How antiferromagnetism drives the magnetization of a ferromagnetic thin film to align out of plane.

Interfacial moments of an antiferromagnet are known for their prominent effects of induced coercivity enhancement and exchange bias in ferromagnetic-antiferromagnetic exchange-coupled systems. Here we report that the unpinned moments of an antiferromagnetic face-centered-cubic Mn layer can drive the magnetization of an adjacent Fe film perpendicular owing to a formation of intrinsic perpendicular anisotropy. X-ray magnetic circular dichroism and hysteresis loops show establishment of perpendicular magnetization on Fe/Mn bilayers while temperature was decreased. The fact that the magnitude of perpendicular anisotropy of the Fe layer is enhanced proportionally to the out-of-plane oriented orbital moment of the Mn unpinned layer, rather than that of Fe itself, gives evidence for the Mn unpinned moments to be the origin of the established perpendicular magnetization.

Single-Atomic-Layer Stanene on Ferromagnetic Co Nanoislands with Topological Band Structures.

Introducing magnetism to two-dimensional topological insulators is a central issue in the pursuit of magnetic topological materials in low dimensionality. By means of low-temperature growth at 80 K, we succeeded in fabricating a monolayer stanene on Co/Cu(111) and resolving ferromagnetic spin contrast by field-dependent spin-polarized scanning tunneling microscopy (SP-STM). Increases of both remanence to saturation magnetization ratio (Mr/Ms) and coercive field (Hc) due to an enhanced perpendicular magnetic anisotropy (PMA) are further identified by out-of-plane magneto-optical Kerr effect (MOKE). In addition to ultraflat stanene fully relaxed on bilayer Co/Cu(111) from density functional theory (DFT), characteristic topological properties including an in-plane s-p band inversion and a spin-orbit coupling (SOC) induced gap about 0.25 eV at the Γ̅ point have also been verified in the Sn-projected band structure. Interfacial coupling of single-atomic-layer stanene with ferromagnetic Co biatomic layers allows topological band features to coexist with ferromagnetism, facilitating a conceptual design of atomically thin magnetic topological heterostructures.

[BCG cell wall proteins enhance clearance of Pseudomonas aeruginosa in rat lungs].

OBJECTIVE: To test the function of BCG cell wall proteins in enhancing the clearance of Pseudomonas aeruginosa in rat lungs. METHODS: The BCG cells were broken by supersonic technique. The cell wall proteins were isolated by discontinues sucrose density-gradient centrifugation, and fractionated by Sephadex G-150 chromatography. The relative molecular mass of the isolated proteins was analyzed by SDS-PAGE. The hBD-1 gene mRNA expression in the SPC-A-1 cells was identified by Northern blot and RT-PCR. The cell wall component was injected intraperitoneally to the rats. Forty eight hours later, 5 X 10(6) CFU of P. aeruginosa ATCC27853 or Staphylococcus aureus ATCC25923 were inoculated via trachea. The bacteria colony in the supernatant of the homogenated lungs of the rats was counted 24 hours after the inoculation. RESULTS: Two protein components of BCG cell wall were fractionated. The relative molecular mass of component 2 was in the range of 18 X 10(3) -30 X 10(3). The hBD-1 mRNA expression detected by Northern blot was markedly enhanced by the stimulation of heat-inactivated BCG whole cells. The BCG-induced hBD-1 mRNA expression in the SPC-A-1 cells detected by RT-PCR was mainly contributed by fraction 2 of the BCG cell wall proteins. The bacteria decreased significantly in the lungs of the rats with the injection of BCG component 2 (n=8, P<0. 01). CONCLUSION: The fraction (relative molecular mass is 18 X 10(3) -30 X 10(3)) of BCG cell wall proteins improve the defense of rat lungs against P. aeruginosa infection.

[Antimicrobial peptide may originate from hemoglobin and play a role in the antibacterial mechanisms of endometrium].

