Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice

Rotavirus is the most important cause of infantile gastroenteritis. Since in vivo mucosal responses to a rotavirus infection thus far have not been extensively studied, we related viral replication in the murine small intestine to alterations in mucosal structure, epithelial cell homeostasis, cellular kinetics, and differentiation. Seven-day-old suckling BALB/c mice were inoculated with 2 x 10(4) focus-forming units of murine rotavirus and were compared to mock-infected controls. Diarrheal illness and viral shedding were recorded, and small intestinal tissue was evaluated for rotavirus (NSP4 and structural proteins)- and enterocyte-specific (lactase, SGLT1, and L-FABP) mRNA and protein expression. Morphology, apoptosis, proliferation, and migration were evaluated (immuno)histochemically. Diarrhea was observed from days 1 to 5 postinfection, and viral shedding was observed from days 1 to 10. Two peaks of rotavirus replication were observed at 1 and 4 days postinfection. Histological changes were characterized by the accumulation of vacuolated enterocytes. Strikingly, the number of vacuolated cells exceeded the number of cells in which viral replication was detectable. Apoptosis and proliferation were increased from days 1 to 7, resulting in villous atrophy. Epithelial cell turnover was significantly higher (<4 days) than that observed in controls (7 days). Since epithelial renewal occurred within 4 days, the second peak of viral replication was most likely caused by infection of newly synthesized cells. Expression of enterocyte-specific genes was downregulated in infected cells at mRNA and protein levels starting as early as 6 h after infection. In conclusion, we show for the first time that rotavirus infection induces apoptosis in vivo, an increase in epithelial cell turnover, and a shutoff of gene expression in enterocytes showing viral replication. The shutoff of enterocyte-specific gene expression, together with the loss of mature enterocytes through apoptosis and the replacement of these cells by less differentiated dividing cells, likely leads to a defective absorptive function of the intestinal epithelium, which contributes to rotavirus pathogenesis.     

Functional symmetries in the schmelzmuster and morphology of rootless rodent molars

Morphology and schmelzmuster of rootless cheek teeth of 25 extant rodent genera were studied in relation to jaw movement. A differentiation between leading and trailing edges is observed regularly in enamel thickness and schmelzmuster. Similarities between antagonists are interpreted as 'functional symmetries'. Differences in the enamel thickness, the schmelzmuster and orientation of cutting edges are controlled by functional and phylogenetic constraints. The heterogenous sample allows discrimination between these two constraints. The most obvious functional constraint leads to the almost regular occurrence of radial enamel on the push sides of cutting edges. The degree of functional symmetry seems to be determined by phylogenetic limitations.     

Pheomelanin as a Binding Site for Drugs and Chemicals

Certain drugs and chemicals, such as chloroquine, chlorpromazine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), are bound to melanin and retained in pigment cells for long periods. This specific retention in pigmented tissues can cause adverse effects in the skin, eye, inner ear, and pigmented nerve cells of the substantia nigra of the brain. To date, all studies have been focused on eu- and neuromelanin. In the present study, we show that chloroquine, chlorpromazine, chlomipramine, paraquat, acridine orange, and nickel, which are bound to eumelanin, also bind to synthetic pheomelanin, but the binding to pheomelanin is lower. The binding varied with the cysteine content and pH, and the results indicate that the binding is complex and includes ionic interactions. In addition, we have shown that these substances also bind to synthetic thiourea-containing melanin, but to quite a low extent. We also present a microautoradiographic study on the binding of ¹⁴C-chloroquine to natural pheomelanin in vivo in yellow mice C57BL (A^y/a). Black (C57/BL) and albino (NMRI) mice were used as controls. The autoradiography demonstrated a pronounced uptake of chloroquine in the hair follicles and the dermal melanocytes in the ear of yellow mice, which was comparable to the corresponding accumulation of label in black mice. In the albino mouse, the uptake was lower and more homogeneously distributed in the skin. These results suggest that the toxicologi-cal risks of melanin-related adverse effects are applicable to persons with a high content of pheomelanin in the skin and hair.     

Respiratory allergy to Blomia tropicalis: Immune response in four syngeneic mouse strains and assessment of a low allergen-dose,short-term experimental model

Background

The dust mite Blomia tropicalis is an important source of aeroallergens in tropical areas. Although a mouse model for B. tropicalis extract (BtE)-induced asthma has been described, no study comparing different mouse strains in this asthma model has been reported. The relevance and reproducibility of experimental animal models of allergy depends on the genetic background of the animal, the molecular composition of the allergen and the experimental protocol.

