Molecular crowding and viscosity as determinants of translational diffusion of metabolites in subcellular organelles

The role of molecular crowding and viscosity on the apparent translational diffusion coefficient (ADC) of small metabolites was investigated in different subcellular organelles using the pulse-field gradient spin-echo 1H NMR technique. ADCs of metabolites with increasing radius of gyration (0.7 A < RG < 4.5 A) were measured in the cytoplasm of rat or chicken erythrocytes, in the nucleus of chicken erythrocytes, and in isolated rat liver mitochondria. Metabolite ADCs in these systems were compared with the corresponding ADCs determined in model solutions of increasing bulk viscosity but different molecular crowding. For solutions having the same viscosity, metabolite ADCs decreased with increasing concentration of cosolutes. This effect is adequately described by the modified Stokes-Einstein relationship, ADC = k/RG (1 + 2.5Phi), where k is a constant for a given temperature and Phi is an obstruction factor reporting the fractional volume of solution occupied by cosolutes, a measure of the molecular crowding in the solution. Cytoplasmic values of Phi for metabolites of different sizes did not depend exclusively on metabolite RG but on additional factors including the chemical nature of the metabolite, the presence of diffusional barriers, and metabolite-specific binding sites. In the case of water, nuclear Phi values approached those of the extracellular space while mitochondrial Phi values were significantly higher than those of the cytoplasm. Taken together, these results reveal important differences in molecular crowding within the different subcellular compartments, suggesting considerable diffusional heterogeneity for small metabolites within the different intracellular organelles.     

Distribution of nitrazepam and its metabolites in the subcellular fractions of several white rat organs

After intraperitoneal administration into rats at a dose of 100 mg per kg of body weight nitrazepame (mogadone, eunoktine) was enzymatically reduced with the subsequent acetylation. Derivatives of nitrazepame were found in cellular fragments and nuclei, in mitochondrial, microsomal and soluble fractions of liver, lungs, heart and brain tissues. Reduction of the substrate was shown to occur in soluble and microsomal fractions of liver tissue and acetylation--in mitochondria of lungs and liver tissue. Nitrazepame metabolites were quite uniformly distributed over cell organelles of heart and brain tissues; this suggests that they originate in the organs from other tissues, where the processes of reduction and acetylation take place. Nitrazepame and its derivatives penetrated into brain very effectively; this phenomenon is considered as an essential one for their pharmocological activity.     

Immunohistochemical distribution and subcellular localization of three distinct specific molecular structures of advanced glycation end products in human tissues

Using three mouse anti-human monoclonal antibodies for advanced glycation end products (AGEs), 6D12, 1F6, and 2A2, we examined the immunohistochemical distribution and localization of AGEs in various organs and tissues obtained from nondiabetic autopsy or biopsy cases (men and women, 41 to 86 years of age). 6D12 recognizes Nepsilon-(carboxymethyl)lysine (CML), a nonfluorescent and non-cross-linked AGE structure, and 1F6 recognizes fluorolink, a fluorescent and cross-linked AGE structure. The epitope of 2A2 is unknown but is different from that of CML and fluorolink or other known AGE structures such as pyrraline, pentosidine, and crosslines. Immunohistochemistry with these monoclonal antibodies revealed the intra- and extracellular accumulation of AGEs in these organs and tissues. By double immunohistochemical staining with two of the three monoclonal antibodies in different combinations, positive reaction products for all three monoclonal antibodies were demonstrated in macrophages widely distributed in various organs and tissues; endothelial cells of endocardium, arteries, veins, and blood capillaries; mesenchymal cells; epithelial or parenchymal cells; blood cells; and extracellular matrix. This result indicates that these three different AGE-specific molecules are formed intracellularly and extracellularly. In some cell types, however, one or two of these specific molecules were not always found together, suggesting that the molecular structures of AGEs and their formation are heterogeneous. Immunoelectron microscopy demonstrated the localization of AGE-labeled immunogold particles in the nuclei, nuclear envelope, mitochondria, endoplasmic reticula, Golgi complexes, endocytic vesicles, lysosomal vacuoles or granules, secretory granules, cytosol, and cell membranes, as well as in the extracellular matrix. In addition, the double histochemical staining method for ceroid/lipofuscin and immunohistochemistry for AGEs demonstrated intralysosomal formation and accumulation of AGEs in ceroid/lipofuscin pigments. These results suggest that the extracellularly produced AGEs are taken up by receptors into the cells and accumulate in secondary lysosomes and that AGEs are formed intranuclearly and/or intracellularly, probably via different metabolic pathways.     

