Ultrastructural effects in potato leaves due to antisense-inhibition of the sucrose transporter indicate an apoplasmic mode of phloem loading

To study the export of sugars from leaves and their long-distance transport, sucrose-proton/co-transporter activity of potato was inhibited by antisense repression of StSUT1 under control of either a ubiquitously active (CaMV 35S ) or a companion-cell-specific (rolC) promotor in transgenic plants. Transformants exhibiting reduced levels of the sucrose-transporter mRNA and showing a dramatic reduction in root and tuber growth, were chosen to investigate the ultrastructure of their source leaves. The transformants had a regular leaf anatomy with a single-layered palisade parenchyma, and bicollateral minor veins within the spongy parenchyma. Regardless of the promoter used, source leaves from transformants showed an altered leaf phenotype and a permanent accumulation of assimilates as indicated by the number and size of starch grains, and by the occurrence of lipid-storing oleosomes. Starch accumulated throughout the leaf: in epidermis, mesophyll and, to a smaller degree, in phloem parenchyma cells of minor veins. Oleosomes were observed equally in mesophyll and phloem parenchyma cells. Companion cells were not involved in lipid accmulation and their chloroplasts developed only small starch grains. The similarity of ultrastructural symptoms under both promotors corresponds to, rather than contradicts, the hypothesis that assimilates can move symplasmically from mesophyll, via the bundle sheath, up to the phloem. The microscopical symptoms of a constitutively high sugar level in the transformant leaves were compared with those in wild-type plants after cold-girdling of the petiole. Inhibition of sugar export, both by a reduction of sucrose carriers in the sieve element/companion cell complex (se/cc complex), or further downstream by cold-girdling, equally evokes the accumulation of assimilates in all leaf tissues up to the se/cc complex border. However, microscopy revealed that antisense inhibition of loading produces a persistently high sugar level throughout the leaf, while cold-girdling leads only to local patches containing high levels of sugar. Received: 4 March 1998 / Accepted: 7 April 1998     

Use of saguaro fruit by white-winged doves: isotopic evidence of a tight ecological association

We report the use of stable isotope and crop content analyses to quantify the use of saguaro (Carnegiea gigantea) nectar and fruit by migratory desert white-winged doves (Zenaida asiatica mearsnii). Saguaro resources had characteristically ¹³C-enriched CAM values (δ¹³C=–12.8±0.7‰ SD VPDB and –13.1±0.5‰ SD VPDB for nectar and fruit, respectively) relative to other food plants used by doves (δ¹³C_C3=–24.9±3.3‰ SD VPDB). The water contained in saguaro nectar and fruit was deuterium enriched (δD=19.6±2.0‰ SD VSMOW and 48.4±1.6‰ SD VSMOW for nectar and fruit, respectively) relative to other water sources (ranging from –41 to –19‰ VSMOW). During the fruiting season, there was a positive correlation between δ¹³C in dove liver tissues and percent of saguaro in crop contents. A two-point mixing model indicated that during the peak of saguaro fruit use, most of the carbon incorporated in dove tissues was from saguaro. Desert white-winged doves appear to be saguaro specialists. Averaged over the period when doves were resident, saguaro comprised about 60% of the total carbon incorporated into dove tissues. Tissue δ¹³C and δD of body water showed a significant positive correlation, indicating that doves were using saguaro as a source of both nutrients and water. However, at the peak of saguaro utilization, the doves’ body-water δD was more positive (by about 20‰) than saguaro fruit water. We hypothesize that this enrichment is due to fractionated evaporative water losses by doves. Using dove carbon isotope data and a two end-point mixing model we estimate that, on average, doves consume the equivalent of 128 saguaro fruits per season; each fruit contains on average 26.0±14.8 g SD of pulp (wet mass) of which 19.4 g is water. Stable isotopes have been used to produce qualitative re-constructions of animal diets. Our study shows that they can be used to provide quantitative estimates of the flow of nutrients from resources into consumers as well. Received: 30 September 1999 / Accepted: 23 March 2000     

Insulin receptor substrate 1-induced inhibition of endoplasmic reticulum Ca2+ uptake in beta-cells. Autocrine regulation of intracellular ca2+ homeostasis and insulin secretion.

