Expression of ATP7B in normal human liver

ATP7B is a copper transporting P-type ATPase, also known as Wilson disease protein, which plays a key role in copper distribution inside cells. Recent experimental data in cell culture have shown that ATP7B putatively serves a dual function in hepatocytes: when localized to the Golgi apparatus, it has a biosynthetic role, delivering copper atoms to apoceruloplasmin; when the hepatocytes are under copper stress, ATP7B translocates to the biliary pole to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile. The above data on ATP7B localization have been mainly obtained in tumor cell systems in vitro. The aim of the present work was to assess the presence and localization of the Wilson disease protein in the human liver. We tested immunoreactivity for ATP7B in 10 human liver biopsies, in which no significant pathological lesion was found using a polyclonal antiserum specific for ATP7B. In the normal liver, immunoreactivity for ATP7B was observed in hepatocytes and in biliary cells. In the hepatocytes, immunoreactivity for ATP7B was observed close to the plasma membrane, both at the sinusoidal and at the biliary pole. In the biliary cells, ATP7B was localized close to the cell membrane, mainly concentrated at the basal pole of the cells. The data suggest that, in human liver, ATP7B is localized to the plasma membrane of both hepatocytes and biliary epithelial cells.     

Cholesterol dictates the freedom of EGF receptors and HER2 in the plane of the membrane

The flow of information through the epidermal growth factor receptor (EGFR) is shaped by molecular interactions in the plasma membrane. The EGFR is associated with lipid rafts, but their role in modulating receptor mobility and subsequent interactions is unclear. To investigate the role of nanoscale rafts in EGFR dynamics, we used single-molecule fluorescence imaging to track individual receptors and their dimerization partner, human epidermal growth factor receptor 2 (HER2), in the membrane of human mammary epithelial cells. We found that the motion of both receptors was interrupted by dwellings within nanodomains. EGFR was significantly less mobile than HER2. This difference was likely due to F-actin because its depolymerization led to similar diffusion patterns between the EGFR and HER2. Manipulations of membrane cholesterol content dramatically altered the diffusion pattern of both receptors. Cholesterol depletion led to almost complete confinement of the receptors, whereas cholesterol enrichment extended the boundaries of the restricted areas. Interestingly, F-actin depolymerization partially restored receptor mobility in cholesterol-depleted membranes. Our observations suggest that membrane cholesterol provides a dynamic environment that facilitates the free motion of EGFR and HER2, possibly by modulating the dynamic state of F-actin. The association of the receptors with lipid rafts could therefore promote their rapid interactions only upon ligand stimulation.     

Design and evaluation of a novel flow chamber for measuring cell adhesion to absorbable polymer films

There is great interest in improving cellular attachment to synthetic materials, particularly for developing small diameter tissue-engineered vascular grafts. However, limited research has been conducted to evaluate the adhesion characteristics of different cell types to absorbable substrates. Tissue engineered vessels typically fail as a result of delamination of the endothelial cell layer when exposed to fluid or blood flow. The focus of this research was to design and evaluate a flow chamber, using fibroblasts, smooth muscle cells, and endothelial cells, to probe the bounds of the system. A flow chamber was designed and fabricated to compare the relative adhesion characteristics of cells to absorbable polymer films. A preliminary investigation of mouse fibroblast (3T3M) adhesion to semicrystalline poly-L-lactide (PLL) films was conducted to determine general operating specifications. Cell coverage on films was evaluated using a live-dead assay and image analysis; following exposure to flow, tests were similarly conducted. Based on these results, additional studies were conducted to compare the adhesion of rat aortic smooth muscle cells (SMC) and endothelial cells (EC) on PLL films.     