OBJECTIVE: To isolate and purify antimicrobial peptides from human uterus mucus. METHODS: Acid-soluble extract was obtained from the specimens of endometrium mucus from 3 hysteromyomas patients during total hysterectomy. Acidic urea-polyacrylamide gel electrophoresis was used to analyze the acidic extract. The corresponding antimicrobial band HUP was further isolated and purified by electrophoretic elution and reversed-phase high-performance liquid chromatography (RP-HPLC). The antimicrobial activity of the fractions was analyzed by agarose radial diffusion assay. The molecular weight was determined by Tricine-SDS-PAGE. According to the results of the N-terminal sequencing and Mass Spectrometry analysis, the amino acid sequences of the purified molecules were deduced. The deduced amino acid sequence of the antibacterial fragment was further analyzed by ExPASy and OMIGA softwares. RESULTS: An antibacterial peptide named HUP-39 was purified from the human uterine mucus with a molecular weight of 6.777 Ku. N-terminal sequencing and Mass Spectrometry analysis suggested that this antimicrobial peptide should be hHEM-alpha 33-95 amino acid fragment. ExPASy and OMIGA analysis showed that it contained 3alpha-helical transmembrane domains and its pI was 8.38. The fragment was mainly against Escherichia coli ML-35p, E. coli ATCC 25 922, and the clinically isolated strain E. coli 54 080. CONCLUSION: An antimicrobial peptide has been isolated and purified from human uterine mucus, a hHEM-alpha 33-95 amino acid fragment. The antimicrobial molecule in the uterine mucus originates not only from epithelial cells and leucocytes, but also from erythrocytes.

[Effect of human beta defensin 2 on Staphylococcus aureus infection: in vivo study with transgenic mice].

OBJECTIVE: To investigate the effect of human beta defensin 2 (HBD-2) on Staphylococcus aureus infection. METHODS: A minigene of HBD-2 containing pCMV promoter, full length of HBD-2 cDNA, and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57/ICR hybridized mouse by microinjection. After gestation of 3 - 4 weeks, immunohistochemistry was used to detect the expression of HBD-2 peptide in different tissues of the transgenic young mice. Staphylococcus aureus was cultured and injected intraperitoneally to wild type mice and transgenic mice to observe their surviving status. RESULTS: PCR showed that the HBD-2 fragment had been successfully integrated into the chromosome of the mice. A widespread expression of HBD-2 gene was found in many tissues of the transgenic mice: trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium, brain, etc. Four of the 7 transgenic mice survived the Staphylococcus aureus infection, and 10 wild type mice all died within 24 hours. CONCLUSION: HBD-2 may play an important role ion the host defense against Staphylococcus aureus infection.

[Transcriptional activation of interleukin-8 gene induced by shear stress in human umbilical vein endothelial cells].

OBJECTIVE: To examine the transcriptional activation of IL-8 gene induced by 4.2 dyne/cm2 shear stress in human vascular endothelial cells. METHODS: The one-step RT-PCR was used for detection of IL-8 mRNAs expression in human umbilical vein endothelial cells (HUVECs) after shear stress for 0.5, 1, 2 hours. To construct IL-8 green fluorescent protein reporter gene plasmid pEGFP1-IL8USCS. The endothelial cells were transfected with the pEGFP1-IL8USCS, and stimulated with 4.2 dyne/cm2 shear stress for 3 hours. The green fluorescent protein expression was analyzed by flow cytometry. NF-kappaB nuclear translocation was observed by immunocytofluorescent staining in HUVECs stimulated by shear stress for 0.5, 1, 1.5, 2 hours. Western blotting was used to examine kappaB phosphorylation and degradation after shear stress for 10, 20, 30, 60 minutes respectively. RESULTS: The results of RT-PCR showed that low laminar shear stress could induce IL-8 mRNA expression in HUVECs, its increased effect was time-dependent. Flow cytometric analysis showed that when exposed to 4.2 dyne/cm2 shear stress for 3 hours, there was a marked increase in the green fluorescent protein expression in the pEGFP1-IL8USCS-transfected cells. NF-kappaB p65 immunocytofluorescent staining of HUVECs showed that flow shear stress could induce nuclear translocation of NF-kappaB. Flow shear stress could induce IkappaB phosphorylation and degradation in HUVECs detected by Western blotting. CONCLUSION: These experiments suggested that the NF-kappaB signaling pathway would probably be involved in flow shear stress-induced IL-8 gene transcriptional activation, It may be involved in the development of atherosclerosis.

[Study of antmicrobial mechanisms of human cervical mucus: isolation and characterization of antibacterial polypeptides].