Interference of bronchographic agents with lung surfactant

Dionosil Oily (a suspension of propyliodone crystals in peanut oil and powdered tantalum were introduced into the right principal bronchi of rabbit lungs. The left lungs were used as controls. Pressure-volume characteristics of excised lungs with Dionosil Oily or peanut oil demonstrated significantly reduced compliance on inflation at a pressure of 3-4 cm H2O. These lungs also retained less air on deflation and therefore demonstrated a significantly reduced stability index. Histological sections revealed microatelectasis closely associated with crystals and/or peanut oil. Lungs with tantalum powder were not measurably influenced by the bronchographic agents. Surface balance experiments with lung surfactant and synthetic dipalmitoyl phosphatidylcholine (DPPC) demonstrated an increased minimum surface tension due to the oil suspension of propyliodone, peanut oil and particles (propyliodone and tantalum). There is good evidence that the oil suspension of propyliodone reduced the surface activity of lung surfactant in situ. Particles also may prevent the minimum surface tension from reaching relatively low values if they enter the alveoli in sufficient quantities.     

The structures of pyridovericin and pyridomacrolidin, new metabolites from the entomopathogenic fungus, Beauveria bassiana

Lung cancer is the leading cause of cancer-related deaths. The development of sensitive screening methods to identify at-risk individuals before emergence of clinical disease would permit early intervention that could decrease this mortality. Our previous studies have shown that cells with trisomy 7 can be detected in bronchial epithelium from cancer-free smokers and former uranium miners. However, the use of more than one molecular marker could increase the chance of identifying at-risk individuals. Trisomy 20, which is found in 43-57% of non-small cell lung cancers, is a candidate marker. The purpose of the current investigation was to determine the percentage of cells with trisomy 20 in persons with a high risk for lung cancer. Bronchial epithelial cells that had been assayed for trisomy 7 were assayed for trisomy 20 by fluorescence in situ hybridization. Trisomy 20 was detected in bronchial epithelial cells from lung cancer patients and from smokers and ex-uranium miners without lung cancer. In some cases, patients who were negative for trisomy 7 exhibited trisomy 20. Consequently, more people with field cancerization were identified using both markers. However, the two markers combined did not appear to stratify the risk for lung cancer.     

Functional alterations of cardiac subcellular structures during energy deficiency in relation to the metabolic state of the heart muscle cell

The functional behaviour of membrane systems of the cardiac cell during oxygen deficiency was analyzed and the alterations were related to the metabolic state of the tissue as an index of injury. 1. The retention function of the cell membrane for proteins. With increasing energy deficiency the cardiac sarcolemma loses its ability to retain macromolecules (myoglobin, enzymes) within the cell. Close correlations exist between protein release and oxygen supply as well as ATP content of the tissue. 2. Function of isolated mitochondria after ischemia. In parallel with a strong impairment of oxidative phosphorylation (decrease of QO2, RCI values, phosphorylation rates) the Ca++-transporting activity of mitochondria is continuously depressed with decreasing myocardial ATP. 3. Function of isolated sarcoplasmic reticulum after ischemia. With breakdown of high energy phosphates during ischemia rate and extent of Ca++ binding with decrease markedly.     

Measuring group processes: A comparison of the GCQ and CCI.

This study verified and compared the factor structures of two frequently used measures of small group climate, the Group Climate Questionnaire (GCQ-S; MacKenzie, 1983) and the Curative Climate Instrument (CCI; Fuhriman, Drescher, Hanson, Henrie, & Rybicki, 1986) at both group and individual levels. Data included third session assessments of 124 group members in 20 university counseling center groups. Confirmatory factor analyses partially supported the GCQ factor structure, but indicated that the Catharsis subscale of the CCI was not independent of the other two CCI subscales. Factor analysis of the six subscales from both measures yielded two higher order factors, representing positive (Engagement, Cohesion, Insight, and Catharsis) and negative (Conflict and Avoidance) group processes. Findings provide guidance for interpreting and comparing group processes using these measures. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Interference of bcl-2 with intercellular control of carcinogenesis

Induction of apoptosis in transformed fibroblasts by surrounding normal cells has been discussed as a potent early control step in carcinogenesis. According to this hypothesis, tumor progression should require resistance of transformed cells against this TGF-beta-triggered control mechanism. Here we show that Bcl-2, a protein involved in inhibition of apoptosis, can protect transformed cells from induction of apoptosis by surrounding cells. Rather than acting on the transformation process itself, Bcl-2 may thus represent an efficient modulator of carcinogenesis at an intercellular level.     

