Influence of ions on two-dimensional and three-dimensional atomic force microscopy at fluorite-water interfaces.

Recent advancement in liquid-environment atomic force microscopy (AFM) has enabled us to visualize three-dimensional (3D) hydration structures as well as two-dimensional (2D) surface structures with subnanometer-scale resolution at solid-water interfaces. However, the influence of ions present in solution on the 2D- and 3D-AFM measurements has not been well understood. In this study, we perform atomic-scale 2D- and 3D-AFM measurements at fluorite-water interfaces in pure water and a supersaturated solution of fluorite. The images obtained in these two environments are compared to understand the influence of the ions in solution on these measurements. In the 2D images, we found clear difference in the nanoscale structures but no significant difference in the atomic-scale contrasts. However, the 3D force images show clear difference in the subnanometer-scale contrasts. The force contrasts measured in pure water largely agree with those expected from the molecular dynamics simulation and the solvent tip approximation model. In the supersaturated solution, an additional force peak is observed over the negatively charged fluorine ion site. This location suggests that the observed force peak may originate from cations adsorbed on the fluorite surface. These results demonstrate that the ions can significantly alter the subnanometer-scale force contrasts in the 3D-AFM images.

Fabrication of electron beam deposited tip for atomic-scale atomic force microscopy in liquid.

Recently, possibilities of improving operation speed and force sensitivity in atomic-scale atomic force microscopy (AFM) in liquid using a small cantilever with an electron beam deposited (EBD) tip have been intensively explored. However, the structure and properties of an EBD tip suitable for such an application have not been well-understood and hence its fabrication process has not been established. In this study, we perform atomic-scale AFM measurements with a small cantilever and clarify two major problems: contaminations from a cantilever and tip surface, and insufficient mechanical strength of an EBD tip having a high aspect ratio. To solve these problems, here we propose a fabrication process of an EBD tip, where we attach a 2 µm silica bead at the cantilever end and fabricate a 500-700 nm EBD tip on the bead. The bead height ensures sufficient cantilever-sample distance and enables to suppress long-range interaction between them even with a short EBD tip having high mechanical strength. After the tip fabrication, we coat the whole cantilever and tip surface with Si (30 nm) to prevent the generation of contamination. We perform atomic-scale AFM imaging and hydration force measurements at a mica-water interface using the fabricated tip and demonstrate its applicability to such an atomic-scale application. With a repeated use of the proposed process, we can reuse a small cantilever for atomic-scale measurements for several times. Therefore, the proposed method solves the two major problems and enables the practical use of a small cantilever in atomic-scale studies on various solid-liquid interfacial phenomena.

Molecular-scale surface structures of oligo(ethylene glycol)-terminated self-assembled monolayers investigated by frequency modulation atomic force microscopy in aqueous solution.

The structure and protein resistance of oligo(ethylene glycol)-terminated self-assembled monolayers (OEG-SAMs) have been studied intensively using various techniques. However, their molecular-scale surface structures have not been well understood. In this study, we performed molecular-resolution imaging of OH-terminated SAMs (OH-SAMs) and hexa(ethylene glycol) SAMs (EG(6)OH-SAMs) formed on a Au(111) surface in an aqueous solution by frequency modulation atomic force microscopy (FM-AFM). The results show that most of the ethylene glycol (EG) chains in an EG(6)OH-SAM are closely packed and well-ordered to present a molecularly flat surface even in an aqueous solution. In addition, we found that EG(6)OH-SAMs have nanoscale defects, where molecules take a disordered arrangement with their molecular axes parallel to the substrate surface. We also found that the domain size (50-200 nm) of an EG(6)OH-SAM is much larger than that of OH-SAMs (10-40 nm). These findings should significantly advance molecular-scale understanding about the surface structure of OEG-SAMs.

Significant improvements in stability and reproducibility of atomic-scale atomic force microscopy in liquid.

