The Litsea genome and the evolution of the laurel family.

The laurel family within the Magnoliids has attracted attentions owing to its scents, variable inflorescences, and controversial phylogenetic position. Here, we present a chromosome-level assembly of the Litsea cubeba genome, together with low-coverage genomic and transcriptomic data for many other Lauraceae. Phylogenomic analyses show phylogenetic discordance at the position of Magnoliids, suggesting incomplete lineage sorting during the divergence of monocots, eudicots, and Magnoliids. An ancient whole-genome duplication (WGD) event occurred just before the divergence of Laurales and Magnoliales; subsequently, independent WGDs occurred almost simultaneously in the three Lauralean lineages. The phylogenetic relationships within Lauraceae correspond to the divergence of inflorescences, as evidenced by the phylogeny of FUWA, a conserved gene involved in determining panicle architecture in Lauraceae. Monoterpene synthases responsible for production of specific volatile compounds in Lauraceae are functionally verified. Our work sheds light on the evolution of the Lauraceae, the genetic basis for floral evolution and specific scents.

KILLER CELL IMMUNOGLOBULIN-LIKE RECEPTOR GENES AND THEIR HLA-C LIGANDS IN HASHIMOTO THYROIDITIS IN A CHINESE POPULATION.

OBJECTIVE: Natural killer (NK) cells serve as primary immune surveillance and are partially regulated by combinations of killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen-C (HLA-C) ligands. Alterations in NK cell activity have been associated with Hashimoto thyroiditis (HT). The aim of this study was to determine whether certain KIR/HLA-C genotype combinations play a role in HT pathogenesis. METHODS: The present study enrolled 107 unrelated HT patients and 108 random healthy individuals in a case-control study. Blood was collected for DNA extraction; typing of KIR genes and HLA-C alleles was performed by polymerase chain reaction with sequence specific primers (PCR-SSP), followed by electrophoresis on agarose gels. RESULTS: Among a panel of KIR2D/HLA-C genotype combinations, the frequency of KIR2DS2/HLA-C1 was significantly increased in HT patients compared to controls (33.64% vs. 12.96%, P<.001). To further analyze the precise genotype, we investigated inhibitory or activating KIR/HLA-C gene pairs when their corresponding activating or inhibitory KIR genes were absent in the 2 groups. Only the frequency of KIR2DS2(-)2DL2/3(+)HLA-C1(+) was significantly decreased in HT patients compared to controls (48.60% vs. 70.37%, P = .001). CONCLUSION: Our data suggest that KIR2DS2/HLA-C1 may correlate with HT pathogenesis. On the contrary, the predominance of KIR2DL2/3/HLA-C1 in the absence of KIR2DS2 suggests a potential inhibitory role in HT pathogenesis. In conclusion, our findings may further elucidate the mechanisms underlying the pathogenesis of HT and other autoimmune diseases. ABBREVIATIONS: HLA-C = human leukocyte antigen-C HT = Hashimoto thyroiditis KIR = killer immunoglobulin-like receptor NK = natural killer PCR = polymerase chain reaction.

Short low concentration cisplatin treatment leads to an epithelial mesenchymal transition-like response in DU145 prostate cancer cells.

BACKGROUND: Prostate cancer is one of the main causes of cancer death, and drug resistance is the leading reason for therapy failure. However, how this occurs is largely unknown. We therrfore aimed to study the response of DU145 cells to cisplatin. MATERIALS AND METHODS: Du145 prostate cancer cells were treated with a low dose of cisplatin for 24 h and cell viability and number were determined by MTT assay and trypan blue exclusion assay, respectively. The real time polymerase chain reaction (PCR) was used to assess responses to cisplatin treatment. RESULTS: After 24h 2 µg/ml treatment did not result in significant reduction in cell viability or number. However, it led to enhanced cancer cell invasiveness. E-cadherin mRNA was reduced, and vimentin, Snail, Slug, metalloproteinase 9 (MMP9) mRNA expression increased significantly, a feature of epithelial-mesenchymal transition (EMT). CONCLUSIONS: Short time low concentration cisplatin treatment leads to elevated invasiveness of DU145 cancer cells and this is possibly due to EMT.

Elevated plasma level of HMGB1 is associated with disease activity and combined alterations with IFN-α and TNF-α in systemic lupus erythematosus.

