Scanning Near-field Optical/Atomic Force Microscopy detection of fluorescence in situ hybridization signals beyond the optical limit

Fluorescence in situ hybridization (FISH) is widely used in molecular biological study. However, high-resolution analysis of fluorescent signals is theoretically limited by the 300-nm resolution optical limit of light microscopy. As an alternative to detection by light microscopy, we used Scanning Near-field Optical/Atomic Force Microscopy (SNOM/AFM), which can simultaneously obtain topographic and fluorescent images with nanometer-scale resolution. In this study, we demonstrated high-resolution SNOM/AFM imaging of barley chromosome (Hordeum vulgare, cv. Minorimugi) FISH signals using telomeric DNA probes. Besides detecting the granular structures on chromosomes in a topographic image, we clearly detected fluorescent signals in telomeric regions with low-magnification imaging. The high-resolution analysis suggested that one of the telomeric signals could be observed by expanded imaging as two fluorescent regions separated by approximately 250 nm. This result indicated that the fluorescent signals beyond the optical limit were detected with higher resolution scanning by SNOM/AFM.     

Occurrence of Yersinia enterocolitica in the Tokyo Tama Zoo

Yersinia enterocolitica serogroups O5A and O8 were isolated from fecal samples of one colony of Japanese macaques (Macaca fuscata) from the Tokyo Tama Zoo (Japan). Serogroup O5A was detected in brown bear (Ursus arctos) prior to the isolation from the macaques. Serogroup O5A organisms also were isolated from the Japanese macaques' breeding area. Serogroup O5A and O8 isolates were not pathogenic. Serogroup O8 isolates did not possess the O7 or O19 antigens.     

Morphology of the Hagfish Inner Ear

The inner ear of five species of hagfishes was examined with different light and electron microscopical techniques. In all species, the labyrinth contains a single macula and two cristae, in a single semicircular canal. The macula consists of a horizontal, a middle vertical and a posterior horizontal component. Each component is covered by numerous round statoconia. The ring-shaped cristae have very long kinocilia, but lack a proper cupula. The sensory epithelia show signs of regeneration, indicated by the presence of mitoses and apoptotic hair cells.     

Cross-linking of apolipoproteins is involved in a loss of the ligand activity of high density lipoprotein upon Cu(2+)-mediated oxidation.

A recent study demonstrated that Cu(2+)-mediated oxidation of high density lipoprotein (HDL) resulted in a loss of the capacity to reduce cholesterol from macrophage foam cells [(1991) Proc. Natl. Acad. Sci. USA 88, 6457-6461]. In the present study we characterized the physicochemical properties of oxidized HDL and correlated them with the ligand activity toward the HDL receptor. Among them, the cross-linking of apolipoproteins and an increase in lipid peroxides were characteristic and closely similar to those of tetranitromethane-treated HDL, an abortive ligand for the HDL receptor. Cellular experiments with murine peritoneal macrophages revealed that both the cellular binding activity of HDL and its capacity to enhance cholesterol efflux from macrophage foam cells were markedly reduced upon oxidation. These results suggest that cross-linking of HDL apolipoproteins is involved in the loss of the ligand activity of oxidized HDL.     

Cadmium toxicity and liver mitochondria. I. Different effects of cadmium administered in vivo to adult, young, and ethionine-fed rats

The changes in liver mitochondrial respiratory activities and cytochrome concentrations were investigated when cadmium chloride was administered orally to adult, young, and ethionine-fed rats. Following a seven-day administration of 30 ppm cadmium in drinking water, adult rats showed no change, while young rats and ethionine-fed rats exhibited a marked increase in mitochondrial respiration with concomitant decrease of respiratory control index and P/O ratio. The concentrations of cytochromes aa3, b, and c + c1 in liver mitochondria were unchanged in adult rats, but increased significantly in ethionine-fed rats. In young rats receiving cadmium the liver mitochondrial protein increased with a slight change in the cytochrome concentration in mitochondria. It was further found that in adult rats a higher concentration (300 ppm) of cadmium in drinking water was toxic to the liver mitochondrial functions. Thus, the effect of oral administration of cadmium on the liver mitochondrial function depends on the condition of the animals.     

