Factors controlling denitrification of mudflat sediments in Ariake Bay, Japan.

To investigate the seasonal variation of denitrification rate (DR) and clarify the controlling factors of denitrification in the mudflat sediments of Ariake Bay, we conducted field surveys biweekly each month from April 2006 to January 2008. NH4(+)-N porewater concentration increased from summer to autumn due to the organic material mineralization under higher sediment temperatures. The seasonal pattern of NH4 (+)-N flux between sediments and overlying water interface indicated that the mudflat sediments were a source of NH4(+)-N in summer. NO3(-)+NO2(-)-N porewater concentrations were low, ranging from 0.53 to 11.46 µM, and mudflat sediments were sinks of NO3(-)+NO2(-)-N throughout the year. The mean number of denitrifiers tended to increase in July-September (2188-75,057 MPN g(-1)) and to decrease in March-May (500-3740 MPN g(-1)). DR tended to increase in summer, ranging from 76.03 to 990.21 µmol m(-2) day(-1), and to decrease in winter, ranging from 25.01 to 206.07 µmol m(-2) day(-1). There was no significant correlation between DR and denitrifier number. Environmental factors influencing DR during the investigation period were determined by multiple regression analysis with the stepwise method. The results indicated that NO3(-)+NO2(-)-N flux was an important factor in denitrification of mudflat sediments in Ariake Bay. Denitrification was depended on nitrate diffusing from overlying water into sediments under reduced sediment conditions during summer-mid-autumn. On the other hand, in late autumn-winter at Eh>+200 mV and sediment temperature >10 °C, nitrate produced by sediment nitrification was thought to be denitrified subsequently; that is, the coupled nitrification-denitrification may have taken place in the upper layer of mudflat sediments.

Characterization of kidney marrow in zebrafish (Danio rerio) by using a new surgical technique.

Zebrafish kidney marrow (ZKM), which is equivalent to the haematopoietic bone marrow of mammals, produces all major blood cell types, which morphologically resemble their mammalian counterparts. To be able to exploit the advantages of zebrafish genetics for analysis of the general mechanisms controlling self-renewal, proliferation and lineage decisions of vertebrate haematopoetic cell populations, it is essential to develop a simple surgical technique in order to identify, dissect and take out the ZKM without contamination with other surrounding tissues and cells. However, the size of adult zebrafish is small (average size: 2.5 cm) and the ZKM is an extremely protected organ and not easy to localize, which makes this procedure a great microsurgical challenge. Here we report a new microsurgical technique to identify, localize and dissect ZKM in adult zebrafish using a new approach. The potential advantages of this technique are summarized here: it allows purity of the sample, which is critical for performing flow cytometry analysis and/or cell number count; it enables visualization of the ZKM without a parenchimal incision, which simplifies the further dissection; the learning curve is short, requiring only basic microsurgical skills, and it is reliable and highly reproducible. To further characterize the kidney marrow cells obtained by this technique, we performed histology, flow cytometry, cytospin experiments and cell counts.

Availability of lobe-selective bronchial blockade for video-assisted thoracic surgery: an initial experience with three cases.

BACKGROUND: Stable oxygenation and sufficient collapse of the lung are essential for video-assisted thoracic surgery (VATS). We performed the lobe-selective lung collapse technique with VATS for patients who had deteriorated lung function. METHODS AND RESULTS: CASE 1: A 75-year-old man who had undergone thoracoplasty showed spontaneous pneumothoraces in the contralateral side. Bullae were stapled successfully under complete isolation and collapse of diseased lobe. CASE 2: A 57-year-old woman who had undergone left lower lobectomy for lung cancer presented with another lung cancer in the right lower lobe. The right lower lobe bronchus was closed selectively, and basal segmentectomy was performed. CASE 3: A 60-year-old woman who had lost left lung function presented with right-side spontaneous pneumothoraces. The right upper lobe was collapsed selectively, and bullectomy was performed. CONCLUSION: Lobe-selective bronchial blockade may be beneficial when VATS is performed for selected patients with deteriorated lung function.

Effects of Ca2+ sensitizers on contraction, [Ca2+]i transient and myofilament Ca2+ sensitivity in diabetic rat myocardium: potential usefulness as inotropic agents.

