A spontaneous CD8 T cell-dependent autoimmune disease to an antigen expressed under the human keratin 14 promoter

Using a previously described human keratin 14 (K14) promoter, we created mice expressing a peptide Ag (OVAp) in epithelial cells of the skin, tongue, esophagus, and thymus. Double transgenic mice that also express a TCR specific for this Ag (OT-I) showed evidence for Ag-driven receptor editing in the thymus. Surprisingly, such mice exhibited a severe autoimmune disease. In this work we describe the features of this disease and demonstrate that it is dependent on CD8 T cells. Consistent with the Ag expression pattern dictated by the human K14 promoter, an inflammatory infiltrate was observed in skin and esophagus and around bile ducts of the liver. We also observed a high level of TNF-alpha in the serum. Given that Ag expression in the thymus induced development of T cells with dual TCR reactivity, and that dual-reactive cells have been suggested to have autoimmune potential, we tested whether they were a causal factor in the disease observed here. We found that OT-I/K14-OVAp animals on a recombinase-activating gene-deficient background still suffered from disease. In addition, OT-I animals expressing OVA broadly in all tissues under a different promoter did not experience disease, despite having a similar number of dual-specific T cells. Thus, in this model it would appear that dual-reactive T cells do not underlie autoimmune pathology. Finally, we extended these observations to a second transgenic system involving 2C TCR-transgenic animals expressing the SIY peptide Ag with the hK14 promoter. We discuss the potential relationship between autoimmunity and self-Ags that are expressed in stratified epithelium.     

Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

Background

Previous studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44^mapk) and ERK2 (p42^mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca²⁺ and Ca²⁺-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F_2α.

Results

Pretreatment of the cells with the Ca²⁺ chelators BAPTA-AM or EGTA, as well as the Ca²⁺ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca²⁺/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF_2α.

Conclusion

The present data indicate that Ca²⁺ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.     

Identification and characterization of two novel low-molecular-weight dual specificity phosphatases

We have cloned and characterized two novel human low molecular weight dual specificity phosphatases (LMW-DSPs). Both genes are expressed exclusively in the testis, but are not altered in any of several disease states examined. Transfection into COS cells indicates that both proteins are expressed in the nucleus and the cytoplasm. Both proteins are able to dephosphorylate the phosphotyrosine analog pNPP in vitro and can be inhibited by sodium orthovanadate. In vitro experiments also demonstrate that both DSPs can dephosphorylate single and diphosphorylated synthetic MAPK peptides, with preference for the phosphotyrosine and diphosphorylated forms over phosphothreonine. However, when co-transfected with MAPKs into COS cells, the novel DSPs exhibited no detectable in vivo activity against MAPKs under our conditions. Our data suggest that these novel LMW-DSPs might belong to a new subclass of testis-specific proteins that act independently of the MAPK signal transduction cascade and do not depend on N-terminal docking regions for substrate binding.     

Cooperativity and flexibility of cystic fibrosis transmembrane conductance regulator transmembrane segments participate in membrane localization of a charged residue

Polytopic protein topology is established in the endoplasmic reticulum (ER) by sequence determinants encoded throughout the nascent polypeptide. Here we characterize 12 topogenic determinants in the cystic fibrosis transmembrane conductance regulator, and identify a novel mechanism by which a charged residue is positioned within the plane of the lipid bilayer. During cystic fibrosis transmembrane conductance regulator biogenesis, topology of the C-terminal transmembrane domain (TMs 7-12) is directed by alternating signal (TMs 7, 9, and 11) and stop transfer (TMs 8, 10, and 12) sequences. Unlike conventional stop transfer sequences, however, TM8 is unable to independently terminate translocation due to the presence of a single charged residue, Asp(924), within the TM segment. Instead, TM8 stop transfer activity is specifically dependent on TM7, which functions both to initiate translocation and to compensate for the charged residue within TM8. Moreover, even in the presence of TM7, the N terminus of TM8 extends significantly into the ER lumen, suggesting a high degree of flexibility in establishing TM8 transmembrane boundaries. These studies demonstrate that signal sequences can markedly influence stop transfer behavior and indicate that ER translocation machinery simultaneously integrates information from multiple topogenic determinants as they are presented in rapid succession during polytopic protein biogenesis.     

Carbon and nitrogen distribution in the green algal lichens<Emphasis Type="Italic"> Hypogymnia physodes</Emphasis> and<Emphasis Type="Italic"> Platismatia glauca</Emphasis> in relation to nutrient supply

With the aim of understanding how some lichens can survive intensive fertilization we investigated two green algal ( Trebouxia) lichens, Hypogymnia physodes (L.) Nyl. and Platismatia glauca (L.) W. Culb., and compared control (Ctr), and intensively fertilized (F) thalli. We measured total N, proteins and amino acids to assess lichen N status. Chlorophyll a indicated photosynthetic capacity and photobiont mass, ergosterol the metabolic demands of the fungus, and chitin the fungal biomass. For carbon status we measured glucose, the photobiont ( Trebouxia) export product ribitol, and the mycobiont-specific carbohydrates arabitol and mannitol. The F-thalli had 2-3 times higher protein and N concentrations, 5-10 times higher chlorophyll a concentrations, while ergosterol and chitin were doubled. The ribitol concentrations were 4-5 times higher in the F-thalli, while the fungal carbohydrates did not increase to the same extent. The amino acid arginine had increased 60-fold. The F-thalli also had a relatively higher N investment in the photobiont in relation to mycobiont tissue compared to the Ctr-thalli, probably resulting in an increased capacity for carbon assimilation, most possibly required for maintaining the higher nutrient status of the F-thalli. Arginine accumulation possibly avoided toxic effects of accumulated NH4+, albeit binding a significant fraction of assimilated carbon.     

