Macrophage deficiency in osteopetrotic (op/op) mice inhibits activation of satellite cells and prevents hypertrophy in single soleus fibers.

Effects of macrophage on the responses of soleus fiber size to hind limb unloading and reloading were studied in osteopetrotic homozygous (op/op) mice with inactivated mutation of macrophage colony-stimulating factor (M-CSF) gene and in wild-type (+/+) and heterozygous (+/op) mice. The basal levels of mitotically active and quiescent satellite cell (-46 and -39% vs. +/+, and -40 and -30% vs. +/op) and myonuclear number (-29% vs. +/+ and -28% vs. +/op) in fibers of op/op mice were significantly less than controls. Fiber length and sarcomere number in op/op were also less than +/+ (-22%) and +/op (-21%) mice. Similar trend was noted in fiber cross-sectional area (CSA, -15% vs. +/+, P = 0.06, and -14% vs. +/op, P = 0.07). The sizes of myonuclear domain, cytoplasmic volume per myonucleus, were identical in all types of mice. The CSA, length, and the whole number of sarcomeres, myonuclei, and mitotically active and quiescent satellite cells, as well as myonuclear domain, in single muscle fibers were decreased after 10 days of unloading in all types of mice, although all of these parameters in +/+ and +/op mice were increased toward the control values after 10 days of reloading. However, none of these levels in op/op mice were recovered. Data suggest that M-CSF and/or macrophages are important to activate satellite cells, which cause increase of myonuclear number during fiber hypertrophy. However, it is unclear why their responses to general growth and reloading after unloading are different.

Angiomyofibroblastoma of the vulva.

BACKGROUND: Angiomyofibroblastoma (AMF) is a rare benign mesenchymal neoplasm that arises in the pelviperial region. CASE: A patient presented with a painless mass in the right vulva. Under the preoperative diagnosis of Bartholin cyst, she underwent a simple tumor excision. Pathological examination revealed an AMF. Immunohistochemical examination showed that tumor cells were positive for estrogen receptor, progesterone receptor, vimentin, and CD34. She has been with no evidence of local recurrence for ten months after surgery. CONCLUSION: AMF of the vulva is a distinctive mesenchymal tumor that is curable with a simple excision.

Transition of low-grade to high-grade endometrial stromal sarcoma: a case report.

BACKGROUND: The transition of low-grade endometrial stromal sarcoma (ESS) to high-grade ESS remains a rare clinical event. CASE: A patient presented with abdominal pain and abnormal genital bleeding. She underwent a supracervical hysterectomy with bilateral salpingo-oophorectomy, omentectomy, and resection of peritoneal disseminated lesions. Pathological examination revealed low-grade ESS in the uterus and omentum. Immunohistochemical examination showed immunoreactivity for CD10 and Ki-67 (MIB1) in the uterus and omentum. However, estrogen receptor, progesterone receptor, alpha-SMA, desmin, h-caldesmon, and CAM5-2 were negative. P53 immunoreactivity was noted only in the omental lesion. Despite performing six courses of adjuvant chemotherapy, she recurred in the abdomen. She underwent ileostomy and resection of peritoneal disseminated lesions. Pathology showed high-grade ESS in the recurrent lesion of the ileum, which was characterized by severe cytologic atypia, high mitotic index, multifocal necrosis, increased Ki-67 index, and immunoreactivity for p53. CONCLUSION: Although rare, the transition of low-grade ESS to high-grade ESS may occur and suggests the worsening of the prognosis. Pathological examination and immunohistochemistry are useful for the diagnosis of the transition of low-grade ESS to high-grade ESS.

Effect of exercise training on the density of endothelial cells in the white adipose tissue of rats.

