Atomic-force microscopy imaging of plasma membranes purified from spinach leaves

Summary: Plasma membranes purified from spinach leaves by aqueous two-phase partitioning were examined by atomic-force microscopy (AFM) in phosphate buffer, and details on their structure were reported at nanometric scale. Examination of the fresh membrane preparation deposited on mica revealed a complex organization of the surface. It appeared composed of a first layer of material, about 8 nm in thickness, that practically covered all the mica surface and on which stand structures highly heterogeneous in shape and size. High-resolution imaging showed that the surface of the first layer appeared relatively smooth in some regions, whereas different characteristic features were observed in other regions. They consisted of globular-to-elliptical protruding particles of various sizes, from 4-5 nm x-y size for the smallest to 40-70 nm for the largest, and of channel-like structures 25-30 nm in diameter with a central hole. Macromolecular assemblies of protruding particles of various shapes were imaged. Addition of the proteolytic enzyme pronase led to a net roughness decrease in regions covered with particles, indicating their proteinaceous nature. The results open fascinating perspectives in the investigation of membrane surfaces in plant cells with the possibility to get structural information at the nanometric rang

Polarization state of the optical near-field

The polarization state of the optical electromagnetic field lying several nanometers above complex dielectric structures reveals the intricate light-matter interaction that occurs in this near-field zone. This information can only be extracted from an analysis of the polarization state of the detected light in the near-field. These polarization states can be calculated by different numerical methods well-suited to near--field optics. In this paper, we apply two different techniques (Localized Green Function Method and Differential Theory of Gratings) to separate each polarisation component associated with both electric and magnetic optical near-fields produced by nanometer sized objects. The analysis is carried out in two stages: in the first stage, we use a simple dipolar model to achieve insight into the physical origin of the near-field polarization state. In the second stage, we calculate accurate numerical field maps, simulating experimental near-field light detection, to supplement the data produced by analytical models. We conclude this study by demonstrating the role played by the near-field polarization in the formation of the local density of states.Comment: 9 pages, 11 figures, accepted for publication in Phys. Rev.

Creating Well-Defined Hot Spots for Surface-Enhanced Raman Scattering by Single-Crystalline Noble Metal Nanowire Pairs

Well-defined surface-enhanced Raman scattering (SERS) active systems were fabricated by single-crystal line noble metal nanowires. Crossed and parallel nanowire pairs were constructed by using a nanomanipulator to create SERS hot spots in the form of nanowire junction. SERS spectra of brilliant cresyl blue (BCB), p-mercaptoaniline (pMA), and p-mercaptobenzoic acid (pMBA) were observed at the junction of two nanowires. The SERS enhancement and polarization dependence are correlated well with the enhanced electric field intensities calculated by the finite difference time domain (FDTD) method for specific nanowire geometries. These simple and effective SERS active systems have a practical advantage that the hot spots can be readily located and visualized by an optical microscope. These well-defined SERS active systems based on noble metal nanowires can be further developed to find applications in a variety of biological and chemical sensingclose38404

Differential effect of hypophysectomy and growth hormone treatment on hepatic glucuronosyltransferases in male rats: evidence for an action at a pretranslational level for isoforms glucuronidating bilirubin.

International audienceThe influence of growth hormone (GH) on 4-nitrophenol, bilirubin, testosterone, androsterone and estrone glucuronidation activities was studied in fully activated male rat hepatic microsomes. Sham-operated and hypophysectomized animals were injected with two different dosages of GH, mimicking either the male or female GH secretion pattern. Half the animals received thyroxine and cortisol in concentrations chosen to compensate for the lack of thyroid hormones and glucocorticoids in hypophysectomized rats. GH induced a decrease in several glucuronidation activities: bilirubin glucuronidation in both sham-operated and cortisol/ thyroxine-treated hypophysectomized rats in a dose-dependent manner, testosterone glucuronidation in hypophysectomized animals, and androsterone and estrone glucuronidation in cortisol/thyroxin-treated hypophysectomized rats. 4-nitrophenol glucuronidation was not affected by GH treatment. A hypothetical "feminizing" effect of GH (due to an almost continuous secretion) could not be invoked to explain these results, contrary to what has been observed elsewhere for other hepatic enzyme activities. Hypophysectomy altered all the activities tested, with bilirubin the most modified (a 200% enhancement). Restoration of control values was achieved in hypophysectomized animals with cortisol/thyroxine replacement together with a low dosage of GH (mimicking a male GH secretion pattern), except for androsterone glucuronidation activity where both GH and cortisol/thyroxine treatments reinforced the decreasing effect of hypophysectomy. Variations in protein amounts were correlated to variations in bilirubin, testosterone and androsterone conjugation activities induced by hypophysectomy and GH treatment. Reverse transcription-polymerase chain reaction (RT-PCR) mRNA analysis of bilirubin cluster isoforms or uridine diphosphate glucuronosyltransferase 1B1 (UGT1B1), UGT1B2 and UGT1B5 showed that GH controlled the different isoforms involved in bilirubin glucuronidation differentially at a pretranslational level