OBJECTIVE: To isolate and purify antibacterial polypeptides from human cervical mucus. METHODS: Human cervical mucus was collected from human healthy subjects and acid-soluble extract was obtained by solving the mucus with 5% acetic acid in the presence of protease inhibitors. The antibacterial components were identified by Agar radial diffusion assay and gel overlay technique. For further purification, Preparative acid-urea gel electrophoresis and Reverse Phase HPLC were performed. The N-terminal sequencing and degenerate PCR-directered cDNA cloning were performed. The E. coli-based recombinant product was prepared and its antibacterial property was determined by minimal inhibitory concentration and minimal bactericidal concentration. RESULTS: A purified antibacterial polypeptide was obtained. Agar radial diffusion assay showed that the purified polypeptide had antibacterial activities against E. coli ML-35P and Pseudomanas aeruginosa ATCC 27853. The N-terminal amino acid sequence and its full length of cDNA were identical to High Mobility Group Chromosal protein N2 (HMGN2). The purified recombinant HMGN2 was obtained. Its MIC against E. coli ML-35p and P. aeruginosa ATCC27853 were 10.42 microg/ml +/- 3.13 microg/ml and 27.78 microg/ml +/- 8.33 microg/ml respectively, which were equal to human neutrophil defensins HNP, and the MBC were 20.83 microg/ml +/- 6.25 microg/ml and 55.56 microg/ml +/- 16.67 microg/ml respectively. CONCLUSION: HMGN2 may be another antibacterial effecter in the defense mechanisms of human cervical mucus.

[Production of HMGN2 polyclonal antibody by immunization with recombinant GST-HMGN2 fusion protein and its application to analysis of HMGN2 distribution in human monocytes].

OBJECTIVE: To prepare high mobility group chromosomal protein N2 (HMGN2) polyclonal antibodies and determine the subcellular localization of HMGN2 in human monocytes. METHODS: The recombinant prokaryotic expression vector pGEX-1lambdaT-HMGN2 was constructed and E. coli-based product of GST-HMGN2 fusion protein was prepared and used to immunize rabbit for producing the anti-serum against HMGN2. The polyclonal antibodies were partially purified by caprylic acid and ammonium sulfate precipitation. The titter of specific polyclonal antibodies against HMGN2 was detected by ELISA. The immunocytochemical staining was performed to determine the distribution of HMGN2 in THP-1 cells. RESULTS: Gel electrophoresis of the enzyme-digested recombinant plasmid and the DNA sequencing confirmed that the recombinant prokaryotic expression vector pGEX-1lambdaT-HMGN2 was correctly constructed. After IPTG induction, the recombinant-transformed E. coli produced a bulk of GST-HMGN2 fusion protein. The polyclonal antibodies to HMGN2 was obtained from the serum of rabbit immunized with GST-HMGN2 fusion protein and its ELISA titer was 1:2000. The immunocytochemistry staining indicated that when stimulated with LPS, HMGN2 was present not only in THP-1 nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant. CONCLUSION: This result suggests that recombinant peptide fusion protein could be used to produce peptide antibody. HMGN2 could be present in the cytoplasm of monocytes and release to the extracellular environment when stimulated with lipopolysaccharide (LPS).

[Charactierization of a new antibacterial polypeptide isolated from human cervical mucas].

OBJECTIVE: To identify a new antibactrial polypeptide HCP-1 isolated from human cervical mucus. METHODS: HCP-1 was isolated and purified from human cervical mucus, and N-terminal sequence of HCP-1 was determined. A degenerate primer was designed according to CodeHop methods, and an "anchor-oliga-dT primer" was used for the synthesis of cDNA. Using the degenerate primer and anchor-primer, cDNAs were amplified by PCR. The PCR products were cloned, sequenced, and analyzed by biological software. RESULTS: N-terminat sequence of HCP-1 was PKRKAEGDAK. The full length of HCP-1 cDNA was isolated and of which the sequence was the same as HMG-17. OMIGA software analysis indicated that this molecule contained an alpha-helix region. CONCLUSION: The new antibacterial polypeptide isolated from human cervical mucus is HMG-17. It may play a role in the human cervical mucus and the alpha-helix domain may be its antibacterial activity region.

[Involvement of MAPKs and NF-kappaB pathways in Pseudomonas pyocyanin-induced interleukin-8 expression by human airway epithelial cells].

To investigate the molecular mechanisms of signaling transduction by which Pseudomonas pyocyanin induces IL-8 expression in human airway epithelial cells, A549 and SPC-A-1 cells were challenged with P. aeruginosa conditioned medium or pyocyanin. Chemokine interleukin-8 (IL-8) release from the challenged cells was measured by ELISA, and Western blot was performed to analyze the degradation of IkappaB-alpha and the phosphorylation of MAPKs (mitogen-activated protein kinases) in the extracts from cells stimulated with pyocyanin. Both of P. aeruginosa conditioned medium and pyocyanin remarkably increased IL-8 expression by human airway epithelial cells. Degradation of IkappaB-alpha was found shortly after A549 cells were stimulated with pyocyanin. Western hybridization analysis also demonstrated that pyocyanin caused phosphorylation of MAPKs including ERK1/2, p38 and JNK in A549 cells. Pretreatment of A549 cells with U0126 (10 micromol/L), a selective inhibitor of MEK1/2 (ERK1/2 kinase) or with SB203580 (10 micromol/L), a specific inhibitor of p38 MAPK, diminished the pyocyanin-induced IL-8 production. These findings suggest that Pseudomonas pyocyanin can increase IL-8 expression by human airway epithelial cells through MAPKs signaling pathways and the activation of NF-kappaB is also involved in this process.