We examined the effects of a 9-week exercise training (TR) in Wistar male rats, beginning at 4 weeks of age, on the density of endothelial cells (ECs) in epididymal white adipose tissue (WAT) and the mRNA expression of angiogenic factors in adipose tissue stromal vascular fraction (SVF) cells. The number of ECs and mRNA expressions were assessed by lectin staining and real-time reverse transcriptase-polymerase chain reaction, respectively. Compared with control (CR) rats, TR rats gained weight more slowly and had significantly lower final weight of WAT due to the reduction in the size and the number of adipocytes. TR significantly increased the number of ECs per square millimeter and per adipocyte (1.37- and 1.23-fold, respectively) in WAT. This is probably because the number of adipocytes is fewer while the number of ECs is constant in the WAT of TR rats, because the regression line of TR rats for adipocyte number-dependent EC number was shifted toward the left without significant differences in the slopes between groups. TR also induced the upregulation of mRNA expression of vascular endothelial growth factor (Vegf)-A and Vegf-receptor-2 in SVF cells, thereby retaining a constant number of ECs in the WAT.

Effects of exercise training on adipogenesis of stromal-vascular fraction cells in rat epididymal white adipose tissue.

AIM: Previous studies have shown that exercise training reduced white adipose tissue (WAT) mass compared to that in sedentary controls, and that the smaller mass contained fewer adipocytes. However, the effect of exercise training on adipogenesis is not completely clear. Therefore, we re-examined the effect of exercise training on adipocyte numbers in WAT and, if such an effect was found tested the adipogenic responses of stromal-vascular fraction (SVF) cells containing adipose tissue-derived stem cells (ADSC) in epididymal WAT from exercise-trained (TR) rats. METHODS: Wistar male rats were divided into two groups: control (C) and TR. The TR rats were subjected to exercise on a treadmill for 9 weeks. SVF cells containing ADSC were separated from epididymal WAT by centrifugation. Expression of adipocyte differentiation-related genes and adipogenesis of SVF cells were examined. RESULTS: In SVF cells of TR rats, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and that of PPARγ target lipogenic genes was dramatically downregulated, whereas that of preadipocyte factor-1 gene was significantly upregulated. Lipid accumulation in SVF cells of TR rats after the induction of adipocyte differentiation was significantly suppressed in comparison with that of C rats. Moreover, increased expression of hypoxia-inducible factor-1α (HIF-1α) protein was observed in SVF cells of TR rats. Pre-treatment of YC-1, a potent HIF-1α inhibitor, in SVF cells of TR rats restored adipogenesis. CONCLUSION: These results suggest that exercise training suppresses the ability of SVF cells to differentiate into adipocytes, and that underlying mechanisms involve the upregulation of HIF-1α expression.

Muscle type-specific response of PGC-1 alpha and oxidative enzymes during voluntary wheel running in mouse skeletal muscle.

AIM: It is generally accepted that endurance exercise increases the expression of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), which governs the expression of oxidative metabolic enzymes. A previous report demonstrated that the regulation of mitochondrial protein expression in skeletal muscles in response to cold exposure depends on muscle fibre type. Cold exposure and endurance exercise are both metabolic challenges that require adjustments in mitochondrial energy metabolism, we hypothesized that the exercise-induced increase in oxidative enzymes and PGC-1alpha expression is higher in fast-type than in slow-type muscle. METHODS: Female ICR mice were individually housed in cages equipped with running wheel for 1, 2, 4, 6 or 8 weeks. The soleus, plantaris (PLA) and tibialis anterior (TA) muscles were then prepared from each mouse. The expression levels of PGC-1alpha, mitochondrial proteins and GLUT4 were evaluated by Western blotting. RESULTS: The expression level of PGC-1alpha was increased only in the PLA muscle. Furthermore, the expression levels of all mitochondrial proteins and GLUT4 in the PLA muscle were increased. In the TA muscle, although there was no increase in PGC-1alpha expression, the expression levels of mitochondrial proteins and GLUT4 were increased. CONCLUSIONS: These results suggest that muscle type-specific responses occur during endurance exercise, and that the increase in PGC-1alpha expression is not the only factor that promotes oxidative capacity as a result of endurance exercise.

Contribution of the calcineurin signaling pathway to overload-induced skeletal muscle fiber-type transition.

