A spontaneous mutation in the nicotinamide nucleotide transhydrogenase gene of C57BL/6J mice results in mitochondrial redox abnormalities

NADPH is the reducing agent for mitochondrial H₂O₂ detoxification systems. Nicotinamide nucleotide transhydrogenase (NNT), an integral protein located in the inner mitochondrial membrane, contributes to an elevated mitochondrial NADPH/NADP⁺ ratio. This enzyme catalyzes the reduction of NADP⁺ at the expense of NADH oxidation and H⁺ reentry to the mitochondrial matrix. A spontaneous Nnt mutation in C57BL/6J (B6J-Nnt^MUT) mice arose nearly 3 decades ago but was only discovered in 2005. Here, we characterize the consequences of the Nnt mutation on the mitochondrial redox functions of B6J-Nnt^MUT mice. Liver mitochondria were isolated both from an Nnt wild-type C57BL/6 substrain (B6JUnib-Nnt^W) and from B6J-Nnt^MUT mice. The functional evaluation of respiring mitochondria revealed major redox alterations in B6J-Nnt^MUT mice, including an absence of transhydrogenation between NAD and NADP, higher rates of H₂O₂ release, the spontaneous oxidation of NADPH, the poor ability to metabolize organic peroxide, and a higher susceptibility to undergo Ca²⁺-induced mitochondrial permeability transition. In addition, the mitochondria of B6J-Nnt^MUT mice exhibited increased oxidized/reduced glutathione ratios as compared to B6JUnib-Nnt^W mice. Nonetheless, the maximal activity of NADP-dependent isocitrate dehydrogenase, which is a coexisting source of mitochondrial NADPH, was similar between both groups. Altogether, our data suggest that NNT functions as a high-capacity source of mitochondrial NADPH and that its functional loss due to the Nnt mutation results in mitochondrial redox abnormalities, most notably a poor ability to sustain NADP and glutathione in their reduced states. In light of these alterations, the potential drawbacks of using B6J-Nnt^MUT mice in biomedical research should not be overlooked.     

Double-strand breaks of DNA of C57BL and mdx mouse cardiomyocytes after dynamic stress

One of the approaches to analysis of survival of cardiomyocytes during oxidative stress can be the use of animals with genetic defects—mdx mice. In mdx mice, disturbance of dystrophine synthesis is known to be accompanied by development of oxydative stress in contractile cells that in turn produces cell death. Earlier we established that dynamic stress leads to the formation of low molecular DNA fragments in the mdx mouse myocardium. It is beyond any doubt that the DNA fragmentation develops via formation of double-strand DNA breaks (DB). To record the dynamics of the appearance and disappearance of DB in the mdx mouse cardiomyocytes after dynamic stress, we used an antibody to the phosphorylated form of the γ-H2Ax histone. In the absence of stress, DB in myocardial cell nuclei are revealed both in C57Bl and in mdx mice. The percentage of cardiomyocyte nuclei with DB in C57Bl and in mdx mice was 0.05 ± 0.07% and 6.7 ± 0.2%, respectively (Table 1). In the C57Bl mice 1 h after dynamic stress the fraction of labeled cardiomyocyte nuclei rose to 1.0 ± 0.02%, while in the mdx mice—to 41.7 ± 11.4% (Table 1). At 24 h after the dynamic stress 5.7 ± 0.2% cardiomyocyte nuclei remained labeled in the mdx mouse myocardium (Table 1), whereas in C57Bl mice no labeled cardiomyocyte nuclei were revealed. One hour after the dynamic stress, 0.3 ± 0.2% of cardiomyocyte nuclei of the C57Bl mice incorporated ³H-thymidine. In the mdx mice, 2.9 ± 0.5% of cardiomyocyte nuclei incorporated ³H-thymidine. At 24 h after the stress and ³H-thymidine administration the percentage of cardiomyocyte nuclei in the mdx mice fell to 0.4 ± 0.2%. In the C57Bl mice primarily labeled nuclei were not revealed. The ³H-thymidine incorporation is not associated with entrance of cardiomyocytes into the mitotic cycle; we consider it as a manifestation of reparative DNA synthesis. We conclude is that the disappearance of DB in DNA from the mdx mouse myocardium 24 h after the dynamic stress is associated both with DNA reparation and the loss of cardiomyocytes.     

