Human endo-alpha1,2-mannosidase is a Golgi-resident type II membrane protein

The cDNA for human endo-alpha1,2-mannosidase was reconstructed using two independent EST-clones and its properties characterized. The 2837 bp cDNA construct contained a 1389 bp open reading frame (ORF) encoding for 462 amino acids and an approximately 53.6 kDa protein, respectively. Hydrophobicity analysis of this amino acid sequence, as well as proteolytic degradation studies, indicate that the enzyme is a type II protein, anchored in the membrane via a 19 amino-acid long apolar sequence close to the N-terminus. Human endo-alpha1,2-mannosidase displays a high degree of sequence identity with the catalytic domain of the homologous rat liver endo-enzyme, but differs substantially in the N-terminal peptide region, which includes the transmembrane domain. No sequence similarity exists with other processing alpha-glycosidases. Based on sequence information provided by the 2837 bp construct, the cDNA consisting of the complete 1389 bp ORF was amplified by RT-PCR using human fibroblast RNA. Incubation of E. coli lysates with this cDNA, previously modified for boost translation by codon optimization, resulted in the synthesis of an approximately 52 kDa protein which degraded [(14)C]Glc(3)-Man(9)-GlcNAc(2) efficiently, indicating that the catalytic domain of the enzyme folds correctly under cell-free conditions. Transfection of the endo-alpha1,2-mannosidase wild-type cDNA into COS 1 cells resulted in a moderate (approximately 1.5-fold) but reproducible increase of activity compared with control cells, whereas >18-fold increase in activity was measured after expression of a chimera containing green-fluorescent-protein (GFP) attached to the N-terminus of the endo-alpha1,2-mannosidase polypeptide. This, together with the observation that GFP-endo-alpha1,2-mannosidase is expressed as a Golgi-resident type II protein, points to enzyme-specific parameters directing folding and membrane anchoring, as well as Golgi-targeting, not being affected by fusion of GFP to the endo-alpha1,2-mannosidase N-terminus.     

InlA- but not InlB-mediated internalization of Listeria monocytogenes by non-phagocytic mammalian cells needs the support of other internalins

To determine the contribution of the previously identified internalins, InlA, InlB, InlC, InlE, InlG, and InlH, to internalization of Listeria monocytogenes by non-professional phagocytic mammalian cells, we constructed mutants with various combinations of deletions in the respective inl genes. Internalization of these mutants into the epithelial-like Caco-2 and the microvascular endothelial HBMEC cell lines were studied. Deletion of the inlGHE gene cluster, or of the single genes, led to a two to fourfold increased internalization by HBMEC and other non-phagocytic mammalian cells. Invasion into HBMEC was totally blocked in the absence of InlB, and InlB-dependent internalization did not require the presence of any of the other internalins. Internalization by Caco-2 cells was reduced to a level of about 1% in the absence of InlA and InlB, and was most efficient in the presence of InlA, InlB and InlC and in the absence of InlG, InlH and InlE. InlB and InlA, in each case in the absence of the other internalins, led (compared with the wild-type strain) to reduced internalization of about 20% and less than 10% respectively. InlA-dependent internalization (in the absence of InlB) required the additional function of InlC and InlGHE. The deletion of inlGHE enhanced the expression of InlA and InlB. The increased amount of InlA led to an increase in early association of L. monocytogenes with Caco-2 cells without enhancing its uptake in the absence of the other internalins, whereas the larger amount of InlB did not enhance early association of L. monocytogenes with HBMEC but led to an increase in internalization of L. monocytogenes. The results suggest that InlB is able to induce phagocytosis in HBMEC and (at a lower efficiency) in Caco-2 cells by itself, but InlA needs the supportive functions of the other internalins to trigger phagocytosis. None of these internalins seems to be required for cell-to-cell spread by L. monocytogenes, as shown by microinjection of Caco-2 cells with appropriate inl mutants.     