Skeletal muscle is highly adaptable, being capable of undergoing changes in its structural and functional properties in response to physiological stimuli. The fast-to-slow muscle fiber-type transition is evoked by increased motor nerve activity. Recently, the calcineurin (CaN) signaling pathway has been implicated in the transcriptional regulation of slow muscle fiber genes. Here we investigated the effect of treatment with a CaN-specific inhibitor, FK506, on skeletal muscle fiber-type transition in functionally overloaded muscles. The overloaded plantaris muscle showed fast-to-slow muscle fiber type transition, i.e., a decrease in myosin heavy chain (MHC) IIb, an increase in MHCIIa+d/x, and new expression of MHCI. In the FK506-administered group, however, overload-induced muscle fiber-type transition was completely prevented. We have demonstrated, therefore, that the CaN signaling pathway is required for fast-to-slow skeletal muscle fiber-type transition. Furthermore, we also confirmed that the protein expression levels of downstream effectors of CaN signaling exhibit a transient increase in the early phase of the overloaded condition.

Intermittent hypobaric hypoxia increases the ability of neutrophils to generate superoxide anion in humans.

1. We investigated the effect of intermittent exposure to hypobaric hypoxia on the ability of neutrophils to generate.O2-. 2. Seven male volunteers were exposed intermittently to hypobaric hypoxia, equivalent to an altitude of 4500 m, for 7 successive days. Peripheral blood samples were collected before and after the 2 h course of hypobaric hypoxia on days 1 and 7 and neutrophils were subjected to a chemiluminescence assay for.O2- production. 3. On day 1, 2 h exposure to hypobaric hypoxia induced granulocytosis (P < 0.01), but the ability of neutrophils to generate.O2- was unchanged. 4. On day 7, such granulocytosis was not observed, suggesting acclimatization to hypobaric hypoxia. 5. The ability of neutrophils to generate.O2- was significantly increased on day 7 (P < 0.01), although there was no definite change in the mRNA expression of NADPH oxidase subunits in the cells. 6. The results suggest that the ability of neutrophils to generate.O2- may be gradually potentiated by intermittent exposure to hypobaric hypoxia, even after the number of neutrophils in peripheral blood stabilizes.

Genetic variation in hypoxia-inducible factor 1alpha and its possible association with high altitude adaptation in Sherpas.

Hypoxic stress at high altitude requires adaptations in several physiological functions to ensure the optimal oxygenation of all cells. Several lines of evidence suggested that high-altitude native populations such as Sherpas have been genetically adapted to their stressful environment. We investigated the genetic variation in the hypoxia-inducible factor (HIF)-1alpha gene in Sherpas as compared with Japanese, native lowlanders, and found a novel dinucleotide repeat polymorphism in intron 13 of the HIF-1alpha gene. GT15 allele was more frequent in Japanese than in Sherpas with statistical significance, while GT14 allele was significantly more frequent in Sherpas as compared with Japanese. A possible genetic variation in the HIF-1alpha gene might function in adaptation to living at high altitude. Because the activity of HIF-1 is regulated by multiple steps including the transcriptional level, the effect of the polymorphism in intron 13 on the cellular hypoxic responses remains to be elucidated.

Negative regulation of LPS-stimulated expression of inducible nitric oxide synthase by AP-1 in macrophage cell line J774A.1.

The level of NOS II mRNA was markedly increased during 24 h lipopolysaccharide (LPS) stimulation, but showed no further increase thereafter. On the other hand, the level of NOS II mRNA in J774A.1 cells transfected with an expression vector containing the rat csk cDNA (J.Csk) was significantly increased during 3 h LPS stimulation, but rather decreased thereafter. Although no significant difference was observed in the activation of NF-kappaB by LPS among parental J774A.1, J774A.1 transfected with promoterless vector (J.pBK), and J.Csk cells, activity of c-Jun N-terminal kinase (JNK) and nuclear translocation of nuclear factor activator protein-1 (AP-1) were markedly upregulated in the J.Csk cells. Then luciferase reporter vectors containing NOS II promoter with mutations in two AP-1-like sites (U site, -1126 approximately -1120; L site, -524 approximately -518) were transiently transfected in J774A.1 cells. The promoter activity following LPS stimulation for 24 h was significantly increased by mutation at the L site, but not by mutation at the U site, suggesting that NOS II expression is negatively regulated, at least in part, through the AP-1-like L site in response to LPS.

