Interaction between gastrocnemius muscle weakness and moderate exercise deteriorates joint integrity in rat knee.

The aim of this study was to determine the effect on the knee joint of the interaction between ankle muscle weakness and moderate exercise. Gastrocnemius muscle weakness was induced by intramuscular injection of botulinum toxin type A (BTX) in rats. Low-speed treadmill running (12 m/min for 60 min) was applied for 6 weeks in rats with and without BTX. Untreated animals were used as controls. After BTX injection, the gastrocnemius muscle weakness was confirmed by 3-D motion analysis in kinematic features of the hindlimb during locomotion as an increased maximal dorsiflexion angle during the stance phase. Serum biomarker analysis by enzyme-linked immunosorbent assay revealed that low-speed running decreased the catabolic effect on type II collagen. However, the inhibition of catabolism induced by running exercise was significantly counteracted by BTX injection. In addition, thinning of the cartilage layer and a reduction in the chondrocyte density was also found in the tibial plateau of the knee in the BTX-injected rats after running for 6 weeks. These data suggest that moderate exercise have a positive effect on joint homeostasis. However, ankle muscle weakness may alter the mechanical environment of the knee and impair the integrity of joint cartilage with moderate exercise.

Inhibition of the CXCR3-mediated pathway suppresses ultraviolet B-induced pigmentation and erythema in skin.

BACKGROUND: Skin pigmentation by ultraviolet (UV) B radiation is caused in part by inflammation mediated by chemokines and cytokines secreted by keratinocytes in the irradiated area. However, such inflammatory processes have not been well documented. OBJECTIVES: To elucidate the inflammation processes caused by UVB irradiation using skin-lightening agents that suppress melanin synthesis after UVB irradiation. METHODS: Utilizing a three-dimensional (3D) skin model, agents that suppressed formation of sunburn cells (SBC) after UVB irradiation were screened. Molecules whose expression was upregulated by UVB irradiation and attenuated by pretreatment with the agent were then screened by gene microarray to explore the mechanism of UVB irradiation. Messenger RNA expression of the molecules identified to be responsible for melanin biosynthesis was knocked down with a Tet-off shRNA lentivirus construct to confirm the involvement of the molecule in the pigmentation pathway following UVB irradiation. RESULTS: Paeonia suffruticosa Andrews (PSA) pretreatment suppressed SBC formation in the 3D skin model, and erythema formation and pigmentation in volunteers exposed to UVB irradiation. Comprehensive gene analysis after UVB irradiation showed upregulation of CXCR3 and its ligands, CXCL9/monokine induced by interferon (IFN)-γ (MIG), CXCL10/10-kDa IFN-γ-induced protein (IP-10) and CXCL11/inducible T-cell α-chemoattractant (I-TAC). Upregulation of these genes was partially suppressed by PSA pretreatment. Melanin biosynthesis increased upon stimulation of CXCR3 ligands (MIG, IP-10 or I-TAC) and decreased following CXCR3 downregulation by shRNA knockdown. CONCLUSIONS: UVB irradiation activates CXCR3-mediated signalling that leads to melanin synthesis. PSA pretreatment shows a lightening effect partly by attenuating CXCR3-mediated signalling at the transcriptional level.

Morphological changes in hind limb muscles elicited by adjuvant-induced arthritis of the rat knee.

We investigated qualitative and quantitative changes in rat hind limb muscles caused by complete Freund's adjuvant (CFA)-induced knee joint pain. One week after CFA injection, muscle atrophy was induced only on the CFA-injected side. Wet weight of the rectus femoris (RF) and soleus (SOL) muscles were significantly decreased by 20% and 19%, respectively. The reduction in cross-sectional areas by CFA was similar for fast and slow muscle fibers in the RF (10% vs 15%, respectively) and SOL muscles (16% vs 16%, respectively). At the light microscopic level, pathological changes were not found in the RF muscles on both sides, although the infiltration of mononuclear cells and muscle regeneration were found in the SOL muscles on CFA-injected and contralateral control sides. On the other hand, electron microscopy revealed degenerative changes in the RF and SOL muscles on the CFA-injected side. Interestingly, sarcomere hypercontraction, indicating overexercise, was observed to a limited extent in the SOL muscles on the control side. In conclusions, knee joint pain can trigger the rapid development of muscle atrophy with degenerative changes not only in thigh but also calf muscles. This indicates that early interventions to inhibit joint pain or inflammation may prevent muscle atrophy.