Predicting molecular properties of α-synuclein using force fields for intrinsically disordered proteins

Independent force field validation is an essential practice to keep track of developments and for performing meaningful Molecular Dynamics simulations. In this work, atomistic force fields for intrinsically disordered proteins (IDP) are tested by simulating the archetypical IDP α-synuclein in solution for 2.5 μs. Four combinations of protein and water force fields were tested: ff19SB/OPC , ff19SB/TIP4P-D , ff03CMAP/TIP4P-D , and a99SB-disp/TIP4P-disp , with four independent repeat simulations for each combination. We compare our simulations to the results of a 73 μs simulation using the a99SB-disp/TIP4P-disp combination, provided by D. E. Shaw Research. From the trajectories, we predict a range of experimental observations of α-synuclein and compare them to literature data. This includes protein radius of gyration and hydration, intramolecular distances, NMR chemical shifts, and ³J-couplings. Both ff19SB/TIP4P-D and a99SB-disp/TIP4P-disp produce extended conformational ensembles of α-synuclein that agree well with experimental radius of gyration and intramolecular distances while a99SB-disp/TIP4P-disp reproduces a balanced α-synuclein secondary structure content. It was found that ff19SB/OPC and ff03CMAP/TIP4P-D produce overly compact conformational ensembles and show discrepancies in the secondary structure content compared to the experimental data.     

Impacts of storms on Recent planktic foraminiferal test production and CaCO3 flux in the North Atlantic at 47 °N, 20 °W (JGOFS)

Planktic foraminiferal assemblages are well known to vary in accordance with seasonal fluctuations in ocean properties, periodic reproduction cycles, and variations between water masses. Here we report that storms also can significantly influence foraminiferal assemblages. During the RV Meteor cruise 21 to the Northeast Atlantic Ocean ( area), from March to May 1992, planktic foraminifera were sampled using a multiple opening-closing net. While sampling, two storms with wind forces up to 12 Beaufort caused intensified surface layer mixing with shifts in the depth of the upper ocean mixed-layer from 20–40 m to 170–240 m. Subsequently, planktic foraminiferal growth rates increased, resulting in an elevated quantity of small (100–150 μm) tests (Phase 1). When the wind strength increased a second time, the mixed-layer deepened to a depth below the former position of the pycnocline, and again the abundance of small tests increased (Phase 2). During Phase 2, the weight of calcite in specimens of the productive zone reached its maximum. In the export zone, an associated increase in empty tests occurred with a lag time depending on the test sinking velocity. In the upper export zone, down to 700 m water depth, CaCO₃ flux increased from 9.3 to 49.8 mg CaCO₃ m⁻² d⁻¹ after the first storm and from 8.9 to 19.9 mg CaCO₃ m⁻²d⁻¹ after the second storm. In the 700 to 2500 m depth interval, the flux increased from 5.1 mg CaCO₃ m⁻² d⁻¹ to about 9.2 mg CaCO, m⁻² d⁻¹. Thus, the standing stock of living foraminifera and export of empty tests from the productive zone increased after the storms, leading to pulses of CaCO₃ exported from the surface to deep water.     

Removal of the Membrane-anchoring Domain of Epidermal Growth Factor Leads to Intracrine Signaling and Disruption of Mammary Epithelial Cell Organization

Autocrine EGF-receptor (EGFR) ligands are normally made as membrane-anchored precursors that are proteolytically processed to yield mature, soluble peptides. To explore the function of the membrane-anchoring domain of EGF, we expressed artificial EGF genes either with or without this structure in human mammary epithelial cells (HMEC). These cells require activation of the EGFR for cell proliferation. We found that HMEC expressing high levels of membrane- anchored EGF grew at a maximal rate that was not increased by exogenous EGF, but could be inhibited by anti–EGFR antibodies. In contrast, when cells expressed EGF lacking the membrane-anchoring domain (sEGF), their proliferation rate, growth at clonal densities, and receptor substrate phosphorylation were not affected by anti–EGFR antibodies. The sEGF was found to be colocalized with the EGFR within small cytoplasmic vesicles. It thus appears that removal of the membrane-anchoring domain converts autocrine to intracrine signaling. Significantly, sEGF inhibited the organization of HMEC on Matrigel, suggesting that spatial restriction of EGF access to its receptor is necessary for organization. Our results indicate that an important role of the membrane-anchoring domain of EGFR ligands is to restrict the cellular compartments in which the receptor is activated.     

