High-speed atomic force microscope based on an astigmatic detection system.

High-speed atomic force microscopy (HS-AFM) enables visualizing dynamic behaviors of biological molecules under physiological conditions at a temporal resolution of 1s or shorter. A small cantilever with a high resonance frequency is crucial in increasing the scan speed. However, detecting mechanical resonances of small cantilevers is technically challenging. In this study, we constructed an atomic force microscope using a digital versatile disc (DVD) pickup head to detect cantilever deflections. In addition, a flexure-guided scanner and a sinusoidal scan method were implemented. In this work, we imaged a grating sample in air by using a regular cantilever and a small cantilever with a resonance frequency of 5.5 MHz. Poor tracking was seen at the scan rate of 50 line/s when a cantilever for regular AFM imaging was used. Using a small cantilever at the scan rate of 100 line/s revealed no significant degradation in the topographic images. The results indicate that a smaller cantilever can achieve a higher scan rate and superior force sensitivity. This work shows the potential for using a DVD pickup head in future HS-AFM technology.

Operation of astigmatic-detection atomic force microscopy in liquid environments.

The astigmatic detection system (ADS) based on commercial optical pickup head was demonstrated to achieve a sub-nanometer sensitivity in detecting the vertical movement of an object surface in air. The detection laser spot of the ADS was sub-µm and the detection bandwidth was over 80 MHz. These advantages allow detection of high-frequency mechanical resonance of very small objects, which would have many important applications in nanotechnology. In this work, we optimized the operation conditions of ADS to achieve good sensitivity in aqueous solutions. We demonstrated good contrast and good spatial resolution of cancer cells in water with the optical profilometry mode. We also built an ADS-AFM (atomic force microscopy) for imaging in water. A novel cantilever holder was designed, and the spurious peaks were suppressed down to 26.0% of the real resonance peak. Most importantly, we demonstrated that the ADS-AFM could resolve single atomic steps on a graphite substrate and image soft DNA molecules on mica in water.

Rotational positioning system adapted to atomic force microscope for measuring anisotropic surface properties.

The diverse atomic configurations induce the anisotropic surface properties. For investigating anisotropic phenomena, we developed a rotational positioning system adapted to atomic force microscope (AFM). This rotational positioning system is applied to revolve the measured sample to defined angular direction, and it composed of an inertial rotational stepper and a visual angular measurement. The inertial rotational stepper with diameter 30 mm and height 7.6 mm can be easily attached to the AFM-system built in any general optical microscope. Based on a clearance less bearing and the inertial driving method, its bidirectional angular resolution reaches 0.005° per step. For realizing a close-loop controlled angular positioning function, the visual measurement method is utilized. Through the feedback control, the angular positioning error is less than 0.01°. For verifying the system performance, we used it to investigate the anisotropic surface properties of graphite. Through a modified cantilever tip, the atomic-scale stick-slip, and the anisotropic friction phenomena can be distinctly detected.

Nkx2.5 and Nkx2.6, homologs of Drosophila tinman, are required for development of the pharynx.

Nkx2.5 and Nkx2.6 are murine homologs of Drosophila tinman. Their genes are expressed in the ventral region of the pharynx at early stages of embryogenesis. However, no abnormalities in the pharynges of embryos with mutations in either Nkx2.5 or Nkx2.6 have been reported. To examine the function of Nkx2.5 and Nkx2.6 in the formation of the pharynx, we generated and analyzed Nkx2.5 and Nkx2.6 double-mutant mice. Interestingly, in the double-mutant embryos, the pharynx did not form properly. Pharyngeal endodermal cells were largely missing, and the mutant pharynx was markedly dilated. Moreover, we observed enhanced apoptosis and reduced proliferation in pharyngeal endodermal cells of the double-mutant embryos. These results demonstrated a critical role of the NK-2 homeobox genes in the differentiation, proliferation, and survival of pharyngeal endodermal cells. Furthermore, the development of the atrium was less advanced in the double-mutant embryos, indicating that these two genes are essential for both pharyngeal and cardiac development.

Cardiac-specific overexpression of cyclin-dependent kinase 2 increases smaller mononuclear cardiomyocytes.