Recent advancement of dynamic-mode atomic force microscopy (AFM) for liquid-environment applications enabled atomic-scale studies on various interfacial phenomena. However, instabilities and poor reproducibility of the measurements often prevent systematic studies. To solve this problem, we have investigated the effect of various tip treatment methods for atomic-scale imaging and force measurements in liquid. The tested methods include Si coating, Ar plasma, Ar sputtering and UV/O3 cleaning. We found that all the methods provide significant improvements in both the imaging and force measurements in spite of the tip transfer through the air. Among the methods, we found that the Si coating provides the best stability and reproducibility in the measurements. To understand the origin of the fouling resistance of the cleaned tip surface and the difference between the cleaning methods, we have investigated the tip surface properties by x-ray photoelectron spectroscopy and contact angle measurements. The results show that the contaminations adsorbed on the tip during the tip transfer through the air should desorb from the surface when it is immersed in aqueous solution due to the enhanced hydrophilicity by the tip treatments. The tip surface prepared by the Si coating is oxidized when it is immersed in aqueous solution. This creates local spots where stable hydration structures are formed. For the other methods, there is no active mechanism to create such local hydration sites. Thus, the hydration structure formed under the tip apex is not necessarily stable. These results reveal the desirable tip properties for atomic-scale AFM measurements in liquid, which should serve as a guideline for further improvements of the tip treatment methods.

Atomic-resolution imaging in liquid by frequency modulation atomic force microscopy using small cantilevers with megahertz-order resonance frequencies.

In this study, we have investigated the performance of liquid-environment FM-AFM with various cantilevers having different dimensions from theoretical and experimental aspects. The results show that reduction of the cantilever dimensions provides improvement in the minimum detectable force as long as the tip height is sufficiently long compared with the width of the cantilever. However, we also found two important issues to be overcome to achieve this theoretically expected performance. The stable photothermal excitation of a small cantilever requires much higher pointing stability of the exciting laser beam than that for a long cantilever. We present a way to satisfy this stringent requirement using a temperature controlled laser diode module and a polarization-maintaining optical fiber. Another issue is associated with the tip. While a small carbon tip formed by electron beam deposition (EBD) is desirable for small cantilevers, we found that an EBD tip is not suitable for atomic-scale applications due to the weak tip-sample interaction. Here we show that the tip-sample interaction can be greatly enhanced by coating the tip with Si. With these improvements, we demonstrate atomic-resolution imaging of mica in liquid using a small cantilever with a megahertz-order resonance frequency. In addition, we experimentally demonstrate the improvement in the minimum detectable force obtained by the small cantilever in measurements of oscillatory hydration forces.

Cardioprotective mechanism of ischemic preconditioning is impaired by postinfarct ventricular remodeling through angiotensin II type 1 receptor activation.

BACKGROUND: Activation of protein kinase C-linked receptors and subsequent opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel are crucial in preconditioning (PC). This study examined whether postinfarct ventricular remodeling interferes with the PC mechanism. METHODS AND RESULTS: Two weeks before isolation of hearts, rabbits underwent a sham operation or coronary ligation (COL) to induce remodeling. Isolated buffer-perfused hearts were subjected to 30-minute global ischemia/2-hour reperfusion, and infarct size was expressed as a percentage of the left ventricle (%I/LV), from which the scarred infarct by COL was excluded. Although %I/LV was similar in sham-operated and remodeled hearts (52.9+/-6.5% versus 45.8+/-5.2%), PC with 2 episodes of 5-minute ischemia protected sham-operated but not remodeled hearts (%I/LV=18.1+/-2.5% versus 54.8+/-2.9%, P<0.05). Infusion of valsartan (10 mg x kg(-1). d(-1), an angiotensin II type 1 (AT(1)) receptor blocker, for 2 weeks after COL prevented the ventricular remodeling and preserved the response to PC (%I/LV=27.4+/-3.8%), although valsartan alone did not change %I/LV. Diazoxide, a mitoK(ATP) channel opener, protected both sham-operated and remodeled hearts (%I/LV=14.1+/-3.1% and 8.3+/-3.6%). CONCLUSIONS: The myocardium remodeled after infarction is refractory to PC, which is probably due to interruption of cellular signaling by PC upstream of mitoK(ATP) channels. An AT(1) receptor blocker is beneficial not only for suppression of ventricular remodeling but also for preservation of the PC mechanism.