Foxp3+ Regulatory T Cells Delay Expulsion of Intestinal Nematodes by Suppression of IL-9-Driven Mast Cell Activation in BALB/c but Not in C57BL/6 Mice

Accumulating evidence suggests that IL-9-mediated immunity plays a fundamental role in control of intestinal nematode infection. Here we report a different impact of Foxp3⁺ regulatory T cells (Treg) in nematode-induced evasion of IL-9-mediated immunity in BALB/c and C57BL/6 mice. Infection with Strongyloides ratti induced Treg expansion with similar kinetics and phenotype in both strains. Strikingly, Treg depletion reduced parasite burden selectively in BALB/c but not in C57BL/6 mice. Treg function was apparent in both strains as Treg depletion increased nematode-specific humoral and cellular Th2 response in BALB/c and C57BL/6 mice to the same extent. Improved resistance in Treg-depleted BALB/c mice was accompanied by increased production of IL-9 and accelerated degranulation of mast cells. In contrast, IL-9 production was not significantly elevated and kinetics of mast cell degranulation were unaffected by Treg depletion in C57BL/6 mice. By in vivo neutralization, we demonstrate that increased IL-9 production during the first days of infection caused accelerated mast cell degranulation and rapid expulsion of S. ratti adults from the small intestine of Treg-depleted BALB/c mice. In genetically mast cell-deficient (Cpa3-Cre) BALB/c mice, Treg depletion still resulted in increased IL-9 production but resistance to S. ratti infection was lost, suggesting that IL-9-driven mast cell activation mediated accelerated expulsion of S. ratti in Treg-depleted BALB/c mice. This IL-9-driven mast cell degranulation is a central mechanism of S. ratti expulsion in both, BALB/c and C57BL/6 mice, because IL-9 injection reduced and IL-9 neutralization increased parasite burden in the presence of Treg in both strains. Therefore our results suggest that Foxp3⁺ Treg suppress sufficient IL-9 production for subsequent mast cell degranulation during S. ratti infection in a non-redundant manner in BALB/c mice, whereas additional regulatory pathways are functional in Treg-depleted C57BL/6 mice.     

T-cell neoplasm induced by subcutaneous transplantation of a plasmacytoma: characterization of tumor cells.

Thymocytes, isolated 6 days following subcutaneous (sc) transplantation of BALB/c MOPC-315 plasmacytoma into F₁ (BALB/c × C57BL) hybrid mice, when injected sc into normal syngeneic mice, caused the development of a solid sc tumor. The cells of the newly developed tumor were of a mixed population of θ⁺F₁ (BALB/c × C57BL) and θ^? BALB/c cells (approximately 1:1), which represents a new type of mixed T cell-plasma cell neoplasm. Efforts were made to isolate the transformed thymocytes (θ⁺) from the plasmacytoma (θ^?) cells in the new tumor, exploiting differences in their surface properties. Treatment of the mixed tumor cell population with peanut agglutinin (PNA) revealed that only the T tumor cells were agglutinated. The agglutinated cells were recovered after dispersing the clumps with d-galactose (0.15 M) and consisted of 95% θ⁺ cells. The PNA-agglutinated cells were found to induce a similar tumor (85% θ⁺ cells) when injected sc into F₁ (BALB/c × C57BL) mice.     

Regulatory T cells enhance susceptibility to experimental trypanosoma congolense infection independent of mouse genetic background

Background

BALB/c mice are highly susceptible while C57BL/6 are relatively resistant to experimental Trypanosoma congolense infection. Although regulatory T cells (Tregs) have been shown to regulate the pathogenesis of experimental T. congolense infection, their exact role remains controversial. We wished to determine whether Tregs contribute to distinct phenotypic outcomes in BALB/c and C57BL/6 mice and if so how they operate with respect to control of parasitemia and production of disease-exacerbating proinflammatory cytokines.