Cyclin-dependent kinase 2 (cdk2) plays a critical role in the G1- to S-phase checkpoint of the cell cycle. Adult cardiomyocytes are believed to withdraw from the cell cycle. To determine whether forced overexpression of cdk2 results in altered cell-cycle regulation in the adult heart, we generated transgenic mice specifically overexpressing cdk2 in hearts. Transgenic hearts expressed high levels of both cdk2 mRNA and catalytically active cdk2 proteins. Cdk2 overexpression significantly increased the levels of cdk4 and cyclins A, D3, and E. There was an increase in both DNA synthesis and proliferating cell nuclear antigen levels in the adult transgenic hearts. The ratio of heart weight to body weight in cdk2 transgenic mice was significantly increased in neonatal day 2 but not in adults compared with that of wild-type mice. Analysis of dispersed individual adult cardiomyocytes showed a 5.6-fold increase in the proportion of smaller mononuclear cardiomyocytes in the transgenic mice. Echocardiography revealed that transgenic heart was functionally normal. However, adult transgenic ventricles expressed beta-myosin heavy chain and atrial natriuretic factor. Surgically induced pressure overload caused an exaggerated maladaptive hypertrophic response in transgenic mice but did not change the proportion of mononuclear cardiomyocytes. The data suggest that overexpression of cdk2 promotes smaller, less-differentiated mononuclear cardiomyocytes in adult hearts that respond in an exaggerated manner to pressure overload.

Expression of class A scavenger receptor inhibits apoptosis of macrophages triggered by oxidized low density lipoprotein and oxysterol.

The class A macrophage scavenger receptor (MSR-A) is a multifunctional trimeric glycoprotein involved in innate immune response as well as the development of lipid-laden foam cells during atherosclerosis. The MSR ligand, oxidized low density lipoprotein (oxLDL), is known to be cytotoxic to macrophages and other cell types. This study examined whether MSR mediates or modulates oxLDL-induced apoptosis. Treatment with oxLDL and its cytotoxic oxysterol, 7-ketocholesterol (7-KC), reduced viability and increased DNA fragmentation in human THP-1 cells, Chinese hamster ovary cells, and mouse peritoneal macrophages. However, cell death and DNA fragmentation were markedly diminished in the phorbol ester-differentiated MSR-expressing THP-1 cells and Chinese hamster ovary cells, with stable expression of MSR-AI after cDNA transfection when exposed to the same concentrations of oxLDL and 7-KC. Moreover, treatment with oxLDL and 7-KC induced much greater death and DNA fragmentation in MSR-A-deficient peritoneal macrophages compared with wild-type macrophages. Thus, MSR-A does not act as a receptor responsible for the apoptotic effect of oxLDL, and instead, expression of this receptor confers resistance of macrophages to the apoptotic stimulation by oxLDL and its cytotoxic lipid component. These results suggest that by preventing apoptosis, MSR-A may contribute to the long-term survival of macrophages and macrophage-derived lipid-laden foam cells in atherosclerotic lesions.

Transcriptional inhibition by interleukin-6 of the class A macrophage scavenger receptor in macrophages derived from human peripheral monocytes and the THP-1 monocytic cell line.

Expression of the class A macrophage scavenger receptor (MSR) contributes to the uptake of modified low density lipoproteins (LDL) by macrophages and transformation of these cells into lipid-laden foam cells, which characterize atherosclerosis. Many environmental factors, in particular, proinflammatory cytokines and growth factors, can exert regulatory effects on MSR expression, whereas intracellular accumulation of cholesterol itself does not influence MSR levels to any considerable extent. In the present study, by using an in vitro model, we examined whether stimulation with interleukin-6 (IL-6), an immunoregulatory, multipotential cytokine, modulates the expression and activities of the MSR in macrophages. When treated with IL-6, macrophages derived from peripheral monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 monocytic cells showed significantly reduced uptake and/or binding of the MSR ligand, acetylated LDL. This effect was paralleled by a reduction in the expression of MSR protein and mRNA. Analysis of MSR promoter activity in THP-1 cells transfected with an MSR promoter-reporter gene construct demonstrated decreased activity of the MSR promoter in IL-6-treated THP-1 macrophages. Electrophoretic mobility gel shift assay also showed a reduction in the binding of a transcription factor to the MSR promoter AP-1/ets elements in IL-6-treated cells. Thus, exposure to IL-6 may inhibit expression of the class A MSR in differentiated macrophages at transcriptional levels. This result suggests that this cytokine may modulate foam cell formation during atherogenesis.

De novo expression of the class-A macrophage scavenger receptor conferring resistance to apoptosis in differentiated human THP-1 monocytic cells.