The purpose of the present study was to investigate the effects of Ca2+ sensitizers EMD 57033, MCI-154, and EGIS-9377 in cardiac preparations from streptozotocin-induced diabetic rats. In enzymatically dissociated ventricular myocytes loaded with the Ca2+ probe indo 1, these Ca2+ sensitizers caused an increase in cell shortening without a significant effect on the intracellular Ca2+ ([Ca2+]i) transient. The contractile responses were substantially similar in myocytes from diabetic and age-matched control rats. In contrast, the contractile and [Ca2+]i responses to pimobendan and isoproterenol were significantly less in diabetic myocytes. The Ca2+ sensitivity of tension in beta-escin-skinned trabeculae from diabetic hearts was not significantly different from that of controls. The effect of EMD 57033 on myofilament responsiveness to Ca2+ was identical in control and diabetic preparations. The slower time course of relaxation observed in diabetic papillary muscles was further prolonged in the presence of EMD 57033. However, the extent of the increase in relaxation produced by EMD 57033 did not differ between control and diabetic muscles, and the detrimental effect on resting tension was less pronounced in the two groups. In anesthetized rats, echocardiography showed that intra-duodenal administration of EMD 57033 increased left ventricular systolic function without affecting variables of diastolic filling in both groups. Taken together, the present results suggest that Ca2+ sensitizers, unlike conventional inotropic agents, have the potential to increase in force of contraction to the same extent in nondiabetic and diabetic myocardium, possibly without exaggerating extremely the impairment of diastolic function in diabetes.

Beam characteristics for various sizes of annular aperture on scanning electron microscope.

Using an analogy between light optics and electron optics, we have calculated beam characteristics such as the beam profile and the optical transfer function for several sizes of annular and circular apertures on a scanning electron microscope (SEM). It has been found that an annular aperture improves the image quality with regard to several kinds of image resolution and the depth of focus at the price of good low-frequency (nu) contrast. In contrast with conventional circular-aperture SEM images, a combination of a low-nu-pass filtered, circular-aperture SEM image with a high-nu-pass filtered, annular-aperture SEM image has the potential to enhance the image quality in terms of both the image resolution and the depth of focus.

Tissue factor pathway inhibitor response does not correlate with tissue factor-induced disseminated intravascular coagulation and multiple organ dysfunction syndrome in trauma patients.

OBJECTIVE: To determine the precise relationship between tissue factor and tissue factor pathway inhibitor (TFPI) after trauma, as well as to test the hypothesis that low TFPI levels are not sufficient to prevent tissue factor-dependent intravascular coagulation, leading to multiple organ dysfunction syndrome (MODS). DESIGN: Prospective, observational cohort study. SETTING: Emergency room and intensive care unit in a university hospital. PATIENTS: Thirty-three trauma patients, 18 with disseminated intravascular coagulation (DIC) and 15 without DIC were studied. Ten normal, healthy volunteers served as control subjects. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Antigen concentration of tissue factor and TFPI, and global parameters of coagulation and fibrinolysis were measured on the day of admission, and on days 1-4 after admission. The number of systemic inflammatory response syndrome (SIRS) criteria that patients met and the DIC score were determined, simultaneously. The results of these measurements, incidence of MODS, and outcome were compared between the DIC patients and those without DIC. In the DIC patients, significantly higher tissue factor levels (p =.0049) and lower platelet counts (p =.0016) were found compared with the non-DIC patients and control subjects. However, the TFPI values remained at normal levels during the study period. No correlation was found between the peak levels of tissue factor and TFPI. The mean duration of SIRS and the maximum number of the SIRS criteria being met by the patients in the DIC group were statistically longer and higher than those in the non-DIC patients. The incidence of MODS and the number of the dysfunctioning organs were higher in the DIC patients compared with those in the non-DIC patients, and the DIC patients had a poor outcome. CONCLUSIONS: We systematically elucidated the relationship between tissue factor and TFPI in post-trauma patients. Highly activated tissue factor-dependent coagulation pathway is not sufficiently prevented by the normal TFPI levels in patients with DIC. The DIC associated with thrombotic and inflammatory responses causes MODS, and leads to poor outcome in post-trauma patients.

Diminished function and expression of the cardiac Na+-Ca2+ exchanger in diabetic rats: implication in Ca2+ overload.