Methodology/Findings

BALB/c and C57BL/6 mice were infected intraperitoneally (i.p) with 10³ T. congolense clone TC13 and both the kinetics of Tregs expansion and intracellular cytokine profiles in the spleens and livers were monitored directly ex vivo by flow cytometry. In some experiments, mice were injected with anti-CD25 mAb prior or post T. congolense infection or adoptively (by intravenous route) given highly enriched naïve CD25⁺ T lymphocytes prior to T. congolense infection and the inflammatory cytokine/chemokine levels and survival were monitored. In contrast to a transient and non significant increase in the percentages and absolute numbers of CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) in C57BL/6 mouse spleens and livers, a significant increase in the percentage and absolute numbers of Tregs was observed in spleens of infected BALB/c mice. Ablation or increasing the number of CD25⁺ cells in the relatively resistant C57BL/6 mice by anti-CD25 mAb treatment or by adoptive transfer of CD25⁺ T cells, respectively, ameliorates or exacerbates parasitemia and production of proinflammatory cytokines.

Conclusion

Collectively, our results show that regulatory T cells contribute to susceptibility in experimental murine trypanosomiasis in both the highly susceptible BALB/c and relatively resistant C57BL/6 mice.     

Aldose reductase,NADPH and NADP+ in normal,galactose-fed and diabetic rat lens

NADPH and NADP⁺ levels were measured in rat lens from normal controls, from galactose-fed and diabetic rats during the first week of cataract formation.The level of NADPH in normal rat lens was determined to be 12.3 ± 0.4 nmol/g wet weight, and that of NADP⁺ 4.6 ± 0.2 nmol/g wet weight. In early cataract formation NADPH levels decreased rapidly during the first 2 days and then remained stable at 76% of control for galactose-fed and 84% for diabetic rats. NADP⁺ levels increased by 38% of control for galactose-fed and 54% for diabetic rats. Calculated NADPH/NADP⁺ ratios dropped from 3.36 ± 0.21 to 1.86 ± 0.16 in galactose fed rats, and from 2.81 ± 0.15 to 1.61 ± 0.16 in diabetic rats (P < 0.001 for both experimental groups). These data are consistent with rapid NADPH oxidation during onset of lens cataracts. No significant changes in aldose reductase enzymatic activity levels were observed in either the galactosemic or the diabetic rats during the times measured.     

Natural regulatory T cells mediate the development of cerebral malaria by modifying the pro-inflammatory response

Cerebral malaria (CM) is a severe neurologic complication that arises predominantly in children and non-immune adults infected with Plasmodium falciparum. In the current study, the dynamics of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) and pro- and anti-inflammatory cytokines were analyzed in P. berghei ANKA (P.bANKA)-infected C57BL/6, BALB/c, and DBA/2 mice. We showed that C57BL/6 mice were susceptible to CM, while BALB/c and DBA/2 mice were resistant to CM and succumbed to hyperparasitemia and severe anemia. The proportion and absolute numbers of Tregs in BALB/c and DBA/2 mice were significantly higher than in C57BL/6 mice. The levels of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-6, IL-17 and NO in CM-susceptible C57BL/6 mice were obviously higher than in CM-resistant BALB/c and DBA/2 mice, while the level of the anti-inflammatory cytokine IL-10 was the opposite to that of pro-inflammatory cytokines, confirming that an appropriate balance between pro- and anti-inflammatory immune responses is essential to control the pathogenesis of severe malaria, and Tregs are important regulators if this balance is to be maintained. In vivo depletion of Tregs significantly protected C57BL/6 mice from experimental CM and the production of pro- and anti-inflammatory cytokines was reversed, indicating that this cell population contributes to pathogenesis by modulating the balance of pro- and anti-inflammatory responses. Our data demonstrate that Tregs mediate the incidence and outcome of CM in P.bANKA-infected mice by modifying the pro-inflammatory response.     

The substrate of the glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa provides structural stability

In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP⁺. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the K_d values determined for the PaG6PDH-G6P complex (78.0 ± 7.9 μM) and the K_0.5 values determined for G6P (57.9 ± 2.5 and 104.5 ± 9.3 μM in the NADP⁺- and NAD⁺-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD⁺ and NADP⁺ from those that use NADP⁺ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.     

Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability

Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD⁺ and their low operational stability in the presence of reactive organic compounds such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD⁺ binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k _cat/K _m) with NADP⁺. The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00?±?0.13 U?mg^?1 and a K _{m, NADP} ⁺ of 0.147?±?0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP⁺-accepting FDH (v _max, 10.25?±?1.63 U?mg^?1). However, the half-saturation constant for NADP⁺ (K _{m, NADP} ⁺ , 0.92?±?0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

Lecithin/cholesterol acyltransferase modulates diet-induced hepatic deposition of triglycerides in mice

Lecithin/cholesterol acyltransferase (LCAT) is responsible for the esterification of the free cholesterol of plasma lipoproteins. Here, we investigated the involvement of LCAT in mechanisms associated with diet-induced hepatic triglyceride accumulation in mice. LCAT-deficient (LCAT^?/?) and control C57BL/6 mice were placed on a Western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% cholesterol, 4.5 kcal/g) for 24 weeks, then histopathological and biochemical analyses were performed. We report that, in our experimental setup, male LCAT^?/? mice are characterized by increased diet-induced hepatic triglyceride deposition and impaired hepatic histology and architecture. Mechanistic analyses indicated that LCAT deficiency was associated with enhanced intestinal absorption of dietary triglycerides (3.6±0.5 mg/dl per minute for LCAT^?/? vs. 2.0±0.7 mg/dl per minute for C57BL/6 mice; P<.05), accelerated clearance of postprandial triglycerides and a reduced rate of hepatic very low density lipoprotein triglyceride secretion (9.8±1.1 mg/dl per minute for LCAT^?/? vs. 12.5±1.3 mg/dl per minute for C57BL/6 mice, P<.05). No statistical difference in the average daily food consumption between mouse strains was observed. Adenovirus-mediated gene transfer of LCAT in LCAT^?/? mice that were fed a Western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content (121.2±5.9 mg/g for control infected mice vs. 95.1±5.8 mg/g for mice infected with Ad-LCAT, P<.05) and a great improvement of hepatic histology and architecture. Our data extend the current knowledge on the functions of LCAT, indicating that LCAT activity is an important modulator of processes associated with diet-induced hepatic lipid deposition.     

CD45-positive cells are not an essential component in cardiosphere formation

The cardiosphere (CS) is composed of a heterogeneous population of cells, including CD45⁺ cells that are bone marrow (BM)-derived. However, whether the CD45⁺ cells are an essential cell component in CS formation is unknown. The current study was undertaken to address this question. Cardiospheres (CSs) were harvested from 1-week post-myocardial infarction (MI) or non-MI hearts of C57BL/6 J mice. The process of CS formation was observed by timelapse photography. To analyze the role of BM-derived CD45⁺ cells in CS formation, CD45⁺ cells were depleted from populations of CS-forming cells by immunomagnetic beads. We recorded the number of CSs formed in culture from the same amount (10⁵) of intact CS-forming cells, from CD45⁺-cell-depleted CS-forming cells and from CD45⁺ cells alone (n=6–9/cell type). CS-forming cells selectively aggregated together to form CSs by 35 h after plating. The depletion of CD45⁺ cells from CS-forming cells actually increased the formation of CSs (67±10 CSs/10⁵ cells) compared with non-depleted CS-forming cells (51±6 CSs/10⁵ cells, P<0.0001). Purified CD45⁺ cells from CS-forming cells did not form CSs in culture. Thus, BM-derived CD45⁺ cells including BM progenitors are neither necessary nor sufficient for CS formation.     

Immunogenetic differences between lymph node cell and bone marrow cell grafts in irradiated mice