[Transfection and expression of recombinant human beta-defensin-2 gene in insect cells].

OBJECTIVE: This study was conducted to determine the feasibility and relevant technique itinerary for the production of hBD-2 with Baculovirus Expression Vector System (BEVS). METHODS: The full hBD-2/His cDNA was amplified from rpcDNA3.1/hBD-2/myc-His by using PCR with a pair of primers (hBD2p10 and hBD2p11) and was inserted into the MCS of transfer vector: pAcGHLT-A. AcNPV DNA and rpAcGHLT-A/hBD-2/His were co-transfected into Sf21 cells. Recombination would take place within the Sf21 cells between the homologous regions in the transfer vector and AcNPV DNA. After 5 days of co-transfer, both supernatant of the experimental cells and positive control cells were collected. Sf21 cells were infected with virus rAcNPVhBD-2/His and then determined by end-point dilution assay. The expression of hBD-2/His in both cell lysate and supernatant was analyzed by western blot with specific 6 poly-histamines antibody. RESULTS: Both enzyme cutting result and sequence analysis showed that recombinant hBD-2 with C terminal of bi-tags of myc and 6xHis had been inserted into the transfer vector of BEVS system correctly, and recombinant transfer vector rpAcGHLT-A/hBD-2/His had been constructed successfully. End-point dilution assay proved that recombinant virus rAcNPVhBD-2/His had been acquired. Western blot revealed that lysate of Sf21 cells transfected by rAcNPVhBD-2/His showed a band of relative moleculal mass about 47.5 x 10(3) which implied that a fuse peptide of hBD-2/His with up-stream of GST tag, 6xHistag, protein kinase A site and thrombin cleavage had expressed. The culture supernatant showed two bands of relative moleculal mass about 40 x 10(3) and 30 x 10(3), which were inferred to be the proceeded products of the fuse peptide during secretion process from cell into culture supernatant. CONCLUSION: These results suggested that it may be feasible to use BEVS system as a high efficient biologic reactor for producing recombinant hBD-2.

Band gap engineering of chemical vapor deposited graphene by in situ BN doping.

Band gap opening and engineering is one of the high priority goals in the development of graphene electronics. Here, we report on the opening and scaling of band gap in BN doped graphene (BNG) films grown by low-pressure chemical vapor deposition method. High resolution transmission electron microscopy is employed to resolve the graphene and h-BN domain formation in great detail. X-ray photoelectron, micro-Raman, and UV-vis spectroscopy studies revealed a distinct structural and phase evolution in BNG films at low BN concentration. Synchrotron radiation based XAS-XES measurements concluded a gap opening in BNG films, which is also confirmed by field effect transistor measurements. For the first time, a significant band gap as high as 600 meV is observed for low BN concentrations and is attributed to the opening of the π-π* band gap of graphene due to isoelectronic BN doping. As-grown films exhibit structural evolution from homogeneously dispersed small BN clusters to large sized BN domains with embedded diminutive graphene domains. The evolution is described in terms of competitive growth among h-BN and graphene domains with increasing BN concentration. The present results pave way for the development of band gap engineered BN doped graphene-based devices.

Alpha-helical domain is essential for antimicrobial activity of high mobility group nucleosomal binding domain 2 (HMGN2).