To understand the role of the insulin receptor pathway in beta-cell function, we have generated stable beta-cells (betaIRS1-A) that overexpress by 2-fold the insulin receptor substrate-1 (IRS-1) and compared them to vector-expressing controls. IRS-1 overexpression dramatically increased basal cytosolic Ca2+ levels from 81 to 278 nM, but it did not affect Ca2+ response to glucose. Overexpression of the insulin receptor also caused an increase in cytosolic Ca2+. Increased cytosolic Ca2+ was due to inhibition of Ca2+ uptake by the endoplasmic reticulum, because endoplasmic reticulum Ca2+ uptake and content were reduced in betaIRS1-A cells. Fractional insulin secretion was significantly increased 2-fold, and there was a decrease in betaIRS1-A insulin content and insulin biosynthesis. Steady-state insulin mRNA levels and glucose-stimulated ATP were unchanged. High IRS-1 levels also reduced beta-cell proliferation. These data demonstrate a direct link between the insulin receptor signaling pathway and the Ca2+-dependent pathways regulating insulin secretion of beta-cells. We postulate that during regulated insulin secretion, released insulin binds the beta-cell insulin receptor and activates IRS-1, thus further increasing cytosolic Ca2+ by reducing Ca2+ uptake. We suggest the existence of a novel pathway of autocrine regulation of intracellular Ca2+ homeostasis and insulin secretion in the beta-cell of the endocrine pancreas.     

A quartz crystal biosensor for measurement in liquids.

The detection of anti-human immunodeficiency virus (HIV) antibodies by means of synthetic HIV peptide immobilized on a piezoelectric quartz sensor is demonstrated. The measurement set-up consists of an oscillator circuit, a suitably modified AT-cut thickness-shear-mode quartz crystal with gold electrodes, which is housed in a special reaction vessel, and a computer-controlled frequency counter for the registration of the measured frequency values. The quartz crystal is adapted for a steady operation in liquids at a frequency of 20 MHz. In phosphate-buffered saline solution the oscillator reaches a stability of about 0.5 Hz within a few seconds, of about 2 Hz within 10 min and about 30 Hz within 1 h. The frequency shift due to the adsorption of various proteins to the uncoated sensor surface has been investigated. It can be shown that a stable adsorptive binding of proteins to an oscillating gold surface is feasible and can be used for the immobilization of a receptor layer (e.g. HIV peptide). Specific binding of the anti-HIV monoclonal antibody to the HIV peptide immobilized on the quartz sensor is demonstrated. Control experiments show, however, additional unspecific binding. According to the experiments, the Sauerbrey formula gives a sufficiently accurate value for the decrease of the resonant frequency due to adsorption or binding of macromolecular proteins on the quartz crystal surface.     

Nerve growth factor receptors are preaggregated and immobile on responsive cells.

It has been hypothesized that signal transduction occurs by ligand-induced receptor clustering and immobilization. For many peptide receptors, cross-linking by anti-receptor antibodies is sufficient for receptor activation. This is not, however, the case for nerve growth factor receptor (NGFR). Using fluorescence microscopy and fluorescence recovery after photobleaching (FRAP), we have analyzed the distribution and diffusibility of NGFR on a series of cell lines. We have found the following: (1) Cells expressing high-affinity responsive NGFR's display clustered NGFR's even in the absence of ligand. In contrast, NGFR's in nonresponsive cell lines are diffusely distributed. (2) Receptors on responsive cell lines are largely nondiffusing while most receptors on nonresponsive cell lines are relatively free to diffuse. (3) NGF does not greatly alter the distribution or diffusion properties of the NGFR on either nonresponsive or responsive cell lines. Thus, NGFR is preclustered and immobile on responsive cells, which suggests that immobilization of NGFR prior to ligand binding is required for signal transduction.     

Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.     

Feature-based,automated segmentation of cerebral infarct patterns using T2- and diffusion-weighted imaging

Diffusion-weighted imaging enables the diagnosis of cerebral ischemias very early, thus supporting therapies such as thrombolysis. However, morphology and tissue-characterizing parameters (e.g. relaxation times or water diffusion) may vary strongly in ischemic regions, indicating different underlying pathologic processes. As the determination of the parameters by a supervised segmentation is very time consuming, we evaluated whether different infarct patterns may be segmented by an automated, multidimensional feature-based method using a unified segmentation procedure. Ischemias were classified into 5 characteristic patterns. For each class, a 3D histogram based on T(2)- and diffusion-weighted images as well as calculated apparent diffusion coefficients (ADC) was generated from a representative data set. Healthy and pathologic tissue classes were segmented in the histogram as separate, local density maxima with freely shaped borders. Segmentation control parameters were optimized in a 3-step procedure. The method was evaluated using synthetic images as well as results of a supervised segmentation. For the analysis of cerebral ischemias, the optimal control parameter set led to sensitivities and specificities between 1.0 and 0.9.