Fibronectin suppresses lipopolysaccharide-induced liver damage and promotes the cytoprotection abilities of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells.

We isolated mesenchymal stem cells (MSCs) from adult human bone marrow. By using reverse-transcription polymerase chain reactions, we confirmed that MSCs possessed the potential to differentiate into hepatocyte-like cells (MSC-HLCs) with the expression of hepatocyte-specific marker genes. We further observed that fibronectin (FN) treatment significantly inhibited lipopolysaccharide (LPS)-induced apoptotic activities in FN-treated MSC-HLCs, as detected by caspase 3 enzyme-linked immunosorbent (ELISA) and terminal dUTP nick-end labeling (TUNEL) assays (P<.05). The FN-treated MSC-HLCs were transplanted into SCID mice with or without LPS injection. This study demonstrated that FN treatment improved liver function repair and survival rates among LPS-treated SCID mice.

Evaluation of wound healing effect on skin-defect nude mice by using human dermis-derived mesenchymal stem cells.

We report that human dermis-derived mesenchymal stem cells (hDMSCs) possess differentiation potential of epidermis facilitating wound healing in skin-defect nude mice in combination with the treatment using gelatin/thermosensitive poly N-isopropylacrylamide (pNIPAAm)/polypropylene (PP). The results showed that the rate of cell growth and wound recovery in the hDMSC and gelatin/pNIPAAm/PP-treated group was significantly greater than those in the gelatin/pNIPAAm/PP-treated only group (P < .01). The reepithelialization marker of human pan-cytokeratin was also significantly increased on days 14 and day 21 in the wound site of hDMSCs and gelatin/pNIPAAm/PP-treated group. Furthermore, the stem cell marker of human CD13 gradually decreased during the period of wound healing. In sum, this novel method provided a transferring system for stem cell therapy, maintaining its temperature-sensitive property of easy peeling by lower temperature treatment.

Using gelatin scaffold with coated basic fibroblast growth factor as a transfer system for transplantation of human neural stem cells.

Gelatin scaffolds for ex vivo cell cultures are a promising development. These scaffolds can be used as three-dimensional skeletons for cell attachment and culture before transplantation. In this study, we isolated and cultivated neural stem cells from human brain tissues in serum-free medium (DMEM+F12 nutrient). Better neuron growth was observed using the tetrazolium assay (MTT) in the group when basic fibroblast growth factor (bFGF) was coated on the gelatin polymer scaffold. Further development of this nontoxic system may help the future development of transplantation of human neural stem cells.

Using human plasma supplemented medium to cultivate human bone marrow-derived mesenchymal stem cell and evaluation of its multiple-lineage potential.

The objective of this study was to evaluate the proliferation and the multiple-lineage differentiation capacity when bone marrow mesenchymal stem cells (BMSCs) were cultured short-term in autologous serum/plasma instead of fetal calf serum (FCS). The BMSCs from 12 donors were cultivated individually in 10% autogenic plasma or serum, with or without bFGF and EGF growth factors. Cell proliferation was examined by a Tetrazolium assay (MTT) after passages 1, 3, and 5. A medium supplemented with 10% human plasma or serum was sufficient to propagate BMSCs. However, no significant proliferation was shown when bFGF and EGF (20 ng/mL each) were added into the medium with autologous serum/plasma. We examined, inductions of adipogenesis, osteogenesis, and chondrocytogenesis, as capacities of multiple-lineage differentiation of cultivated BMSCs (passages 8). Differentiation was investigated by both RT-PCR and immunohistochemistry staining (IHC). Qualitative evidence demonstrated the differentiation capacity was preserved in cultivated BMSCs with autologous serum/plasma.

Relationship between mitochondrial superoxide and hydrogen peroxide production and longevity of mammalian species.

The objective of this study was to examine the possible involvement of oxygen free radicals in the aging process. Rates of mitochondrial O2.- and H2O2 production and oxygen consumption in the kidney and the heart were compared among seven different mammalian species namely, mouse, hamster, rat, guinea pig, rabbit, pig, and cow, whose maximum life span potential (MLSP) varies from 3.5 to 30 years. The rates of mitochondrial O2.- and H2O2 generation were inversely correlated to MLSP, and directly related to specific metabolic rate and state 4 mitochondrial respiration. Results of this study indicate that under identical conditions, mitochondria from shorter-lived species produce relatively higher amounts of reactive oxygen species than those from the longer-lived species, and, thus, support the free radical hypothesis of aging.

Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells.