Alteration in articular cartilage of rat knee joints after spinal cord injury.

OBJECTIVE: Mechanical forces are crucial for the maintenance of the morphologic and functional integrity of articular cartilage. The alteration of the articular cartilage after spinal cord injury (SCI) has been described in relation to a suppression of mechanical forces, since the joint is unloaded and restricted in movement. However, the morphological and biochemical characteristics of the cartilage after SCI are still poorly understood. We identified the localization of cartilage alterations after SCI and verified the influence of mechanical forces on the articular cartilage. METHOD: A total of 32 Wistar rats were used. Sixteen animals underwent an SCI and 16 animals served as control. The articular cartilage of the knee joint was assessed, respectively, at 4, 8, 10, and 12 weeks after intervention by histochemical, histomorphometric, immunohistochemical, and biochemical analyses. RESULTS: Cartilage thickness of spinal cord-injured knees decreased at the tibial and posterior femoral (FP) regions and increased at the anterior femoral (FA) region. Spinal cord injuries decreased the number of chondrocytes at the anterior regions and decreased the cartilage matrix staining only at the tibial regions. Immunolabeling to collagen type II was noted comparably in the superficial layer but noted weakly from the middle to deep layer. Collagen type I existed excessively at the cartilage surface and the pericellular regions. CONCLUSION: Cartilage alterations after SCI would not be explained by only a suppression of mechanical forces by unloading and immobilization, but there may be influences on the cartilage in addition to the change in mechanical forces.

Factors responsible for glucose intolerance in Japanese subjects with impaired fasting glucose.

Impaired fasting glucose (IFG) represents risk of development of diabetes (DM) and its complications. We investigated insulin secretion and insulin sensitivity in 403 IFG subjects divided into three levels of 2-hour postchallenge glucose (2-h PG) to clarify the factors responsible in the development of glucose intolerance in Japanese IFG. Nearly 60% of the subjects at annual medical check-up with FPG of 6.1-7.0 mmol/l at the first screening were diagnosed by 75 g oral glucose tolerance test (OGTT) to have impaired glucose tolerance (IGT; FPG <7.0 mmol/l and 7.8 mmol/l <2-h PG <11.1 mmol/l) or DM (isolated postchallenge hyperglycemia (IPH); FPG <7.0 mmol/l and 11.1 mmol/l <2-h PG level). The primary factor in the decreased glucose tolerance was a decrease in early-phase insulin, with some contribution of increasing insulin resistance. In addition, IFG/IGT and IFG/IPH subjects showed a compensatory increase in basal insulin secretion sufficient to keep FPG levels within the non-diabetic range. IFG is composed of three different categories in basal, early-phase insulin secretion, and insulin sensitivity.

The lacrimal fascia redefined.

It has been commonly accepted that the lacrimal fascia is an intact septum, composed of connective tissue, that separates the orbital cavity into a large compartment, which contains the eyeball, and a small compartment, which contains the lacrimal sac and nasolacrimal duct. the septum, however, is not necessarily always intact. We found a circular or oval opening in the lacrimal fascia in 37 of 52 specimens (71.2%) examined. The opening, which we shall refer to as the lacrimal fascial foramen, was located at variable levels in the lacrimal fossa. The lacrimal fascial foramen was buried in fatty tissue through which passed a branch of either the inferior palpebral artery or the infraorbital artery and a vein of the nasolacrimal duct. The clinical implications of the lacrimal fascial foramen in obstruction of the nasolacrimal duct are discussed.

The elongation domain of ELL is dispensable but its ELL-associated factor 1 interaction domain is essential for MLL-ELL-induced leukemogenesis.