C3H lymph node cell (LNC) grafts, but not bone marrow cell (BMC) grafts, were resisted by lethally irradiated NZB, (C57BL × NZB)F1, and (C57BL/6 × DBA/2)F1 mice. BALB/ c hosts did not resist C3H LNC, suggesting that Ir-like genes regulate resistance to such grafts. Cyclophosphamide, silica particles, and ⁸⁹Sr pretreatments of prospective host mice resulted in successful proliferation of C3H LNC in most instances. These agents were known to abrogate resistance to incompatible BMC grafts. The determinants for antigens recognized on LNC appear to map in or near the D region of H-2. LNC grafts of all H-2^k strains tested (C3H, CBA, C58, C57BR) were strongly resisted while A, C3H.A, B10.A(5R), A.TL, and A.Tla^b LNC grafts were not strongly resisted by NZB hosts. Grafts of H-2^b (C57BL/6, C57BL/10, 129) LNC, or BMC are resisted by NZB or (C57BL/6 × DBA/2)F1 hosts. (C3H × C57BL)F1 LNC but not BMC were resisted by similar hosts. (C57BL/6 × DBA/2)F1 mice were injected with C57BL/6 spleen cells four times to induce specific “unresponsiveness” to parental-strain Hemopoietic histocompatibility (Hh) antigens. Unresponsiveness was induced to C57BL/6 BMC, as expected, but C57BL/6 and C3H LNC grafts were resisted despite the spleen cell injections. The data suggest that the antigens recognized during rejection of C3H LNC are not expressed on C3H BMC. It is even conceivable that Hh antigens on C57BL/6 BMC and LNC have separate determinants. Alternatively, the injections of C57BL/6 spleen cells may have induced an anti-idiotypic response that was capable of eliminating C57BL/6 LNC by a different effector mechanism.     

Discovery of an acidic,thermostable and highly NADP<Superscript>+</Superscript> dependent formate dehydrogenase from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> NRRL B-30929

Objectives

To identify a robust NADP⁺ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties.

Results

A new NADP⁺ dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6?×?histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8–6.2). Its optimum temperature was 60 °C and 50 °C with NADP⁺ and NAD⁺, respectively and its T_m value was 78 °C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The K_M constants were 49.8, 0.12 and 1.68 mM for formate (with NADP⁺), NADP⁺ and NAD⁺, respectively.

Conclusions

An NADP⁺ dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration through biocatalysis requiring acidic conditions and high temperatures.

     

Electrochemical titrations of a ferredoxin-ferredoxin:NADP+ oxidoreductase complex

Potentiometric titrations employing an electrochemical thin-layer cell indicate that complex formation between ferredoxin and ferredoxin:NADP⁺ oxidoreductase alters the midpoint oxidation-reduction potentials of both proteins. The midpoint potential of ferredoxin in the complex becomes 22 ± 6 mV more negative compared to ferredoxin alone while the midpoint potential of ferredoxin:NADP⁺ oxidoreductase becomes 23 ± 4 mV more positive on complex formation.     

Effects of cadmium and zinc ions on purified lamb kidney cortex glucose-6-phosphate dehydrogenase activity

Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme in the pentose phosphate pathway. Cadmium is a toxic heavy metal that inhibits several enzymes. Zinc is an essential metal but overdoses of zinc have toxic effects on enzyme activities. In this study G-6-PD from lamb kidney cortex was competitively inhibited by zinc both with respect to glucose-6-phosphate (G-6-P) and NADP⁺ with Ki values of 1.066 ± 0.106 and 0.111 ± 0.007 mM respectively whereas cadmium was a non-competitive inhibitor with respect to both G-6-P and NADP⁺ Ki values of 2.028 ± 0.175 and 2.044 ± 0.289 mM respectively.     

Activation of AKT signaling promotes cell growth and survival in α7β1 integrin-mediated alleviation of muscular dystrophy

Transgenic expression of the α7 integrin can ameliorate muscle pathology in a mouse model of Duchenne muscular dystrophy (mdx/utr^−/−) and thus can compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the α7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remains to be established. The purpose of this study was to determine a role for signaling in the amelioration of muscular dystrophy by α7 integrin. Activation of PI3K, ILK, AKT, mTOR, p70S6K, BAD, ERK, and p38 was measured in the muscle from wild type (WT), mdx/utr^−/− and α7BX2-mdx/utr^−/− mice using in vitro activity assays or phosphospecific antibodies and western blotting. Significant increases in PI3K activity (47%), ILK activity (2.0-fold), mTOR (Ser2448) (57%), p70S6K (Thr389) (11.7-fold), and ERK (Thr202/Tyr204) (66%) were demonstrated in dystrophic mdx/utr^−/− muscle compared to WT. A significant decrease in p38 phosphorylation (2.9-fold) was also observed. Although most of these signaling events were similar in dystrophic mdx/utr^−/− mice overexpressing the α7 integrin, the AKT (Ser473):AKT ratio (2-fold vs. WT) and p70S6K phosphorylation (18-fold vs. WT) were higher in α7BX2-mdx/utr^−/− compared to mdx/utr^−/− mice. In addition, increased phosphorylation of BAD Serine 112 may contribute to the significant reduction in TUNEL⁺ cells observed in α7BX2-mdx/utr^−/− mice. We conclude that the α7β1 integrin confers a protective effect in dystrophic muscle through the activation of the ILK, AKT, p70S6K and BAD signaling to promote muscle cell survival.     

