Wnt/��-catenin signaling in hepatic organogenesis

Wnt/β-catenin signaling has come to the forefront of liver biology in recent years. This pathway regulates key pathophysiological events inherent to the liver including development, regeneration and cancer, by dictating several biological processes such as proliferation, apoptosis, differentiation, adhesion, zonation and metabolism in various cells of the liver. This review will examine the studies that have uncovered the relevant roles of Wnt/β-catenin signaling during the process of liver development. We will discuss the potential roles of Wnt/β-catenin signaling during the phases of development, including competence, hepatic induction, expansion and morphogenesis. In addition, we will discuss the role of negative and positive regulation of this pathway and how the temporal expression of Wnt/β-catenin can direct key processes during hepatic development. We will also identify some of the major deficits in the current understanding of the role of Wnt/β-catenin signaling in liver development in order to provide a perspective for future studies. Thus, this review will provide a contextual overview of the role of Wnt/β-catenin signaling during hepatic organogenesis.Key words: liver development, liver cancer, liver regeneration, Wnt signaling, proliferation, differentiationThe Wnt/β-catenin pathway is an evolutionarily well-conserved pathway that has proven to be essential to normal cellular processes such as development, growth, survival, regeneration and self-renewal.^1–5 Its diverse functions also include the initiation and progression of cancer.⁶ In fact, one area in which this pathway has been extensively studied is in liver cancer.Mutations of Wnt/β-catenin pathway members in hepatocarcinogenesis are common. For example, 90–100% of hepatoblastomas contain mutations in adenomatous polyposis coli (APC), CTNNB1 and/or Axin1/2, which causes cytoplasmic and nuclear localization of β-catenin.^7–9 Axin1 and β-catenin mutations have also been identified in approximately 25% of hepatocellular carcinomas,^10–12 while overexpression of the frizzled-7 receptor¹³ and glycogen synthase kinase-3 (GSK-3) inactivation¹⁴ can also lead to aberrant β-catenin pathway activation. The dysregulation of this pathway in hepatic cancers makes it an attractive target for potential therapies, and experimental treatment in vivo has shown promising results. For example, inhibiting β-catenin expression by siRNA or R-Etodolac decreases proliferation and survival of human hepatoma cell lines.^15,16 Since cancer recapitulates development, determining the timing of β-catenin activation during hepatogenesis will help us to better understand the inappropriate activation of this pathway in hepatocarcinogenesis.Recent work has elucidated the role of β-catenin signaling in the liver, and has highlighted its essential role in liver health and disease.¹⁷ In addition, emerging evidence suggests that this pathway plays a key role in liver organogenesis.     

Identification of ATP binding residues of a protein from its primary sequence

Background  

One of the major challenges in post-genomic era is to provide functional annotations for large number of proteins arising from genome sequencing projects. The function of many proteins depends on their interaction with small molecules or ligands. ATP is one such important ligand that plays critical role as a coenzyme in the functionality of many proteins. There is a need to develop method for identifying ATP interacting residues in a ATP binding proteins (ABPs), in order to understand mechanism of protein-ligands interaction.     

Similarity of rpoB gene sequences of sucrose-fermenting and non-fermenting Corynebacterium diphtheriae strains

During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.     

Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

Myosin light-chain expression during avian muscle development

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.     

Regulation of cell-mediated cytotoxicity. II. Synergism of two types of thymus dependent cells in vivo.

Sublethal (500 rads) doses of radiation given to mice before the intravenous injection of allogeneic spleen cells induced the development of an increased cell-mediated cytotoxicity (CMC) of the recipients' spleen cells. The effector cells from the irradiated animals were shown to carry the θ alloantigenic marker and to be capable of transferring adoptive immunity in vivo. On the other hand, irradiation of mice with the same dose before the administration of skin or tumor allografts induced a suppression of CMC. The response of irradiated mice treated with tumor allografts was restored with small numbers of spleen or lymph node cells from syngeneic or semi-allogeneic F₁ hybrid donors. With the use of the appropriate cytotoxic alloantisera, it was demonstrated that the majority of the effector cells generated in the spleens of mice restored with semiallogeneic cells were of host origin. These results demonstrate that the precursors of the cytotoxic lymphocytes are radioresistant and indicate that for their stimulation some radiosensitive T cells are necessary to amplify their reaction to nonlymphoid allografts. Allogeneic lymphoid cells, on the other hand, supply a stimulus which does not require the intervention of such amplifier cells. In this case, irradiation induces a stronger CMC response probably by inactivating radiosensitive cells with suppressor activity.     

Common structural domains in the sarcoplasmic reticulum Ca-ATPase and the transverse tubule Mg-ATPase

Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.     

Detection of nonspecific cytotoxicity in graft-versus-host reaction as a function of target cell type

The cytotoxic activity of spleen cells from mice undergoing graft-versus-host (GVH) reaction on ⁵¹Cr-labeled target cells was studied under in vitro conditions. Among normal tissues used as target cells, skin fibroblasts proved to be most sensitive to the nonspecific cytotoxic effects of spleen cells from mice undergoing GVH reaction, whereas kidney cells or macrophages were insensitive to these nonspecific cytotoxic effects. Of the two murine neoplastic target cells used, Sarcoma 1 cells were susceptible to these nonspecific cytotoxic effects whereas mastocyoma cells were resistant. However, the target cells which were insensitive to the nonspecific cytolytic effects, were lysed specifically by the spleen cells from animals specifically sensitized. Therefore, both specific and nonspecific cytotoxic effects of spleen cells from mice undergoing GVH reaction could be detected with appropriate targets. These results provide a basis for reconciliation of several apparently contradictory results, reported in the literature, concerning the specificity of the cytotoxic effects of specifically sensitized lymphocytes.