Recent studies indicate that high-mobility group box protein 1 (HMGB1) contributes to the pathogenesis of diverse autoimmune disorders. It induces the production of interferon-alpha (IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) in vitro. In the present study, plasma HMGB1, TNF-alpha, and IFN-alpha were determined with ELISA in 37 patients with systemic lupus erythematosus (SLE) and 39 age- and sex-matched healthy controls (HC). The possible associations of these cytokines with disease activities, autoantibodies, and certain laboratory parameters were also explored. The plasma levels of HMGB1, TNF-alpha, and IFN-alpha were increased in SLE patients compared with those of HC (P < 0.05). Moreover, the levels of HMGB1 and TNF-alpha in the active SLE patients were elevated compared with those in inactive patients and HC. Additionally, plasma HMGB1 was positively correlated with peripheral neutrophils, and plasma TNF-alpha was positively correlated with anti-Sm, ESR and CRP, while plasma IFN-alpha was inversely correlated with the age and platelet level in SLE patients. Our data indicated that increased plasma HMGB1 was associated with disease activity in SLE, which was similar to TNF-alpha. High level of plasma IFN-alpha may be related to nephritis and thrombocytopenia in SLE.

Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study.

OBJECTIVE: This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor (DT388sBAFF) in Escherichia coli (E. coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1). METHODS: A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl ß-D-1-thiogalactopyranoside (IPTG). The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and then purified by Ni(2+)-NTA affinity chromatography. The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. CONCLUSION: The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

Polymorphisms of KIR gene and HLA-C alleles: possible association with susceptibility to HLA-B27-positive patients with ankylosing spondylitis.

Accumulating evidences indicate that killer cell immunoglobulin-like receptors (KIRs) and their corresponding specific HLA-C ligands contribute to the pathogenesis of multiple autoimmune diseases via the modulation of natural killer (NK) cell and T cell functions. The present study was performed to investigate whether the polymorphism of KIR genes and HLA ligands associates with the susceptibility of ankylosing spondylitis (AS). Previous studies have demonstrated a strong association between HLA-B27 gene and the pathogenesis of AS. In this study, 115 unrelated HLA-B27-positive AS patients and 119 HLA-B27-positive healthy controls were recruited. Polymerase chain reaction using sequence-specific primers was used to determine the genotypes of KIR genes and HLA-C alleles. The results showed that the frequencies of KIR2DL1 and KIR2DL5 were significantly higher in the AS patient group than those in the control group (p = 0.012 and p = 0.009, respectively). Meanwhile, individuals with AS showed an increased frequency of HLA-Cw*08 (p = 0.001, p (c) = 0.008) compared with that in controls. Our findings indicate that with the genetic background of HLA-B27, variation at the KIRs and their corresponding specific HLA-C ligands may influence the ability of NK cells and T cells to recognize and lyse targets in immune responses, which thereby contributes to pathogenesis of AS.

[Expression of human HMGB1 A box in E. coli and its inhibitory effects on monocytes].

AIM: To clone human high mobility guoup box1 A box (HMGB1 A box) and express it in escherichia coli effectly, investigate the inhibit effection of the purpose protern to the activation of monocytes stimulated by immunocomplex. METHODS: According to human HMGB1 gene order which was optimized by our laboratory the PCR primer was designed which containing restriction enzyme cutting site. The HMGB1 A box gene was cloned following the whole gene synthesis template of human HMGB1, then the PCR product was inserted into clone vector pMD19-T. The positive colone was identified by colony PCR, zymography analysis and DNA sequencing. Recombinant colne vector was digested by restriction enzymes Nde I and Xho I and separated by agarose gel electrophoresis, then the fragment was inserted into the corresponding sites of expression vector pQE-T7-2. The positive recombinant expression vector was identified by colony PCR and the recombinant strains was induced by IPTG, then the purpose protein was identified by SDS-PAGE and Western blot. The recombinant protein of human HMGB1 A box was purificated by Ni(2+)-NTA chromatography and the inhibit effection of the purpose protern to the activation of monocyte stimulated by immunocomplex was identified by RT-PCR. RESULTS: We acquired expression strains of recombinant human HMGB1 A box, the target protein account for up to 40% of the whole protein of E.coli. Western blot showed recombinant protein can specificly reacted with anti-human HMGB1 polyclonal antibody and anti-His-Tag polyclonal antibody.The purpose protein was found more than 90% after purified, and can effectively inhibit the production of BAFF, IFN-gamma and TNF-alpha in monocyte which were induced by IC. CONCLUSION: A recombinant bacterial strain for expressing human HMGB1A box with biological activities was constructed successfully.

