High-sensitivity sequence determination of proteins quantitatively recovered from sodium dodecyl sulfate gels using an improved electrodialysis procedure

An improved electrodialysis procedure has been developed to recover quantitatively proteins from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of the method has been demonstrated successfully on H-2K^k, β₂-microglobulin, complement factor D, and viral structural protein p27. The results indicate that yields exceeding 93% are obtainable, and that extended amino acid sequences of the eluted proteins in microgram quantities can be obtained in the presence of SDS without intrinsic and/or extrinsic labeling with radioisotopes.     

Co-expression of pendrin, vacuolar H+-ATPase alpha4-subunit and carbonic anhydrase II in epithelial cells of the murine endolymphatic sac.

The endolymph in the endolymphatic sac (ES) is acidic (pH 6.6-7). Maintaining this acidic lumen is believed to be important for the normal function of the ES. The acid-base regulation mechanisms of the ES are unknown. Here we investigated the expression patterns of acid-base regulators, including vacuolar (v)H+-ATPase (proton pump), carbonic anhydrase (CA) II, and pendrin in the murine ES epithelium by immunohistochemistry (IHC) and compared their expression patterns by double immunostaining. We found that pendrin and vH+-ATPase were co-localized in the apical membrane of a specific type of ES epithelial cell. Pendrin- and vH+-ATPase-positive cells also expressed cytoplasmic CA II. Co-expression of pendrin, vH+-ATPase, and CA II in the same subgroup of ES cells suggests that this specific type of ES cell is responsible for the acid-base balance processes in the ES and pendrin, vH+-ATPase, and CA II are involved in these processes.     

Visual, haptic and cross-modal recognition of objects and scenes.

In this article we review current literature on cross-modal recognition and present new findings from our studies on object and scene recognition. Specifically, we address the questions of what is the nature of the representation underlying each sensory system that facilitates convergence across the senses and how perception is modified by the interaction of the senses. In the first set of our experiments, the recognition of unfamiliar objects within and across the visual and haptic modalities was investigated under conditions of changes in orientation (0 degrees or 180 degrees ). An orientation change increased recognition errors within each modality but this effect was reduced across modalities. Our results suggest that cross-modal object representations of objects are mediated by surface-dependent representations. In a second series of experiments, we investigated how spatial information is integrated across modalities and viewpoint using scenes of familiar, 3D objects as stimuli. We found that scene recognition performance was less efficient when there was either a change in modality, or in orientation, between learning and test. Furthermore, haptic learning was selectively disrupted by a verbal interpolation task. Our findings are discussed with reference to separate spatial encoding of visual and haptic scenes. We conclude by discussing a number of constraints under which cross-modal integration is optimal for object recognition. These constraints include the nature of the task, and the amount of spatial and temporal congruency of information across the modalities.     

Roles of the Carboxy-Terminal Half of Pseudomonas aeruginosa Major Outer Membrane Protein OprF in Cell Shape, Growth in Low-Osmolarity Medium, and Peptidoglycan Association

OprF, the major outer membrane protein of Pseudomonas aeruginosa, is multifunctional in that it can act as a nonspecific porin, plays a role in the maintenance of cell shape, and is required for growth in a low-osmolarity environment. The latter two structural roles of OprF, and OprF’s association with the peptidoglycan, have been proposed to be localized in the carboxy terminus of the protein, based on this region’s similarity to members of the OmpA family of proteins. To determine if this is correct, we constructed a series of C-terminally truncated OprF derivatives and examined their effects on P. aeruginosa cell length and growth in low-osmolarity medium. While the C terminus of OprF was required for wild-type cell length and growth in low-osmolarity medium, expression of the N terminus (first 163 amino acids [aa]) also influenced these phenotypes (compared with OprF deficiency). The first 154 to 164 aa of OprF seemed required for stable protein expression, consistent with the existence of a β-barrel domain in the N terminus of OprF. Greater than 215 aa of the protein were required for strong peptidoglycan association, confirming that residues in the C-terminal end of OprF are required for peptidoglycan binding. OprF deficiency did not affect the in vivo growth of an OprF-deficient strain in a mouse chamber model. Collectively, these data suggest that the C terminus of OprF plays a role in cell length, growth of P. aeruginosa in low-osmolarity media (but not in vivo), and peptidoglycan association, while the N terminus has an influence on the first two characteristics and is additionally important for stable protein expression.     

Separation of autoinhibitor from cytokinin activity in spores of Dictyostelium discoideum

Spores of Dictyostelium discoideum NC-4H contain a spore germination autoinhibitor (SGI) that, on purification using Sephadex LH-20 eluted with 35% ethanol, can be separated from the bulk of cytokinin activity. Inhibitor studies and chromatographic analysis indicate that SGI is not a known cytokinin such as zeatin or IPA (N⁶-(δ²-isopentenyl)-adenine), N²-dimethylguanosine or discadenine. Considering these findings and conflicting views in the literature concerning the structure of the autoinhibitor, the identity of SGI is discussed.     