Mercury pollution in the Tapajos River basin, Amazon: mercury level of head hair and health effects.

There is increasing concern about the potential neurotoxic effects of exposure to methylmercury for the 6 million people living in the Amazon, even in regions situated far away from the gold mines (garimpos), considered to be the major source of mercury pollution. In November 1998, a spot investigation on mercury contamination was conducted in three fishing villages (Barreiras, Rainha, and Sao Luiz do Tapajos) on the Tapajos River, an effluent of the Amazon, situated several hundred kilometers downstream from the gold-mining areas. A total of 132 fishermen and their families volunteered for the current study. As was anticipated, the total mercury levels in the head hair collected from the fishing villages were relatively high (14.1-20.8 ppm on the average) and the number of subjects with a high total mercury level over 10 ppm (the least upper bound of a normal value) was 103 (78.0%) in total, along with various symptoms, thereby suggesting wide mercury contamination in the Tapajos River basin. Moreover, in view of the absence of other diseases (e.g., alcoholism or malaria), a high intake of fish containing a methylmercury level, and high hair mercury levels in addition to the various symptoms such as sensory disturbance (especially glove-and-stocking type, which is characteristic of Minamata disease), tremor, failure in two-point discrimination, and slight balancing failure, several subjects examined were diagnosed with mild Minamata disease. The findings obtained suggest, thus, that the mercury pollution in the Amazon should be crucially observed for head hair mercury level and health in a much broader region.

Strenuous endurance training in humans reduces oxidative stress following exhausting exercise.

The aim of this study was to evaluate whether high-intensity endurance training would alleviate exercise-induced oxidative stress. Nine untrained male subjects (aged 19-21 years) participated in a 12-week training programme, and performed an acute period of exhausting exercise on a cycle ergometer before and after training. The training programme consisted of running at 80% maximal exercise heart rate for 60 min.day-1, 5 days.week-1 for 12 weeks. Blood samples were collected at rest and immediately after exhausting exercise for measurements of indices of oxidative stress, and antioxidant enzyme activities [superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT)] in the erythrocytes. Maximal oxygen uptake (VO2max) increased significantly (P < 0.001) after training, indicating an improvement in aerobic capacity. A period of exhausting exercise caused an increase (P < 0.01) in the ability to produce neutrophil superoxide anion (O2.-) both before and after endurance training, but the magnitude of the increase was smaller after training (P < 0.05). There was a significant increase in lipid peroxidation in the erythrocyte membrane, but not in oxidative protein, after exhausting exercise, however training attenuated this effect. At rest, SOD and GPX activities were increased after training. However, there was no evidence that exhausting exercise enhanced the levels of any antioxidant enzyme activity. The CAT activity was unchanged either by training or by exhausting exercise. These results indicate that high-intensity endurance training can elevate antioxidant enzyme activities in erythrocytes, and decrease neutrophil O2.- production in response to exhausting exercise. Furthermore, this up-regulation in antioxidant defences was accompanied by a reduction in exercise-induced lipid peroxidation in erythrocyte membrane.

Activation and apoptosis of murine peritoneal macrophages by acute cold stress.

Effects of acute cold stress (5 degrees C for 24 h) on the functions of peritoneal macrophages and the mechanisms for controlling host homeostasis were investigated in mice. Phagocytic activity and expression of the cell surface adhesion molecule CD11b/CD18 were markedly increased in peritoneal exudate cells by acute cold stress. These alterations were attributable to an increased number and phenotypical changes of adherent cells from acute cold-stressed mice. On the other hand, a lipopolysaccharide-induced activity of src-family tyrosine kinase Fgr, an expression of interleukin-1beta (IL-1 beta) mRNA, and a bioactivity of IL-1 in the culture supernatants of adherent cells from acute cold-stressed mice were markedly lower than those from control mice. A time course study revealed that the number of adherent cells in peritoneal exudate cells was markedly increased in mice exposed to cold for 24 h but returned to normal numbers when mice were exposed to cold for 72 h. DNA fragmentation and Annexin-V(+) cells were observed in peritoneal exudate cells from acute-cold stressed mice. Thus, cold stress activated macrophages but these macrophages were destined to be eliminated by apoptosis.