[Genotype and haplotype analysis of killer cell immunoglobulin-like receptors in ankylosing spondylitis].

OBJECTIVE: To investigate the association of killer cell immunoglobulin-like receptors (KIR) genotype and haplotype with ankylosing spondylitis (AS). METHODS: Peripheral blood samples were collected from 105 AS patients, 62 patients of osteoarthritis (OA), and 412 randomly selected healthy controls. Polymerase chain reaction with sequence-specific primers (PCR-SSP) was used to detect the KIR genotype and haplotype. RESULTS: The genotype frequency of 3DL3-2DL3-2DL1-2DP1-2DL4-3DL1-2DS4-3DL2 (6.67%) was significantly lower in the AS patients than in the control subjects (20.15%) and OA patients (17.74%, P = 0.001, 0.037 respectively). The genotype frequency of 3DL3-2DL3-2DL2-2DL1-2DP1-2DLA-3DL1-2DL5-2DS1-2DS2-2DS3-2DS4-2DS5-3DS1-3DL2 and 3DL3-2DL3-2DL2- 2DL1-2DP1-2DL4-3DL1-2DL5-2DS1-2DS4-3DL2 of the AS patients (9.52%, 5.71%)was significantly higher than that of the controls(2.18%, 0.49%; P = 0.001, 0.001), and these two genotypes were not detected in the OA patients. There were not significant differences in the haplotypes A and B among the AS patients, OA patients, and healthy controls. CONCLUSION: KIR genotypes may be associated with the susceptibility to AS.

Association of estrogen receptor alpha gene polymorphisms with cytokine genes expression in systemic lupus erythematosus.

AIM: To analyze the association of estrogen receptor alpha (OR alpha) gene polymorphisms with cytokine genes expression in patients with systemic lupus erythematosus (SLE) and controls. METHODS: Genomic DNA was extracted and polymorphisms of XbaI, Ukrainian (XX, Xx, or xx genotype) and PvuII (PP, Pp, or pp) in intron 1 of OR alpha gene were detected by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method. The messenger RNA (mRNA) levels of interleukin (IL)-10, IL-4, interferon (IFN)-gamma, and IL-2 were assessed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: In patients with SLE with PpXx genotype, IL-10 and IL-4 mRNA expression was higher (P < 0.001 and P = 0.013, respectively), while in patients with SLE with Ppxx genotype IFN-gamma and IL-2 mRNA expression was lower than in controls (P < 0.001). There was no significant difference in mRNA expression of 4 cytokines among controls with various genotypes. CONCLUSION: OR alpha gene polymorphism may be associated with the expression of IL-10, IL-4, IL-2, and IFN-gamma in patients with SLE.

Polymorphisms of KIRs gene and HLA-C alleles in patients with ankylosing spondylitis: possible association with susceptibility to the disease.

INTRODUCTION: An emerging body of evidence is accumulating to suggest that killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I ligands contribute to the pathogenesis of diverse kinds of autoimmune diseases. However, the functional effects of their polymorphism remain largely unknown to date. Thus, the present study was undertaken to determine the association of the polymorphisms KIRs gene and HLA-C alleles with the susceptibility to ankylosing spondylitis (AS) by means of polymerase chain reaction/sequence-specific primers for genotyping KIRs from genomic DNA of 119 patients with AS together with 128 healthy donors as a control group. RESULTS AND DISCUSSION: We found that the frequencies of KIR3DS1 and KIR2DL5 were statistically significantly higher in the patient group than those in the control group (P = 0.016 and P = 0.003, respectively). Meanwhile, the percentage of patients, who were carrying two or more of the activating KIRs, was higher than that of control group. With respect to HLA-C alleles, individuals with AS showed an increased frequency of HLA-Cw02. If HLA-C was divided into group 1 or group 2 based on whether there was an asparagine or lysine present at position 80 of the alpha-chain, HLA-C group 2 was more common in subjects with AS compared to control subjects. The genotype 2DS1+/HLA-C lys(80)+ was more common in subjects with AS. Moreover, the CD69 expression, a NK activation marker, remarkably increased in patient with AS. CONCLUSION: In conclusions, this study suggests that KIR3DS1 may severe as AS susceptive genes to trigger continuous injury of arthrosis. The imbalance of activating and inhibitory KIR as well as HLA-C group 1 and group 2 may be the key factor, which influences the pathogenesis of AS. Moreover, KIR2DS1 might associate with the susceptibility of AS by influencing NK cell activity once group 2 HLA-C ligands are present.