Objectives

This work had two objectives. The first was to study the anti-B. tropicalis allergic responses in different mouse strains using a short-term model of respiratory allergy to BtE. This study included the comparison of the allergic responses elicited by BtE with those elicited by ovalbumin in mice of the strain that responded better to BtE sensitization. The second objective was to investigate whether the best responder mouse strain could be used in an experimental model of allergy employing relatively low BtE doses.

Methods

Groups of mice of four different syngeneic strains were sensitized subcutaneously with 100 μg of BtE on days 0 and 7 and challenged four times intranasally, at days 8, 10, 12, and 14, with 10 μg of BtE. A/J mice, that were the best responders to BtE sensitization, were used to compare the B. tropicalis-specific asthma experimental model with the conventional experimental model of ovalbumin (OVA)-specific asthma. A/J mice were also sensitized with a lower dose of BtE.

Results

Mice of all strains had lung inflammatory-cell infiltration and increased levels of anti-BtE IgE antibodies, but these responses were significantly more intense in A/J mice than in CBA/J, BALB/c or C57BL/6J mice. Immunization of A/J mice with BtE induced a more intense airway eosinophil influx, higher levels of total IgE, similar airway hyperreactivity to methacholine but less intense mucous production, and lower levels of specific IgE, IgG1 and IgG2 antibodies than sensitization with OVA. Finally, immunization with a relatively low BtE dose (10 μg per subcutaneous injection per mouse) was able to sensitize A/J mice, which were the best responders to high-dose BtE immunization, for the development of allergy-associated immune and lung inflammatory responses.

Conclusions

The described short-term model of BtE-induced allergic lung disease is reproducible in different syngeneic mouse strains, and mice of the A/J strain was the most responsive to it. In addition, it was shown that OVA and BtE induce quantitatively different immune responses in A/J mice and that the experimental model can be set up with low amounts of BtE.     

Two-rung model of a left-handed beta-helix for prions explains species barrier and strain variation in transmissible spongiform encephalopathies

In this study, a new beta-helical model is proposed that explains the species barrier and strain variation in transmissible spongiform encephalopathies. The left-handed beta-helix serves as a structural model that can explain the seeded growth characteristics of beta-sheet structure in PrP(Sc) fibrils. Molecular dynamics simulations demonstrate that the left-handed beta-helix is structurally more stable than the right-handed beta-helix, with a higher beta-sheet content during the simulation and a better distributed network of inter-strand backbone-backbone hydrogen bonds between parallel beta-strands of different rungs. Multiple sequence alignments and homology modelling of prion sequences with different rungs of left-handed beta-helices illustrate that the PrP region with the highest beta-helical propensity (residues 105-143) can fold in just two rungs of a left-handed beta-helix. Even if no other flanking sequence participates in the beta-helix, the two rungs of a beta-helix can give the growing fibril enough elevation to accommodate the rest of the PrP protein in a tight packing at the periphery of a trimeric beta-helix. The folding of beta-helices is driven by backbone-backbone hydrogen bonding and stacking of side-chains in adjacent rungs. The sequence and structure of the last rung at the fibril end with unprotected beta-sheet edges selects the sequence of a complementary rung and dictates the folding of the new rung with optimal backbone hydrogen bonding and side-chain stacking. An important side-chain stack that facilitates the beta-helical folding is between methionine residues 109 and 129, which explains their importance in the species barrier of prions. Because the PrP sequence is not evolutionarily optimised to fold in a beta-helix, and because the beta-helical fold shows very little sequence preference, alternative alignments are possible that result in a different rung able to select for an alternative complementary rung. A different top rung results in a new strain with different growth characteristics. Hence, in the present model, sequence variation and alternative alignments clarify the basis of the species barrier and strain specificity in PrP-based diseases.     

Glutamine synthetase in the phloem plays a major role in controlling proline production

To inhibit expression specifically in the phloem, a 274-bp fragment of a cDNA (Gln1-5) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn superoxide dismutase promoter of Nicotiana plumbaginifolia. After Agrobacterium-mediated transformation, two transgenic N. tabacum lines exhibiting reduced levels of GS1 mRNA and GS activity in midribs, stems, and roots were obtained. Immunogold labeling experiments allowed us to verify that the GS protein content was markedly decreased in the phloem companion cells of transformed plants. Moreover, a general decrease in proline content in the transgenic plants in comparison with wild-type tobacco was observed when plants were forced to assimilate large amounts of ammonium. In contrast, no major changes in the concentration of amino acids used for nitrogen transport were apparent. A (15)NH(4)(+)-labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was directly related to glutamine availability. After 2 weeks of salt treatment, the transgenic plants had a pronounced stress phenotype, consisting of wilting and bleaching in the older leaves. We conclude that GS in the phloem plays a major role in regulating proline production consistent with the function of proline as a nitrogen source and as a key metabolite synthesized in response to water stress.