Experimental regulation of granulomatous reactions around Schistosoma japonicum eggs implanted into the liver of mice.

Using a liver model, various granulomatous responses against Schistosoma japonicum eggs were studied in C57BL/6 mice immunized with tissue-extracted eggs prior to challenge implantation with freshly laid eggs. In mice receiving two ip injections of 20,000 eggs, there was little effect on early granuloma formation. Three weeks after implantation, however, tissue reaction accompanied by marked fibrosis was significantly augmented, compared to that in the untreated mice. In contrast, when mice were given four ip injections, the early reaction was accelerated and the subsequent fibrosis came to an end earlier than in the twice immunized or untreated mice. Different routes of injection produced differing effects on 2-week granulomas, with an augmented reaction following two sc injections and a diminished reaction following the same number of ip injections. Histologically, the diminished reaction was characterized by less cellularity, especially in the case of eosinophil infiltration.

Nucleic acid binding by zinc finger-like motif of human papillomavirus type 16 E7 oncoprotein.

Human papillomavirus (HPV) types 16 and 6b E7 proteins and their chimeric or mutant proteins were analyzed for oligonucleotide-binding activity by surface plasmon resonance-based biomolecular interaction analysis. The results indicated that type 16 E7 protein has stronger nucleic acid-binding activity than that of type 6b E7 protein. In addition, the results also indicated that the zinc finger-like motif in the C-terminal region of the type 16 E7 protein plays an important role in this activity.

V1-receptor mediated GSH efflux by vasopressin from rat hepatocytes.

Vasopression increases sinusoidal efflux of GSH in the perfused rat liver. The mechanism of this effect was studied in the perfused rat liver and in isolated rat hepatocytes. Vasopressin stimulated GSH efflux in both systems and a V1-receptor antagonist (OPC-21268) significantly inhibited the effect of vasopressin suggesting that vasopressin stimulates GSH efflux from rat hepatocytes via V1-receptor.

Different courses of granulomatous reactions around Schistosoma japonicum eggs in three strains of mice.

Tissue reaction around Schistosoma japonicum eggs was studied after implantation into 3 strains of mice. When freshly laid eggs were implanted into the livers of C57BL/6, CBA/J, and BALB/c mice, the tissue reaction, which could be divided into 3 stages, i.e., abscess formation, inflammatory stage, and fibrous stage, differed in persistence and reactivity according to the strain. This was true especially during the later 2 stages. In BALB/c mice, the inflammatory stage ended earlier. In CBA/J mice, it continued for the longest period and the subsequent fibrosis was the most marked. In C57BL/6 mice, the period of persistence of this inflammatory stage was intermediate, but these mice showed the highest degree of inflammatory reaction. When footpad reactions were tested, the delayed-type hypersensitivity reaction was generally comparable with the morphometric analysis. In presensitized mice implanted with lyophilized eggs, tissue reactivity was analogous to the fibrous stage of freshly laid egg-implanted mice. In addition, there was an inverse relationship between antibody level and granuloma size, particularly at later reactions. The analysis of experimental granuloma formation presented in this report contributes to the study of stage-specific regulation.

Inhibitory effect of circulating egg antigens on Schistosoma japonicum egg-induced granuloma formation.

Schistosoma japonicum produces an enormous quantity of eggs during infection. This study was conducted to examine the effect of egg-derived antigens on the development of granuloma formation around S. japonicum eggs in the livers of mice. When soluble egg antigen (SEA) (75 micrograms/mouse/day) was injected 3-4 times via a vein into mice implanted with laid eggs, the magnitude of tissue lesion was drastically inhibited when assessed at maximal occurrence (14 days after implantation of eggs), whereas adult worm antigen (AWA), rabbit hyperimmune serum against SEA, or bovine serum albumin (BSA) did not show any effect on either the cellularity or the magnitude. In contrast to intravenous injection, there was no effect from subcutaneous injections of SEA. When serum taken from heavily infected mice or rabbit was transferred, there was a considerable extent of inhibition. In addition, an immune complex fraction of infected rabbit serum was found to have a stronger inhibitory effect than the supernatant fraction. This study indicates that the amount of egg-derived circulating antigens has a crucial effect on the development of schistosome granuloma formation.