AIM: To examine the antimicrobial spectrum and functional structure of high mobility group nucleosomal binding domain 2 (HMGN2). METHODS: OMIGA protein structure software was used to analyze the two-dimensional structure of HMGN2. Synthetic short peptides were generated for studying the relationship between function and structure. Prokaryotic expression vectors were constructed for the holo-HMGN2 and its helical domain. Their E coli-based products were also prepared for antimicrobial testing. The antimicrobial assay included minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration. RESULTS: OMIGA protein structure software analysis revealed a transmembrane alpha-helical structure (the putative antimicrobial domain) located from position 18 to 48 of the HMGN2 protein sequence. The antimicrobial assay showed that the MIC of the recombinant holo-HMGN2 against E coli ML-35p (an ampicillin-resistance strain), Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231 were 12.5, 25, and 100 mg/L, respectively. Against the same microorganisms, the MIC of the synthetic HMGN2 alpha-helical domain were 12.5, 25, and 100 mg/L, respectively, that is, the same as with the recombinant form of HMGN2. In contrast, recombinant holo-HMGN2 was inactive against Staphylococcus aureus ATCC 25923. The synthetic N-terminal and C-terminal fragments of HMGN2 had no antimicrobial activity against E coli ML-35p, P aeruginosa ATCC 27853 or C albicans ATCC 10231. CONCLUSION: HMGN2 showed potent antimicrobial activity against E coli ML-35p, P aeruginosa ATCC 27853 and, to some extent, against C albicans ATCC 10231, but was inactive against S aureus ATCC 25923 in these assay systems. Itos alpha-helical structure may be essential for the antimicrobial activity of HMGN2.

PCR-based site-specific mutagenesis of peptide antibiotics FALL-39 and its biologic activities.

AIM: To construct PGEX-1lambdaT-FALL-39 expression vector and its mutant vector, and study the relationship of function and structure. METHODS: A cDNA encoding mature FALL-39 was cloned from SPCA-1 cell mRNA and the prokaryotic expression vector PGEX-1lambdaT-FALL-39 was constructed. Two kinds of polymerase chain reaction (PCR) for the site-direction mutagenesis were used to construct FALL-39 mutant expression vector, FALL-39-Lys-32 and FALL-39-Lys-24. Minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration were used to assay the antibacterial activities of these peptides. Effects of different solution on the antibacterial activity of FALL-39 and FALL-39-Lys-32 were observed by CFU determination. The hemolytic effects of these peptides were also examined on human red blood cells. RESULTS: Two site-specific mutants FALL-39-Lys-32 and FALL-39-Lys24 were obtained by PCR-induced mutagenesis. In comparison with two-step PCR which required two pairs of primers, one step PCR which required one pair of primers is a simple and efficient method for the PCR based site-specific mutagenesis. Using the prokaryotic expression system, the E coli-based products of recombinant FALL39 and its mutant peptides were also obtained. The antibacterial assay showed that FALL-39-Lys-32 and FALL-39-Lys24 were more potential in the antibacterial activity against E coli ML35p and Pseudomonas aeruginosa ATCC27853 than that of FALL-39, and no increase in hemolysis was observed at the antibacterial concentrations. The antibacterial activity of FALL-39-Lys-32 against E coli was more potent than that of FALL-39 in NaCl-containing LB medium, while its activity was almost the same as FALL-39 in SO4(2-) containing Medium E. CONCLUSION: PCR-based mutagenesis is a useful model system for studying the structure and function relationship of antimicrobial peptides. Keeping a-helical conformation of FALL-39 and increasing net positive charge can increase the antibacterial activity of FALL-39 without increasing hemolysis at the antibacterial concentrations.

Mycobacterium bovis bacille Calmette-Guérin (BCG) enhances human beta-defensin-1 gene transcription in human pulmonary gland epithelial cells.

AIM: To examine the stimulatory effect of bacille Calmette-Gu rin (BCG) cell wall components on human beta-defensin-1 (hBD-1) gene expression and analyze the response element in the 5'-flanking region of the gene. METHODS: BCG cell wall proteins were fractionated by Sephadex G-150 chromatography. Using reverse-transcription polymerase chain reaction (RT-PCR) and Northern hybridization analysis, hBD-1 mRNA expression was detected in a human pulmonary gland epithelial cell line SPC-A-1 cells. Progressive deletions of 5'-flanking region of hBD-1 gene were produced by PCR and ligated into promoterless chloramphenicol acetyltransferase (CAT) expression plasmid to construct pCAT reporter plasmids. Reporter gene expression was determined by ELISA. RESULTS: There was an obvious enhancement of hBD-1 mRNA expression after stimulation with heat-inactivated BCG whole cells (50 mg/L), or the cell wall components with a molecular weight of 18-30 kDa (3 mg/L) for 8 h. The upstream sequence between -314 bp and +54 bp had the inducible activity by BCG, which contained CCAAT/enhancer binding protein-beta (C/EBP beta), activator protein-1 (AP-1), and CP2 cis element. CONCLUSION: BCG cell wall components (18-30 kDa) can stimulate hBD-1 mRNA expression in pulmonary gland epithelial cells. The sequence (-314/+54) containing C/EBP beta, AP-1, and CP2 binding sites in the upstream of hBD-1 is involved in this induction.