCT 116 cells that expressed recombinant protein displayed a much lower relative growth ratio of 8% (p<0.01) against the control tumor cells. From these results, we conclude that human apolipoprotein(a) carboxyl-terminal kringle IV-10-kringle V fusion protein is an effective inhibitor of angiogenesis and angiogenesis-dependent tumor growth.

Cysteine mutants of human apolipoprotein A-I: a study of secondary structural and functional properties.

Apolipoprotein A-I(Milano) (A-I(M)) (R173C), a natural mutant of human apolipoprotein A-I (apoA-I), and five other cysteine variants of apoA-I at residues 52 (S52C), 74 (N74C), 107 (K107C), 129 (G129C), and 195 (K195C) were generated. Cysteine residues were incorporated in each of the various helices at the same helical wheel position as for the substitution in A-I(M). The secondary structural properties of the monomeric mutants, their abilities to bind lipid and to promote cholesterol efflux from THP-1 macrophages, and the possibility of antiperoxidation were investigated. Results showed that the alpha helical contents of all of the cysteine mutants were similar to that of wild-type apoA-I (wtapoA-I). The cysteine variant of A-I(M) at residue 173 [A-I(M)(R173C)] exhibited weakened structural stability, whereas A-I(G129C) a more stable structure than wtapoA-I. A-I(G129C) and A-I(K195C) exhibited significantly impaired capabilities to bind lipid compared with wtapoA-I. A-I(K107C) possessed a higher capacity to promote cholesterol efflux from macrophages than wtapoA-I, and A-I(M)(R173C) and A-I(K195C) exhibited an impaired efflux capability. Neither A-I(M)(R173C) nor any other cysteine mutant could resist oxidation against lipoxygenase. In summary, in spite of the similar mutant position on the helix, these variants exhibited different structural features or biological activities, suggesting the potential influence of the local environment of mutations on the whole polypeptide chain.

[Study on the relationship between smoking, alcohol intake and hyperlipidemia in fishermen].

OBJECTIVE: To identify the relationship between smoking, alcohol intake and hyperlipidemia in fishermen. METHODS: 115 fishermen were randomly recruited and divided into case and control groups according to the result of blood lipoprotein. A questionnaire was used to record general information and the history of smoking and alcohol intake. Statistics were gathered to compare the difference of lipoprotein and apolipoprotein level between exposure and control groups and to calculate the OR value of smoking and alcohol intake. RESULTS: The OR of smoking was 3.417 (95% CI: 1.132 - 10.308), with significant dosage-effect relationship between smoking index and hyperlipidemia. The serum low density lipoprotein-cholesterol (LDL-C) and apolipoprotein (apo) B levels in smoking group was higher than that of control group. The OR value of alcohol intake at early age (early than 20) were 3.275 (95% CI: 1.249 - 8.580) and 4.016 (95% CI: 1.475 - 10.952) respectively. The LDL-C, apoB, the serum total cholesterol (TC)/high density lipoprotein-cholesterol (HDL-C) levels in alcohol abuse group were higher than that of control group. CONCLUSION: Smoking and alcohol abuse were important risk factors of hyperlipidemia, through changing the level of LDL-C and apoB. There was synergistic action between smoking and alcohol abuse in the development of hyperlipidemia.