Lipopolysaccharide-induced hepatic oxidative injury is not potentiated by knockout of GPX1 and SOD1 in mice

Knockout of copper, zinc-superoxide dismutase (SOD1) and (or) cellular glutathione peroxidase (GPX1) has been reported to have dual impacts on coping with free radical-induced oxidative injury. Because bacterial endotoxin lipopolysaccharide (LPS) triggers inflammatory responses involving the release of cytokines, nitric oxide and superoxide in targeted organs such as liver, in this study we used SOD1 knockout (SOD1−/−), GPX1 knockout (GPX1−/−), GPX1 and SOD1 double-knockout (DKO) and their wild-type (WT) mice to investigate the role of these two antioxidant enzymes in LPS-induced oxidative injury in liver. Mice of the four genotypes (2 month old) were killed at 0, 3, 6 or 12 h after an i.p. injection of saline or 5 mg LPS/kg body weight. The LPS injection caused similar increase in plasma alanine aminotransferase among the four genotypes. Hepatic total glutathione (GSH) was decreased (P < 0.05) compared with the initial values by the LPS injection at all time points in the WT mice, but only at 6 and 12 h in the other three genotypes. The GSH level in the DKO mice was higher (P < 0.05) than in the WT at 6 h. Although the LPS injection resulted in substantial increases in plasma NO in a time-dependent manner in all genotypes, the NO level in the DKO mice was lower (P < 0.05) at 3, 6 and 12 h than in the WT. The level in the GPX1−/− and SOD1−/− mice was also lower (P < 0.05) than in the WT at 3 h. The LPS-mediated hepatic protein nitration was detected in the WT and GPX1−/− mice at 3, 6 or 12 h, but not in the SOD1−/−. In conclusion, knockout of SOD1 and (or) GPX1 did not potentiate the LPS-induced liver injury, but delayed the induced hepatic GSH depletion and plasma NO production.     

Age-dependent changes in the mechanical properties of tail tendons in TGF-beta inducible early gene-1 knockout mice.

The purpose of this study is to investigate age-dependent changes in the architecture and mechanical properties of tendon in TGF-beta inducible early gene-1 (TIEG) knockout mice. Wild-type and TIEG knockout mice, aged 1, 2, and 15 mo, were used. The mechanical properties of tail tendons isolated from these mice were determined using uniaxial tensile ramp (0.05 mm/s) and relaxation (5 mm/s) tests, with a strain of 10%. Mechanical parameters (Young's modulus from the ramp test; fast and static stresses from the relaxation test) were measured and recorded. The structure of the tail tendon fascicle was characterized by transmission electron microscopy. The results of the mechanical testing revealed no significant difference between the knockout and wild-type groups at 1 or 15 mo of age. However, the fascicles of the knockout mice at 3 mo of age exhibited decreased fast and static stresses compared with those of the wild-type mice. Electron microscopy revealed an increase in fibril size in the knockout mouse tendons relative to wild-type controls at 1 and 3 mo of age. These data indicate an important role for TIEG in tendon microarchitecture and strength in adult mice.     

Evidence against proteoglycan mediated collagen fibril load transmission and dynamic viscoelasticity in tendon

The glycosaminoglycan (GAG) dermatan sulfate and chondroitin sulfate side-chains of small leucine-rich proteoglycans have been increasingly posited to act as molecular cross links between adjacent collagen fibrils and to directly contribute to tendon elasticity. GAGs have also been implicated in tendon viscoelasticity, supposedly affecting frictional loss during elongation or fluid flow through the extra cellular matrix. The current study sought to systematically test these theories of tendon structure–function by investigating the mechanical repercussions of enzymatic depletion of GAG complexes by chondroitinase ABC in a reproducible tendon structure–function model (rat tail tendon fascicles). The extent of GAG removal (at least 93%) was verified by relevant spectrophotometric assays and transmission electron microscopy. Dynamic viscoelastic tensile tests on GAG depleted rat tail tendon fascicle were not mechanically different from controls in storage modulus (elastic behavior) over a wide range of strain-rates (0.05, 0.5, and 5% change in length per second) in either the linear or nonlinear regions of the material curve. Loss modulus (viscoelastic behavior) was only affected in the nonlinear region at the highest strain-rate, and even this effect was marginal (19% increased loss modulus, p = 0.035). Thus glycosaminoglycan chains of small leucine-rich proteoglycans do not appear to mediate dynamic elastic behavior nor do they appear to regulate the dynamic viscoelastic properties in rat tail tendon fascicles.     

