Exploring rare cellular activity in more than one million cells by a transscale scope.

In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.

Deletion of JMJD2B in neurons leads to defective spine maturation, hyperactive behavior and memory deficits in mouse.

JMJD2B is a histone demethylase enzyme that regulates gene expression through demethylation of H3K9me3. Although mutations of JMJD2B have been suggested to be responsible for neurodevelopmental disorders, the function of JMJD2B in the central nervous system (CNS) remains to be elucidated. Here we show that JMJD2B has a critical role in the development of the CNS. We observed JMJD2B expression, which was especially strong in the hippocampus, throughout the CNS from embryonic periods through adulthood. We generated neuron-specific JMJD2B-deficient mice using the cre-loxP system. We found an increase in total spine number, but a decrease in mature spines, in the CA1 region of the hippocampus. JMJD2B-deficient mice exhibited hyperactive behavior, sustained hyperactivity in a novel environment, deficits in working memory and spontaneous epileptic-like seizures. Together these observations indicate that JMJD2B mutant mice display symptoms reminiscent of neurodevelopmental disorders. Our findings provide evidence for the involvement of histone demethylation in the formation of functional neural networks during development.

Exendin-4, glucagon-like peptide-1 receptor agonist, enhances isoflurane-induced preconditioning against myocardial infarction via caveolin-3 expression.

OBJECTIVE: To investigate the cardioprotective effects of isoflurane and exendin-4 against myocardial ischemia/reperfusion injury and the signaling pathways through which these effects are mediated. MATERIALS AND METHODS: For infarct size measurements, anesthetized mice were subjected to 30 min of coronary artery occlusion followed by 2 h of reperfusion. Wild-type or caveolin-3 knockout mice received isoflurane, exendin-4, or isoflurane with exendin-4 before ischemia index determination. Caveolin-3 expression in the heart was measured by immunoblotting. RESULTS: Myocardial infarct size was smaller in the isoflurane- [1.0 minimum alveolar concentration (MAC)] or exendin-4- (30 ng/kg i.v.) treated groups than the controls. Infarct size was not affected by isoflurane at 0.5 MAC or 3 ng/kg i.v. exendin-4, but the combination of these treatments reduced infarct size. Pharmacological preconditioning (isoflurane at 1.0 MAC, 30 ng/kg i.v. exendin-4, or isoflurane at 0.5 MAC with 3 ng/kg i.v. exendin-4) increased caveolin-3 protein expression in the heart after infarct induction. The cardioprotective effects of isoflurane, exendin-4, and isoflurane with exendin-4 were abolished in caveolin-3 knockout mice. CONCLUSIONS: The combination of isoflurane and exendin-4 reduced infarct size, but it was not more effective than either agent alone, and the cardioprotective effects of these agents are mediated by caveolin-3 expression.

Atomoxetine reverses locomotor hyperactivity, impaired novel object recognition, and prepulse inhibition impairment in mice lacking pituitary adenylate cyclase-activating polypeptide.

Attention-deficit/hyperactivity disorder (ADHD) is a complex neurobehavioral disorder that is characterized by attention difficulties, impulsivity, and hyperactivity. A non-stimulant drug, atomoxetine (ATX), which is a selective noradrenaline reuptake inhibitor, is widely used for ADHD because it exhibits fewer adverse effects compared to conventional psychostimulants. However, little is known about the therapeutic mechanisms of ATX. ATX treatment significantly alleviated hyperactivity of pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP(-/-)) mice with C57BL/6J and 129S6/SvEvTac hybrid background. ATX also improved impaired novel object recognition memory and prepulse inhibition in PACAP(-/-) mice with CD1 background. The ATX-induced increases in extracellular noradrenaline and dopamine levels were significantly higher in the prefrontal cortex of PACAP(-/-) mice compared to wild-type mice with C57BL/6J and 129S6/SvEvTac hybrid background. These results suggest that ATX treatment-induced increases in central monoamine metabolism may be involved in the rescue of ADHD-related abnormalities in PACAP(-/-) mice. Our current study suggests that PACAP(-/-) mice are an ideal rodent model with predictive validity for the study of ADHD etiology and drug development. Additionally, the potential effects of differences in genetic background of PACAP(-/-) mice on behaviors are discussed.

Migration ecology of juvenile temperate seabass Lateolabrax japonicus: a carbon stable-isotope approach.