1. The present work was carried out in order to determine whether a decrease in cardiac Na+-Ca2+ exchanger (NCX) activity observed in diabetes is caused by a reduction in NCX protein and mRNA levels and to elucidate the significance of this decrease in alterations in [Ca2+]i homeostasis in diabetic cardiomyocytes. 2. The NCX current was significantly reduced in ventricular myocytes freshly isolated from streptozotocin-induced diabetic rat hearts, and its current density was about 55 % of age-matched controls. 3. Diabetes resulted in a 30 % decrease in cardiac protein and mRNA levels of NCX1, a NCX isoform which is expressed at high levels in the heart. 4. The reduced NCX current and the decreased protein and mRNA levels of NCX1 in diabetes were prevented by insulin therapy. 5. Although both diastolic and peak systolic [Ca2+]i were not different between the two groups of myocytes, increasing external Ca2+ concentration to high levels greatly elevated diastolic [Ca2+]i in diabetic myocytes. Inhibition of NCX by reduction in extracellular Na+ by 50 % could produce a marked rise in diastolic [Ca2+]i in control myocytes in response to high Ca2+, as seen in diabetic myocytes. However, cyclopiazonic acid, an inhibitor of sarcoplasmic reticulum Ca2+ pump ATPase, did not modify the high Ca2+-induced changes in diastolic [Ca2+]i in either control or diabetic myocytes. 6. Only in papillary muscles from diabetic rats did the addition of high Ca2+ cause a marked rise in resting tension signifying a partial contracture that was possibly due to an increase in diastolic [Ca2+]i. 7. In conclusion, the diminished NCX function in diabetic myocytes shown in this study results in part from the decreased levels of cardiac NCX protein and mRNA. We suggest that this impaired NCX function may play an important role in alterations in Ca2+ handling when [Ca2+]i rises to pathological levels.

New concept of information resolution in scanning electron microscope images

As a new means to evaluate SEM images from the viewpoint of image information, we have proposed information resolution, Rinf, defined as a spatial length per bit in information-passing capacity (IPC). The IPC densities (per basic area) were carried out by both taking into account the signal-to-noise ratio of the statistical mean power densities and applying a previously reported fitting functions to an optical system with an arbitrary size of aberration, diffraction (including lambda = 0), and source. Here, the nu-independent specimen contrast of tau(s)(nu)= 1 [used in Eq. (9)] is assumed. General characteristics on Rinf are typically plotted as a curve of log (Rinf/R(inf, ideal)) vs. log T, where T is (Bm2 + Cm4)(1/2), Bm and Cm are the modified spherical and chromatic aberrations, respectively. The subscript "ideal" represents an aberration-free optical system. The values of Rinf [calculated by Eq. (11)] are compared to those of the conventional beam size d [calculated by Eq. (1)] and the previously reported modified resolution Res [calculated by Eq. (7)]. It was found that R(inf, min) value is 0.3-0.9 times as small as the d(min) value and its corresponding beam semi-angle alpha(inf, min) is 1.2-1.8 times as large as the alpha(conv, min) value (depending on S/N).

Cardiac profile of EGIS-9377, a novel cardiotonic agent as a Ca2+ sensitizer with bradycardiac activity.

The cardiac profile of EGIS-9377 ¿2-(1-methylthio)-5-(2-morpholinoethylamino)-8,9-dihydro-7H-thi opyrano[3,2-d][1,2,4]triazolo[1,5-a]pyrimidine dihydrochloride¿, a newly synthesized cardiotonic agent, was compared with those of pimobendan and isoprenaline in cardiac preparations isolated from guinea pigs. The positive inotropic potency and efficacy of EGIS-9377 were equal to those of pimobendan in electrically paced papillary muscles, with each agent maximally increasing force of contraction by 30-35% of the maximum effect of isoprenaline. The positive inotropic effects of EGIS-9377 and pimobendan were accompanied by an increase in the relaxation time of the isometric contraction curve, whereas that of isoprenaline was associated with an abbreviation of this parameter. Pimobendan significantly increased the spontaneously beating frequency of right atria, and its positive chronotropic effect amounted to 40% of the maximum effect of isoprenaline. In contrast, EGIS-9377 exerted a significant negative chronotropic action, which resulted in a 30% decrease in the basal frequency. In beta-escin-skinned trabecular muscles, both EGIS-9377 and pimobendan substantially enhanced contractions induced by Ca2+. EGIS-9377 at concentrations to cause a significant negative chronotropic action produced a marked prolongation of action potential duration (APD) in guinea pig papillary muscle and greatly inhibited the delayed rectifier potassium current (I(K)) in guinea pig ventricular single cells. This suggests that the negative chronotropic effect of EGIS-9377 may, at least in part, be due to the prolongation of APD as a result of the I(K) inhibition. The present results indicate that EGIS-9377 efficiently increases myocardial contractile force possibly due to its Ca2+ -sensitizing activity, and yet produces a substantial negative chronotropic action. This cardiac profile of EGIS-9377 is suggested to be a clinically favorable feature compared with the inotropic agents having cyclic AMP generation or phosphodiesterase inhibition as their action mechanisms.