The class-A macrophage scavenger receptor (MSR) is a trimeric multifunctional protein expressed selectively in differentiated monomyeloid phagocytes which mediates uptake of chemically modified lipoproteins and bacterial products. This study investigated whether MSR plays a role in the regulation of apoptosis, a model of genetically programmed cell death. De novo expression of MSR occurred in human THP-1 monocytic cells differentiated with phorbol esters, which activated a nuclear transcription factor binding to the Ap1/ets-like domain of the MSR promoter. The phorbol ester-stimulated THP-1 cells also expressed increased levels of the pro-apoptotic gene products, caspase-3 and Fas ligand, but the cells exhibited no change in apoptosis. Global activation of GTP-binding proteins with fluoride anions triggered apoptosis of THP-1 cells in a time- and concentration-dependent manner, demonstrated by nuclear shrinkage and fragmentation and internucleosomal DNA fragmentation. However, the MSR-expressing THP-1 macrophage-like cells showed a significant reduction in apoptosis compared to undifferentiated control THP-1 cells, which produce MSR at undetectable levels. Fluoride stimulation also triggered apoptosis of human Jurkat T cells. Stimulation with phorbol ester made no difference in apoptosis between treated and untreated Jurkat cells. Finally, Chinese hamster ovary (CHO) cells overexpressing the class-A MSR type I by cDNA transfection showed markedly increased resistance to G-protein-coupled apoptosis. Thus, de novo expression of MSR associated with monocyte maturation into macrophages appears to confer the resistance of macrophages to apoptotic stimulation by G-protein activation.

Novel elements located at -504 to -399 bp of the promoter region regulated the expression of the human macrophage scavenger receptor gene in murine macrophages.

The expressions of type I and type II macrophage scavenger receptors (MSRs) are highly specific in macrophages and related cell types. Although some reports have described the regulation of MSR gene expression and proposed some cis-elements related to cell-specific expression, the regulation of MSR remains largely unclear. This is due, in part, to an unacceptably low efficiency of transfection into monocyte/ macrophage cells. In the present study, we optimized the conditions of electroporation in murine macrophage (P388D1) cells. The efficiency of electroporation was increased 20-fold compared with previous methods. Using the optimized method, we focused on studying the regulation of the human MSR promoter in macrophages. We presently demonstrate that: a) the proximal -10 to +50 bp human MSR promoter region is necessary for the cell type-specific expression of human MSR; b) the 6.5 kbp upstream sequence suppresses the expression of human MSR; c) a promoter region extending from -504 to -399 bp produced the greatest increase in transcriptional activity; d) macrophage cell-specific transcription factors bind to the region as determined by electrophoretic mobility shift assay (EMSA) and a footprint assay; and e) mutations of the region reduced about 40-75% of the promoter activity in a transfecting assay. We concluded that novel elements located at the -504 to -399 bp region may play an important role in the regulation of the MSR gene expression in macrophages. We speculate that macrophage-specific factors binding to those elements may be responsible for the transcription regulation of the MSR gene in macrophages.

Multiple function of macrophage scavenger receptors mediated by fibrous coiled coil domains.

Type I and type II macrophage scavenger receptors (MSR) have six structurally distinct domains. MSR are known to mediate a wide range of ligand recognition, endocytosis, phagocytosis and macrophage adhesion. Expression of mutated receptors in various cultured cells and analysis using synthetic peptides indicate that two coiled-coil domains, alpha-helical coiled-coil domain (domain IV) and collagen-like domain (domain V) mediate these functions. Domain IV is essential for the trimerization of MSR and EDTA-resistant adhesion function. Domain V is essential for the wide range of ligand recognition. Cooperation of these two domains is also essential for the cellular function of MSR including pH-dependent ligand dissociation.

Histocompatibility antigens associated with Behçet's disease in northern Han Chinese.

Among Han nationality Chinese and living in the northern area of the Yellow River, 120 patients suffering from Behçet's disease and 100 unrelated healthy individuals were typed for histocompatibility antigens (HLA)-A, -B, -C, and -DR and -DQ antigens. HLA-DR and DQ typing was performed on B-lymphocyte separated with Lympho-B-Kwik. The HLA-antisera were provided by 11th IHWC. Bf alleles and C4 allotypes were determined by immunofixation agarose-gel electrophoresis. HLA-B51 was found in 67/120 (55.83%) patients and in 12/100 (12%) controls, the Chi-square and relative risk values were 45.54 and 9.27, respectively (p < 0.0005). C4AQ0 frequency was significantly increased in the patient group. In the complete form group HLA-B51 was observed more frequently (62.79%). No significant differences of other HLA antigens, frequencies, Bf or B4 alleles were found between the groups.