Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis

Tumor necrosis factor alpha (TNF) plays an important role in mediating hepatocyte injury in various liver pathologies. TNF treatment alone does not cause the death of primary cultured hepatocytes, suggesting other factors are necessary to mediate TNF-induced injury. In this work the question of whether reactive oxygen species can sensitize primary cultured hepatocytes to TNF-induced apoptosis and necrosis was investigated. Sublethal levels of H(2)O(2), either as bolus doses or steady-state levels generated by glucose oxidase, were found to sensitize cultured hepatocytes to TNF-induced apoptosis. High levels of H(2)O(2) also triggered necrosis in hepatocytes regardless of whether TNF was present. Similarly, antimycin, a complex III inhibitor that increases reactive oxygen species generation from mitochondria, sensitized hepatocytes to TNF-induced apoptosis at low doses but caused necrosis at high doses. Redox changes seem to be important in sensitizing primary hepatocytes, because diamide, a thiol-oxidizing agent, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase, also increased TNF-induced apoptosis in cultured primary hepatocytes at sublethal doses. High doses of diamide and BCNU predominantly triggered necrotic cell death. Agents that sensitized hepatocytes to TNF-induced apoptosis -- H(2)O(2), antimycin, diamide, BCNU -- all caused a dramatic fall in the GSH/GSSG ratio. These redox alterations were found to inhibit TNF-induced IkappaB-alpha phosphorylation and NF-kappaB translocation to the nucleus, thus presumably inhibiting expression of genes necessary to inhibit the cytotoxic effects of TNF. Taken together, these results suggest that oxidation of the intracellular environment of hepatocytes by reactive oxygen species or redox-modulating agents interferes with NF-kappaB signaling pathways to sensitize hepatocytes to TNF-induced apoptosis. The TNF-induced apoptosis seems to occur only in a certain redox range -- in which redox changes can inhibit NF-kappaB activity but not completely inhibit caspase activity. The implication for liver disease is that concomitant TNF exposure and reactive oxygen species, either extrinsically generated (e.g., nonparenchymal or inflammatory cells) or intrinsically generated in hepatocytes (e.g., mitochondria), may act in concert to promote apoptosis and liver injury.     

The role of endogenous opioids in the protective effects of local sublethal hyperthermia against the progression of burn injury

The aim of the present study was to evaluate the role of endogenous opioids in local sublethal hyperthermia-induced protection against burn injury.Second-degree burn wounds were induced on the back of Balb/c mice. Progression of burn injury and expression of heat shock protein (HSP)-70 were evaluated after 24 h.Both inhibition of HSP synthesis and blocking opioid receptors before applying local sublethal hyperthermia decreased the protective effects of sublethal hyperthermia against the progression of burn injury. Blocking opioid receptors attenuated induction of HSP-70 by sublethal hyperthermia.     