Is Enterobius gregorii Hugot, 1983 (Nematoda: Oxyuridae) a distinct species?

A series of 849 male pinworms collected from a 64-yr-old Japanese male was examined. They were classified by the spicule morphology into 87 Enterobius vermicularis (Linnaeus, 1758), 754 Enterobius gregorii Hugot, 1983, and 6 immature adults, whereas 2 worms lacked spicules. The worm length of E. vermicularis was significantly larger than E. gregorii. The shape and length of the distal tubular portion of the spicule were identical in these forms, whereas the basal portion was different. The immature adults just after the final molt or still within the cuticle of the fourth stage had only a distal tubular portion, indicating that the basal portion is added during subsequent development. Moreover, various transitional forms were observed in the spicule morphology in the worms with intermediate body size between E. vermicularis and E. gregorii, showing that the basal portion of the spicule of E. vermicularis develops after the completion of E. gregorii-type basal portion. It is suggested that E. gregorii is a young stage of E. vermicularis.

Beta(+)-thalassemia with hemochromatosis.

A 64-year-old man was admitted due to ascites. Laboratory data showed hemoglobin 6.7 g/dl, mean corpuscular volume 82 fl, and ferritin 2,360 ng/ml. Liver biopsy showed hemochromatosis. The diagnosis of beta-thalassemia was suggested by a decreased ratio of beta/alpha-globin synthesis in vitro (0.26). Cloning of the beta-globin gene showed A-to-G mutation in the first base of the ATA box. He was confirmed to be homozygous for this specific allele by beta-gene complex analysis and analysis of Southern blot hybridization of the alpha- and beta-globin genes. His two sons were confirmed to be heterozygous for this allele.

Rapid uptake and phosphorylation of D-mannose, and limited D-mannose 6-phosphate isomerization in the glycolytic pathway of bloodstream forms of Trypanosoma brucei gambiense.

Bloodstream forms of the parasitic protozoa Trypanosoma brucei gambiense derive all of needed energy through an unusual glycolysis. In an earlier study, we showed that D-mannose specifically inhibited the growth of bloodstream forms of T. b. gambiense. We investigated D-mannose transport into the T. b. gambiense bloodstream forms and its metabolism in the initial phase of the glycolytic pathway. D-Mannose was transported rapidly into the bloodstream forms of T. b. gambiense (K(m) = 378 microM), and D-glucose competitively inhibited D-mannose uptake. D-Mannose and D-glucose are transported into bloodstream trypanosomes by the same carrier. Hexokinase from the bloodstream trypanosomes could convert D-mannose to D-mannose 6-phosphate (K(m) = 155.8 microM; Vmax = 0.93 mumol/min/mg protein); with kinetic properties very similar to D-glucose phosphorylation (K(m) = 199.4 microM; Vmax = 1.15 mumol/min/mg protein). D-Mannose 6-phosphate could be further metabolized in the glycolytic pathway. However, the metabolic rate was extremely slow, and D-mannose 6-phosphate accumulated in the glycosomes. D-Mannose may cause growth inhibition of bloodstream trypanosomes through an extremely high concentration of D-mannose 6-phosphate in the glycosomes.

[Experimental infection of Naegleria fowleri in mice].