Cytochrome c release from mitochondria is central to apoptosis, but the events leading up to it are disputed. The mitochondrial membrane potential has been reported to decrease, increase or remain unchanged during cytochrome c release. We measured mitochondrial membrane potential in Jurkat cells undergoing apoptosis by the uptake of the radiolabelled lipophilic cation TPMP, enabling small changes in potential to be determined. The ATP/ADP ratio, mitochondrial and cell volumes, plasma membrane potential and the mitochondrial membrane potential in permeabilised cells were also measured. Before cytochrome c release the mitochondrial membrane potential increased, followed by a decrease in potential associated with mitochondrial swelling and the release of cytochrome c and DDP-1, an intermembrane space house keeping protein. Mitochondrial swelling and cytochrome c release were both blocked by bongkrekic acid, an inhibitor of the permeability transition. We conclude that during apoptosis mitochondria undergo an initial priming phase associated with hyperpolarisation which leads to an effector phase, during which mitochondria swell and release cytochrome c.

Comparison of mitochondrial pro-oxidant generation and anti-oxidant defenses between rat and pigeon: possible basis of variation in longevity and metabolic potential.

Non-passerine birds and mammals of similar body weight have a roughly comparable metabolic rate, but the life span and the metabolic potential, i.e. the total amount of energy consumed per unit of body mass during life, is several times higher in the birds. The objective of this study was to explore the possible basis of this characteristic in the context of the predictions of the free radical hypothesis of aging. Accordingly, pigeon and rat, which have a similar body weight, were compared by examining the mitochondrial rates of O2.- and H2O2 generation and activities of superoxide dismutase, catalase and glutathione peroxidase and concentration of glutathione in the brain, heart and kidney. Compared with the rat, the rate of mitochondrial O2.- generation in the pigeon ranged between 50 and 67%, and H2O2 production between 31 and 77%. Activity of superoxide dismutase was uniformly higher and catalase activity consistently lower in the tissues of the pigeon compared with the rat. Glutathione peroxidase activity and glutathione concentration were higher in the pigeon in two out of the three organs studied, and comparable in the third organ. The magnitude of the differences between the two species was greater in the rates of O2.- and H2O2 generation than in anti-oxidant defenses. Results indicate that the relatively greater longevity and metabolic potential of the pigeon may be related to significantly lower rates of O2.- and H2O2 generation and higher overall level of anti-oxidant defenses.

Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse.

This study was conducted in order to test the concept that oxidative damage is associated with aging and may be a factor in the modulation of life span in response to variations in caloric intake. Mice fed a diet that was 40% lower in calories (DR) than the ad libitum fed (AL) animals exhibited a 43% extension in average life span and a 61% prolongation in mortality rate doubling time. A comparison of AL and DR mice at 9, 17 and 23 months of age indicated that the protein carbonyl content in the brain, heart and kidney increased with age and was significantly greater in the AL than DR group in each organ at each of the three ages. Mitochondrial state 4 or resting respiratory rate increased with age in the AL, but not the DR group, and was also relatively higher in the former. The rates of mitochondrial superoxide and hydrogen peroxide generation increased with age and were higher in the AL than DR mice in all the three organs at each age. In contrast, there was no clear-cut overall pattern of age-related or dietary-related changes in antioxidant defenses provided by superoxide dismutase, catalase and glutathione peroxidase. Results suggest that mechanisms of aging and life span shortening by enhanced caloric intake are associated with oxidative damage arising from corresponding changes in mitochondrial oxidant production. Protein carbonyl content, and mitochondrial O2.- and H2O2 generation may act as indices of aging.

Oxidative stress is insignificant in N1S1-transplanted hepatoma despite markedly declined activities of the antioxidant enzymes.

It has been proposed that persistent oxidative stress accounts for the increased levels of DNA damage in cancer tissues. We have examined the profile of anti-oxidant enzymes in a transplanted hepatic tumor model by injecting N1S1 rat hepatoma cells into the liver of Sprague-Dawley rats. The transplanted N1S1 tumors displayed characteristics resembling human hepatocellular carcinoma. The immunoreactivities of catalase (CAT), manganese-superoxide dismutase (Mn SOD), copper/zinc-SOD (Cu/Zn SOD), and glutathione peroxidase (GPx) were found to decrease significantly. The enzyme activity in tumors decreased 26.2-, 4.2-, 4.5-, and 5.4-fold for CAT, Mn SOD, Cu/Zn SOD, and GPx, respectively, relative to those in normal liver tissue from the same animals. In contrast, the mRNA levels of CAT and GPx in tumors decreased only 5- and 2-fold, respectively, and the mRNA levels of Cu/Zn SOD and Mn SOD showed either no change or an increase as compared to those of normal liver tissue. The contents of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and thiobarbituric acid-reactive substances (TBARS) were comparable to those of normal controls. Furthermore, mitochondrial production of superoxide in tumors was 4 times lower than that in normal tissues. In conclusion, the data indicate that the reduced activities of anti-oxidant enzymes in the N1S1 tumor did not cause significant oxidative stress.

Hepatic portal venous gas associated with acute mesenteric infarction--a case report.

