Analysis of response mechanism of a proton-pumping gate FET hydrogen gas sensor in air

Two different types of hydrogen response signals (DC and AC) of a proton-pumping gate FET with triple layer gate structure (Pd/proton conducting polymer/Pt) were obtained. The proton-pumping gate FET showed good selectivity against other gases (CH4, C2H6, NH3, and O2). For practical use, the hydrogen response characteristics of the proton-pumping gate FET were investigated in air (a gaseous mixture of oxygen and nitrogen). The proton-pumping gate FET showed different hydrogen response characteristics in nitrogen as well as in air, despite the lack of oxygen interference independently. To clarify the response mechanism of the proton-pumping gate FET, a hydrogen response measurement was performed, using a gas flow system and electrochemical impedance spectroscopy. Consequently, the difference in response between nitrogen and air was found to be due to the hydrogen dissociation reaction and the interference with the proton transfer caused by the adsorbed oxygen on the upper Pd gate electrode</p

Alteration of proliferation and apoptotic markers in normal and premalignant tissue associated with prostate cancer

BACKGROUND: Molecular markers identifying alterations in proliferation and apoptotic pathways could be particularly important in characterizing high-risk normal or pre-neoplastic tissue. We evaluated the following markers: Ki67, Minichromosome Maintenance Protein-2 (Mcm-2), activated caspase-3 (a-casp3) and Bcl-2 to determine if they showed differential expression across progressive degrees of intraepithelial neoplasia and cancer in the prostate. To identify field effects, we also evaluated whether high-risk expression patterns in normal tissue were more common in prostates containing cancer compared to those without cancer (supernormal), and in histologically normal glands adjacent to a cancer focus as opposed to equivalent glands that were more distant. METHODS: The aforementioned markers were studied in 13 radical prostatectomy (RP) and 6 cystoprostatectomy (CP) specimens. Tissue compartments representing normal, low grade prostatic intraepithelial neoplasia (LGPIN), high grade prostatic intraepithelial neoplasia (HGPIN), as well as different grades of cancer were mapped on H&E slides and adjacent sections were analyzed using immunohistochemistry. Normal glands within 1 mm distance of a tumor focus and glands beyond 5 mm were considered "near" and "far", respectively. Randomly selected nuclei and 40 × fields were scored by a single observer; basal and luminal epithelial layers were scored separately. RESULTS: Both Ki-67 and Mcm-2 showed an upward trend from normal tissue through HGPIN and cancer with a shift in proliferation from basal to luminal compartment. Activated caspase-3 showed a significant decrease in HGPIN and cancer compartments. Supernormal glands had significantly lower proliferation indices and higher a-casp3 expression compared to normal glands. "Near" normal glands had higher Mcm-2 indices compared to "far" glands; however, they also had higher a-casp3 expression. Bcl-2, which varied minimally in normal tissue, did not show any trend across compartments or evidence for field effects. CONCLUSION: These results demonstrate that proliferation and apoptosis are altered not only in preneoplastic lesions but also in apparently normal looking epithelium associated with cancer. Luminal cell expression of Mcm-2 appears to be particularly promising as a marker of high-risk normal epithelium. The role of apoptotic markers such as activated caspase-3 is more complex, and might depend on the proliferation status of the tissue in question

Occlusion of calcium in the ADP-sensitive phosphoenzyme of the adenosine triphosphatase of sarcoplasmic reticulum

In order to characterize the form of the phosphorylated Ca2+-ATPase of sarcoplasmic reticulum which occludes the calcium bound in the enzyme (Takisawa, H., and Makinose, M. (1981) Nature (Lond.) 290, 271-273), a kinetic method was developed allowing quantitation of the amount of ADP-sensitive and ADP-insensitive phosphoenzyme. The relationships between occluded Ca2+ in the enzyme and the two forms of phosphoenzyme were studied at various concentrations of CaCl2 and MgCl2. The amount of tightly bound Ca2+ in the phosphoenzyme increases concordantly with the increase in the amount of ADP-sensitive phosphoenzyme, suggesting that occlusion of Ca2+ occurs in the ADP-sensitive phosphoenzyme. These results suggest that 1 mol of ADP-sensitive phosphoenzyme occludes 2 mol of Ca2+. Ca2+ is released from the enzyme under conditions which favor the formation of the ADP-insensitive phosphoenzyme (e.g. 5 mM MgCl2 and 50 microM CaCl2). Ca2+ release correlates approximately with the formation of the ADP-insensitive phosphoenzyme. The simulated time course of Ca2+ release, based on the Ca2+-binding properties of the two forms of phosphoenzyme, shows a good fit with the Ca2+ release curves observed, indicating that the ADP-insensitive phosphoenzyme binds no Ca2+ under these conditions

Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk.