Ten SPF mice (ddY, 4w-old, female) were infected by nasal instillation with an isolate of Naegleria fowleri that was first isolated from a patient with primary amoebic meningoencephalitis (PAM) in Japan. Of these mice, 2 showed clinical signs typical for PAM on the 4th day. On the next day, 5 mice became very ill and remained immobile; their movement and response to painful stimuli diminished progressively. All the infected mice were then examined histopathologically on the same day regardless of their clinical signs. Pathological changes due to invasion and/or proliferation of amoebae were observed in 5 mice with clinical signs. Swelling of the nasal mucosa and ulcerated nasal epithelium with inflammatory cells were observed. Proliferation of amoebae was detected to a lesser extent in nasal cavity including mucous membrane and nasal epithelium. Olfactory lobes and arteriolar hemisphere were necrotic with haemorrhage and filled with amoebae. From these findings the pathogenicity of the isolate was confirmed to develop PAM in experimental animals. It was also observed that the olfactory neuroepithelium was the route of invasion in PAM due to N. fowleri and consequently migration occurred through olfactory lobes into the cerebrum.

The application of molecular biological tools to epidemiology of African trypanosomosis.

Difficulties have often been encountered in the field surveys due to a lack of definitive morphological characters, particularly where mixed infections are expected. To address this problem, some molecular biological techniques such as DNA probe hybridization, restriction fragment length polymorphism (RFLP) analysis, the polymerase chain reaction (PCR), analyses of ribosomal DNA, and pulsed-field gel electrophoresis (PFGE), have been applied to the analysis of field samples collected during epidemiological surveys of African trypanosomosis. Concurrent natural infection of different individual tsetse flies and mammalian hosts with different species of the trypanosomes have been demonstrated, through the use of a combination of specific DNA probe hybridization and the PCR. Molecular karyotypes of Trypanosoma brucei species were analyzed by PFGE in 45 - 2,000 kb range. There are distinctive differences in intermediate and mini-chromosomes among the strains. We have compared the nucleotide sequences of ribosomal DNAs of the parasites by PCR techniques. From this data new phylogenetic tree can be inferred. It is apparent that these technologies can provide powerful tools for identification and diagnosis of trypanosomes in their hosts and vectors, and for their more accurate phylogenetic classification.

Expression of TGF-beta-like molecules in the life cycle of Schistosoma japonicum.

The transforming growth factor beta (TGF-beta) family controls an extremely wide range of biological activities, such as the growth and differentiation of cells, and immunological events against infectious agents. Although TGF-beta homologs appear to be widely present in metazoan animals, studies of parasite-derived molecules are relatively few. Using antibodies against anti-mouse TGF-beta1, -beta2, and -beta3, we show the expression of TGF-beta-like molecules in Schistosoma japonicum cercariae, schistosomula, eggs and adult worms. Intense immunoreactivity was found on the surface of free-living cercarial bodies. In transverse sections of cercariae, the molecules were localized in the tegument and subtegumental cells, and the number and distribution of producing cells significantly differed with each antibody. In the skin-migrating stage, the expression in the tegumental surface gradually decreased and became almost negative within 48 h of exposure. In adult worms and eggs, the reactivity was found in subtegumental cells and in cells of a tubular structure, respectively. In western blot analysis, the detection of conventional TGF-beta molecules failed. The expression of TGF-beta-like molecules was distinctly regulated at each developmental stage.

Characteristics of eosinophilic inclusions within Schistosoma japonicum eggs.

Although eosinophilic bar- or droplet-like inclusions are frequently detectable inside eggs deposited in the livers of Schistosoma japonicum-infected animals, little is known of their exact nature. In the livers of mice implanted with freshly laid eggs, inclusion-positive eggs were found in 28.7 and 46.2% of deposited eggs at 2 and 4 weeks, respectively, after implantation, but in 4.3% at 5 weeks when most of the eggs had already degenerated. When the extent of granuloma formation was investigated, granulomas around inclusion-positive eggs were smaller than those around negative eggs. Host factors associated with the formation of inclusion were sought using in vivo and in vitro studies. Following the administration of anti-egg antigen serum into egg-implanted mice, no increase in occurrence of inclusion-positive eggs was seen. In a co-culture of mature eggs with infected rabbit or mouse serum, inclusions were rarely found. In contrast, they were found in 17.9% of eggs in the presence of splenic cells. The present study is the first to show that there is decreased granuloma formation in the presence of eosinophilic inclusions inside eggs and our in vitro study suggests that host cell-egg interaction is responsible for the formation of inclusions.