Mechanical properties of collagen fascicles from the rabbit patellar tendon

Tensile and viscoelastic properties of collagen fascicles of approximately 300 microns in diameter, which were obtained from rabbit patellar tendons, were studied using a newly designed micro-tensile tester. Their cross-sectional areas were determined with a video dimension analyzer combined with a CCD camera and a low magnification microscope. There were no statistically significant differences in tensile properties among the fascicles obtained from six medial-to-lateral locations of the patellar tendon. Tangent modulus, tensile strength, and strain at failure of the fascicles determined at about 1.5 percent/s strain rate were 216 +/- 68 MPa, 17.2 +/- 4.1 MPa, and 10.9 +/- 1.6 percent (mean +/- S.D.), respectively. These properties were much different from those of bulk patellar tendons; for example, the tensile strength and strain at failure of these fascicles were 42 percent and 179 percent of those of bulk tendons, respectively. Tangent modulus and tensile strength of collagen fascicles determined at 1 percent/s strain rate were 35 percent larger than those at 0.01 percent/s. The strain at failure was independent of strain rate. Relaxation tests showed that the reduction of stress was approximately 25 percent at 300 seconds. These stress relaxation behavior and strain rate effects of collagen fascicles differed greatly from those of bulk tendons. The differences in tensile and viscoelastic properties between fascicles and bulk tendons may be attributable to ground substances, mechanical interaction between fascicles, and the difference of crimp structure of collagen fibrils.     

Comparison of elastic,viscoelastic and failure tensile material properties of knee ligaments and patellar tendon

The knee ligaments and patellar tendon function in concert with each other and other joint tissues, and are adapted to their specific physiological function via geometry and material properties. However, it is not well known how the viscoelastic and quasi-static material properties compare between the ligaments. The purpose of this study was to characterize and compare these material properties between the knee ligaments and patellar tendon.Dumbbell-shaped tensile test samples were cut from bovine knee ligaments (ACL, LCL, MCL, PCL) and patellar tendon (PT) and subjected to tensile testing (n = 10 per ligament type). A sinusoidal loading test was performed at 8% strain with 0.5% strain amplitude using 0.1, 0.5 and 1 Hz frequencies. Subsequently, an ultimate tensile test was performed to investigate the stress-strain characteristics.At 0.1 Hz, the phase difference between stress and strain was higher in LCL compared with ACL, PCL and PT (p < 0.05), and at 0.5 Hz that was higher in LCL compared with all other ligaments and PT (p < 0.05). PT had the longest toe-region strain (p < 0.05 compared with PCL and MCL) and MCL had the highest linear and strain-dependent modulus, and toughness (p < 0.05 compared with ACL, LCL and PT).The results indicate that LCL is more viscous than other ligaments at low-frequency loads. MCL was the stiffest and toughest, and its modulus increased most steeply at the toe-region, possibly implying a greater amount of collagen. This study improves the knowledge about elastic, viscoelastic and failure properties of the knee ligaments and PT.     

Keratan sulfate and related murine glycosylation can suppress murine cartilage damage in vitro and in vivo