The migration pattern of juvenile temperate seabass Lateolabrax japonicus in the stratified estuary of the Yura River was examined using carbon stable-isotope ratios (δ(13)C). δ(13)C values of mysids, which are the most important prey items for this species, were consistently enriched in the lower estuary and surf zone (LES), while depleted in the freshwater zone (FW). δ(13)C values of juveniles in LES were enriched, while those in FW were depleted, consistent with the δ(13)C difference in prey items. The results of δ(13)C showed that many juveniles migrated upstream from April to June and most of them stayed in FW until at least July. Juveniles that stayed in FW and LES for a relatively longer period (> c. 20 days) showed higher condition factors than those that stayed in FW for a short period (several days). This indicates that residence in FW enabled juveniles to achieve as good a body condition as residence in LES.

ACO1, a gene for aminocyclopropane-1-carboxylate oxidase: effects on internode elongation at the heading stage in rice.

Although reports on a gene for 1-amino-cyclopropane-1-carboxylate (ACC) oxidase (ACO1) in rice (Oryza sativa L.) suggest that high levels of its transcript are associated with internode elongation of deep-water rice during submergence, the role of ACO1 in rice development is largely unknown. The tissue-specificity of ACO1 expression indicated that its transcript significantly accumulated in lower parts of elongating internodes at the heading stage. Histochemical analysis and in situ hybridization showed that the ACO1 expression was localized in the basal parts of leaf sheaths immediately above nodes or the lower parts of elongating internodes. To further examine the role of ACO1, ACO1-deficient (aco1) and overexpressing (ACO1-OX) mutants were characterized. The total length of the elongated internodes of aco1 mutants was slightly shorter than that of wild-type plants and that of ACO1-OX mutants was longer. Interestingly, expression of the ACC synthase gene ACS1 and ethylene signalling gene OsEIN2 was up-regulated in the aco1 mutants. This study suggests that the ACO1 has a little effect on internode elongation at the heading stage, and that up-regulation of the ACS1 and OsEIN2 expression may attenuate inhibition of internode elongation.

Aerodynamic advantages of upside down take-off for aerial dispersal in Tetranychus spider mites.

Aerial dispersal may be important for redistribution of spider mites into new habitats. Evidence for behavioral control of aerial take-off has been well documented for Tetranychus urticae Koch. Before aerial dispersal they exhibit the aerial take-off posture that involves lifting the forelegs upright and raising the forebody. However, whether the aerial take-off posture functions to increase drag has remained unclear. The objectives of this study were to clarify: (i) aerodynamic effects of the aerial take-off posture; and (ii) actual aerial take-off behavior in T. urticae. To evaluate the aerodynamic forces experienced by grounded spider mites in different postures, we constructed three-dimensional models of T. urticae, exhibiting the aerial take-off posture and the normal posture, using computer graphics. We found that the aerial take-off posture was effective in receiving greater rearward forces from wind rather than upward forces. As a result, aerial take-off from a horizontal platform is unlikely. Instead, inverted departure surfaces, e.g., lower leaf surfaces, with inclines are likely to be effective sites for take-off. Laboratory experiments and field observations indicated that the mites preferentially adopted such a position for orientation and take-off. Our findings provided a rationale for the take-off behavior of Tetranychus spider mites.

Atypical, bidirectional regulation of cadmium-induced apoptosis via distinct signaling of unfolded protein response.

Cadmium is a widely distributed nephrotoxic metal that causes renal tubular injury. In this report, we investigated involvement of endoplasmic reticulum (ER) stress and individual unfolded protein responses in cadmium-initiated apoptosis of tubular epithelial cells. Cadmium chloride (CdCl(2)) induced expression of endogenous ER stress markers, GRP78, GRP94 and CHOP in vitro and in vivo, and subsequently caused cytological changes typical of apoptosis. Attenuation of ER stress by transfection with ER chaperone GRP78 or ORP150 suppressed CdCl(2)-triggered apoptosis. In response to CdCl(2), phosphorylation of RNA-dependent protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2alpha (eIF2alpha) was observed. Enhanced phosphorylation of eIF2alpha attenuated, whereas inhibition of eIF2alpha exacerbated CdCl(2)-induced apoptosis. Activating transcription factor 6 (ATF6) was also activated by CdCl(2) and blockade of this process suppressed induction of CHOP and thereby improved cell survival. CdCl(2) also triggered activation of the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1)-X-box-binding protein 1 (XBP1) pathway and inhibition of XBP1 attenuated apoptosis independent of GRP78 and CHOP. c-Jun N-terminal kinase (JNK), another molecule downstream of IRE1, was also phosphorylated by CdCl(2) and its inhibition attenuated apoptosis. These results evidenced bidirectional regulation of apoptosis in cadmium-exposed cells. The ATF6 and IRE1 pathways cooperatively caused apoptosis via induction of CHOP, activation of XBP1 and phosphorylation of JNK, and the PERK-eIF2alpha pathway counteracted the proapoptotic processes.

