Exo-endocytosis in isolated peptidergic nerve terminals occurs in the sub-second range

Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horserdish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in subsecond time peroids after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.     

Besprechungen

Ohne Zusammenfassung     

Long Range Surface Plasmons for Observation of Biomolecular Binding Events at Metallic Surfaces

A long range surface plasmon (LRSP) is an electromagnetic wave propagating along a thin metal film with an order of magnitude lower damping than conventional surface plasmon (SP) waves. Thus, the excitation of LRSP is associated with a narrower resonance and it provides larger enhancement of intensity of the electromagnetic field. In surface plasmon resonance (SPR) biosensors, these features allow a more precise observation of the binding of biomolecules in the proximity to the metal surface by using the (label-free) measurement of refractive index (RI) variations and by SP-enhanced fluorescence spectroscopy. In this contribution, we investigate LRSPs excited on a layer structure consisting of a fluoropolymer buffer layer, a thin gold film, and an aqueous sample. By implementing such structure in an SPR sensor, we achieved a 2.4- and 4.4-fold improvement of the resolution in the label-free and fluorescence-based detection, respectively, of the binding of biomolecules in the close proximity to the surface. Moreover, we demonstrate that the sensor resolution can be improved by a factor of 14 and 12 for the label-free and fluorescence-based detection, respectively, if the biomolecular binding events occur within the whole evanescent field of LRSP.     

Temperature stress interferes with male reproductive system development in clementine (Citrus clementina Hort. ex. Tan.)

Male gametophyte development is a critical phase of the plant life cycle due to its high sensitivity to environmental stresses. The rise in the average global temperature, often accompanied by extreme fluctuations, has an important impact on biological processes. Among those, male gametophytes are particularly sensitive to temperature stress during flower bud development and anthesis. Male gametophyte development was extensively studied in several plant species, but little information is available about the effects of temperature stress on male gametophyte development in the genus Citrus. We evaluated the effects of cold and hot temperatures during microsporogenesis and microgametogenesis on one of the most economically valuable citrus species, the “Comune” clementine (Citrus clementina Hort. ex. Tan.). The effect of constant temperature on the androecium was evaluated by a time course histological analysis performed on the anthers and by monitoring in vitro pollen germination. The results revealed how suboptimal hot and cold temperatures induce drastic alterations on the morphology of the tapetal cells, microspores and mature pollen grains. Shifting from the optimal temperature affected the timing of starch depletion in the anther walls, such as epidermis, endothecium and middle layer, influencing the pollen germination rate and pollen tube growth. To the best of our knowledge this is the first study attempting to assess how temperature stress affects male reproductive development in citrus. A better understanding of the mechanisms underlining male sterility will provide novel insights to elucidate the physiology of this agronomical important quality trait.     

Identification of a region in G protein γ subunits conserved across species but hypervariable among subunit isoforms

The heterotrimeric GTP binding proteins, G proteins, consist of three distinct subunits: alpha, beta, and gamma. There are 12 known mammalian gamma subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain gamma(10) protein by electrospray mass spectrometry revealed that it differs from the human protein by an Ala to Val substitution near the N-terminus. Comparison of gamma isoform subunit sequences indicated that they vary substantially more at the N-terminus than at other parts of the protein. Thus, species variation of this region might reflect the lack of conservation of a functionally unimportant part of the protein. Analysis of 38 gamma subunit sequences from four different species shows that the N-terminus of a given gamma subunit isoform is as conserved between different species as any other part of the protein, including highly conserved regions. These data suggest that the N-terminus of gamma is a functionally important part of the protein exhibiting substantial isoform-specific variation.     

Microchromatography of hemoglobins : VII. Detection of some uncommon hemoglobin variants and two rapid methods for the quantitation of Hb-A2 in the presence of Hb-C

Microchromatographic procedures on columns of DEAE- or CM-cellulose are described for the separation of the more common abnormal hemoglobins S and C from other, often uncommon, variants such as J, N, I, K-Woolwich, Hope, D, E, O, and Deer Lodge. Two procedures on conventionally sized columns of CM-cellulose permit the quantitative determination of Hb-A₂ in the presence of Hb-C in a day.     

Spectroscopic and surface-analytical characterization of self-assembled layers on Au

Future devices for electronic, photonic or other “intelligent” application involving (bio-) organic materials require nano-fabrication, -manipulation, -patterning and -functionalization techniques. Supramolecular assemblies, aggregates, small molecules and ions have to be controlled with regard to their structure, order and dynamic behaviour down to the molecular or even atomic level.

This contribution summarizes some of our activities aiming at a better understanding of the physical and chemical properties of functionalized and patterned surfaces. We focus on structure/order-property/function relations in such complex systems as interfaces and thin film architectures. Optical techniques (surface plasmon-spectroscopy) as well as surface analytical techniques (cyclic voltammetry and contact angle investigations) are introduced and demonstrated as powerful tools for the characterization of these interfaces and thin films.

Examples will be given covering self-assembly monolayers and molecular recognition—as well as complexation-reactions.