Covalent interactions of reactive naphthalene metabolites with proteins

Naphthalene produces selective necrosis of Clara cells in the mouse but not in the rat. The pulmonary toxicity depends on cytochrome P450-mediated metabolism; however, the selective pulmonary toxicity of naphthalene in the mouse does not correspond to tissue-selective covalent binding of reactive naphthalene metabolites in vivo. These studies compare reactive metabolite binding in target and nontarget cells and in various subcompartments of mouse lung and characterize, by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the proteins to which arylating metabolites are bound. Reactive metabolite binding was substantially higher in incubations of [3H]-naphthalene with distal bronchioles and isolated Clara cells than with explants of trachea or bronchus from the mouse. Likewise, binding was substantially higher in incubations of murine Clara cells than in identical incubations with mouse hepatocytes (nontarget cells) or rat trachea cells (nonsusceptible species). These data show a good correlation between cellular susceptibility to toxicity and the amount of reactive metabolite bound in vitro. Concentrations of adduct were highest in the medium and the nuclear/cell debris fraction (1000 x g pellet) of isolated Clara cells incubated with naphthalene; very small amounts of adduct were noted in pellets isolated at 20,000 or at 100,000 x g (mitochondrial and microsomal fractions) or in cytosol. These observations were consistent with the finding that adduct concentrations in bronchoalveolar lavage were substantially higher than in the lung at low doses of naphthalene and suggest that monitoring adducts in lavage may serve as a useful biomarker of exposure and effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of biliary metabolites of 4-n-nonylphenol in rainbow trout (Oncorhynchus mykiss)

1. [R-2,6-3H]-4-n-nonylphenol was synthesized and a single dose (5 mg, 1850 KBq) orally administered to rainbow trout. After 48 h, the radioactivity present in the bile amounted 5.5%. More than ten biliary metabolites were separated by hplc and collected for subsequent mass spectrometry analysis. The metabolic profile was totally modified by beta-glucuronidase hydrolysis, showing that most of the metabolites were glucuronic acid conjugates. 2. Conjugated metabolites were identified by lc-ms analysis and their aglycones were analysed by gc-ms analysis as TMS and acetyl derivatives. 3. The major metabolite accounted for 52+/-11% of the biliary radioactivity and was identified as nonylphenol-glucuronide. 4. Nonylphenol was hydroxylated at both omega and omega-1 positions of the alkyl chain, giving 9-hydroxynonylphenol and 8-hydroxynonylphenol. 5. 9-Hydroxynonylphenol was oxidized to the corresponding acid, and subsequently beta-oxidized, yielding 7-(4-hydroxyphenyl)heptanoic acid, 5-(4-hydroxyphenyl)pentanoic acid, 3-(4-hydroxyphenyl)propionic acid and 3-(4-hydroxyphenyl)-2-propenoic acid.     

PLC控制系统的电磁干扰及抗干扰措施

通过对PLC控制系统常见干扰源的分析,结合工程实践,阐述了解决抗电磁干扰的技术,具体从电源、电缆敷设、滤波、接地系统等方面论述了设计原理与方法,提高了控制系统的可靠性。     

Changes in the subcellular localization of the Brn4 gene product precede mesenchymal remodeling of the otic capsule

Listeria monocytogenes blood agar (LMBA) was compared to Listeria selective agar based on lithium chloride and ceftazidime (LA) and to the Oxford and Palcam media recommended by ISO and IDF for the detection and enumeration of L. monocytogenes from foodstuffs and food-processing environments. LMBA is based on trypticase soy agar with the following additions: sheep blood (5%) and as selective agents lithium chloride (10 g/l), polymyxin B sulphate (10 mg/l) and ceftazidime (20 mg/l), whereas the selectivity of LA is based on lithium chloride (15 g/l) and ceftazidime (15 g/l). The media were compared in the detection of L. monocytogenes after enrichment from naturally contaminated foodstuffs (n = 423) and from food-processing environments (n = 93), and in the enumeration of the species from naturally contaminated foodstuffs (n = 287). LMBA was superior both to the standard media and to LA in detection after enrichment and also in enumeration, except in the case of fresh broiler cut samples. The overall sensitivities of the Palcam, Oxford, LA and LMBA media were 68%, 67%, 74% and 96% in detection after enrichment and 64%, 73%, 76% and 80% in the enumeration of the species from ready to eat foods. The superiority of LMBA is based on distinguishing L. monocytogenes from other Listeria species by detection of beta-hemolysis, whereas the other media gave false-negative results because of the overgrowth of Listeria spp. other than L. monocytogenes, especially in detection after enrichment. A more selective medium than LMBA would have been required for the enumeration of the species from samples with high levels of competitive bacteria other than Listeria spp. The results indicate the need for a more specific isolation medium for L. monocytogenes in addition to those recommended by ISO and IDF for both detection and enumeration. LMBA offers an alternative to be used in combination with either Palcam or Oxford as well as with LA.     