Tonotopic organization and functional characterization of the auditory thalamus in a songbird,the European starling

1. The diencephalic auditory nucleus of the European starling, the nucleus ovoidalis, shows rostrocaudal and dorsoventral diameters of 500-800 microns and a mediolateral diameter of 800-1000 microns. This small and sharply delimited nucleus is composed of densely packed neurons. 2. Its tonotopic organization consists of evenly spaced isofrequency contours, with best frequencies decreasing ventrally. The frequency range was found to be 150 Hz to 7030 Hz. 3. Apart from tonotopic organization, other characteristics of single units demonstrate the uniformity of the neuronal population. Units have high spontaneous activities (mean 61 pps; Fig. 4a), and show mainly stimulus correlated tonic discharge patterns. In most cases, excitatory frequency bands are enclosed by inhibitory frequency bands. 4. Single units were tested, applying various stimulus classes differing in time structure (BPN, sine, FM up, FM down, SFM, SAM) but sharing a common frequency band. All neurons tested responded to all classes. Evaluation of stimulus class preference, however, revealed that BPN and SFM caused the strongest responses, whereas FM and SAM were less effective. 5. Comparison of the single unit responses in the ovoid nucleus with those known for avian auditory forebrain and midbrain centres strongly suggests a relay function for the diencephalic nucleus.     

Microsclerotial germination of Verticillium dahliae as affected by rape rhizosphere

Abstract The germination of nylon net-trapped microsclerotia of Verticillium dahliae pathogenic to rape ( Brassica napus ) was assessed in different systems by fluorescence microscopy using fluorescein diacetate. The influence of the culture's age and the size of the microsclerotia on germination percentages was assessed in water, mineral salts solution and mineral salts solution plus sucrose for 3 V. dahliae isolates. Large microsclerotia germinated better than smaller ones. The microsclerotia of 2 isolates showed decreased germination percentages with culture age over a 4–11-week period. Microsclerotial germination percentages were always higher in mineral salts solution plus sucrose than in mineral salts solution alone or water. In a sand culture system with the intact rape plant, microsclerotial germination percentages were high close to the root and decreased in a steep gradient to background levels within 5 mm from the root.     

Taste discrimination vs hedonic response to sucrose in coffee beverage. An interlaboratory study

Degree of liking, using a 17-point hedonic scale, and discriminationtaste thresholds, using a paired-comparison technique, weredetermined in a coffee beverage containing 0, 2.5, 5.0, 7.5,and 10.0% sucrose at laboratories in Brazil, Japan, Poland,Sweden and USA. Hedonic responses from the 122 subjects weresubdivided into four distinct sub-groups, according to differentpatterns as a function of sucrose concentration. Different frequenciesof these hedonic patterns resulted from the five laboratories.With few exceptions, repeated hedonic testing at the terminationof the experiment matched those from the beginning, indicatingstability of response during the lengthy study. No differenceswere observed in hedonic responses (nor in discrimination ability)between the male and female subjects at each laboratory. Discrimination thresholds at the five standard sucrose concentrations,and corresponding Weber ratios, were reported for the pooleddata within each laboratory. In general, the Weber ratios werehigher at the lower concentrations, indicating dependence ofdiscrimination upon the standard concentration. Notable differencesin discrimination ability were evident among the five laboratories,but were unrelated to degree of liking for sweetness in thecoffee. Subjects with low or with high degree of liking forall coffee samples, as well as those with increasing or decreasinghedonic responses as a function of sucrose concentration, discriminatedequally well among the concentration levels. The data from alllaboratories showed that ability to discriminate among sucroselevels and degree of liking for sucrose levels in coffee areindependent behavioral responses. ¹Present address: Department of Nutrition, Cornell University,Ithaca, New York, USA