Electron microscopic structural analysis of Photosystem I,Photosystem II,and the cytochromeb6/f complex from green plants and cyanobacteria

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochromeb6/f complex are discussed.     

Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.     

Managing anabolic steroids in pre-hibernating Arctic ground squirrels: obtaining their benefits and avoiding their costs

Androgens have benefits, such as promoting muscle growth, but also significant costs, including suppression of immune function. In many species, these trade-offs in androgen action are reflected in regulated androgen production, which is typically highest only in reproductive males. However, all non-reproductive Arctic ground squirrels, irrespective of age and sex, have high levels of androgens prior to hibernating at sub-zero temperatures. Androgens appear to be required to make muscle in summer, which, together with lipid, is then catabolized during overwinter. By contrast, most hibernating mammals catabolize only lipid. We tested the hypothesis that androgen action is selectively enhanced in Arctic ground squirrel muscle because of an upregulation of androgen receptors (ARs). Using Western blot analysis, we found that Arctic ground squirrels have AR in skeletal muscle more than four times that of Columbian ground squirrels, a related southern species that overwinters at approximately 0°C and has low pre-hibernation androgen levels. By contrast, AR in lymph nodes was equivalent in both species. Brain AR was also modestly but significantly increased in Arctic ground squirrel relative to Columbian ground squirrel. These results are consistent with the hypothesis that tissue-specific AR regulation prior to hibernation provides a mechanism whereby Arctic ground squirrels obtain the life-history benefits and mitigate the costs associated with high androgen production.     

Residues 10-18 within the C5a receptor N terminus compose a binding domain for chemotaxis inhibitory protein of Staphylococcus aureus

Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is excreted by the majority of S. aureus strains and is a potent inhibitor of C5a- and formylated peptide-mediated chemotaxis of neutrophils and monocytes. Recently, we reported that CHIPS binds to the C5a receptor (C5aR) and the formylated peptide receptor, thereby blocking activation by C5a and formylated peptides, respectively. The anaphylatoxin C5a plays an important role in host immunity and pathological inflammatory processes. For C5a a two-site binding model is proposed in which C5a initially binds the C5aR N terminus, followed by interaction of the C5a C-terminal tail with an effector domain on the receptor. We have shown here that CHIPS does not affect activation of the C5aR by a peptide mimic of the C5a C terminus. Moreover, CHIPS was found to bind human embryonic kidney 293 cells expressing only the C5aR N terminus. Deletion and mutation experiments within this C5aR N-terminal expression system revealed that the binding site of CHIPS is contained in a short stretch of 9 amino acids (amino acids 10-18), of which the aspartic acid residues at positions 10, 15, and 18 plus the glycine at position 12 are crucial. Binding studies with C5aR/C3aR and C5aR/IL8RA chimeras confirmed that CHIPS binds only to the C5aR N terminus without involvement of its extracellular loops. CHIPS may provide new strategies to block the C5aR, which may lead to the development of new C5aR antagonists.     

Rapid susceptibility testing and microcolony analysis of Candida spp. cultured and imaged on porous aluminum oxide

Background

Acquired resistance to antifungal agents now supports the introduction of susceptibility testing for species-drug combinations for which this was previously thought unnecessary. For pathogenic yeasts, conventional phenotypic testing needs at least 24 h. Culture on a porous aluminum oxide (PAO) support combined with microscopy offers a route to more rapid results.

Methods

Microcolonies of Candida species grown on PAO were stained with the fluorogenic dyes Fun-1 and Calcofluor White and then imaged by fluorescence microscopy. Images were captured by a charge-coupled device camera and processed by publicly available software. By this method, the growth of yeasts could be detected and quantified within 2 h. Microcolony imaging was then used to assess the susceptibility of the yeasts to amphotericin B, anidulafungin and caspofungin (3.5 h culture), and voriconazole and itraconazole (7 h culture).

Significance

Overall, the results showed good agreement with EUCAST (86.5% agreement; n = 170) and E-test (85.9% agreement; n = 170). The closest agreement to standard tests was found when testing susceptibility to amphotericin B and echinocandins (88.2 to 91.2%) and the least good for the triazoles (79.4 to 82.4%). Furthermore, large datasets on population variation could be rapidly obtained. An analysis of microcolonies revealed subtle effects of antimycotics on resistant strains and below the MIC of sensitive strains, particularly an increase in population heterogeneity and cell density-dependent effects of triazoles. Additionally, the method could be adapted to strain identification via germ tube extension. We suggest PAO culture is a rapid and versatile method that may be usefully adapted to clinical mycology and has research applications.     

Rapid effects of nerve growth factor on the Na,K-pump in rat pheochromocytoma cells

Nerve growth factor (NGF) induces neuronal differentiation of rat pheochromocytoma cells (PC12). Here we show that NGF causes a stimulation of Na⁺,K⁺-pump mediated K⁺ influx, with a maximum at 30 min after addition of NGF. The stimulation of the Na⁺,K⁺-pump is completely blocked by the Na⁺-flux inhibitor amiloride (0.2 mM) and can be mimicked by the Na⁺ ionophore monensin. These results suggest that NGF causes a rapid enhancement of Na⁺ influx leading to an activation of the Na⁺,K⁺-pump, a mechanism similar to the action of other growth factors.     

Identification of an intracellular domain of the EGF receptor required for high-affinity binding of EGF

Although all EGF receptors in EGF receptor-expressing cells are molecularly identical, they can be subdivided in two different classes that have either a high or a low affinity for EGF. Specifically the high-affinity class is associated with filamentous actin. To determine whether the interaction of the EGF receptor with actin induces its high-affinity state, we studied EGF-binding properties of an EGF receptor mutant that lacks the actin-binding site. Interestingly, we found that cells expressing this mutant receptor still display both high- and low-affinity classes of EGF receptors, indicating that the actin-binding domain does not determine the high-affinity binding state. By further mutational analysis we identified a receptor domain, within the tyrosine kinase domain, that regulates the affinity for EGF.     

Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.     

The EGF receptor is an actin-binding protein

In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996.