Identification and characterization of structural domains of human ERp57: association with calreticulin requires several domains

The amino acid sequence of ERp57, which functions in the endoplasmic reticulum together with the lectins calreticulin and calnexin to achieve folding of newly synthesized glycoproteins, is highly similar to that of protein disulfide isomerase (PDI), but they have their own distinct roles in protein folding. We have characterized the domain structure of ERp57 by limited proteolysis and N-terminal sequencing and have found it to be similar but not identical to that of PDI. ERp57 had three major protease-sensitive regions, the first of which was located between residues 120 and 150, the second between 201 and 215, and the third between 313 and 341, the data thus being consistent with a four-domain structure abb'a'. Recombinant expression in Escherichia coli was used to verify the domain boundaries. Each single domain and a b'a' double domain could be produced in the form of soluble, folded polypeptides, as verified by circular dichroism spectra and urea gradient gel electrophoresis. When the ability of ERp57 and its a and a' domains to fold denatured RNase A was studied by electrospray mass analyses, ERp57 markedly enhanced the folding rate at early time points, although less effectively than PDI, but was an ineffective catalyst of the overall process. The a and a' domains produced only minor, if any, increases in the folding rate at the early stages and no increase at the late stages. Interaction of the soluble ERp57 domains with the P domain of calreticulin was studied by chemical cross-linking in vitro. None of the single ERp57 domains nor the b'a' double domain could be cross-linked to the P domain, whereas cross-linking was obtained with a hybrid ERpabb'PDIa'c polypeptide but not with ERpabPDIb'a'c, indicating that multiple domains are involved in this protein-protein interaction and that the b' domain of ERp57 cannot be replaced by that of PDI.     

Inflorescence development in a new teosinte: Zea nicaraguensis (Poaceae)

Inflorescence development in a newly discovered teosinte, Zea nicaraguensis (Poaceae), from Nicaragua has been investigated using scanning electron microscopy (SEM). The SEM examination revealed that the pattern of both male and female inflorescence development was similar to previously described inflorescence in other Zea taxa. Branch primordia were initiated acropetally in a distichous pattern along the rachis of male and female inflorescences. Spikelet pair primordia bifurcated into pedicellate and sessile spikelet primordia. Predictably, pedicellate spikelet development was arrested and aborted in the female teosinte inflorescence. Organogenesis of functional spikelets and florets was similar to that previously described in maize and teosintes. The results were consistent with our hypothesis that both femininity and masculinity share a common mechanism of inflorescence development in Zea and Tripsacum and are in accord with a putative common mechanism of sex determination in the Andropogoneae. Interestingly, this population of teosinte, unique in its ability to grow in water-logged soils, showed a stable pattern of early inflorescence development. Our results also revealed the uncharacteristic presence of inflorescence polystichy in this population of Zea nicaraguensis. We propose this novel phenotypic variation raises the possibility that a domestic evolution of polystichy in maize was enabled by an occasional polystichous phenotypic in teosinte.     

Single-nucleotide polymorphism detection using nanomolar nucleotides and single-molecule fluorescence

We have exploited three methods for discriminating single-nucleotide polymorphisms (SNPs) by detecting the incorporation or otherwise of labeled dideoxy nucleotides at the end of a primer chain using single-molecule fluorescence detection methods. Good discrimination of incorporated vs free nucleotide may be obtained in a homogeneous assay (without washing steps) via confocal fluorescence correlation spectroscopy or by polarization anisotropy obtained from confocal fluorescence intensity distribution analysis. Moreover, the ratio of the fluorescence intensities on each polarization channel may be used directly to discriminate the nucleotides incorporated. Each measurement took just a few seconds and was done in microliter volumes with nanomolar concentrations of labeled nucleotides. Since the confocal volumes interrogated are approximately 1fL and the reaction volume could easily be lowered to nanoliters, the possibility of SNP analysis with attomoles of reagents opens up a route to very rapid and inexpensive SNP detection. The method was applied with success to the detections of SNPs that are known to occur in the BRCA1 and CFTR genes.     

Quality assessment of selected commercially available whitefly and aphid biological control agents in the United States

This study assessed the quality of three commercially available natural enemies used for pest management in greenhouses: the whitefly parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae), the aphid parasitoid Aphidius colemani Viereck (Hymenoptera: Braconidae), and the aphid predatory midge Aphidoletes aphidimlyza (Rondani) (Diptera: Cecidomyiidae). Shipment packaging was consistent for all natural enemies. However, there was high variability in delivery punctuality, product cost, and product information provided by each of the six selected companies. Product quantity, percentage of emergence upon arrival, percentage of total emergence, percentage of females, and percentage of flying insects were assessed using International Organization for Biological Control (IOBC) recommended procedures. The parameters with greatest variability between companies were percentage of emergence upon arrival (0.9-10.5%) and percentage of flying insects (35.4-85.0%) for E. formnosa; product quantity (623.3-833.8 aphid mummies), percentage of emergence upon arrival (6.1-41.2%) and percentage of females (51.1-54.8%) for A. colemani; and percentage of emergence upon arrival (0.0-7.7%) and percentage of females (54.6-76.2%) for A. aphlidimyza. Results are discussed in terms of the value to consumers and compared with IOBC standards.     

