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AbstractThe mechanical properties of the lunar soil, e.g. the bearing capacity and the deformation modulus, are of great importance for the design of the foundations of permanent lunar outposts. To measure these two parameters, the plate loading test (PLT) is a reliable and the most popular method on the Earth, but is not directly applicable on the lunar surface due to the difficulties in setting up the test equipment. An alternative and indirect method is the cone penetration test (CPT) mainly due to its simplicity in operation. The current study concentrates on the relationships between the two mechanical parameters obtained from PLTs and the penetration resistance obtained from CPTs. Both PLTs and CPTs were carried out on Tongji-1 lunar soil simulant (TJ-1 simulant) in a calibration chamber to establish their relationships and then on a large-scale man-made ground to validate the relationships. The test results show that: the link between the acceptable bearing capacity qa, the deformation modulus E0and the average penetration resistance qavgwas established and validated as: qa= 0.25/D0.63qavgand E0= 7.16qavgfor TJ-1 simulant ground. They can be used directly in the foundation design for lunar outposts.

Concanavalin A (Con A) is known to induce acute hepatitis that is mediated by activation of NKT- and T-cell and cytokine production in immunocompetent mice. The observation of Con A-induced autophagic cell death of hepatoma cells via a Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 mediated autophagic pathway made us re-evaluate the effect of Con A-induced hepatitis in mice. Con A was administrated intravenously to BABL/c, SCID, or SCID/NOD mice at doses of 20, 30 or 40 mg/kg, respectively, to induce acute hepatitis. The levels of hepatitis and autophagy induction were both analyzed. We found that Con A can induce acute hepatitis in SCID or SCID/NOD mice with a kinetics similar to that of BALB/c, but requiring a higher dose of Con A. No lymphocyte infiltrations were found in SCID or SCID/NOD mice, and the cytokine productions were different. An autophagy with microtubule-associated protein light chain 3-II conversion was demonstrated in the liver post-Con A injection in SCID/NOD mice. Due to the mannose/glucose-specific binding on cell membrane, Con A can induce a T-cell-independent acute hepatitis with autophagy in SCID/NOD mice.

Lectins are carbohydrate-binding proteins that can modulate cellular activity through cell membrane glycoproteins. A cell-based binding was set up to screen the lectin-like substances in natural products. Lectins, after binding to tumor cells, are detected with a biotinylated carbohydrate-polyacrylamide complex by ELISA. Sugar specificity can also be determined within the assay.

Treatment of U937 human monocyte-like cells with Streptococcus pyogenes led to an induction of apoptosis in these cells. A comparison between the wild-type strain and its isogenic protease-negative mutant indicated that the production of streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, caused a greater extent of apoptosis in U937 cells. Further study using purified SPE B showed that this protease alone could induce U937 cells to undergo apoptosis, which was characterized by morphologic changes, DNA fragmentation laddering on the gel, and an increase in the percentages of hypodiploid cells. The protease activity of SPE B was required for apoptosis to proceed, since treatment with cysteine protease inhibitor E64 or heat inactivation abrogated this death-inducing effect. The SPE B-induced apoptosis pathway was interleukin-1beta converting enzyme (ICE) family protease dependent. Further experiments showed that the phagocytic activity of U937 cells was reduced by SPE B. Treatment with E64 and heat inactivation both abrogated this phagocytosis-inhibitory effect. Taken together, the present data show that SPE B not only possesses the ability to induce apoptosis in monocytic cells but also helps bacteria to resist phagocytosis by host cells.

Internalization of group A streptococcus (GAS) by epithelial cells may have a role in causing invasive diseases. The purpose of this study was to examine the fate of GAS-infected epithelial cells. GAS has the ability to invade A-549 and HEp-2 cells. Both A-549 and HEp-2 cells were killed by infection with GAS. Epithelial cell death mediated by GAS was at least in part through apoptosis, as shown by changes in cellular morphology, DNA fragmentation laddering, and propidium iodide staining for hypodiploid cells. A total of 20% of A-549 cells and 11 to 13% of HEp-2 cells underwent apoptosis after 20 h of GAS infection, whereas only 1 to 2% of these cells exhibited spontaneous apoptosis. We further examined whether streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease produced by GAS, was involved in the apoptosis of epithelial cells. The speB isogenic mutants had less ability to induce cell death than wild-type strains. When A-549 cells were cocultured with the mutant and SPE B for 2 h, the percentage of apoptotic cells did not increase although the number of intracellular bacteria increased to the level of wild-type strains. In addition, apoptosis was blocked by cytochalasin D treatment, which interfered with cytoskeleton function. The caspase inhibitors Z-VAD.FMK, Ac-YVAD.CMK, and Ac-DEVD.FMK inhibited GAS-induced apoptosis. These results demonstrate for the first time that GAS induces apoptosis of epithelial cells and internalization is required for apoptosis. The caspase pathway is involved in GAS-induced apoptosis, and the expression of SPE B in the cells enhances apoptosis.