Hepatic portal venous gas (HPVG) is considered as an ominous prognostic sign and indicates the need for urgent surgical intervention. It has been associated with a wide variety of abdominal catastrophes, but the commonest and most serious one is infarcted bowel. This disease entity is difficult to be detected by conventional radiography. Ultrasonography and computed tomography have been reported to be more valuable methods in detecting HPVG and its cause. We report a case of HPVG associated with acute mesenteric infarction, which was detected early by ultrasonography. The computed tomography done later not only showed typical features of HPVG, but also disclosed intramural gas in the small intestine and gas in the superior mesenteric veins, which were diagnostic clues for acute mesenteric infarction. The patient survived after urgent surgery.

Biochemical correlates of longevity in two closely related rodent species.

The objective of this study was to explore the basis of variations in the life span and metabolic potential, i.e., total amount of energy consumed during life, between different species, in context of the free radical hypothesis of aging. A comparison was made between the house mouse (Mus musculus) and the white-footed mouse (Peromyscus leucopus): the latter has > 2-fold greater life span and metabolic potential than the former. Longer life span and higher metabolic potential of Peromyscus were associated with low rates of mitochondrial O2.- and H2O2 generation, higher activities of catalase and glutathione peroxidase and low levels of protein oxidative damage as well as low susceptibility to oxidative damage in response to experimental oxidative stress. Results support the role of oxidative stress in aging.

Monoclonal antibodies to adenosine receptor by an auto-anti-idiotypic approach.

Monoclonal antiadenosine receptor antibodies have been raised by an auto-anti-idiotypic approach. BALB/c mice were immunized with adenosine 6-aminocaproyl-bovine serum albumin. Hybridoma cell lines were raised and lines that secreted antibodies that bound to rabbit antiadenosine antibodies were obtained. Two such monoclonal antibodies, AA18 and AA21, were studied in detail and found to be directed at adenosine receptors by the following criteria. They inhibited the binding of [3H] adenosine to rabbit antiadenosine antibodies that had binding characteristics similar to those of adenosine receptors. They bound to rat brain membranes and binding could be inhibited by N6-cyclohexyladenosine and L-N6-phenylisopropyladenosine, both adenosine receptor agonists. They also inhibited the binding of [3H]L-N6-phenylisopropyladenosine to rat brain membranes. In functional assays, they inhibited adenylate cyclase of rat brain membranes, but had no effect on adenylate cyclase of rat hepatic membranes, indicating that they mimic agonists of the A1 receptor, therefore, carrying an "internal image" of the adenosine molecule. When adenosine receptors of rat brain membranes were solubilized with 1% cholic acid, partially purified on an adenosine 6-aminocaproyl AH-Sepharose column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, both AA18 and AA21 recognized a 62,000 band under nonreducing conditions, and a major band of 36,000 under reducing conditions. We conclude that the auto-anti-idiotypic route has yielded specific antibodies that recognize the A1 adenosine receptor.

In vitro estradiol synthesis and secretion by tadpole ovaries of different developmental stages.

The tadpole ovaries at TK stage XIX can synthesize and secrete estradiol (E2), yet it is unknown when this ovarian function starts and how it develops. To this end, the present work has been carried out. The ovaries of different developmental stages of tadpoles and young frogs of Rana catesbeiana were taken, cut into small pieces, and incubated for 6 hr at 20 degrees in Krebs-Ringer bicarbonate buffer. The media were then analyzed for E2 by radioimmunoassay. The results indicate that the tadpoles synthesize and release E2 as early as stage X, that E2 increases slowly and gradually through stage XXV, and that E2 increases rapidly in juvenile frogs. This trend of growth of the ovarian function in estradiol secretion in vitro is in accordance with that reported for female chick embryos. There exists the possibility that the growth of E2 secretion could be biphasic, one with slow increment of autonomous E2 secretion at early stages and the other with quick increase due to pituitary stimulation at the stage of metamorphic climax.

Contrasting effects of ACTH and cyanoketone on delta 5-3 beta-hydroxysteroid dehydrogenase activity in interrenal glands of tadpoles of Rana catesbeiana in vitro.

The present communication describes an investigation of stimulation and inhibition of delta 5-3 beta-hydroxysteroid dehydrogenase in interrenal glands of tadpoles of Rana catesbeiana. Frozen sections of interrenal glands, together with kidneys, were prepared histochemically for assay of delta 5-3 beta-HSD activity. Concentrations of 0.01, 0.1, 1, and 10 IU/ml of ACTH or of 0.01, 0.1, 1, and 10 micrograms/ml of cyanoketone were added to the incubation media. The reaction products of the histochemically prepared slides, in terms of absorbance, were scanned at a defined area with a computerized microscope spectrophotometer. The results indicate that ACTH causes a significant dose-response stimulation of delta 5-3 beta-HSD activity in tadpole interrenals; cyanoketone, on the other hand, causes significant dose-dependent inhibition.