Keratan sulfate (KS) proteoglycan side chains are abundant in the human cartilage matrix, but these chains have been said to be absent in murine skeletal tissues. We previously showed that KS suppresses cartilage damage and ameliorates inflammation in mice arthritis model. Because mice deficient of N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) (KS biosynthesis enzyme) are now available, we decided to do further examinations.We examined, in culture, the difference between GlcNAc6ST-1^−/− and wild-type (WT) mice for interleukin (IL)-1α-induced glycosaminoglycan (GAG) release from the articular cartilage. Arthritis was induced by intravenous administration of an anti-type II collagen antibody cocktail and subsequent intraperitoneal injection of lipopolysaccharide. We examined the differences in arthritis severities in the two genotypes. After intraperitoneal KS administration in phosphate-buffered saline (PBS) or PBS alone, we evaluated the potential of KS in ameliorating arthritis and protecting against cartilage damage in deficient mice.GAG release induced by IL-1α in the explants, and severity of arthritis were greater in GlcNAc6ST-1^−/− mice than their WT littermates. Intraperitoneal KS administration effectively suppressed arthritis induction in GlcNAc6ST-1^−/− mice. Thus, GlcNAc6ST-1^−/− mice cartilage is more fragile than WT mice cartilage, and exogenous KS can suppress arthritis induction in GlcNAc6ST-1^−/− mice. Vestigial KS chain or altered glycosylation in articular cartilage in GlcNAc6ST-1^−/− mice may be protective against arthritis and associated cartilage damage as well as cartilage damage in culture. KS may offer therapeutic opportunities for chondroprotection and suppression of joint damage in inflammatory arthritis and may become a therapeutic agent for treating rheumatoid arthritis.     

Strain-rate sensitive mechanical properties of tendon fascicles from mice with genetically engineered alterations in collagen and decorin

Tendons have complex mechanical behaviors that are nonlinear and time dependent. It is widely held that these behaviors are provided by the tissue composition and structure. It is generally thought that type I collagen provides the primary elastic strength to tendon while proteoglycans, such as decorin, play a role in failure and viscoelastic properties. This study sought to quantify such structure-function relationships by comparing tendon mechanical properties between normal mice and mice genetically engineered for altered type I collagen content and absence of decorin. Uniaxial tensile ramp to failure experiments were performed on tail tendon fascicles at two strain rates, 0.5%/s and 50%/s. Mutations in type I collagen led to reduced failure load and stiffness with no changes in failure stress, modulus or strain rate sensitivity. Fascicles without decorin had similar elastic properties to normal fascicles, but reduced strain rate sensitivity. Fascicles from immature mice, with increased decorin content compared to adult fascicles, had inferior elastic properties but higher strain rate sensitivity. These results showed that tendon viscoelasticity is affected by decorin content but not by collagen alterations. This study provides quantitative evidence for structure-function relationships in tendon, including the role of proteoglycan in viscoelasticity.     

Inhibition of autophagy stimulate molecular iodine-induced apoptosis in hormone independent breast tumors

Estrogen receptor negative (ER^−ve) and p53 mutant breast tumors are highly aggressive and have fewer treatment options. Previously, we showed that molecular Iodine (I₂) induces apoptosis in hormone responsive MCF-7 breast cancer cells, and non-apoptotic cell death in ER^−ve–p53 mutant MDA-MB231 cells (Shrivastava, 2006). Here we show that I₂ (3 μM) treatment enhanced the features of autophagy in MDA-MB231 cells. Since autophagy is a cell survival response to most anti-cancer therapies, we used both in vitro and in vivo systems to determine whether ER^−ve mammary tumors could be sensitized to I₂-induced apoptosis by inhibiting autophagy. Autophagy inhibition with chloroquine (CQ) and inhibitors for PI3K (3MA, LY294002) and H+/ATPase (baflomycin) resulted in enhanced cell death in I₂ treated MDA-MB231 cells. Further, CQ (20 μM) in combination with I₂, showed apoptotic features such as increased sub-G1 fraction (∼5-fold), expression of cleaved caspase-9 and -3 compared to I₂ treatment alone. Flowcytometry of I₂ and CQ co-treated cells revealed increase in mitochondrial membrane permeability (p < 0.01) and translocation of cathepsin D activity to cytosol relative to I₂ treatment. For in vivo studies ICRC mice were transplanted subcutaneously with MMTV-induced mammary tumors. A significant reduction in tumor volumes, as measured by MRI, was found in I₂ and CQ co-treated mice relative to I₂ or vehicle treated mice. These data indicate that inhibition of autophagy renders ER^−ve breast tumor cells more sensitive to I₂ induced apoptosis. Thus, I₂ together with autophagy inhibitor could have a potential tumorostatic role in ER^−ve aggressive breast tumors that may be evaluated in future studies.     

