IL-7 exacerbates chronic colitis with expansion of memory IL-7Rhigh CD4+ mucosal T cells in mice

We have previously demonstrated that mucosal CD4(+) T cells expressing high levels of IL-7 receptor (IL-7R(high)) are pathogenic cells responsible for chronic colitis. Here we investigate whether IL-7 is directly involved in the expansion of IL-7R(high) memory CD4(+) mucosal T cells and the exacerbation of colitis. We first showed that CD4(+) lamina propria lymphocytes (LPLs) from wild-type, T cell receptor-alpha-deficient (TCR-alpha(-/-)), and recombinase-activating gene (RAG)-2(-/-)-transferred mice with or without colitis showed phenotypes of memory cells, but only CD4(+) LPLs from colitic mice showed IL-7R(high). In vitro stimulation by IL-7, but not by IL-15 and thymic stromal lymphopoietin, enhanced significant proliferative responses and survival of colitic CD4(+), but not normal CD4(+) LPLs. Importantly, in vivo administration of IL-7 mice accelerated the expansion of IL-7R(high) memory CD4(+) LPLs and thereby exacerbated chronic colitis in RAG-2(-/-) mice transferred with CD4(+) LPLs from colitic TCR-alpha(-/-) mice. Conversely, the administration of anti-IL-7R monoclonal antibody significantly inhibited the development of TCR-alpha(-/-) colitis with decreased expansion of CD4(+) LPLs. Collectively, the present data indicate that IL-7 is essential for the expansion of pathogenic memory CD4(+) T cells under pathological conditions. Therefore, therapeutic approaches targeting the IL-7R pathway may be feasible in the treatment of human inflammatory bowel disease.     

Design and properties of [Met35(O)]Aβ42-lactam(Asp23/Lys28) possessing a lactam tether as a salt-bridge surrogate

A characteristic feature of higher-order structures of amyloid β peptide (Aβ) aggregates observed in Alzheimer disease is the salt-bridge between the side-chains of Asp²³ (carboxylate) and Lys²⁸ (ammonium). We synthesized an [Met³⁵(O)]Aβ42 possessing a covalently bound lactam tether as an Asp²³/Lys²⁸ salt-bridge surrogate (compound 3). The lactam tether of 3 markedly promoted the formation of stable protofibril-like species that exhibited amyloidogenic properties such as a cross-β-sheet structure and cytotoxicity. This finding is consistent with reports that the Asp²³/Lys²⁸ salt-bridge of Aβ42 is transiently formed in aggregation intermediates.     

Age related increased threshold for electroshock seizure in BDF1 mice

The thresholds for inducing the minimal and maximal electroshock seizures were examined in relation to age in BDF1 mice of both sexes. The 50 percent effective intensities for the maximal electroshock seizure (tonic hindlimb extensor component) were lowest in the youngest age groups (6-month-old) for both male (10.68 mA) and female (9.18 mA) animals. The threshold increased with age and became significantly higher at 24 months (14.00 mA, 12.70 mA for male and female mice respectively). There was also a further increase in threshold at 30 months for both sexes. Similarly, the threshold for inducing the minimal seizure also increased with age but the differences in mean threshold levels between the youngest and oldest groups were much smaller in comparison to the maximal seizure. It was concluded that the threshold for inducing electroshock seizures significantly increases with age in mice of both sexes.     

Endotoxin of Pseudomonas pseudomallei detected by the body weight-decreasing reaction in mice and comparison of it with those of P. cepacia and P. aeruginosa.

The heat-treated whole cells, culture supernatants, and extracted endotoxin preparations of Pseudomonas pseudomallei were examined for endotoxin by the mouse body weight-decreasing (BWD) test. The experiments were conducted also with those of P. cepacia and P aeruginosa. Endotoxin was detected in all the samples of P. pseudomallei. Endotoxin of P. cepacia was detected in whole cells, but not in culture supernatant. The BWD activity of P. aeruginosa was 30 times as high as that of P. pseudomallei. This result was confirmed by the experiments with endotoxin preparations. In the limulus amebocyte lysate gelation (LAL) test, however, the endotoxin preparations of the two species showed the same level of activity.     

Microtubule bundling by tau proteins in vivo: analysis of functional domains.

Tau varies both in the N-terminal region (three types) and in the C-terminal repeated microtubule binding domain (two types), generating six isoforms through alternative splicing. To understand the differences between the isoforms and to determine which domains are important for microtubule bundling, we performed transfection studies on fibroblasts using tau isoforms and deletion mutants to quantify their ability to bundle microtubules. By comparing the isoforms, we found that a longer N-terminal region induced microtubule bundling more efficiently, but changes in the microtubule binding domain did not. Mutants lacking the proline rich region or the repeated domain did not bind to microtubules. Although all the other mutants could bind to and bundle microtubules, deletion in the N-terminal neutral region or the first half of the C-terminal tail caused a significant decrease in microtubule bundling, indicating the importance of these regions in microtubule bundling.     

Purification and Properties of Extracellular Amylase from the Hyperthermophilic Archaeon Thermococcus profundus DT5432

A hyperthermophilic archaeon, Thermococcus profundus DT5432, produced extracellular thermostable amylases. One of the amylases (amylase S) was purified to homogeneity by ammonium sulfate precipitation, DEAE-Toyopearl chromatography, and gel filtration on Superdex 200HR. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 5.5 to 6.0 and was stable in the range of pH 5.9 to 9.8. The optimum temperature for the activity was 80(deg)C. Half-life of the enzyme was 3 h at 80(deg)C and 15 min at 90(deg)C. Thermostability of the enzyme was enhanced in the presence of 5 mM Ca(sup2+) or 0.5% soluble starch at temperatures above 80(deg)C. The enzyme activity was inhibited in the presence of 5 mM iodoacetic acid or 1 mM N-bromosuccinimide, suggesting that cysteine and tryptophan residues play an important role in the catalytic action. The amylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen to produce maltose and maltotriose of (alpha)-configuration as the main products. Smaller amounts of larger maltooligosaccharides were also produced with a trace amount of glucose. Pullulan; (alpha)-, (beta)-, and (gamma)-cyclodextrins; maltose; and maltotriose were not hydrolyzed.     

Molecular Evolution of the Photolyase–Blue-Light Photoreceptor Family

The photolyase–blue-light photoreceptor family is composed of cyclobutane pyrimidine dimer (CPD) photolyases, (6-4) photolyases, and blue-light photoreceptors. CPD photolyase and (6-4) photolyase are involved in photoreactivation for CPD and (6-4) photoproducts, respectively. CPD photolyase is classified into two subclasses, class I and II, based on amino acid sequence similarity. Blue-light photoreceptors are essential light detectors for the early development of plants. The amino acid sequence of the receptor is similar to those of the photolyases, although the receptor does not show the activity of photoreactivation. To investigate the functional divergence of the family, the amino acid sequences of the proteins were aligned. The alignment suggested that the recognition mechanisms of the cofactors and the substrate of class I CPD photolyases (class I photolyases) are different from those of class II CPD photolyases (class II photolyases). We reconstructed the phylogenetic trees based on the alignment by the NJ method and the ML method. The phylogenetic analysis suggested that the ancestral gene of the family had encoded CPD photolyase and that the gene duplication of the ancestral proteins had occurred at least eight times before the divergence between eubacteria and eukaryotes. Received: 23 October 1996 / Accepted: 1 April 1997