Ventilation and control of acid-base status during temperature acclimation in the crab,Cancer magister

Summary Frequencies of scaphognathite (ventilatory,f _sc) and heart (f _h) pumping, oxygen consumption ( ), and hemolymph oxygen, carbon dioxide and pH levels were measured in adult Dungeness crabs (Cancer magister) during 7–10 day periods of exposure to 7, 12, and 17°C seawater. Ventilation volume ( ) was calculated for individual animals fromf _sc and a previously determined relationship between stroke volume and animal mass. increases (Q₁₀=2.3) with temperature were associated with larger increases inf _sc (Q₁₀=3.3) and (Q₁₀=3.5) and smaller increases inf _h (Q₁₀=1.5). The incidence of unilateral scaphognathite pumping and pausing decreased as temperature rose.Postbranchial oxygen tension was maintained in vivo but hemolymph oxygen content decreased both in vivo and in vitro as temperature rose. Postbranchial carbon dioxide tension did not change significantly but relative alkalinity was maintained as temperature rose by loss of hemolymph bicarbonate. The effects of increased ventilation volume and potential mechanisms of bicarbonate regulation are discussed.The responses of the essentially subtidalCancer magister are compared with those of subtidal, intertidal and terrestrial crabs demonstrating that the concepts of acid-base regulation developed for water and air breathing vertebrates are also applicable to water and air breathing crabs, and that intertidal crabs may exhibit transitional states.This work was supported by Grant No. A.5762 National Research Council of Canada     

Expression of TRAIL and TRAIL receptors in normal and malignant tissues

TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, is a member of the TNF family of proteins.Tumour cells were initially found to have increased sensitivity to TRAIL compared with normal cells, raising hopes that TRAIL would prove useful as an anti-tumor agent. The production of reliable monoclonal antibodies against TRAIL and its receptors that can stain fixed specimens will allow a thorough analysis of their expression on normal and malignant tissues. Here we report the generation of monoclonal antibodies against TRAIL and its four membrane-bound receptors(TR1-4), which have been used to stain a range of normal and malignant cells, as routinely fixed specimens. Low levels of TRAIL expression were found to be limited mostly to smooth muscle in lung and spleen as well as glial cells in the cerebellum and follicular cells in the thyroid. Expression of the TRAIL decoy receptors (TR3 and 4) was not as widespread as indicated by Northern blotting, suggesting that they may be less important for the control of TRAIL cytotoxicity than previously thought. TR1 and TR2 expression increases significantly in a number of malignant tissues,but in some common malignancies their expression was low, or patchy, which may limit the therapeutic role of TRAIL.Taken together, we have a panel of monoclonal antibodies that will allow a better assessment of the normal role of TRAIL and allow assessment of biopsy material, possibly allowing the identification of tumors that may be amenable to TRAIL therapy.     

ARABIDOPSIS CRINKLY4 function, internalization, and turnover are dependent on the extracellular crinkly repeat domain

The study of the regulation and cellular dynamics of receptor kinase signaling in plants is a rapidly evolving field that promises to give enormous insights into the molecular control of signal perception. In this study, we have analyzed the behavior of the L1-specific receptor kinase ARABIDOPSIS CRINKLY4 (ACR4) from Arabidopsis thaliana in planta and have shown it to be present in two distinct compartments within cells. These represent protein export bodies and a population of internalized vesicles. In parallel, deletion analysis has shown that a predicted beta-propeller-forming extracellular domain is necessary for ACR4 function. Nonfunctional ACR4 variants with deletions or point mutations in this domain behave differently to wild-type fusion protein in that they are not internalized to the same extent. In addition, in contrast with functional ACR4, which appears to be rapidly turned over, they are stabilized. Thus, for ACR4, internalization and turnover are linked and depend on functionality, suggesting that ACR4 signaling may be subject to damping down via internalization and degradation. The observed rapid turnover of ACR4 sets it apart from other recently studied plant receptor kinases. Finally, ACR4 kinase activity is not required for protein function, leading us to propose, by analogy to animal systems, that ACR4 may hetero-oligomerize with a kinase-active partner during signaling. Plant and animal receptor kinases have distinct evolutionary origins. However, with other recent work, our study suggests that there has been considerable convergent evolution between mechanisms used to regulate their activity.     

EB1 is essential during Drosophila development and plays a crucial role in the integrity of chordotonal mechanosensory organs

EB1 is a conserved microtubule plus end tracking protein considered to play crucial roles in microtubule organization and the interaction of microtubules with the cell cortex. Despite intense studies carried out in yeast and cultured cells, the role of EB1 in multicellular systems remains to be elucidated. Here, we describe the first genetic study of EB1 in developing animals. We show that one of the multiple Drosophila EB1 homologues, DmEB1, is ubiquitously expressed and has essential functions during development. Hypomorphic DmEB1 mutants show neuromuscular defects, including flightlessness and uncoordinated movement, without any general cell division defects. These defects can be partly explained by the malfunction of the chordotonal mechanosensory organs. In fact, electrophysiological measurements indicated that the auditory chordotonal organs show a reduced response to sound stimuli. The internal organization of the chordotonal organs also is affected in the mutant. Consistently, DmEB1 is enriched in those regions important for the structure and function of the organs. Therefore, DmEB1 plays a crucial role in the functional and structural integrity of the chordotonal mechanosensory organs in Drosophila.