A C/EBP-like transcription factor, AGP/EBP, that binds to three distinct motifs in the 5'-flanking region of alpha 1-acid glycoprotein gene (AGP) has been identified. Here we report the cloning and properties of cDNA corresponding to mouse AGP/EBP. AGP/EBP and C/EBP share 87% amino acid sequence homology in the "leucine zipper" and its associated DNA-binding domains, while their sequences outside these domains and the sizes of their mRNAs are different. Unlike the limited expression of C/EBP in tissues and cells, AGP/EBP appears to be ubiquitously expressed in tissues like lung, spleen, kidney, heart, testis, and liver and cell lines like p388D1, 129P (hepatoma cell line of C3H/HeJ), FO (mouse myeloma), and L929. Antibody against cloned and expressed AGP/EBP which was raised in rabbits could recognize AGP/EBP from nuclear extract of a number of cells and tissues. On the basis of our findings about the structural relationship and the similarity of motif recognition, we propose that a family of C/EBP-like transcription factors exists.

The development of inbred strains of mutant mice has proven useful in ascribing specific gene functions to particular genetic loci within the regions and subregions of the H-2 complex. The B6.C-H-2bm12 (bm12) strain is of particular interest in that, compared to parental C57Bl/6Kh (B6) mice, it bears a presumptive single gene mutation altering the Ab beta chain encoded by the I-A subregion. Our data show that bm12 mice have gained the ability to respond to poly(Glu50Tyr50)(GT) and have lost the ability to make plaque-forming cell or delayed-type hypersensitivity responses to the closely related copolymer, poly(Glu60Ala30Tyr10)(GAT), although retaining the ability to mount a GAT-specific T cell proliferative response. This is in sharp contrast to the parental B6 strain, which is a GT nonresponder and a GAT responder. Thus, this study is the first to report the establishment of responder status as a consequence of mutation. Possible mechanisms accounting for the gain/loss of GT/GAT responsiveness in the context of a two-step helper T cell model are discussed.

Glucocorticoid can induce apoptosis of thymocytes, but its mechanism is not clear yet. In this study, we reported that dexamethasone-induced apoptosis was associated with intracellular alkalinization. Dexamethasone induced a higher percentage of apoptosis in 138 mM than in 50 mM NaCl, total abrogation of apoptosis was noted in NaCl-depleted culture medium. Highest apoptotic rate was observed in medium with pH 7.2, whereas it was partially and completely inhibited at pH 6.5 and pH 6.0, respectively. Intracellular pH was higher in pre-apoptotic thymocytes than non-apoptotic ones. The Na+ /H+ antiporter inhibitor of 5-(N,N'-dimethyl)-amiloride inhibited the dexamethasone-induced increase in pHi and apoptosis of thymocytes. Glucocorticoid antagonist RU486 also blocked the dexamethasone-induced effect. Furthermore, the apoptosis and increase in intracellular pH induced by dexamethasone were inhibited by cycloheximide, actinomycin D. It seems that intracellular pH is increased during the development of thymocyte apoptosis and inhibiting its increment would retard the rate of progression to cell death.

Poly(Glu50Tyr50) (GT) is not immunogenic in most inbred mouse strains. GT injection produces an I-J--bearing, GT-specific T-cell--derived suppressor factor (GT-TsF1) in H-2b,d,k haplotype mice. GT-TsF1 generates second-order suppressor T cells (Ts2) in H-2a,d,k haplotype mice. Here, we show that in order for GT-TsF1 to act, the recipient strain must express I-E molecules. This suggests that T cells are not the primary target of GT-TsF1. GT-TsF1 can be presented by Ia+ A20-2J B lymphoma cells. GT-TsF1 presentation is blocked by anti-I-E, but not by anti--I-A, mAb, whereas GAT presentation is blocked by anti-I-A, but not by anti--I-E, mAbs. These data suggest that I-J recognizes (or is recognized by) I-E. The existence and role of I-J molecules in immune regulation are discussed in light of these data.