Npc1 deficiency in the C57BL/6J genetic background enhances Niemann-Pick disease type C spleen pathology

Niemann–Pick type C (NPC) disease is an autosomal recessive neurovisceral lipid storage disorder. The affected genes are NPC1 and NPC2. Mutations in either gene lead to intracellular cholesterol accumulation. There are three forms of the disease, which are categorized based on the onset and severity of the disease: the infantile form, in which the liver and spleen are severely affected, the juvenile form, in which the liver and brain are affected, and the adult form, which affects the brain. In mice, a spontaneous mutation in the Npc1 gene originated in the BALB/c inbred strain mimics the juvenile form of the disease. To study the influence of genetic background on the expression of NPC disease in mice, we transferred the Npc1 mutation from the BALB/c to C57BL/6J inbred background. We found that C57BL/6J-Npc1^−/− mice present with a much more aggressive form of the disease, including a shorter lifespan than BALB/c-Npc1^−/− mice. Surprisingly, there was no difference in the amount of cholesterol in the brains of Npc1^−/− mice of either mouse strain. However, Npc1^−/− mice with the C57BL/6J genetic background showed striking spleen damage with a marked buildup of cholesterol and phospholipids at an early age, which correlated with large foamy cell clusters. In addition, C57BL/6J Npc1^−/− mice presented red cell abnormalities and abundant ghost erythrocytes that correlated with a lower hemoglobin concentration. We also found abnormalities in white cells, such as cytoplasmic granulation and neutrophil hypersegmentation that included lymphopenia and atypias. In conclusion, Npc1 deficiency in the C57BL6/J background is associated with spleen, erythrocyte, and immune system abnormalities that lead to a reduced lifespan.     

Heteromeric TRPV4-C1 channels contribute to store-operated Ca(2+) entry in vascular endothelial cells

There is controversy as to whether TRP channels participate in mediating store-operated current (I_SOC) and store-operated Ca²⁺ entry (SOCE). Our recent study has demonstrated that TRPC1 forms heteromeric channels with TRPV4 in vascular endothelial cells and that Ca²⁺ store depletion enhances the vesicle trafficking of heteromeric TRPV4-C1 channels, causing insertion of more channels into the plasma membrane in vascular endothelial cells. In the present study, we determined whether the enhanced TRPV4-C1 insertion to the plasma membrane could contribute to SOCE and I_SOC. We found that thapsigargin-induced SOCE was much lower in aortic endothelial cells derived from trpv4^−/− or trpc1^−/− knockout mice when compared to that of wild-type mice. In human umbilical vein endothelial cells (HUVECs), thapsigargin-induced SOCE was markedly reduced by knocking down the expression of TRPC1 and/or TRPV4 with respective siRNAs. Brefeldin A, a blocker of vesicular translocation, inhibited the SOCE. These results suggest that an enhanced vesicular trafficking of heteromeric TRPV4-C1 channels contributes to SOCE in vascular endothelial cells. Vascular tension studies suggest that such an enhanced trafficking of TRPV4-C1 channels may play a role in thapsigargin-induced vascular relaxation in rat small mesenteric arteries.     

The Effect of Lubricin on the Gliding Resistance of Mouse Intrasynovial Tendon

The purpose of this study was to investigate the role of lubricin on the gliding resistance of intrasynovial tendons by comparing lubricin knockout, heterozygous, and wild type mice. A total of thirty-six deep digital flexor (DDF) tendons in the third digits of each hind paw from eighteen adult mice were used, including six lubricin knockout mice (Prg4 –/–), six heterozygous mice (Prg4 +/–), and six wild type mice (Prg4 +/+). The tendon gliding resistance was measured using a custom-made device. Tendon structural changes were evaluated by scanning electron and light microscopy. The gliding resistance of intrasynovial tendons from lubricin knockout mice was significantly higher than the gliding resistance of either wild type or heterozygous mice. The surface of the lubricin knockout tendons appeared to be rougher, compared to the wild type and heterozygous tendons. Synovial hyperplasia was found in the lubricin knockout mice. Cartilage-like tissue was found in the tendon and pulley of the lubricin knockout mice. Our findings confirm the importance of lubricin in intrasynovial tendon lubrication. This knockout model may be useful in determining the effect of lubricin on tendon healing and the response to injury.     

