<Emphasis Type="Italic">Caloplaca testudinea</Emphasis> V. Wirth &; Kärnefelt sp. nov. and <Emphasis Type="Italic">C. rubelliana</Emphasis> (Ach.) Lojka,new to southern Africa

Caloplaca testudinea is newly described from the Namib Desert and C. rubelliana, formerly known from the Mediterranean region and western North America, is reported from the same area and therefore new to southern Africa. Morphology, anatomy, ecology and secondary chemistry of the two species are investigated.     

Synthesis and mesomorphic properties of glycosyl dialkyl- and diacyl-glycerols bearing saturated, unsaturated and methyl branched fatty acid and fatty alcohol chains. Part I. Synthesis

Glycosyl dialkyl- and diacyl-glycerols bearing saturated, unsaturated or chiral methyl branched chains in the tail and disaccharide and trisaccharide carbohydrate headgroups were synthesised. Standard procedures were used for the preparation of the educts and the glyco lipids: trichloracetimidate procedure for the preparation of long-chained compounds, glycosylation using the beta-peracetate and boron trifluoride etherate was successful for the preparation of lipids with a medium-alkyl chain length. Preparation of the ester was afforded in a multi-step synthesis according to published procedures. Thus, several lipids were synthesised in a few synthetic steps in good yields. The introduction of unsaturated or methyl branched chains lead to liquid crystallinity at ambient temperature, because these compounds will be used as model compounds for biological systems. The biophysical properties of these compounds will be reported in a following paper.     

Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton

We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip by usually one precipitous step of P-selectin:PSGL-1 dissociation. In this first article I, we focus on the initial elastic response and its termination by membrane separation from the cytoskeleton, initiating tether formation. Quantifying membrane unbinding forces for rates of loading (force/time) in the elastic regime from 240 pN/s to 38,000 pN/s, we discovered that the force distributions agreed well with the theory for kinetically limited failure of a weak bond. The kinetic rate for membrane unbinding was found to increase as an exponential function of the pulling force, characterized by an e-fold scale in force of approximately 17 pN and a preexponential factor, or apparent unstressed off rate, of approximately 1/s. The rheological properties of tether growth subsequent to the membrane unbinding events are presented in a companion article II.     

Production of a heterologous endo-1,4-beta-xylanase in the yeast Pichia stipitis with an O(2)-regulated promoter

The Cryptococcus albidus XLN-gene (encoding endo-1,4-β-xylanase) was expressed in the yeast Pichia stipitis under the control of the PsADH2-promoter, which is activated under O₂ limitation. The resulting transformant produced endo-1,4-β-xylanase after a shift to anoxic conditions. Endo-1,4-β-xylanase production was enhanced by limited aeration after the shift.     

Aerobic induction of respiro-fermentative growth by decreasing oxygen tensions in the respiratory yeast Pichia stipitis

The fermentative and respiratory metabolism of Pichia stipitis wild-type strain CBS 5774 and the derived auxotrophic transformation recipient PJH53 trp5-10 his3-1 were examined in differentially oxygenated glucose cultures in the hermetically sealed Sensomat system. There was a good agreement of the kinetics of gas metabolism, growth, ethanol formation and glucose utilisation, proving the suitability of the Sensomat system for rapid and inexpensive investigation of strains and mutants for their respiratory and fermentative metabolism. Our study revealed respiro-fermentative growth by the wild-type strain, although the cultures were not oxygen-limited. The induction of respiro-fermentative behaviour was obviously due to the decrease in oxygen tension but not falling below a threshold of oxygen tension. The responses differed depending on the velocity of the decrease in oxygen tension. At high oxygenation (slow decrease in oxygen tension), ethanol production was induced but glucose uptake was not influenced. At low oxygenation, glucose uptake and ethanol formation increased during the first hours of cultivation. The transformation recipient PJH53 most probably carries a mutation that influences the response to a slow decrease in oxygen tension, since almost no ethanol formation was found under these conditions.     