At the onset of S phase, chromosomal replication is initiated by the loading of DNA polymerase alpha onto replication origins. However, the molecular mechanisms for controlling the initiation are poorly understood. Using Xenopus egg extract, we report here the identification of a Xenopus homolog of Cdc45, a yeast protein essential for the initiation of replication, which is shown to be an essential molecule for the initiation of replication via the loading of DNA polymerase alpha onto chromatin. XCdc45, by physically interacting with the polymerase in the extract, became associated with chromatin only after nuclear formation. During S phase, XCdc45 co-localized with the polymerase in the nuclei, and the loading of the polymerase, which depended on endogenous XCdc45, was facilitated by exogenously added recombinant XCdc45. These findings, together with the apparent requirement of S-phase-cdk activity for the loading of XCdc45, suggest that XCdc45, under the control of S-phase cdk, plays a pivotal role in the loading of DNA polymerase alpha onto chromatin

Occluded bound calcium on the phosphorylated sarcoplasmic transport ATPase

The Ca2+ + Mg2+-activated ATPase of the sarcoplasmic reticulum is responsible for the active Ca2+ transport of this membrane system, the key feature of which is the formation of an energy-rich phosphorylated transport enzyme (EP) and its conversion. To understand the Ca2+-transport mechanism, it is essential to clarify the behaviour of this intermediate in relation to such ligands as ATP, ADP, Mg2+ and, particularly, Ca2+. Recent kinetic studies on the phosphate turnover of this system suggested a relatively slow rate of Ca2+ dissociation from the phosphorylated enzyme, which possibly indicated Ca2+ binding in some occluded form with the intermediate. Here we report direct measurements of the binding and release of Ca2+ during phosphorylation of the sarcoplasmic transport enzyme. The results indicate an occlusion of the Ca2+ binding, accompanying an initial configurational change of the enzyme induced by the energy-rich phosphoryl transfer

[16] Occluded Ca2+

This chapter focuses on the occluded Ca2+. Ion-transport enzymes undergo a sequence of reaction steps in which the respective ions and phosphates interact with the enzyme mutually. One enzyme state in the main reaction cycle has been defined as occluded with respect to the ion to be translocated, i.e., the ions bound to the enzyme in this state are slowly exchanged with free ions in the medium. In order to measure the occluded ions bound to the enzyme, free ions are removed from assays as rapidly as possible. Several procedures have been applied to studies of the sodium-potassium and the calcium pump. The centrifuge column procedure is the most suitable method to identify occluded Ca2+-binding site and the corresponding phosphoprotein intermediates. The procedure is rather simple and the sensitivity is high. For extended use of this method, the following precautions should be observed. First, when membranous vesicular preparations are used, the membranes should be completely permeable to the transported solute so that only the bound and not the trapped solute are measured. Second, the rate at which the intermediate decomposes should be relatively slow so that no significant decomposition takes place during the column centrifugation

The RLF-M component of the replication licensing system forms complexes containing all six MCM/P1 polypeptides.

Replication licensing factor (RLF) is involved in preventing re-replication of chromosomal DNA in a single cell cycle, and previously has been separated into two components termed RLF-M and RLF-B. Here we show that Xenopus RLF-M consists of all six members of the MCM/P1 protein family, XMcm2-XMcm7. The six MCM/P1 polypeptides co-eluted on glycerol gradients and gel filtration as complexes with a mol. wt of approximately 400 kDa. In crude Xenopus extract, all six MCM/P1 polypeptides co-precipitated with anti-XMcm3 antibody, although only XMcm5 quantitatively co-precipitated from purified RLF-M. Further fractionation separated RLF-M into two sub-components, one consisting of XMcms 3 and 5, the other consisting of XMcms 2, 4, 6 and 7. Neither of the sub-components provided RLF-M activity. Finally, we show that all six MCM/P1 proteins bind synchronously to chromatin before the onset of S-phase and are displaced as S-phase proceeds. These results strongly suggest that complexes containing all six MCM/P1 proteins are necessary for replication licensing