Dual roles of CagA protein in Helicobacterpylori-induced chronic gastritis in mice

Cytotoxin-associated gene A (CagA) acts directly on gastric epithelial cells. However, the roles of CagA in host adaptive immunity against Helicobacter pylori (H. pylori) infection are not fully understood. In this study, to investigate the roles of CagA in the development of H. pylori-induced chronic gastritis, we used an adoptive-transfer model in which spleen cells from C57BL/6 mice with or without H. pylori infection were transferred into RAG2^−/− mice, with gastric colonization of either CagA⁺H. pylori or CagA⁻H. pylori. Colonization of CagA⁺H. pylori but not CagA⁻H. pylori in the host gastric mucosa induced severe chronic gastritis in RAG2^−/− mice transferred with spleen cells from H. pylori-uninfected mice. In addition, when CagA⁺H. pylori-primed spleen cells were transferred into RAG2^−/− mice, CD4⁺ T cell infiltration in the host gastric mucosa were observed only in RAG2^−/− mice infected with CagA⁺H. pylori but not CagA⁻H. pylori, suggesting that colonization of CagA⁺H. pylori in the host gastric mucosa is essential for the migration of H. pylori-primed CD4⁺ T cells. On the other hand, transfer of CagA⁻H. pylori-primed spleen cells into CagA⁺H. pylori-infected RAG2^−/− mice induced more severe chronic gastritis with less Foxp3⁺ regulatory T-cell infiltration as compared to transfer of CagA⁺H. pylori-primed spleen cells. In conclusion, CagA in the stomach plays an important role in the migration of H. pylori-primed CD4⁺ T cells in the gastric mucosa, whereas CagA-dependent T-cell priming induces regulatory T-cell differentiation, suggesting dual roles for CagA in the pathophysiology of H. pylori-induced chronic gastritis.     

Angiotensin II induced catabolic effect and muscle atrophy are redox dependent

Angiotensin II (Ang II) causes skeletal muscle wasting via an increase in muscle catabolism. To determine whether the wasting effects of Ang II were related to its ability to increase NADPH oxidase-derived reactive oxygen species (ROS) we infused wild-type C57BL/6J or p47^phox−/− mice with vehicle or Ang II for 7 days. Superoxide production was increased 2.4-fold in the skeletal muscle of Ang II infused mice, and this increase was prevented in p47^phox−/− mice. Apocynin treatment prevented Ang II-induced superoxide production in skeletal muscle, consistent with Ang II increasing NADPH oxidase derived ROS. Ang II induced loss of body and skeletal muscle weight in C57BL/6J mice, whereas the reduction was significantly attenuated in p47^phox−/− animals. The reduction of skeletal muscle weight caused by Ang II was associated with an increase of proteasome activity, and this increase was completely prevented in the skeletal muscle of p47^phox−/− mice. In conclusion, Ang II-induced skeletal muscle wasting is in part dependent on NADPH oxidase derived ROS.     

The biomechanical effects of limb lengthening and botulinum toxin type A on rabbit tendon

Numerous studies have examined the effects of distraction osteogenesis (DO) on bone, but relatively fewer have explored muscle adaptation, and even less have addressed the concomitant alterations that occur in the tendon. The purpose herein was to characterize the biomechanical properties of normal and elongated rabbit (N=20) tendons with and without prophylactic botulinum toxin type A (BTX-A) treatment. Elastic and viscoelastic properties of Achilles and Tibialis anterior (TA) tendons were evaluated through pull to failure and stress relaxation tests.All TA tendons displayed nonlinear viscoelastic responses that were strain dependent. A power law formulation was used to model tendon viscoelastic responses and tendon elastic responses were fit with a microstructural model. Distraction-elongated tendons displayed increases in compliance and stress relaxation rates over undistracted tendons; BTX-A administration offset this result. The elastic moduli of distraction-lengthened TA tendons were diminished (p=0.010) when distraction was combined with gastrocnemius (GA) BTX-A administration, elastic moduli were further decreased (p=0.004) and distraction following TA BTX-A administration resulted in TA tendons with moduli not different from contralateral control (p>0.05). Compared to contralateral control, distraction and GA BTX-A administration displayed shortened toe regions, (p=0.031 and 0.038, respectively), while tendons receiving BTX-A in the TA had no differences in the toe region (p>0.05). Ultimate tensile stress was unaltered by DO, but stress at the transition from the toe to the linear region of the stress–stretch curve was diminished in all distraction-elongated TA tendons (p<0.05). The data suggest that prophylactic BTX-A treatment to the TA protects some tendon biomechanical properties.     

