Ultrastructural localization of carbohydrates in Reichert's membrane of the mouse

In the present investigation, we examined the role of trophoblast and parietal endoderm cells in the synthesis of carbohydrate-containing components of Reichert's membrane. To eliminate the function of Reichert's membrane as a filter between maternal and embryonal tissues we carried out our examination under in vitro conditions. Parietal yolk sac from mouse embryos on day 9 post coitum (p.c.) were cultivated for 0 to 5 days. Because tannic acid enables a complex formation between carbohydrates and osmium we chose the fixation with this acid for the ultrastructural study. Electron microscopy showed that for assembly of Reichert's membrane, trophoblast cells produce and then release components that were detected as tannic acid-positive granules both in the Reichert's membrane and in the vacuoles of the trophoblast cells. To localize specific carbohydrates we used postembedding-gold-lectin histochemistry on LR-Gold^R-embedded tissues. Strong binding sites for the lectins WGA (Triticum vulgare), RCA I (Ricinus communis) and Con A (Canavalia ensiformis) were observed in Reichert's membrane and trophoblast cells but not in the parietal endoderm cells. The LTA (Lotus tetragonolobus)-binding pattern was positive in the membrane and its adjacent cells but that of the LFA (Limax flavus) was negative in the parietal endoderm cells and very weak in Reichert's membrane and trophoblast cells. Our results demonstrate that trophoblast cells are involved in the construction of Reichert's membrane through the production and release of specific glycoconjugates.     

Influence of GrpE on DnaK-substrate interactions

The DnaK chaperone of Escherichia coli assists protein folding by an ATP-dependent interaction with short peptide stretches within substrate polypeptides. This interaction is regulated by the DnaJ and GrpE co-chaperones, which stimulate ATP hydrolysis and nucleotide exchange by DnaK, respectively. Furthermore, GrpE has been claimed to trigger substrate release independent of its role as a nucleotide exchange factor. However, we show here that GrpE can accelerate substrate release from DnaK exclusively in the presence of ATP. In addition, GrpE prevented the association of peptide substrates with DnaK through an activity of its N-terminal 33 amino acids. A ternary complex of GrpE, DnaK, and a peptide substrate could be observed only when the peptide binding to DnaK precedes GrpE binding. Furthermore, we demonstrate that GrpE slows down the release of a protein substrate, sigma(32), from DnaK in the absence of ATP. These findings suggest that the ATP-triggered dissociation of GrpE and substrates from DnaK occurs in a concerted fashion.     

Retinal projections in European Salamandridae

Summary The retinal projections to the brain were studied in three species of European Salamandridae using anterograde transport of horseradish peroxidase and autoradiography. The results obtained were basically identical for all species and confirmed earlier findings on the fiber supply to the preoptic nucleus and the basal optic neuropil. In the anterior thalamus projections to three distinct terminal fields are clearly visible: (i) the diffusely stained corpus geniculatum thalamicum, (ii) the neuropil of Bellonci, pars lateralis, and (iii) a dorsomedial terminal field, the neuropil of Bellonci, pars medialis. Caudal to these terminal fields is an almost terminal-free region, the lateral neuropil. In the posterior thalamus a medial terminal field, the uncinate field, and a laterally located terminal field, the posterior thalamic neuropil, are distinguishable. The tectum opticum displays as many as four dense layers of retinofugal fibers and terminals in the rostral part and, in addition, a more densely stained strip of neuropil running from rostral to caudal over the tectum. The extent of ipsilateral fibers is greater than previously reported in other urodele species. They supply the medial and the lateral parts of the neuropil of Bellonci, the uncinate field, and reach the tectum opticum via the medial optic tract. Further, they form terminals in the innermost optic fiber layer throughout the rostral half of the ipsilateral tectum. A small proportion of ipsilateral fibers contributes very sparsely to all other thalamic terminal fields, leaving only the caudal part of the tectum and several layers of the rostral tectum completely free of a direct retinofugal fiber supply.     

Subunit composition and functional properties of G-protein heterotrimers on rat chromaffin granules

Heterotrimeric G-proteins at the plasma membrane serve as switches between heptahelical receptors and intracellular signal cascades. Likewise endomembrane associated G-proteins may transduce signals from intracellular compartments provided they consist of a functional trimer. Using quantitative immunoelectron microscopy we found heterotrimeric G-protein subunits Galpha2, Galpha(q/11), Gbeta2 and Gbeta5 to reside on secretory granules in chromaffin cells of rat adrenal glands.Thus rat chromaffin granules are equipped with functional G-proteins that consist of a specific alpha-, beta- and probably gamma-subunit combination. Serotonin uptake into a crude rat chromaffin granule preparation was inhibited by activated Galphao2 (10 nM) to nearly the same extent as by GMppNp (50 microM) whereas GDPbetaS was ineffective. The data support the idea that vesicular G-proteins directly regulate the transmitter content of secretory vesicles. In this respect Galphao2 appears to be the main regulator of vesicular momoamine transporter activity.     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Square-cut: a segmentation algorithm on the basis of a rectangle shape

We present a rectangle-based segmentation algorithm that sets up a graph and performs a graph cut to separate an object from the background. However, graph-based algorithms distribute the graph's nodes uniformly and equidistantly on the image. Then, a smoothness term is added to force the cut to prefer a particular shape. This strategy does not allow the cut to prefer a certain structure, especially when areas of the object are indistinguishable from the background. We solve this problem by referring to a rectangle shape of the object when sampling the graph nodes, i.e., the nodes are distributed non-uniformly and non-equidistantly on the image. This strategy can be useful, when areas of the object are indistinguishable from the background. For evaluation, we focus on vertebrae images from Magnetic Resonance Imaging (MRI) datasets to support the time consuming manual slice-by-slice segmentation performed by physicians. The ground truth of the vertebrae boundaries were manually extracted by two clinical experts (neurological surgeons) with several years of experience in spine surgery and afterwards compared with the automatic segmentation results of the proposed scheme yielding an average Dice Similarity Coefficient (DSC) of 90.97±2.2%.     