Increased activating killer immunoglobulin-like receptor genes and decreased specific HLA-C alleles in couples with recurrent spontaneous abortion.

Accumulating evidence indicates natural killer (NK) cells play crucial roles in successful pregnancy. To investigate whether the killer cell immunoglobulin-like receptor (KIR) gene polymorphism and the corresponding specific HLA ligands in parent couples possessing a susceptibility to unexplained recurrent spontaneous abortion (RSA), we searched 73 pairs of childless couples with three or more abortions characterized as unexplained RSA and 68 pairs of healthy control couples. Peripheral blood was drawn to obtain genomic DNA which was used for a polymerase chain reaction using sequence-specific primers (PCR-SSP) in order to determine whether 15 selected KIR genes and two groups of HLA-C alleles were present. Our result showed that gene frequency of KIR2DS1 was higher in patients with RSA compared to that of control subjects (P =0.029). Increased numbers of activating KIR genes was observed in patients (P =0.041). Women who possessed more than two activating KIR genes were found more frequently in patients than those in control subjects (P =0.018). From a cohort of husband and wife couples, the women with a KIR2DS1 gene, and with a decreased group 2 HLA-C allele for the homologous inhibitory receptor KIR2DL1, had a tendency to fall into the RSA group (P =0.004). The results suggest that a genetic variation at the KIR locus influences the susceptibility to unexplained RSA in the Chinese Han population. Moreover, decreased ligands for inhibitory KIRs could potentially lower the threshold for NK cell activation, mediated through activating receptors, thereby contributing to pathogenesis of RSA.

[Cloning and expression of human interleukin-26 in Escherichia coli].

To clone human interleukin-26 (hIL-26) and express it in E. coli efficiently. Two pairs of primers were synthesized according to the hIL-26 gene reported on GenBank. The hIL-26 gene was cloned by nest PCR following the first round RT-PCR from human peripherial blood monocytes total RNA, and then the PCR product was cloned into pMD18-T vector. Colony PCR, restriction analysis and sequence analysis showed that the gene cloned was the same as the reported hIL-26. The recombinant was cut with BamHI and EcoR I to obtain the hIL-26 fragment, and then the fragment was inserted into pBV220 which was cut with the same enzymes. The recombinant expression vector was induced to express hIL-26 at 42 degrees C, SDS-PAGE analysis showed that the recombinant protein accounted for up to 20% of the whole protein of E. coli, and the protein was also confirmed by Western blotting. Purity of the protein was found to be above 90% after purified with molecular sieve. After renaturalized with glutathione buffer, the promoting effect of it on the production of IFN-y in PBMC was detected by RT-PCR. A recombinant bacterial strain for expressing hIL-26 with biological activity was constructed successfully.

[Construction of co-expression vector pSLC-IRES-IL-2 and its expression in COS-7 cells].

AIM: To construct an eukaryotic co-expression plasmid pSLC-IRES-IL-2, and to express it in COS-7 cells. METHODS: Human IL-2 and SLC genes were cloned by RT-PCR and PCR, respectively, and then the eukaryotic expression plasmid pSLC-IRES-IL-2 was constructed.The constructed plasmid was transfected into COS-7 cells by electroporation method. The expression of SLC and IL-2 was detected by Western blot. RESULTS: SLC and IL-2 in the culture supernatant and lysate of transfected COS-7 cells were detected by Western blot. The relative molecular masses of the expressed products were consistent with the theoretical values. CONCLUSION: The SLC and IL-2 co-expression plasmid is successfully constructed and expressed in COS-7 cells, which offers a pathway for research on gene therapy of tumors.