Streptococcal pyrogenic exotoxin B (SPE B) is a cysteine protease produced by Streptococcus pyogenes. In this study, the differences in virulence between protease-positive clinical isolates and their protease-negative mutants were examined in a mouse model. Isogenic protease-negative mutants were constructed by homologous recombination, using integrational plasmids to disrupt the speB gene. These mutants caused less mortality and tissue damage than protease-positive strains when inoculated into BALB/c mice via air pouch, suggesting that SPE B cysteine protease plays an important role in the pathogenesis of S. pyogenes infection. Reconstitution of SPE B in the air pouches increased the mortality of mice receiving the speB mutant strain. Infiltrated cell numbers in the exudates from the air pouches of mice infected with SPE B-producing S. pyogenes were higher than those from mice infected with protease-negative mutants at 12 h. However, despite pretreatment with vinblastine to deplete neutrophils, injection of protease-positive bacteria still resulted in severe tissue injury, indicating that neutrophil infiltration may not be the major factor involved in SPE B-enhanced tissue damage. The role of SPE B was further confirmed by demonstrating that SPE B immunization of mice conferred protection from challenge with a lethal dose of protease-positive bacteria.

Cysteine protease of group A streptococci (GAS) is considered an important virulence factor. However, its role in invasiveness of GAS has not been investigated. We demonstrated in this study that two strains of protease-producing GAS had the ability to invade A-549 human respiratory epithelial cells. Isogenic protease mutants were constructed by using integrational plasmids to disrupt the speB gene and confirmed by Southern hybridization and Western immunoblot analyses. No extracellular protease activity was produced by the mutants. The mutants had growth rates similar to those of the wild-type strains and produced normal levels of other extracellular proteins. When invading A-549 cells, the mutants had a two- to threefold decrease in activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect the adhesive ability of GAS. These results suggested that streptococcal cysteine protease can enhance the invasion ability of GAS in human respiratory epithelial cells.

Expression of the acute-phase response genes, such as that for alpha-1 acid glycoprotein (AGP), involves both positive and negative transcription factors. A positive transcription factor, AGP/EBP, and a negative transcription factor, factor B, have been identified as the two most important factors responsible for the induction of the AGP gene. In this paper we report the purification, characterization, and identification of a B-motif-binding factor from the mouse hepatoma cell line 129p. The purified factor has been identified as nucleolin by amino acid sequence analysis. Biochemical and functional studies further established that nucleolin is a transcription repressor for regulation of AGP and possibly other acute-phase response genes. Thus, in addition to the many known functions of nucleolin, such as rRNA transcription, processing, ribosome biogenesis, and the shuttling of proteins between the cytoplasmic and nuclear compartments, it may also function as a transcriptional repressor.

Antigen-specific suppression to poly(Glu50-Tyr50) (GT) is under the control of two complementary immune suppressor (Is) genes located in the major histocompatibility (H-2) complex of the mouse. Suppressor strains of mice produce both suppressor T (Ts) cells and Ts-derived suppressor factors (TsF) that bear antigenic determinants of the I-J subregion of the H-2 complex. Nonsuppressor strains of mice, on the other hand, are not suppressed by GT preimmunization. These nonsuppressor mice, however, can be classified according to those that lack the ability to make GT-specific T cell-derived suppressor factor (GT-TsF) after GT injection (i.e., H-2a, I-Jk mice) and those that lack the ability to be suppressed by the appropriate GT-TsF (i.e., H-2b,g2, I-Jb mice). In the present study, we demonstrate that (H-2a x H-2b,g2)F1 hybrid mice produce distinct GT-specific suppressor factors of both parental I-J haplotypes. Moreover, only the I-Jb-bearing GT-TsF derived from these F1 hybrid mice is able to induce second-order suppressor cells (Ts2). This is consistent with the observation that injection of GT-TsF1 derived from C57BL/6 (I-Jb) mice into A/J (I-Jk) mice leads to the production of an antigen-specific I-Jk GT-TsF2. Our results suggest that Is gene complementation occurs through a different cellular mechanism that was previously observed for Ir gene complementation. Further, we show that complementing (non-suppressor X nonsuppressor)F1 hybrid mice produce an I-Jb (and not an I-Jk) GT-TsF1 and an I-Jk (not an I-Jb) GT-TsF2, thus suggesting a heterogeneity of Ia loci within the I-J subregion. Data presented in the present study suggest that there may be even more heterogeneity within the I-J subregion than has has been heretofore reported with regard to I-J expression on Ts.