The NCI-N87 cell line as a gastric epithelial barrier model for drug permeability assay

The objective of this study was to evaluate the human NCI-N87 cell line as a model for gastric permeability drug studies under pH conditions of the stomach. The optimal conditions that led NCI-N87 cells to form a typical differentiated gastric epithelial barrier were a seeding density of 2.5 × 10⁵ cells/cm² on porous inserts and growth in serum-complemented RPMI-1640 medium until 18–27 days post-confluency. The resulting cell monolayers showed moderately high transepithelial electrical resistance (TEER) values of about 500 Ω cm², cells of polygonal morphology expressing E-cadherin and ZO-1 proteins at their contact surfaces, and production of mucus clusters. The monolayers withstood apical pH of 7.4 down to 3.0 with the basal pH fixed at 7.4. The apparent permeability coefficients (P_app) of model compounds were evaluated in the apical-to-basolateral and basolateral-to-apical directions under different pH gradients. The monolayers were impermeable to the integrity marker Lucifer Yellow (low P_app of 0.3–1.1 × 10⁻⁶ cm/s). The furosemide P_app (0.4–1.5 × 10⁻⁵ cm/s) were slightly dependent on pH but remained moderate. The caffeine P_app (4.2–5.0 × 10⁻⁵ cm/s) were higher and insensitive to pH changes. The NCI-N87 cell line provides a useful in vitro tool to assess gastric drug permeability and absorption under physiologic conditions prevailing in the human stomach.     

NADPH-oxidase but not inducible nitric oxide synthase contributes to resistance in a murine Staphylococcus aureus Newman pneumonia model

Staphylococcus aureus is a pathogen that often causes severe nosocomial infections including pneumonia. The present study was designed to examine innate phagocyte mediated immune mechanisms using a previously described murine S. aureus Newman pneumonia model. We found that BALB/c mice represent a more susceptible mouse strain compared to C57BL/6 mice after intranasal S. aureus Newman challenge. Depletion experiments revealed that neutrophils are a crucial determinant for resistance whereas depletion of alveolar macrophages protected mice to some degree from acute pulmonary S. aureus challenge. C57BL/6 mice lacking the subunit gp91phox of the NADPH-oxidase (gp91phox−/− mice) proved to be highly susceptible against the pathogen. In contrast, C57BL/6 inducible nitric oxidase synthase deficient (iNOS−/−) mice did not differ in their clinical outcome after infection. Neither bone marrow macrophages from iNOS−/− nor from gp91phox−/− mice were impaired in controlling intracellular persistence of S. aureus. Our data suggest that neutrophil and NADPH-oxidase mediated mechanisms are essential components in protecting the host against pulmonary S. aureus Newman challenge. On contrary, macrophages as well as NO mediated mechanisms do not seem to play a critical role for resistance in this model.     

Post-yield relaxation behavior of bovine cancellous bone

Relaxation studies were conducted on specimens of bovine cancellous bone at post-yield strains. Stress and strain were measured for 1000 s and the relaxation modulus was determined. Fifteen cylindrical, cancellous bone specimens were removed from one bovine femur in the anterior–posterior direction. The relaxation modulus was found to be a function of strain. Therefore cancellous bone is non-linearly viscoelastic/viscoplastic in the plastic region. A power law regression was fit to the relaxation modulus data. The multiplicative constant was found to be statistically related through a power law relationship to both strain (p<0.0005) and apparent density (p<0.0005) while the power coefficient was found to be related through a power law relationship, E(t, ε)=A(ε)t^?n(ε), to strain (p<0.0005), but not apparent density.     