Age-associated changes of superoxide dismutase and catalase activities in the rat brain.

Oxygen free radicals have been proposed to be involved in the process of aging. Superoxide dismutase (SOD) and catalase are important for antioxidative defense. In this study, profiles of SOD, catalase, and their mRNA levels were investigated in the frontal, parietal, temporal and occipital lobes, subcortex and cerebellum of male Wistar rats at ages 1-21 months. The total SOD and Mn SOD activities increased with age and exhibited higher levels at 6 and 12 months but decreased thereafter. Activity of catalase showed a similar trend and notably peaked at 12 months. The mRNA levels of Cu/Zn SOD, Mn SOD, and catalase remained constant in all areas tested (frontal, parietal, temporal and occipital lobes, and subcortex) except the cerebellum. Post-transcriptional regulation was involved in modulating the enzymes' activities during aging. Furthermore, the rate of mitochondrial generation of the superoxide anion (O(2)(-).) increased gradually with aging. Taken together, the results suggest that the increase of oxidative potential and the loss of proper antioxidant defense in the rats appear to be highly involved in the aging process of the brain.

The development of ACTH-like substance during tadpole metamorphosis.

The interrenals of tadpoles of Rana catesbeiana at various developmental TK stages were frozen, sectioned, and prepared histochemically for assessment of delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) activity. The interrenals of hypophysectomized (HX) tadpoles from the same batch of eggs as control tadpoles were prepared similarly for delta 5-3 beta-HSD activity following a series of intraperitoneal injections of different concentrations of mammalian ACTH. The histochemically stained sections of control and HX tadpoles were scanned with a computerized microscope spectrophotometer for diformazan absorbance. Two regression equations were obtained: Y = 0.1460 + 0.0015X and Y' = 0.1494 + 0.0893X', where Y or Y' = delta 5-3 beta-HSD activity absorbance (for the control and HX tadpoles, respectively), X = developmental stages of control, and X' = exogenous ACTH dosage administered to the HX tadpoles. Taking absorbance of delta 5-3 beta-HSD activity as the common factor, a curve of concentrations of ACTH-like substance against TK stages was made. The developing concentration of ACTH-like substance was very low at first and then rose during metamorphic climax. The trend of the development of ACTH-like substance was discussed from the view point of thyroid and delta 5-3 beta-HSD activities.

In vitro inhibition of estradiol secretion of tadpole ovaries by cyanoketone.

We have demonstrated the presence of delta 5-3 beta-hydroxysteroid dehydrogenase (HSD) activity in tadpole ovaries of Rana catesbeiana. In the present study, we wish to determine whether estradiol (E2) secretion of tadpole ovaries could be influenced by cyanoketone (CK), a specific inhibitor of delta 5-3 beta-HSD. R catesbeina tadpoles at the premetamorphic climax were used, and pooled ovaries were incubated, 30 mg/tube, with CK at dosages of 0, 0.001, 0.01, 0.1, 1, and 10 micrograms/ml of Krebs-Ringer bicarbonate buffer for 6 hr. Media were collected for assay of E2 by radioimmunoassay (RIA). Results showed an inhibition of E2 secretion by CK that was positively correlated with CK dosage, but plateaued at doses of 0.1 microgram/ml and higher. This finding was comparable to that of G.F. Young, H. Kagawa, and Y. Nagahama (1982, Gen. Comp. Endocrinol. 47, 357-360) on adult fish ovaries. However, adult vertebrates depend on gonadotropins to regulate secretion of E2 while tadpoles, being immature, might secrete E2 independently of pituitaries. When the histochemical test for delta 5-3 beta-HSD activity was performed on in vitro CK-treated ovaries, there was a decrease of enzyme activity by CK. The RIA and histochemical findings may contribute to the concept of sex transformation in which a disturbance of steroidogenesis may induce sex reversal from females to males, at least in tadpoles.

Estradiol secretion by the ovarian tissue, in response to hypophyseal stimulation, during ontogenesis of the bullfrog.

Ovaries of tadpoles, froglets, young frogs, and mature frogs of Rana catesbeiana were cut into small pieces. They were incubated for 6 hr in Krebs-Ringer bicarbonate buffer as controls. Another series of ovaries of the same developmental stages were incubated with pituitary extracts in the buffer as experimentals. Media were then analyzed for estradiol secretion by radioimmunoassay. The results showed that estradiol secretion by tadpole ovaries during development was not affected by the addition of pituitary extracts of mature frogs in the media at any stage while those of young and mature frogs with pituitary extracts secreted more estradiol than those without. These findings indicate that tadpole ovaries are unresponsive to pituitary agents to produce estradiol while frog ovaries are dependent on some pituitary hormones to synthesize estradiol. Thus frog ovaries acquire dependence on the pituitary agent only after metamorphosis.