Screening and identification of substances that regulate nephrin gene expression using engineered reporter podocytes.

Downregulation of nephrin in podocytes leads to development of proteinuria in human and experimental kidney diseases. However, little is understood about pathophysiologic substances that regulate nephrin expression. In this report, we established conditionally immortalized reporter podocytes REPON for sensitive, continuous monitoring of nephrin gene expression. A murine podocyte cell line harboring a temperature-sensitive simian virus 40 large T antigen was stably transfected with a gene encoding secreted alkaline phosphatase (SEAP) under the control of the 5.4 or 8.3 kb nephrin gene promoter. The established reporter cells REPON5.4 and REPON8.3 were exposed to various pathophysiologic substances, and culture media were subjected to SEAP assay to identify regulators of nephrin gene expression. Among the bioactive substances tested, three physiological ligands of nuclear receptors including all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, and dexamethasone significantly activated the nephrin gene promoter in a dose-dependent manner. These effects were observed in both REPON5.4 and REPON8.3 and were associated with upregulation of nephrin mRNA. The effects of these substances were synergistic, and the maximum effect was observed by combination of three agents. In contrast, inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha as well as phorbol ester significantly downregulated the activity of the nephrin promoter as well as nephrin gene expression. These results elucidated the bidirectional regulation of nephrin by distinct pathophysiologic substances and may provide molecular bases for explaining how proteinuria is induced under pathologic situations and why some ligands for nuclear receptors have the anti-proteinuric potential.

Enhancement of NF-kappaB activity by resveratrol in cytokine-exposed mesangial cells.

Resveratrol, a natural polyphenolic phytoalexin, has been considered as a potential anti-inflammatory agent because of its suppressive effect on nuclear factor-kappaB (NF-kappaB). However, we recently found that treatment of glomerular mesangial cells with resveratrol significantly and dose-dependently enhanced NF-kappaB activation triggered by proinflammatory cytokines. This finding was evidenced by different reporter assays as well as by expression of an endogenous NF-kappaB-dependent gene, intercellular adhesion molecule-1. The NF-kappaB promoting effect of resveratrol was also observed in renal tubular LLCPK1 cells, but not in HepG2 hepatoma cells. In all cell types tested, treatment with resveratrol alone did not affect NF-kappaB activity. The enhanced activation of NF-kappaB by resveratrol progressed for at least 24 h and was accompanied by sustained down-regulation of an endogenous NF-kappaB inhibitor, IkappaBbeta, but not IkappaBalpha. Although expression of inducible nitric oxide synthase was suppressed by resveratrol, nitric oxide, a negative regulator of NF-kappaB, was not involved in the regulation of NF-kappaB by resveratrol. These data elucidated, for the first time, that resveratrol may enhance activation of NF-kappaB under certain circumstances.

Hepatic portal venous gas associated with nonocclusive mesenteric ischemia in a hemodialysis patient.

Hepatic portal venous gas (HPVG) has been rarely described in chronic hemodialysis patients. We report a case of HPVG in a 59-year-old female patient with hemodialysis-dependent chronic renal failure due to diabetes who presented with acute onset of abdominal pain. Abdominal CT demonstrated the presence of gas in the portal veins. However, on laparotomy, no evidence of bowel necrosis or perforation could be found. HPVG seemed to be caused by nonocclusive mesenteric ischemia (NOMI), an increasingly recognized complication in hemodialysis patients. The patient responded favorably to intravenous hyperalimentation and antibiotics.

[Implementation and evaluation of public relations activity in a community health program].