Concomitant secretion of big endothelin and its C-terminal fragment from human and bovine endothelial cells

A specific radioimmunoassay (RIA) for the carboxyl-terminal fragment (CTF) of big porcine endothelin (pET), an intermediate form of pET, was established to characterize big ET-like and its CTF-like immunoreactivity (LI) secreted from cultured bovine and human endothelial cells (EC). The antibody used crossreacted equally with big pET(1-39) and its CTF(22-39), but not with pET(1-21). Serial dilution curves of the culture media from bovine and human EC were parallel to that of standard CTF. Reverse-phase HPLC coupled with RIAs for big ET and ET of the culture media from bovine and human EC revealed essentially the same elution profiles: two major CTF-LI components, one corresponding to big pET(1-39) and the other to its CTF(22-39), in addition to one major ET-LI component corresponding to pET(1-21). The amounts of CTF-LI were almost equal to that of ET-LI on a molar basis. These data suggest that big ET is processed by a putative ET converting enzyme to yield its CTF and the mature ET(1-21) in EC.     

Salusin-β accelerates inflammatory responses in vascular endothelial cells via NF-κB signaling in LDL receptor-deficient mice in vivo and HUVECs in vitro

The bioactive peptide salusin-β is highly expressed in human atheromas; additionally, infusion of antiserum against salusin-β suppresses the development of atherosclerosis in atherogenic mice. This study examined the roles of salusin-β in vascular inflammation during atherogenesis. Infusion of antiserum against salusin-β attenuated the induction of VCAM-1, monocyte chemoattractant protein (MCP)-1, and IL-1β and as well as nuclear translocation of NF-κB in aortic endothelial cells (ECs) of LDL receptor-deficient mice, which led to the prevention of monocyte adhesion to aortic ECs. In vitro experiments indicated that salusin-β directly enhances the expression levels of proinflammatory molecules, including VCAM-1, MCP-1, IL-1β, and NADPH oxidase 2, as well as THP-1 monocyte adhesion to cultured human umbilical vein ECs (HUVECs). Both salusin-β-induced VCAM-1 induction and monocyte/HUVEC adhesion were suppressed by pharmacological inhibitors of NF-κB, e.g., Bay 11-7682 and curcumin. Furthermore, the VCAM-1 induction was significantly prevented by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002, whereas it was accelerated by the ERK inhibitor, U-0126. Treatment of HUVECs with salusin-β, but not with salusin-α, accelerated oxidative stress and nuclear translocation of NF-κB as well as phosphorylation and degradation of IκB-α, an endogenous inhibitor of NF-κB. Thus, salusin-β enhanced monocyte adhesion to vascular ECs through NF-κB-mediated inflammatory responses in ECs, which can be modified by PI3K or ERK signals. These findings are suggestive of a novel role of salusin-β in atherogenesis.     

Prolonged effects of intracerebroventricular angiotensin II on drinking, eating and locomotor behavior in mice

The effects of centrally administered Angiotensin II (Ang II) on water and food intake in rodent models are well known. However, most studies have focused on the acute effects of intracranial Ang II. In the current study, we evaluated the effects of intracerebroventricular Ang II on food and water intake as well as locomotor activity over the entire dark phase of the murine diurnal cycle. Consistent with the previous reports, centrally administered Ang II rapidly stimulated water intake over the initial 1-hour period following treatment. However, this acute increase was immediately followed by a marked reduction in water intake resulting in decreased cumulative water intake approximately 7h after Ang II treatment. Pretreating animals with an Ang II type 1 receptor blocker, Losartan, completely antagonized the acute effect of Ang II and abolished initial water intake. In contrast, application of an Ang II type 2 receptor blocker, PD123319, abrogated the prolonged inhibitory effect of Ang II on drinking behavior and partially suppressed the initial increases in water intake. The suppressive effects of Ang II on cumulative food intake and spontaneous physical activity were also evident throughout the entire dark phase of diurnal cycle. These experiments are the first to suggest that the stimulatory effect of central Ang II treatment on water consumption is very temporary and that it causes a sustained suppressive effect on voluntary locomotion and food intake behavior in mice.