Correlation of C4ST-1 and ChGn-2 expression with chondroitin sulfate chain elongation in atherosclerosis

Mxi1, a member of the Myc–Max–Mad network, is an antagonist of the c-Myc oncogene and is associated with excessive cell proliferation. Abnormal cell proliferation and tumorigenesis are observed in organs of Mxi1−/− mice. However, the Mxi1-reltaed mechanism of proliferation is unclear. The present study utilized microarray analysis using Mxi1 mouse embryonic fibroblasts (MEFs) to identify genes associated with cell proliferation. Among these genes, insulin-like growth factor binding protein-3 (IGFBP-3) was selected as a candidate gene for real-time PCR to ascertain whether IGFBP-3 expression is regulated by Mxi1. Expression of IGFBP-3 was decreased in Mxi1−/− MEFs and Mxi1−/− mice, and the gene was regulated by Mxi1 in Mxi1 MEFs. Furthermore, proliferation pathways related to IGFBP-3 were regulated in Mxi1−/− mice compared to Mxi1+/+ mice. To determine the effect of Mxi1 inactivation on the induction of cell proliferation, a proliferation assay is performed in both Mxi1 MEFs and Mxi1 mice. Cell viability was regulated by Mxi1 in Mxi1 MEFs and number of PCNA-positive cells was increased in Mxi1−/− mice compared to Mxi1+/+ mice. Moreover, the IGFBP-3 level was decreased in proliferation defect regions in Mxi1−/− mice. The results support the suggestion that inactivation of Mxi1 has a positive effect on cell proliferation by down-regulating IGFBP-3.     

Structural studies of the lipopolysaccharide from Haemophilus parainfluenzae strain 20

Haemophilus parainfluenzae is a Gram-negative bacterium that colonizes the upper respiratory tract of humans and is a part of normal flora. In this study, we investigated the lipopolysaccharide (LPS) expressed by H. parainfluenzae strain 20. Using NMR and MS techniques on LPS, oligosaccharide samples and lipid A, the structures for O-antigen, core oligosaccharide and lipid A could be established. It was found that the biological repeating unit of the O-antigen is →4)-α-d-GalpNAc-(1→P→6)-β-d-Glcp-(1→3)-α-d-FucpNAc4N-(1→, in which d-FucpNAc4N is 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose. This sugar is in β-configuration when linked to O-4 of the glucose residue of β-d-Galp-(1→2)-l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-Glcp-(1→4)]-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. LPS from a wbaP mutant of H. parainfluenzae strain 20 did not contain an O-antigen, consistent with the wbaP gene product being required for expression of O-antigen in fully extended LPS.     

Redundancy of interleukin-6 in the differentiation of T cell and monocyte subsets during cutaneous leishmaniasis

Leishmania (L.) major is a protozoan parasite that infects mammalian hosts and causes a spectrum of disease manifestations that is strongly associated with the genetic background of the host. Interleukin (IL)-6 is an acute phase proinflammatory cytokine, known in vitro to be involved in the inhibition of the generation of regulatory T cells. IL-6-deficient mice were infected with L. major, and T cell and monocyte subsets were analyzed with flow cytometry. Our data show that at the site of infection in the footpad and in the draining popliteal lymph node, numbers of regulatory T cells remain unchanged between WT and IL-6-deficient mice. However, the spleens of IL-6^−/− mice contained fewer regulatory T cells after infection with L. major. The development of cutaneous lesions is similar between WT and IL-6-deficient mice, while parasite burden in IL-6^−/− mice is reduced compared to WT. The development of IFN-γ or IL-10 producing T cells is similar in IL-6^−/− mice. Despite a comparable adaptive T cell response, IL-6-deficient mice develop an earlier peak of some inflammatory cytokines than WT mice. This data indicate that the role of IL-6 in the differentiation of regulatory T cells is complex in vivo, and the effect of an absence of this cytokine can be counter-intuitive.