The MLL-ELL chimeric gene is the product of the (11;19)(q23p13.1) translocation associated with de novo and therapy-related acute myeloid leukemias (AML). ELL is an RNA polymerase II elongation factor that interacts with the recently identified EAF1 (ELL associated factor 1) protein. EAF1 contains a limited region of homology with the transcriptional activation domains of three other genes fused to MLL in leukemias, AF4, LAF4, and AF5q31. Using an in vitro transformation assay of retrovirally transduced myeloid progenitors, we conducted a structure-function analysis of MLL-ELL. Whereas the elongation domain of ELL was dispensable, the EAF1 interaction domain of ELL was critical to the immortalizing properties of MLL-ELL in vitro. To confirm these results in vivo, we transplanted mice with bone marrow transduced with MLL fused to the minimal EAF1 interaction domain of ELL. These mice all developed AML, with a longer latency than mice transplanted with the wild-type MLL-ELL fusion. Based on these results, we generated a heterologous MLL-EAF1 fusion gene and analyzed its transforming potential. Strikingly, we found that MLL-EAF1 immortalized myeloid progenitors in the same manner as that of MLL-ELL. Furthermore, transplantation of bone marrow transduced with MLL-EAF1 induced AML with a shorter latency than mice transplanted with the MLL-ELL fusion. Taken together, these results indicate that the leukemic activity of MLL-ELL requires the EAF1 interaction domain of ELL, suggesting that the recruitment by MLL of a transactivation domain similar to that in EAF1 or the AF4/LAF4/AF5q31 family may be a critical common feature of multiple 11q23 translocations. In addition, these studies support a critical role for MLL partner genes and their protein-protein interactions in 11q23 leukemogenesis.

Change in expression of GBP28/adiponectin in carbon tetrachloride-administrated mouse liver.

GBP28 (gelatin-binding protein of 28 kDa)/adiponectin is an adipocyte-producing plasma protein proposed to interact with the extracellular matrix. To examine the production of GBP28 in non-adipose tissues, we herein analyzed its expression and localization in mouse livers before and after CCl(4) treatment. In immunohistochemical analyses, the boundary of hepatocytes provided positive signals for GBP28 after 3-6 h and their cytoplasm was intensely stained after 18 h of CCl(4) treatment. Quantitative RT-PCR and in situ hybridization revealed that GBP28 mRNA expression was markedly elevated in CCl(4)-treated mouse livers. These results suggest that the circulating GBP28 binds the extracellular matrices of hepatocytes during the initial stage of CCl(4)-induced hepatic injury and the damaged hepatocytes themselves started to produce GBP28 thereafter. The induced expression of GBP28 was also observed in human hepatoma HepG2 cells after treatment with IL-6. Thus, GBP28 is also produced by the liver, where it undergoes tissue damage-induced transcriptional regulation.

Injury and repair of the soleus muscle after electrical stimulation of the sciatic nerve in the rat.

To study injury and subsequent changes in skeletal muscles, the rat sciatic nerve was electrically stimulated at 50 Hz and muscle contraction was induced for 30 min. Muscle damage was classified into five types (hypercontraction, hyperstretching, Z band disorders, misalignment of myofilament and regions of scarce myofilaments) by electron microscopy and quantified by ultrastructural assessment. After electrical nerve stimulation, the percentages of the injured areas of the soleus muscle were 18.8 +/- 15.8% (mean +/- SD) at 0 h, 9.7 +/- 1.0% at 6 h, 22.0 +/- 23.6% at 12 h, 13.1 +/- 3.2% at 24 h, 4.9 +/- 6.0% at 3 days and 0.5 +/- 0.4% at 7 days. At 0 h, the vast majority of ultrastructural alterations were sarcomere hypercontraction. At 6 h, hypercontraction was not recognizable and sarcomere hyperstretching and Z band disarrangement constituted the major findings. At 12 h, when the injury reached its maximum, myofilament disorganization and hyperstretching were predominant. At 24 h or afterwards, the injury began to decrease and recovered to almost normal conditions by 7 days. There were very few necrotic muscle fibers in all specimens. It is considered that the muscle lesions in the present study were reversible, and recovered through changes in various types of sarcomere alterations. Z band streaming and free ribosomes were frequently found at 12 and 24 h, which may indicate repair processes rather than newly formed lesions.