Appendage-Mediated Surface Adherence of Sulfolobus solfataricus

Attachment of microorganisms to surfaces is a prerequisite for colonization and biofilm formation. The hyperthermophilic crenarchaeote Sulfolobus solfataricus was able to attach to a variety of surfaces, such as glass, mica, pyrite, and carbon-coated gold grids. Deletion mutant analysis showed that for initial attachment the presence of flagella and pili is essential. Attached cells produced extracellular polysaccharides containing mannose, galactose, and N-acetylglucosamine. Genes possibly involved in the production of the extracellular polysaccharides were identified.In microbiology, organisms are isolated from their natural habitats and typically cultivated in the laboratory as planktonic species. Though this method has been essential for understanding the concept of life, it remains unclear how microbial ecosystems operate. For bacteria, it is well known that they are able to form large cellular communities with highly complex cellular interactions and symbioses between different microbial or eukaryotic species. Biofilm formation is an essential component of such communities, and studies have shown that bacteria within biofilms are physiologically different from planktonic ones (20, 21). They can exhibit extensive networks of pili on their surfaces and produce and secrete extracellular polysaccharides (EPS), their growth rate is decreased, and cells are much more resistant to physical stresses and antibiotics (19).The study of surface colonization and cellular communities of archaea is crucial for understanding their ecological properties. The only detailed study showed that the hyperthermophilic organism Archaeoglobus fulgidus produced biofilms when challenged with heavy metals and pentachlorophenol (10). Pyrococcus furiosus was able to adhere to different surfaces, such as mica and carbon-coated gold grids, and cells were connected via cable-like bundles of flagella (12). Methanopyrus kandleri was shown to adhere to glass, but P. furiosus could colonize only by attaching to M. kandleri cells, using flagella and direct cell contacts (16).Here we report on the function of cell surface appendages in initial attachment to surfaces of archaea, using directed gene inactivation mutants. The crenarchaeote Sulfolobus solfataricus P2 is a thermoacidophile which grows optimally at 80°C and pH values of 2 to 4 (22). S. solfataricus possesses cell surface structures such as flagella and UV-induced pili (1, 2). The flagellum operon of S. solfataricus encodes, in addition to the structural subunit FlaB, four proteins of unknown function, the ATPase FlaI, and the only integral membrane protein, FlaJ. Previously, we isolated a ΔflaJ mutant which was nonflagellated and had lost its ability for surface motility on Gelrite plates (17). Recently, we described UV-inducible pili in S. solfataricus that directed cellular aggregation after UV stress (8). Deletion of the central ATPase UpsE, responsible for pilus assembly, rendered cells devoid of pili and defective in cellular aggregation after UV treatment (8). In this study, wild-type cells and deletion strains were tested for the ability to attach to a variety of surfaces and the formed structures and extracellular material were analyzed.     

Directed assembly of cell-laden hydrogels for engineering functional tissues

Tissue engineering aims to develop functionalized tissues for organ replacement or restoration. Biodegradable scaffolds have been used in tissue engineering to support cell growth and maintain mechanical and biological properties of tissue constructs. Ideally cells on these scaffolds adhere, proliferate, and deposit matrix at a rate that is consistent with scaffold degradation. However, the cellular rearrangement within these scaffolds often does not recapitulate the architecture of the native tissues. Directed assembly of tissue-like structures is an attractive alternative to scaffold-based approach for tissue engineering which potentially can build tissue constructs with biomimetic architecture and function. In directed assembly, shape-controlled microstructures are fabricated in which organized structures of different cell types can be used as tissue building blocks. To fabricate tissue building blocks, hydrogels are commonly used as biomaterials for cell encapsulation to mimic the matrix in vivo. The hydrogel-based tissue building blocks can be arranged in pre-defined architectures by various directed tissue assembly techniques. In this paper, recent advances in directed assembly-based tissue engineering are summarized as an emerging alternative to meet challenges associated with scaffold-based tissue engineering and future directions are addressed.     

Rho kinase, myosin-II, and p42/44 MAPK control extracellular matrix-mediated apical bile canalicular lumen morphogenesis in HepG2 cells

The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a pair of cells. Prolonged cell culture results in extensive organizational changes, including cell clustering, multilayering, and apical lumen morphogenesis. The latter includes the development of large acinar structures and subsequent elongated canalicular lumens that span multiple cells. These morphological changes closely resemble the early organizational pattern during development, regeneration, and neoplasia of the liver and are rapidly induced when cells are cultured on predeposited extracellular matrix (ECM). Inhibition of Rho kinase or its target myosin-II ATPase in cells cultured on glass coverslips mimics the morphogenic response to ECM. Consistently, stimulation of Rho kinase and subsequent myosin-II ATPase activity by lipoxygenase-controlled eicosatetranoic acid metabolism inhibits ECM-mediated cell multilayering and apical lumen morphogenesis but not initial apical lumen formation. Furthermore, apical lumen remodeling but not cell multilayering requires basal p42/44 MAPK activity. Together, the data suggest a role for hepatocyte-derived ECM in the spatial organization of hepatocytes and apical lumen morphogenesis and identify Rho kinase, myosin-II, and MAPK as potentially important players in different aspects of bile canalicular lumen morphogenesis.     