Changes in the tissue and plasma superoxide dismutase (SOD) levels in a burned rat model.

To examine whether thermal injury alters the superoxide dismutase (SOD) concentrations in various types of tissue or plasma, we studied the plasma and tissue Mn- and Cu/Zn-SOD levels in a rodent burn model. The animals were resuscitated with saline (50 mg/kg, i.p.) immediately following thermal injury and thereafter were sacrificed at either 6 or 24 hours post-burn. The Mn- and Cu/Zn-SOD levels were measured using an enzyme-linked immunosorbent assay (ELISA). The plasma Mn- and Cu/Zn-SOD concentrations significantly increased 6 hours after the injury and positively correlated with the burn size. The kidney Mn-SOD concentrations were significantly higher 24 hours after the injury in the animals with 30% burns than in those with either sham or 50% burn injuries. The lung Cu/Zn-SOD concentrations were also significantly higher 6 hours after the injury in animals with 30% burns than in the other two types above. These findings suggest that the changes in the SOD concentrations after burn injury vary according to the type of SOD and also the type of tissue. As a result, the SOD concentrations may play some role in the early response to thermal trauma.

Wide use of skin-lightening soap may cause mercury poisoning in Kenya.

In a previous study, we speculated that some of the high mercury levels observed in head hair from a total of 14 subjects who resided around Lake Victoria, Tanzania, might be attributable to the habitual use of toilet soap containing considerable amounts of mercury (Harada et al. Sci Total Environ 1999;227:249-256). In August 1998, the current study was conducted to investigate if such mercury-containing soap was also available in the surroundings of Lake Victoria, Kenya, and if so, its toxic effects. A total of nine goldminers, 44 fishermen and their families, and 12 residents of Kisumu City, Kenya, volunteered for the study. Fourteen types of toilet soap were collected in Kisumu. Total mercury content was very significantly higher than in European-made soap (0.47-1.7%, as mercury iodide) compared with Kenya-made soap (0.41 x 10(-4)-6.2 x 10(-4)%). Indeed, all the subjects with a high hair mercury level (> 36.1 ppm) had made habitual use of European-made soap, accompanied by various symptoms, such as tremor, lassitude, vertigo, neurosthenia, and black and white blots, suggesting inorganic-mercury poisoning. On the other hand, any subject who had used soap other than the European-made soap, did not exceed a mercury level of 10 ppm in hair that is well within normal limits (Harada et al. Sci Total Environ 1999:227:249-256). The findings obtained suggest that the mercury-containing soap must be barred from circulation without delay, and that the residents' health in addition to the environmental pollution in Lake Victoria (Kenya as well as Tanzania) should be kept under close observation.

Epstein-Barr virus-negative high grade B cell lymphoma of donor origin developing 19 months after unrelated allogeneic bone marrow transplantation.

A 22-year-old man, in first complete remission of acute myelogenous leukemia, developed a high grade B cell lymphoma 19 months after an allogeneic bone marrow transplant (allo-BMT) from an HLA-identical unrelated donor. Biopsy of a cervical lymph node revealed a lymphoma that was negative for Epstein-Barr virus-encoded small nuclear RNAs (EBERs) in situ hybridization. Genotypic analyses identified the lymphoma to be of donor origin, and there was no evidence of the Epstein-Barr virus (EBV) DNA in the lymphoma by Southern blot analysis. The lymphoma went into complete remission, following four courses of combination chemotherapy, but relapsed after a month and the patient died of congestive heart failure. The patient was thought to be persistently immunosuppressed 11 months after cessation of immunosuppressants, and the lymphoma was thought to be induced by one or more factors other than EBV.

Extracellular superoxide dismutase in tissues from obese (ob/ob) mice.

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-alpha and interleukin-1beta contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.