Histochemistry of food tissue by colour scanning electron microscopy.

A low-vacuum scanning electron microscope (SEM) allows microscopy of insulating specimens without metal coating, and so preserves the intact colour information on the specimen surface. We have attempted a new approach to characterize constituent distribution of food tissues by a histochemical method utilizing a colour SEM with an optical microscope and the low-vacuum SEM through digital image processing. To observe food tissues such as brown rice and adzuki bean, a colour SEM image of the specimen that has been stained by a modified method used in optical microscope histochemistry has proved to provide information of both the microscopic structure and constituent distribution on the specimen surface.

Both amino- and carboxyl-terminal domains of TRAF3 negatively regulate NF-kappaB activation induced by OX40 signaling.

OX40 is a member of the tumor necrosis factor receptor (TNF-R) superfamily. We observed that overexpression of OX40 activated NF-kappaB, which was inhibited by dominant negative forms of TRAF2, NF-kappaB-inducing kinase (NIK), and IkappaB kinase (IKK) alpha. This indicates that OX40 signaling leads to NF-kappaB activation through the same cascade as TNF-R2. We then investigated the negative regulatory function of TRAF3 on OX40-induced NF-kappaB activation. TRAF3 blocked OX40-, TRAF2-induced NF-kappaB activation, but not NIK- and IKKalpha-induced NF-kappaB activation, indicating that TRAF3 blocks the pathway between TRAF2 and NIK. C-terminal deletion mutants as well as the N-terminal deletion mutant of TRAF3 inhibited NF-kappaB activation induced by OX40 or TRAF2. Since TRAF3 bound to OX40 through the C-terminal TRAF domain, the C-terminal domain is likely to work as a dominant negative mutant to compete the recruitment of TRAF2 to the receptor, which transmits the signal from OX40 to the downstream, NIK kinase. On the other hand, the N-terminal domain of TRAF3 seems to affect the downstream of TRAF2 binding. Thus, it is suggested that TRAF3 actively inhibits NF-kappaB activation induced by OX40.

Practical SEM system based on the montage technique applicable to ultralow-magnification observation, while maintaining original functions

A newly developed SEM system has been utilized for obtaining ultralow-magnification SEM images. It is a successful combination of the modern SEM equipped with a motor drive stage fully controlled with PC and digital image processing techniques for automatic montage. In order to accomplish a practical system, several problems peculiar to the field of SEM, i.e. raster rotation, peripheral distortion and charging effects, are discussed and solved. The function of ultralow-magnification (whole area) observation is important during a scanning electron microscopy session.

Effects of short-term denervation and subsequent reinnervation on motor endplates and the soleus muscle in the rat.

The rat sciatic nerve was locally frozen, and changes in the nerve, motor endplates, and the soleus muscle were examined for up to 6 weeks by light and electron microscopy. The wet weights of denervated soleus muscles compared with contralateral values progressively declined to a minimum at 2 weeks after injury (60.7 +/- 2.5%) and began to reverse following 3 weeks. The sciatic nerve thoroughly degenerated after freezing. However, numerous regenerated myelinated and thin nerve fibers were observed at 3 weeks. They were considerably enlarged but still smaller than normal counterparts at 6 weeks postoperatively. Nerve terminals containing synaptic vesicles of endplates disappeared at day 1 and mostly reappeared at 3 weeks (about 70% of the endplates). All endplates examined were reinnervated at 4, 5, and 6 weeks. On the other hand, postsynaptic folds of muscle fibers seemed to be only slightly influenced by denervation or reinnervation. Ultrastructural alterations of myofibrils, in particular the loss of register, immediately appeared after denervation, spread progressively, peaked at 2 weeks, ameliorated following reinnervation, and became significantly normalized at 6 weeks after freezing. The proportion of type II fibers in the soleus muscle similary showed an increase and a decrease with a short delay in response to denervation and reinnervation, respectively. This study clearly demonstrated that the nerve supply affects the ultrastructural integrity of skeletal muscles. In addition, changes in the endplates and the soleus muscle evaluated in this study after short-term denervation are largely reversible following reinnervation.