Method for Cross-sectional Transmission Electron Microscopy Specimen Preparation of Composite Materials Using a Dedicated Focused Ion Beam System.

: A new method for transmission electron microscope (TEM) specimen preparation using a focused ion beam (FIB) system that results in a lower rate of gallium (Ga) implantation has been developed. The method was applied to structural and analytical studies of composite materials such as silicon (Si)-devices and magneto-optical disk. To protect the specimens against Ga ion irradiation, amorphous tungsten (W) was deposited on the surface of the specimen prior to FIB milling. The deposition was quite effective in reducing the Ga implantation rate, and energy-dispersive X-ray (EDX) analysis of these specimens detected 0.3-1.5% Ga incorporated in the thinned area. FIB milling times for these specimens were 1.5-2 hr, with a starting thickness of about 50 µm. Although the milling rate was high, all the materials were properly prepared for TEM study, and clear crystal lattice images were observed on all specimens.

The TAK1-NLK-MAPK-related pathway antagonizes signalling between beta-catenin and transcription factor TCF.

The Wnt signalling pathway regulates many developmental processes through a complex of beta-catenin and the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of high-mobility-group transcription factors. Wnt stabilizes cytosolic beta-catenin, which then binds to TCF and activates gene transcription. This signalling cascade is conserved in vertebrates, Drosophila and Caenorhabditis elegans. In C. elegans, the proteins MOM-4 and LIT-1 regulate Wnt signalling to polarize responding cells during embryogenesis. MOM-4 and LIT-1 are homologous to TAK1 (a kinase activated by transforming growth factor-beta) mitogen-activated protein-kinase-kinase kinase (MAP3K) and MAP kinase (MAPK)-related NEMO-like kinase (NLK), respectively, in mammalian cells. These results raise the possibility that TAK1 and NLK are also involved in Wnt signalling in mammalian cells. Here we show that TAK1 activation stimulates NLK activity and downregulates transcriptional activation mediated by beta-catenin and TCF. Injection of NLK suppresses the induction of axis duplication by microinjected beta-catenin in Xenopus embryos. NLK phosphorylates TCF/LEF factors and inhibits the interaction of the beta-catenin-TCF complex with DNA. Thus, the TAK1-NLK-MAPK-like pathway negatively regulates the Wnt signalling pathway.

MAP kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans.

The signalling protein Wnt regulates transcription factors containing high-mobility-group (HMG) domains to direct decisions on cell fate during animal development. In Caenorhabditis elegans, the HMG-domain-containing repressor POP-1 distinguishes the fates of anterior daughter cells from their posterior sisters throughout development, and Wnt signalling downregulates POP-1 activity in one posterior daughter cell called E. Here we show that the genes mom-4 and lit-1 are also required to downregulate POP-1, not only in E but also in other posterior daughter cells. Consistent with action in a common pathway, mom-4 and lit-1 exhibit similar mutant phenotypes and encode components of the mitogen-activated protein kinase (MAPK) pathway that are homologous to vertebrate transforming-growth-factor-beta-activated kinase (TAK1) and NEMO-like kinase (NLK), respectively. Furthermore, MOM-4 and TAK1 bind related proteins that promote their kinase activities. We conclude that a MAPK-related pathway cooperates with Wnt signal transduction to downregulate POP-1 activity. These functions are likely to be conserved in vertebrates, as TAK1 and NLK can downregulate HMG-domain-containing proteins related to POP-1.

Cross-sectional sample preparation by focused ion beam: a review of ion-sample interaction.

A focused ion beam (FIB) was applied for cross-sectional sample preparation with both transmission electron microscopes (TEM) and scanning electron microscopes (SEM). The FIB sample preparation has the advantage of high positioning accuracy for cross sections. On the other hand, a broad ion beam (BIB) has been conventionally used for thinning TEM samples. Although both FIB and BIB use energetic ion beams, they are essentially different from each other in many aspects such as beam size, beam current density, incident angle of the beam with respect to cross sections, and beam scanning (i.e., dynamic or static beam). In this study, FIB cross-sectioning is compared with BIB thinning. We review inherent characteristics such as positioning accuracy and uniformity of cross section, radiation damage, and beam heating. Discussion is held from a view-point of ion beam and sample interaction.

Focused-ion-beam digging of biological specimens.

The focused ion beam (FIB) technique has been applied for digging damageable biological specimens of human hair and the housefly eye. Sharp cross-sections have been formed for these specimens and their cross-sectional structures have been observed in scanning ion microscope (SIM) images. The applicability of FIB has been confirmed for these biological specimens.