Longitudinal Profiling of the Tissue-Specific Expression of Genes Related with Insulin Sensitivity in Dairy Cows during Lactation Focusing on Different Fat Depots

In dairy cows the milk associated energy output in early lactation exceeds the input via voluntary feed intake. To spare glucose for mammary lactose synthesis, peripheral insulin sensitivity (IS) is reduced and fat mobilization is stimulated. For these processes a link between IS and the endocrine functions of adipose tissue (AT) is likely; we thus aimed to characterise the mRNA expression from bovine AT derived proteins and receptors that are related to IS according to the literature in metabolically active tissues plus systemic IS throughout lactation. Conjugated linoleic acids (CLA) reduce milk fat thus decreasing the milk drain of energy and potentially dampening lipolysis, but may also affect IS. Subcutaneous (s.c.) AT and liver from pluriparous cows receiving either control fat or CLA supplement (100 g/day from 1 to 182 days in milk each) were biopsied covering week −3 to 36 relative to parturition. In an additional trial with primiparous cows treated analogously and slaughtered on days in milk 1, 42 or 105, samples from liver, udder, skeletal muscle and 3 visceral and 3 s.c. AT were obtained and assayed for mRNA abundance of adiponectin, its receptors, leptin, leptin receptor, PPARγ, PPARγ2, IL-6, and TNF-α. In pluriparous animals, the mRNA abundance of most of the target genes decreased after parturition in s.c. AT but increased in liver. In primiparous cows, AT depot specific differences were mostly related to retroperitoneal AT; adiponectin receptor 1 and TNF-α were affected predominantly. CLA effects in primiparous cows were largely limited to decreased PPARγ2 mRNA abundance in udder tissue. In pluriparous cows, insulin secretion was increased by CLA resulting in decreased systemic IS but without consistent changes in tissue target mRNA abundance. The temporal gene expression profiles from the adipokines and related receptors support their coactive function in adapting to the needs of lactation.     

An outcome analysis comparing the thoracodorsal and internal mammary vessels as recipient sites for microvascular breast reconstruction: a prospective study of 100 patients

The thoracodorsal vessels have been the standard recipient vessels for the majority of surgeons performing free transverse rectus abdominis musculocutaneous (TRAM) flap reconstructions. Recently, the internal mammary vessels have been recommended as the first-choice recipient vessels for microvascular breast reconstruction. This approach requires a shorter pedicle length, allows for central placement of flap tissue, and avoids axillary scarring. The use of the internal mammary vessels may provide for a shorter operative time and a higher-quality aesthetic reconstruction. The authors performed a prospective trial examining the differences in operative and aesthetic outcomes between each recipient site. A prospective trial of 108 consecutive free-tissue transfers was conducted in 100 patients. The first 60 TRAM flap patients were randomized so that 30 flaps were anastomosed to the internal mammary vessels and 30 were anastomosed to the thoracodorsal vessels, whereas the recipient vessels for the remaining 40 patients were left to the discretion of the surgeon. Of the 40 nonrandomized patients, 10 patients underwent reconstruction using the internal mammary vessels and 30 patients underwent reconstruction using the thoracodorsal vessels. The patients' medical history and hospital course were noted. To evaluate aesthetic outcome, a group of five blinded nonmedical observers and three blinded plastic surgeons graded the reconstructions in the 60 TRAM flap patients for symmetry and overall aesthetic result on a scale of 1 to 5. Blinded practitioners administered postoperative questionnaires to patients regarding recovery time and satisfaction with the aesthetic result. Forty-three flaps were transferred to the internal mammary vessels and 65 were transferred to the thoracodorsal vessels. No significant differences existed between groups with regard to age of preoperative risk factors. Average operative time was 6 hours in each group. Average hospital stay was 5.8 days in each group. Conversion from initial recipient vessel to a secondary recipient site occurred in 12.5 percent of internal mammary reconstructions and 7 percent of thoracodorsal reconstructions. All converted internal mammary cases occurred in left-sided reconstructions and were attributable to problems with the veins. Overall, 20 percent of left-sided internal mammary reconstructions were found to have an inadequate recipient vein. Unusable thoracodorsal vessels were found only in delayed reconstructions, at a rate of 15 percent in the delayed setting. All flaps from converted procedures survived without complications. Average follow-up was 20 months, during which time there was one flap loss in the thoracodorsal group. There were no significant differences in complication rates between groups. Average aesthetic grade was 3.6 in each group. Postoperative recovery time and overall patient satisfaction were not significantly different between groups. Either recipient site can provide for a safe and acceptable result; however, surgeons should be aware of conversion rates and plan appropriately if recipient vessels appear unusable for free-tissue transfer.