OBJECTIVE: This study evaluated public relations activity in a community health program in order to develop effective strategies to attract the public attention for the program. METHODS: An intervention study was conducted on public relations for "Nutrition Seminar for Citizens," sponsored by the Health Department of Machida City, Tokyo, in October, 1999. One ward in the city was selected as an intervention area, and another ward which had similar demographic and geographic characteristics was chosen as a control area. Two target populations were defined; one was women in their 20s to 60s (#1) and the other was those who had previously never utilized community health programs sponsored by the city (#2). Handbills were used as the medium for public relations for the seminar. These announced the time, place and content of the nutrition seminar and were designed by authors with special attention to catchphrases, colors and fonts. Handbills were distributed in the intervention area through elementary schools, local voluntary organizations and local health volunteers. In addition, the authors directly handed them out to people in front of two supermarkets in the town. The sources of seminar information were requested from the participants of the Nutrition Seminar with a self-administered questionnaire. RESULTS: 1. The percentage of participants who received the seminar information from handbills was higher than that of those who used monthly newsletters from the city as a source of information. 2. The percentage of participants from the intervention area was higher than that from the control area. 3. Regarding target populations #1 and #2, there were no differences in participation rates between the intervention and control area. 4. Among the four distribution routes, the local voluntary organization route was the most effective for attracting participants. CONCLUSIONS: The results show that handbills can be an effective medium for pubic relations to increase the number of participants in community health programs. Also, local voluntary organizations can play a crucial role in information transfer in the community because of their high credibility. On the other hand, we could not reach specified target populations with the medium. Further surveys are needed to establish optimal media and routes to reach appropriate target populations in health programs.

Myocardial connections between left atrial myocardium and coronary sinus musculature in man.

INTRODUCTION: Anatomic studies have shown that muscle morphologically identical to that of the atrial myocardium consistently surrounds the coronary sinus (CS). The CS musculature is connected to the left atrial (LA) myocardium in a variable fashion, with fewer connections in its distal portion. The aim of this study was to document the presence of connections between the LA myocardium and the CS musculature, using pacing maneuvers in man, and to study their potential association with natural atrial arrhythmia occurrence. METHODS AND RESULTS: Thirty patients (19 men; mean age 50.5 years) underwent electrophysiologic study, during which a decapolar catheter with 2-mm interelectrode spacing every 10 mm was inserted into the CS, with the proximal electrode pair positioned at the ostium. Associated atrial arrhythmias were paroxysmal atrial fibrillation in 5, typical atrial flutter in 13, LA flutter in 1, and other in 11. Baseline S1 and a single extrastimulus were delivered during distal and proximal CS pacing, while recordings were obtained from the four remaining bipoles. During distal CS pacing, double potentials with increasing interpotential interval from proximal to distal CS as a function of extrastimulus prematurity were detected in nine patients, suggesting block in a discrete local pathway distally connecting the CS to the LA and leading to reversion of low LA activation. Local delay in this pathway without complete CS-LA block resulting in LA activation fusion was observed in eight patients. A single nonfractionated potential at the distal CS, even at the shortest attainable S1-S2 coupling interval, which was interpreted as no block within distal CS-LA connection(s), was observed in the other 13 patients. History of atrial fibrillation or atypical atrial flutter was found in 8 of 9 patients with block at the distal CS-LA connection but in only 3 of 13 patients with no CS-LA connection block (P = 0.004). CONCLUSION: The ability to dissociate the LA from the distal CS suggests the presence of discrete connections between these structures in man. This observation appears to be associated with the clinical occurrence of atrial arrhythmias.

Comparison of effectiveness of an 8-mm versus a 4-mm tip electrode catheter for radiofrequency ablation of typical atrial flutter.

An 8-mm catheter does not appear superior to 4-mm tip electrode for atrial flutter ablation. The potential advantage of allowing higher energy delivery on a larger surface is compensated by the lack of consistent contact with the endocardial surface.

Shortening of conduction time over arborized atrioventricular accessory pathway with Mahaim fibers physiology just before interruption during radiofrequency ablation.

A 21-year-old woman had paroxysmal wide QRS tachycardia with a left bundle branch block configuration and a retrograde conducted P wave just behind the QRS complex. An electrophysiological study revealed antidromic atrioventricular tachycardia involving an atrioventricular connection with decremental conduction as the anterograde limb and normal atrioventricular node as the retrograde limb. During constant pacing from the high right atrium (HRA) at the cycle length (CL) of 600 ms, the QRS configurations were not identical to those during the wide QRS tachycardia or constant pacing at the CL of less than 500 ms. The process by which this arborized atrioventricular accessory pathway with the Mahaim fibers physiology was interrupted by radiofrequency catheter ablation is described. Radiofrequency energy was delivered to the site recording a Mahaim potential at the tricuspid annulus during constant pacing from the HRA at the CL of 429 ms. The stimulus-QRS interval gradually shortened as it reached the power plateau without changing the preexcited QRS configuration. Shortening of the conduction time over the Mahiam pathway might have resulted in changing of the propagation from a slow to fast conduction zone or acceleration in response to thermal effect in a node-like structure on the atrial insertion site.