Tumor-derived Fas ligand induces toxicity in lymphoid organs and plays an important role in successful chemotherapy

 Recent studies have suggested that Fas ligand (FasL⁺) tumor cells can induce apoptosis in Fas⁺ T cells. However, the effect of growth of FasL⁺ tumors in vivo, on lymphoid tissues of the host is not clear and therefore was the subject of this investigation. Injection of FasL⁺ LSA tumor caused a significant decrease in cellularity of the thymus and spleen, resulting from marked apoptosis, in syngeneic C57BL/6+/+ (wild-type) but not C57BL/6-lpr/lpr (Fas-deficient) mice. The tumor-induced toxicity resulted from tumor-derived rather than host-derived FasL, inasmuch as LSA tumor growth in C57BL/6-gld/gld (FasL-defective) mice, induced marked apoptosis and toxicity in the thymus and spleen. The LSA tumor growth induced a significant decrease in the percentage of CD4⁺CD8⁺ T cells in the thymus of C57BL/6+/+ mice and an increase in the percentage of CD4⁺, CD8⁺ and CD4⁻CD8⁻ T cells. Of the four subpopulations tested, the CD4⁺CD8⁺ T cells showed maximum apoptosis. The LSA (FasL⁺) but not P815(FasL⁻) tumor cell lysates and culture supernatants induced marked apoptosis in Fas⁺ thymocytes, when tested both in vitro and in vivo. The LSA-tumor-induced apoptosis in vitro was inhibited by antibodies against FasL or by caspase and other inhibitors of apoptosis. Chemotherapy of LSA-tumor-bearing C57BL/6+/+ mice at advanced stages of tumor growth failed to cure the mice, whereas, more than 80% of LSA-tumor-bearing C57BL/6-lpr/lpr mice, similarly treated, survived. Together, the current study demonstrates that FasL produced by LSA tumor cells is functional in vivo and can cause severe toxicity in lymphoid organs of the host. Also, Fas/FasL interactions may play an important role in the successful chemotherapy of FasL-bearing tumor. Received: 31 August 1999 / Accepted: 12 November 1999     

Pathology of Tnf-deficient mice infected with Plasmodium chabaudi adami 408XZ

Tumor necrosis factor alpha (Tnf) plays a pleiotropic role in murine malaria. Some investigations have correlated Tnf with hypothermia, hyperlactatemia, hypoglycemia, and a suppression of the erythropoietic response, although others have not. In this study, we have evaluated parasitemia, survival rate and several pathological features in C57BL/6JTnf^−/− and C57BL/6JTnf^+/+ mice after infection with Plasmodium chabaudi adami 408XZ. Compared to the C57BL/6JTnf^+/+ mice, C57BL/6JTnf^−/− mice showed increased parasitemia and decreased survival rate, whereas blood glucose, blood lactate and body weight were not significantly different. However, C57BL/6JTnf^−/− mice suffered significantly more from severe anemia and hypothermia than C57BL/6JTnf^+/+ mice. These results suggest that Tnf is an important mediator of parasite control, but not of anemia development. We hypothesize that the high mortality observed in the Tnf knock-out mice is due to increased anemia and pathology as a direct result of increased levels of parasitemia.     

CD8+ T-Cell Epitope Mapping for Pneumonia Virus of Mice in H-2b Mice

The paramyxovirus pneumonia virus of mice (PVM) is a rodent model of human respiratory syncytial virus (hRSV) pathogenesis. Here we characterized the PVM-specific CD8⁺ T-cell repertoire in susceptible C57BL/6 mice. In total, 15 PVM-specific CD8⁺ T-cell epitopes restricted by H-2D^b and/or H-2K^b were identified. These data open the door for using widely profiled, genetically manipulated C57BL/6 mice to study the contribution of epitope-specific CD8⁺ T cells to PVM pathogenesis.