Phenotype of a herpes simplex virus type 1 mutant that fails to express immediate-early regulatory protein ICP0

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein ICP0 is required for efficient progression of infected cells into productive lytic infection, especially in low-multiplicity infections of limited-passage human fibroblasts. We have used single-cell-based assays that allow detailed analysis of the ICP0-null phenotype in low-multiplicity infections of restrictive cell types. The major conclusions are as follows: (i) there is a threshold input multiplicity above which the mutant virus replicates normally; (ii) individual cells infected below the threshold multiplicity have a high probability of establishing a nonproductive infection; (iii) such nonproductively infected cells have a high probability of expressing IE products at 6 h postinfection; (iv) even at 24 h postinfection, IE protein-positive nonproductively infected human fibroblast cells exceed the number of cells that lead to plaque formation by up to 2 orders of magnitude; (v) expression of individual IE proteins in a proportion of the nonproductively infected cells is incompletely coordinated; (vi) the nonproductive cells can also express early gene products at low frequencies and in a stochastic manner; and (vii) significant numbers of human fibroblast cells infected at low multiplicity by an ICP0-deficient virus are lost through cell death. We propose that in the absence of ICP0 expression, HSV-1 infected human fibroblasts can undergo a great variety of fates, including quiescence, stalled infection at a variety of different stages, cell death, and, for a minor population, initiation of formation of a plaque.     

Buoyancy studies in natural communities of square gas-vacuolate archaea in saltern crystallizer ponds

Background

Possession of gas vesicles is generally considered to be advantageous to halophilic archaea: the vesicles are assumed to enable the cells to float, and thus reach high oxygen concentrations at the surface of the brine.

Results

We studied the possible ecological advantage of gas vesicles in a dense community of flat square extremely halophilic archaea in the saltern crystallizer ponds of Eilat, Israel. We found that in this environment, the cells' content of gas vesicles was insufficient to provide positive buoyancy. Instead, sinking/floating velocities were too low to permit vertical redistribution.

Conclusion

The hypothesis that the gas vesicles enable the square archaea to float to the surface of the brines in which they live was not supported by experimental evidence. Presence of the vesicles, which are mainly located close to the cell periphery, may provide an advantage as they may aid the cells to position themselves parallel to the surface, thereby increasing the efficiency of light harvesting by the retinal pigments in the membrane.     

The Linker for Activation of B Cells (LAB)/Non-T Cell Activation Linker (NTAL) Regulates Triggering Receptor Expressed on Myeloid Cells (TREM)-2 Signaling and Macrophage Inflammatory Responses Independently of the Linker for Activation of T Cells

Triggering receptor expressed on myeloid cells-2 (TREM-2) is rapidly emerging as a key regulator of the innate immune response via its regulation of macrophage inflammatory responses. Here we demonstrate that proximal TREM-2 signaling parallels other DAP12-based receptor systems in its use of Syk and Src-family kinases. However, we find that the linker for activation of T cells (LAT) is severely reduced as monocytes differentiate into macrophages and that TREM-2 exclusively uses the linker for activation of B cells (LAB encoded by the gene Lat2^−/−) to mediate downstream signaling. LAB is required for TREM-2-mediated activation of Erk1/2 and dampens proximal TREM-2 signals through a novel LAT-independent mechanism resulting in macrophages with proinflammatory properties. Thus, Lat2^−/− macrophages have increased TREM-2-induced proximal phosphorylation, and lipopolysaccharide stimulation of these cells leads to increased interleukin-10 (IL-10) and decreased IL-12p40 production relative to wild type cells. Together these data identify LAB as a critical, LAT-independent regulator of TREM-2 signaling and macrophage development capable of controlling subsequent inflammatory responses.