Origins and plasticity of neural crest cells and their roles in jaw and craniofacial evolution

The vertebrate head is a complex assemblage of cranial specializations, including the central and peripheral nervous systems, viscero- and neurocranium, musculature and connective tissue. The primary differences that exist between vertebrates and other chordates relate to their craniofacial organization. Therefore, evolution of the head is considered fundamental to the origins of vertebrates (Gans and Northcutt, 1983). The transition from invertebrate to vertebrate chordates was a multistep process, involving the formation and patterning of many new cell types and tissues. The evolution of early vertebrates, such as jawless fish, was accompanied by the emergence of a specialized set of cells, called neural crest cells which have long held a fascination for developmental and evolutionary biologists due to their considerable influence on the complex development of the vertebrate head. Although it has been classically thought that protochordates lacked neural crest counterparts, the recent identification and characterization of amphioxus and ascidian genes homologous to those involved in vertebrate neural crest development challenges this idea. Instead it suggests thatthe neural crest may not be a novel vertebrate cell population, but could have in fact originated from the protochordate dorsal midline epidermis. Consequently, the evolution of the neural crest cells could be reconsidered in terms of the acquisition of new cell properties such as delamination-migration and also multipotency which were key innovations that contributed to craniofacial development. In this review we discuss recent findings concerning the inductive origins of neural crest cells, as well as new insights into the mechanisms patterning this cell population and the subsequent influence this has had on craniofacial evolution.     

Effects of dietary carbohydrate on the development of obesity in heterozygous Zucker rats

Rats carrying one copy of the fa allele are predisposed to diet-induced metabolic disturbances which contribute to hyperinsulinemia, obesity and dyslipidemia. To investigate the role of dietary carbohydrate and fat in the development of these conditions, we fed 6-week old male heterozygous (fa/+) lean rats carbohydrate-free diets containing primarily saturated fat either ad libitum or pair-fed. These diets were compared to standard chow and to a high saturated fat mixed diet containing 10% energy from sucrose for 4 weeks. The carbohydrate-free diet resulted in significantly lower circulating glucose levels compared to all other groups (p = 0.006). Weight gain was negligible in the carbohydrate free groups compared to standard diet and 10% sucrose diet (p = 0.03). This was reflected in energy efficiency which was markedly reduced (90%) in the carbohydrate-free groups compared to the other groups (p = 0.04). Corresponding changes were noted in fat pad mass. The subscapular and epididymal fat pads were increased 42% and 44%, respectively, in animals consuming the 10% sucrose diet compared to all other groups (p < 0.01). Comparable changes in fatty acid synthase (FAS) mRNA were observed in response to the carbohydrate-free diet, which resulted in a 53% decrease in adipocyte FAS mRNA (p < 0.001). Addition of 10% sucrose to the diet completely reversed this effect resulting in a 69% increase in adipocyte FAS mRNA compared to the carbohydrate-free groups (p = 0.01). Similarly, hepatic FAS mRNA was elevated by 51% and 66% in the 10% sucrose and standard diet groups respectively, compared to the carbohydrate-free groups. Therefore, diets that contain minimal carbohydrate may minimize net lipid storage and adiposity.     

Selection of side-chain carbons in a high-molecular-weight, hydrophobic peptide using solid-state spectral editing methods

Solid-state spectral editing techniques have been used by others to simplify ¹³C CPMAS spectra of small organic molecules, synthetic organic polymers, and coals. One approach utilizes experiments such as cross-polarization-with-polarization-inversion and cross-polarization-with-depolarization to generate subspectra. This work shows that this particular methodology is also applicable to natural-abundance ¹³C CPMAS NMR studies of high-molecular-weight biopolymers. The editing experiments are demonstrated first with model peptides and then with -elastin, a high-molecular-weight peptidyl preparation obtained from the elastic fibers in mammalian tissue. The latter has a predominance of small, nonpolar residues, which is evident in the crowded aliphatic region of typical ¹³C CPMAS spectra. Spectral editing is particularly useful for simplifying the aliphatic region of the NMR spectrum of this elastin preparation.     

Computer-Aided Resolution of an Experimental Paradox in Bacterial Chemotaxis

Escherichia coli responds to its environment by means of a network of intracellular reactions which process signals from membrane-bound receptors and relay them to the flagellar motors. Although characterization of the reactions in the chemotaxis signaling pathway is sufficiently complete to construct computer simulations that predict the phenotypes of mutant strains with a high degree of accuracy, two previous experimental investigations of the activity remaining upon genetic deletion of multiple signaling components yielded several contradictory results (M. P. Conley, A. J. Wolfe, D. F. Blair, and H. C. Berg, J. Bacteriol. 171:5190–5193, 1989; J. D. Liu and J. S. Parkinson, Proc. Natl. Acad. Sci. USA 86:8703–8707, 1989). For example, “building up” the pathway by adding back CheA and CheY to a gutted strain lacking chemotaxis genes resulted in counterclockwise flagellar rotation whereas “breaking down” the pathway by deleting chemotaxis genes except cheA and cheY resulted in alternating episodes of clockwise and counterclockwise flagellar rotation. Our computer simulation predicts that trace amounts of CheZ expressed in the gutted strain could account for this difference. We tested this explanation experimentally by constructing a mutant containing a new deletion of the che genes that cannot express CheZ and verified that the behavior of strains built up from the new deletion does in fact conform to both the phenotypes observed for breakdown strains and computer-generated predictions. Our findings consolidate the present view of the chemotaxis signaling pathway and highlight the utility of molecularly based computer models in the analysis of complex biochemical networks.