A fast and robust quantitative time-lapse assay for cell migration

We describe a simple and widely applicable method to measure cell migration in time-lapse sequences of fluorescently labeled cells in culture. Briefly, binarized cell images obtained after thresholding were cumulatively projected, and the covered areas were measured. This procedure determines the time course of the track area successively covered by the cell population. Under conditions where cell growth is negligible, a robust index of cell motility is derived from normalized plots for the displacement of cells over time. We applied this method to quantitatively examine the migration of B35 neuroblastoma cells transiently expressing GFP and to C6 glioma cells after staining with Hoechst 33258. This sensitive assay detected the influence of agents which inhibit actin polymerization (cytochalasin B) or interfere with the maintenance of cell polarity (methyl-beta-cyclodextrin) on cell migration. Thus, this assay is a versatile tool to measure quickly the migration of different cell types using different labeling strategies.     

Collapsin response mediator protein-4 regulates F-actin bundling

Collapsin response mediator proteins (CRMPs) form a family of cytosolic phosphoproteins which are involved in the signal transduction of semaphorin 3A leading to growth cone collapse. These proteins interact with a variety of cytosolic proteins including tubulin heterodimers. Here, we show that CRMP-4 co-localizes with F-actin in regular rib-like structures within lamellipodia of B35 neuroblastoma cells. Furthermore, depolymerization of actin fibers changed the distribution of GFP-CRMP-4 in vivo. In vitro, recombinant CRMP-4 formed homo-oligomers, bound to F-actin and organized F-actin into tight bundles. Both oligomerization and F-actin bundling depended on the C-terminal part of CRMP-4. The stoichiometry of actin and CRMP-4 in bundles was approximately 1:1 and the apparent equilibrium constant of the microfilament-CRMP-4 interaction was estimated from bundling assays as K(app) = 730 mM(-1). CRMP-4 was abundant in the cytosol of B35 neuroblastoma cells and its concentration was measured as approximately 1.7 microM. Overexpression of CRMP-4 inhibited the migration of B35 neuroblastoma cells, while knockdown of CRMP-4 enhanced cell migration and disturbed rib-like actin-structures in lamellipodia. Taken together, our data indicate that CRMP-4 promotes bundling of F-actin in vitro, that it is an important component of rib-like actin bundles in lamellipodia in vivo and that it functionally regulates the actin cytoskeleton in motile cells. These findings suggest a specific regulatory role of CRMP-4 towards the actin cytoskeleton which may by be relevant for growth cone collapse.     

Hepatoma-derived growth factor. Significance of amino acid residues 81-100 in cell surface interaction and proliferative activity

Hepatoma-derived growth factor (HDGF) has proliferative, angiogenic, and neurotrophic activity. It plays a putative role in the development and progression of cancer. When expressed in cells, the mitogenic activity of HDGF depends on its nuclear localization, but it also stimulates proliferation when added to the cell culture medium. A cell surface receptor for HDGF has not been identified so far. We investigated the interaction of various purified recombinant HDGF fusion proteins with the cell surface of NIH 3T3 fibroblasts. We showed that binding of a HDGF-beta-galactosidase fusion protein to the cell surface of NIH 3T3 fibroblasts was saturable, occurred with high affinity (K(D) = 14 nm), and had a proliferative effect. We identified a peptide comprising amino acid residues 81-100 within the amino-terminal part of HDGF that bound to the cell surface of NIH 3T3 cells with saturation and affinity values similar to those of HDGF. When added to primary human fibroblasts, this peptide stimulated proliferation. Substitution of a single amino acid (K96A) within this peptide was sufficient to abolish its binding to the cell surface and its proliferative activity. In contrast, when expressed transiently in NIH 3T3 cells, a HDGF-beta-galactosidase fusion protein in which amino acid residues 81-100 were deleted still had proliferative activity, whereas a fusion protein containing only the 81-100 peptide did not. Our results suggest the existence of a plasma membrane-located HDGF receptor for which signaling depends on amino acid residues 81-100 of HDGF. This region differs from the one that has been recently identified to be essential for mitogenic activity depending on the nuclear localization of HDGF. Thus, HDGF exerts its proliferative activity via two different pathways.