Production of Autopolyploid Lowland Switchgrass Lines Through In Vitro Chromosome Doubling

Switchgrass is considered one of the most promising energy crops. However, breeding of elite switchgrass cultivars is required to meet the challenges of large scale and sustainable biomass production. As a native perennial adapted to North America, switchgrass has lowland and upland ecotypes, where most lowland ecotypes are tetraploid (2n?=?4x?=?36), and most upland ecotypes are predominantly octoploid (2n?=?8x?=?72). Hybridization between lowland and upland switchgrass plants could identify new cultivars with heterosis. However, crossing between tetraploid and octoploid switchgrass is rare in nature. Therefore, in order to break down the cross incompatibility barrier between tetraploid lowland and octoploid upland switchgrass lines, we developed autoployploid switchgrass lines from an anueploid lowland cv. Alamo. In this study, colchicine was used in liquid and solid mediums to chemically induce chromosome doubling in embryogenic calli derived from cv. Alamo. Thirteen autopolyploid switchgrass lines were regenerated from seedlings and identified using flow cytometry. The autoplyploid switchgrass plants exhibited increased stomata aperture and stem size in comparison with the cv. Alamo. The most autooplyploid plants were regenerated from switchgrass calli that were treated with 0.04 % colchicine in liquid medium for 13 days. One autopolyploid switchgrass line, VT8-1, was successfully crossed to the octoploid upland cv. Blackwell. The autoployploid and the derived inter-ecotype hybrids were confirmed by in situ hybridization and molecular marker analysis. Therefore, the results of this study show that an autopolyploid, generated by chemically induced chromosome doubling of lowland cv. Alamo, is cross compatible with upland octoploid switchgrass cultivars. The outcome of this study may have significant applications in switchgrass hybrid breeding.     

Anaerobic Co-Culture of Mesenchymal Stem Cells and Anaerobic Pathogens - A New In Vitro Model System

Background

Human mesenchymal stem cells (hMSCs) are multipotent by nature and are originally isolated from bone marrow. In light of a future application of hMSCs in the oral cavity, a body compartment with varying oxygen partial pressures and an omnipresence of different bacterial species i.e. periodontitis pathogens, we performed this study to gain information about the behavior of hMSC in an anaerobic system and the response in interaction with oral bacterial pathogens.

Methodology/Principal Findings

We established a model system with oral pathogenic bacterial species and eukaryotic cells cultured in anaerobic conditions. The facultative anaerobe bacteria Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were studied. Their effects on hMSCs and primary as well as permanent gingival epithelial cells (Ca9-22, HGPEC) were comparatively analyzed. We show that hMSCs cope with anoxic conditions, since 40% vital cells remain after 72 h of anaerobic culture. The Ca9-22 and HGPEC cells are significantly more sensitive to lack of oxygen. All bacterial species reveal a comparatively low adherence to and internalization into hMSCs (0.2% and 0.01% of the initial inoculum, respectively). In comparison, the Ca9-22 and HGPEC cells present better targets for bacterial adherence and internalization. The production of the pro-inflammatory chemokine IL-8 is higher in both gingival epithelial cell lines compared to hMSCs and Fusobacterium nucleatum induce a time-dependent cytokine secretion in both cell lines. Porphyromonas gingivalis is less effective in stimulating secretion of IL-8 in the co-cultivation experiments.

Conclusions/significance

HMSCs are suitable for use in anoxic regions of the oral cavity. The interaction with local pathogenic bacteria does not result in massive pro-inflammatory cytokine responses. The test system established in this study allowed further investigation of parameters prior to set up of oral hMSC in vivo studies.     

Temperature-dependent calcium-induced calcium release via InsP3 receptors in mouse olfactory ensheathing glial cells

Cooling can induce Ca(2+) signaling via activation of temperature-sensitive ion channels such as TRPM8, TRPA1 and ryanodine receptor channels. Here we have studied the mechanism of cooling-evoked Ca(2+) signaling in mouse olfactory ensheathing cells (OECs), a specialized type of glial cells in the olfactory nerve layer of the olfactory bulb. Reducing the temperature from above 30°C to 28°C and below triggered Ca(2+) transients that persisted in the absence of external Ca(2+), but were suppressed after Ca(2+) store depletion by cyclopiazonic acid. Cooling-evoked Ca(2+) transients were present in mice deficient of TRPM8 and TRPA1, and were not inhibited by ryanodine receptor antagonists. Inhibition of InsP(3) receptors with 2-APB and caffeine entirely blocked cooling-evoked Ca(2+) transients. Moderate Ca(2+) increases, as evoked by flash photolysis of NP-EGTA (caged Ca(2+)) and cyclopiazonic acid, triggered InsP(3) receptor-mediated Ca(2+) release at 22°C, but not at 31°C. The results suggest that InsP(3) receptors mediate Ca(2+)-induced Ca(2+) release in OECs, and that this Ca(2+) release is temperature-sensitive and can be suppressed at temperatures above 28°C.