Interference of nonsteroidal antiinflammatory drugs with very late activation antigen 4/vascular cells adhesion molecule 1-mediated lymphocyte-endothelial cell adhesion

OBJECTIVE: To study the effect of nonsteroidal antiinflammatory drugs (NSAIDs) on the adhesion of peripheral blood lymphocytes (PBL) to activated human umbilical vein endothelial cells (HUVEC) under conditions that resemble blood flow. METHODS: Assays of adhesion of PBL to HUVEC or recombinant vascular cell adhesion molecule 1 (rVCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin were performed under continuous rotation at 37 degrees C. The phenotype of PBL subpopulations attached was characterized by flow cytometry. Lymphocytes were pretreated with different doses (5-100 microg/ml) of aceclofenac, diclofenac, indomethacin, or piroxicam or with inhibitory monoclonal antibodies (MAb) prior to the adhesion assays. The effect of NSAIDs on lymphocyte adhesion molecules was assessed by flow cytometry. To determine whether NSAIDs interfere with the affinity state of very late activation antigen 4 (VLA-4) integrin, we studied the effect of these drugs on the appearance of a beta1 activation-dependent epitope recognized by the HUTS21 MAb both on human T lymphoblasts and on synovial fluid lymphocytes (SFL). RESULTS: In the flow-resembling model, PBL-HUVEC adhesion was mainly mediated by the VLA-4/ VCAM-1 adhesion pathway. The major PBL subset attached was the CD3+, CD45RO+ memory T cell, with CD49d(high) expression. Aceclofenac, diclofenac, and indomethacin, but not piroxicam, were able to inhibit PBL adhesion to HUVEC or rVCAM-1. However, the quantitative expression of VLA-4 was not affected by treatment of PBL with any of the NSAIDs studied. On T lymphoblasts and SFL, mostly CD45RO+ cells, the expression of the beta1 activation-dependent epitope detected by HUTS21 MAb was significantly decreased by aceclofenac, diclofenac, and indomethacin. CONCLUSION: Some NSAIDs are able to inhibit the adhesion of PBL to HUVEC under conditions that resemble blood flow by interfering with the conformational change in VLA-4 that increases its affinity for VCAM-1.     

Creep expansion of porous Ti-6Al-4V sandwich structures

Low-density titanium alloy sandwich structures consisting of a porous core and fully dense face sheets can be produced by consolidating argon gas charged powder compacts followed by not rolling and annealing to expand the gas-filled pores. Little is known about the rate of pore expansion, its dependence upon temperature, and the morphological evolution of the pore shape during expansion. In situ eddy current and laser ultrasonic sensors have been combined with metallographic and texture measurements to measure the relative density, the elastic moduli, and the microstructural evolution of Ti-6Al-4V sandwich panels during the annealing stage of low-density core (LDC) processing. The eddy current data indicated that expansion began during, the heating phase, reached a maximum expansion rate (Δ) of 2 × 10⁻⁵ s⁻¹ at approximately 685 °C, and had almost ceased (Δ < 1 × 10⁻⁶ s⁻¹) after annealing for 4 hours at 920 °C. The elastic moduli were found to decrease with increasing temperature and volume fraction of porosity. The initial (as-rolled) microstructure consisted of a lamellar α + β microstructure with an α-phase lath thickness of 2.0 μm and contained a distribution of oblate-shaped pores with aspect ratios of up to 10. During the expansion process, it recrystallized into an equiaxed α + β structure with an α-phase grain diameter of 7.5 μm with spheroidal pores with aspect ratios of up to 3. The combination of the two sensors was found to enable the in situ determination of both the porous cores relative density and its elastic properties. These are the two material indices that govern the elastic response of a sandwich structure.