Electrophysiological determinants of persistent dual atrioventricular nodal pathway physiology after slow pathway ablation in atrioventricular nodal reentrant tachycardia.

UNLABELLED: The purpose of this study was to examine the electrophysiological determinants of the elimination of recurrent atrioventricular nodal reentrant tachycardia (AVNRT) despite the persistence of dual AV nodal pathway physiology or single echo beats after ablation procedures. The study included 26 patients with common AVNRT who had undergone successful ablation treatment and no long-term recurrence of AVNRT. The slow pathway potential was targeted, and the endpoint of ablation was one echo during atrial extrastimulus testing (ET) with isoproterenol. Persistent dual pathways physiology or single echoes were present in 12 patients (group I) and absent in 16 (group II) after ablation. The number of anterograde AV nodal pathways and maximum AH interval (Max AH) during ET were measured before and after ablation, and ventriculoatrial conduction during ventricular pacing was examined. RESULTS: (1) multiple AV nodal pathways were more frequently observed in group I than in group II (50.0% vs 7%, P < 0.05); (2) Max AH decreased significantly after ablation in both groups (309 +/- 157 vs 171 +/- 53 ms in group II; P < 0.01, and 409 +/- 65 vs 274 +/- 86 ms in group I; P < 0.001); and (3) retrograde dual pathway conduction was more common in group I than in group II. These data suggest the presence of nonuniform conductive properties of the AV node in group I and that ablation targeting the slow pathway potential prevents recurrences of AVNRT by eliminating the pathway with the longest conduction time.

[A case of metastatic liposarcoma originating in the retroperitoneum successfully treated with combination chemotherapy].

We reported a 36-year-old woman with metastatic liposarcoma originating in the retroperitoneum, which responded well to adjuvant chemotherapy. The primary tumor was removed by surgery. Two months later, the patient developed metastasis to the brain, and to the lung four months later. Metastatic liposarcomas to the brain generally are extremely rare. The patient was treated with combination chemotherapy using cyclophosphamide, vincristine, adriamycin, and dacarbazine (CYVADIC). After she was examined, the former two drugs were alternated with vindesine and ifosfamide, and another regimen with cisplatin and etoposide was given after a three-week interval. As a result, both of the metastases totally disappeared. No recurrent lesion has been noted for two years. Although the role of chemotherapy for liposarcoma has not been well defined and little data support its use in an adjuvant setting, this combination chemotherapy seemed to be effective for advanced liposarcoma.

Electrophysiological characteristics during slow pathway ablation of posterior atrioventricular junctional reentrant tachycardia.

The purpose of this study was to compare the electrophysiological characteristics of posterior and anterior atrioventricular junctional reentrant tachycardia (AVJRT) during radiofrequency (RF) catheter ablation of a slow pathway. Twenty-four patients with common AVJRT, including 4 posterior (P) and 20 anterior AVJRT (A) were studied. We analyzed the retrograde atrial activation sequence of junctional rhythm and the presence of transient HA block during slow pathway ablation. When HA block developed, the AH interval before ablation and immediately after the end of energy delivery was measured. Successful ablation sites were divided into three groups; high (H), middle (M), and low (L) from the His bundle to the floor of the coronary sinus orifice. The results were: (1) the number of successful ablation sites were H 0, M 1, L 3 in P and H 1, M 8, L 11 in A; (2) the HA interval during AVJRT in P was longer than that in A (109 +/- 48 ms vs 43 +/- 6 ms, P < 0.01); (3) the retrograde atrial activation sequence during junctional rhythm was strictly concordant with that during AVJRT in both groups, but HA block developed during slow pathway ablation more often in P than in A (100% vs 30%, P < 0.01); and (4) The AH interval did not lengthen after HA block developed in P. These data suggest that another pathway does exist from the AV node to the atrium in addition to anterograde fast pathway and slow pathway, and that this pathway is used as the retrograde limb of P.