Intracellular pH regulatory mechanism in human atrial myocardium: functional evidence for Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) symporter

Intracellular pH (pH(i)) exerts considerable influence on cardiac contractility and rhythm. Over the last few years, extensive progress has been made in understanding the system that controls pH(i) in animal cardiomyocytes. In addition to the housekeeping Na(+)-H(+) exchanger (NHE), the Na(+)-HCO(3)(-) symporter (NHS) has been demonstrated in animal cardiomyocytes as another acid extruder. However, whether the NHE and NHS functions exist in human atrial cardiomyocytes remains unclear. We therefore investigated the mechanism of pH(i) recovery from intracellular acidosis (induced by NH(4)Cl prepulse) using intracellular 2',7'-bis(2-carboxethyl)-5(6)-carboxy-fluorescein fluorescence in human atrial myocardium. In HEPES (nominally HCO(3)(-)-free) Tyrode solution, pH(i) recovery from induced intracellular acidosis could be blocked completely by 30 microM 3-methylsulfonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE 694), a specific NHE inhibitor, or by removing extracellular Na(+). In 3% CO(2)-HCO(3)(-) Tyrode solution, HOE 694 only slowed the pH(i) recovery, while addition of HOE 694 together with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (an NHS inhibitor) or removal of extracellular Na(+) inhibited the acid extrusion entirely. Therefore, in the present study, we provided evidence that two acid extruders involved in acid extrusion in human atrial myocytes, one which is HCO(3)(-) independent and one which is HCO(3)(-) dependent, are mostly likely NHE and NHS, respectively. When we checked the percentage of contribution of these two carriers to pH(i) recovery following induced acidosis, we found that the activity of NHE increased steeply in the acid direction, while that of NHS did not change. Our present data indicate for the first time that two acid extruders, NHE and NHS, exist functionally and pH(i) dependently in human atrial cardiomyocytes.     

内源荧光法在鉴别细菌表征中的应用

实现对细菌种属快速而高选择性的鉴别和表征在生化检测、临床医学和公共卫生领域变得日趋重要.内源荧光法以其灵敏度高、在线检测时间短、样品前处理简单等优势为微生物鉴别和表征提供了新方法.本文介绍了内源荧光法用于鉴别细菌表征的方法和原理;详细综述了近年来该方法用于食品分析、临床检验、环境监测和防生物恐怖等领域的细菌鉴别、代谢及追踪细菌源的现状和研究进展,并对其应用前景作了展望.     

Can we measure terrestrial photosynthesis from space directly,using spectral reflectance and fluorescence?

Attempts to estimate photosynthetic rate or gross primary productivity from remotely sensed absorbed solar radiation depend on knowledge of the light use efficiency (LUE). Early models assumed LUE to be constant, but now most researchers try to adjust it for variations in temperature and moisture stress. However, more exact methods are now required. Hyperspectral remote sensing offers the possibility of sensing the changes in the xanthophyll cycle, which is closely coupled to photosynthesis. Several studies have shown that an index (the photochemical reflectance index) based on the reflectance at 531 nm is strongly correlated with the LUE over hours, days and months. A second hyperspectral approach relies on the remote detection of fluorescence, which is a directly related to the efficiency of photosynthesis. We discuss the state of the art of the two approaches. Both have been demonstrated to be effective, but we specify seven conditions required before the methods can become operational.     

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3’ ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.     

Cooperativity in the calcium ion-induced quenching of the intrinsic fluorescence of a series of normal and GLA-deficient bovine prothrombin fragment 1 molecules

Ca²⁺ titrations of the intrinsic fluorescence of a series of -carboxyglutamic acid (GLA)-deficient bovine prothrombin fragments 1 yield response Hill plot parameters useful for characterization of the metal ion-binding process. 11-, 10-, and 9-GLA fragments 1 exhibitT _m (the (Ca²⁺)_total concentration at which ln (B/F)=0 in the response Hill plot) values between 0.2 and 0.3 mM. A 22-fold increase inT _m to 5.4 mM is observed for 8-GLA fragment 1.T _m decreases to 3.8 mM for the 7- and 6-GLA proteins. The value ofh, about 2.8±0.2 for 11-, 10-, and 9-GLA fragments 1, abruptly decreases to 1.2–1.3 for 8-, 7-, and 6-GLA fragments 1. The observed degree of quenching induced by saturating levels of calcium ions is affected by both changes in the intrinsic fluorescence of the metal ion-free proteins and in the maximum possible degree of quenching in the presence of calcium. The kinetic characteristics of the calcium ion-induced quenching of the intrinsic fluorescence of 6-GLA fragment 1 are identical to those observed in 10-GLA fragment 1, suggesting that the fluorescence quenching observed in the 6- and 10-GLA fragments 1, while different in magnitude, involves similar processes. Observation of an abrupt change in the relative electrophoretic mobilities of 11- to 9-GLA fragments 1 compared to 8- to 6-GLA fragments 1, in the absence or presence of Ca²⁺, suggests the existence of a major protein conformation change which occurs concomitantly with the noted changes inT _m andh response Hill plot parameters. Molecular mechanics calculations suggest a structural hypothesis unifying these observations. Central to this model is the presumption of the existence of hydrogen bond-mediated interactions between metal ion-binding sites.     

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.     

鲜花的光学性质

本文采用反射光谱、荧光光谱、激发光谱研究了迎春花、紫红色芍药、大红月季和橙红月季的光生态。结果表明,不同的鲜花光生态有明显的差异,鲜花中的花青素和类胡萝卜素在紫外光、可见光的激发下发出荧光,使鲜花更加鲜艳靓丽。通过对鲜花光生态的讨论,揭示出阳光对花儿的影响与作用,为鲜花光生态膜的开发以及人工照明的应用提供一定的生态依据。     

Application of Fluorescence Microscopy to Measure Apoptosis in Jurkat T Cells After Treatment with a New Investigational Anticancer Agent (N.C.1213)

1. Apoptosis as the mechanism of cell death induced by a new cytotoxic and anticancer agent (N.C.1213) was investigated by morphological and biochemical criteria in human Jurkat T leukemia cells.2. The effect of N.C.1213 on the survival of Jurkat T, LV-50, H-9, and Molt-3 cells was measured. Jurkat T cells exhibited the highest response, with less than 10% of the cells remaining viable after exposure to 10 M N.C.1213 for a 24 hr period. All other cell cultures were also affected but to a lesser extent.3. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were indemnified in Jurkat T cells after exposure to N.C.1213 and melphalan. The results indicated that melphalan was more cytotoxic than N.C.1213 as shown by the dye exclusion test. However, N.C.1213 showed a greater apoptotic index than melphalan. The IC₅₀ of N.C.1213 in Jurkat T cells was determined to be 3.5 M 4. A DNA ladder (fragmentation of DNA into multimers of approximately 200 base pairs), which is one characteristic feature of apoptosis, was not detected when Jurkat T cells were exposed to N.C.1213. Hence it is probable that the key morphological events in apoptosis observed in the present experimental conditions precede the internucleosomalcleavage of DNA.     

The anammox case-a new experimental manifesto for microbiological eco-physiology

The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater. It is also a new microbial physiology that was previously believed to be impossible. The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach. That approach is the focus of this paper. The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology. It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems. The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation. We argue that this was impossible using molecular ecology or conventional ‘cultivation based techniques’ alone. We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers. This revised version was published online in August 2006 with corrections to the Cover Date.