Intracellular signaling of gp34, the OX40 ligand: induction of c-jun and c-fos mRNA expression through gp34 upon binding of its receptor, OX40.

We investigated the intracellular signaling events of OX40 ligand (gp34), a member of the TNF family. To elucidate the intracellular signaling via gp34, we prepared a model system in which a human gp34-transfected mouse epithelial cell line was stimulated with a recombinant soluble form of OX40. We demonstrated that OX40 binding resulted in increase in c-jun and c-fos mRNA levels in this transfectant by Northern blot analysis, which was blocked by the pretreatment with anti-gp34 Ab. The studies with various gp34 deletion mutants showed that the cytoplasmic portion including the amino acid sequence 16-21 (RPRFER) was required for the induction of c-jun and c-fos mRNA expression. Furthermore, OX40 binding induced c-jun mRNA expression also in HUVECs, which in our previous study have been shown to express gp34 and interact with activated T cells through the OX40/gp34 pathway. On the other hand, c-fos mRNA was detectable neither in unstimulated HUVECs nor in gp34-stimulated HUVECs. These results indicate that the OX40/gp34 system generates two-way signals and may elicit biological effects on vascular endothelial cells.

Activation of OX40 signal transduction pathways leads to tumor necrosis factor receptor-associated factor (TRAF) 2- and TRAF5-mediated NF-kappaB activation.

We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-kappaB in OX40-transfected HSB-2 cells was detected by electrophoretic mobility shift assay within 30 min after the binding of the ligand gp34. In vitro binding experiments showed that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, TRAF3, and TRAF5 but not TRAF4 associated with glutathione S-transferase-OX40 fusion protein. The cotransfection experiments using human embryo kidney cell derived HEK 293T cells showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in vivo. Studies with OX40 deletion mutants demonstrated that the cytoplasmic portion consisting of amino acid sequence 256-263 (GGSFRTPI) was required for the association with TRAFs and NF-kappaB activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-kappaB activation in a dose-dependent manner. In addition, the introduction of TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5 further reduced NF-kappaB activation. These results indicate that the NF-kappaB activation resulting from OX40 stimulation is mediated by both TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3.

V3 loop of human immunodeficiency virus type 1 reduces cyclin E expression and induces G1 arrest in interleukin 2-dependent T cells.

We previously described that V3 loop derived from the HTLV-III BH10 clone V3-BH10 markedly suppressed IL-2-driven T cell proliferation and produced G1 arrest of the cells. Here, we tested the effect of V3-BH10 on the molecules that are involved in transition from the G1 to S phase of the cell cycle. The effect of V3-BH10 on the IL-2-induced expression of G1 cyclins, Cdk inhibitors, and phosphorylation of retinoblastoma protein (pRb) was tested by immunoblotting, using the IL-2-dependent CD4-positive cell line Kit 225. Furthermore, IL-2-dependent kinase activity of the cyclin E-Cdk2 complex was investigated with histone H1 as a substrate. V3-BH10 reduced the IL-2-dependent expression of cyclin E, but not that of cyclin D and Cdk inhibitors such as p21 and p27. As the result of reduction of cyclin E, histone H1 kinase activity of the cyclin E-Cdk2 complex was markedly reduced even in the presence of rIL-2, followed by incomplete phosphorylation of pRb. The reduction in hyperphosphorylation of pRb by V3-BH10 led to G1 arrest of the cell cycle. Thus, V3-BH10 induced G1 arrest in IL-2-dependent cell cycle progression by reducing cyclin E expression, which may be one of the mechanisms underlying the dysfunction of T cells in HIV-1-infected people.