Differential effect of hypophysectomy and growth hormone treatment on hepatic glucuronosyltransferases in male rats: evidence for an action at a pretranslational level for isoforms glucuronidating bilirubin.

International audienceThe influence of growth hormone (GH) on 4-nitrophenol, bilirubin, testosterone, androsterone and estrone glucuronidation activities was studied in fully activated male rat hepatic microsomes. Sham-operated and hypophysectomized animals were injected with two different dosages of GH, mimicking either the male or female GH secretion pattern. Half the animals received thyroxine and cortisol in concentrations chosen to compensate for the lack of thyroid hormones and glucocorticoids in hypophysectomized rats. GH induced a decrease in several glucuronidation activities: bilirubin glucuronidation in both sham-operated and cortisol/ thyroxine-treated hypophysectomized rats in a dose-dependent manner, testosterone glucuronidation in hypophysectomized animals, and androsterone and estrone glucuronidation in cortisol/thyroxin-treated hypophysectomized rats. 4-nitrophenol glucuronidation was not affected by GH treatment. A hypothetical "feminizing" effect of GH (due to an almost continuous secretion) could not be invoked to explain these results, contrary to what has been observed elsewhere for other hepatic enzyme activities. Hypophysectomy altered all the activities tested, with bilirubin the most modified (a 200% enhancement). Restoration of control values was achieved in hypophysectomized animals with cortisol/thyroxine replacement together with a low dosage of GH (mimicking a male GH secretion pattern), except for androsterone glucuronidation activity where both GH and cortisol/thyroxine treatments reinforced the decreasing effect of hypophysectomy. Variations in protein amounts were correlated to variations in bilirubin, testosterone and androsterone conjugation activities induced by hypophysectomy and GH treatment. Reverse transcription-polymerase chain reaction (RT-PCR) mRNA analysis of bilirubin cluster isoforms or uridine diphosphate glucuronosyltransferase 1B1 (UGT1B1), UGT1B2 and UGT1B5 showed that GH controlled the different isoforms involved in bilirubin glucuronidation differentially at a pretranslational level

Activation of the mouse TATA-less and human TATA-containing UDP-glucuronosyltransferase 1A1 promoters by hepatocyte nuclear factor 1.

UDP-glucuronosyltransferase (UGT) 1A1 (UGT1A1) catalyzes the glucuronidation of bilirubin in liver. Among all UGT isoforms identified to date, it is the only relevant bilirubin-glucuronidating enzyme in human. Because glucuronoconjugation is the major route of bilirubin elimination, any genetic alteration that affects bilirubin glucuronosyltransferase activity may result in a more or less severe hyperbilirubinemia. In this study, we report the cloning and characterization of the transcriptional regulation of the mouse UGT1A1 gene. Primary-structure analysis of the mouse Thymidine Adevice promoter revealed marked differences with its human homolog. First, the mouse promoter lacks the highly polymorphic thymidine/adenine repeat occurring in the human promoter, which has been associated with some forms of hyperbilirubinemia. Second, an L1 transposon element, which is absent in the human promoter, is found 480 bp upstream of the transcription start site in mouse. Using the electromobility shift and DNase I footprinting experiments, we have identified a hepatocyte nuclear factor 1-binding site in the mouse UGT1A1 promoter that confers responsiveness to both factors HNF1alpha and HNF1beta in HEK293 cells. Furthermore, we show that this element, which is conserved in the human promoter, also confers strong HNF1 responsiveness to the human UGT1A1 gene. Together, these results provide evidence for a major regulatory function of this liver-enriched transcription factor in UGT1A1 activity in both rodents and human