Gene expression profiling reveals the mechanism and pathophysiology of mouse liver regeneration

Comprehensive analysis of the changes in gene expression during liver regeneration was carried out by using an in-house microarray composed of 2,304 distinct mouse liver cDNA clones. Mice were subjected to partial two-thirds hepatectomy, and changes in mRNA levels were monitored up to 48 h. Of the 2,304 genes analyzed, 496 genes showed expression levels measurable at all time points after the partial hepatectomy. 317 genes were up- or down-regulated 2-fold or more at least at one time point during liver regeneration and were classified into eight clusters based on their expression patterns. With a more stringent cut-off value of +/-2 S.D., 68 genes were listed and were classified into five clusters. In these two analyses with different clustering criteria, functionally categorized genes showed similar cluster distributions. Genes involved in protein synthesis and posttranslational processing were significantly enriched in the cluster characterized by rapid gene activation and subsequent persistence. This suggests the importance of modulating the efficiency of protein supply and/or altering the composition of protein population from the early phase of hepatocyte proliferation. Genes for two major liver functions, i.e. plasma protein secretion and intermediate metabolism were enriched in distinct clusters exhibiting the features of gradual gene activation and sustained repression, respectively. Therefore, these genes are differentially regulated during the regeneration, possibly leading to changes in the flow of amino acids and energy from enzyme proteins to plasma proteins in their synthesis. Thus, clustering analysis of expression patterns of functionally classified genes gave insights into mechanism and pathophysiology of liver regeneration.     

Mammalian translesion DNA synthesis across an acrolein-derived deoxyguanosine adduct. Participation of DNA polymerase eta in error-prone synthesis in human cells

alpha-OH-PdG, an acrolein-derived deoxyguanosine adduct, inhibits DNA synthesis and miscodes significantly in human cells. To probe the cellular mechanism underlying the error-free and error-prone translesion DNA syntheses, in vitro primer extension experiments using purified DNA polymerases and site-specific alpha-OH-PdG were conducted. The results suggest the involvement of pol eta in the cellular error-prone translesion synthesis. Experiments with xeroderma pigmentosum variant cells, which lack pol eta, confirmed this hypothesis. The in vitro results also suggested the involvement of pol iota and/or REV1 in inserting correct dCMP opposite alpha-OH-PdG during error-free synthesis. However, none of translesion-specialized DNA polymerases catalyzed significant extension from a dC terminus when paired opposite alpha-OH-PdG. Thus, our results indicate the following. (i) Multiple DNA polymerases are involved in the bypass of alpha-OH-PdG in human cells. (ii) The accurate and inaccurate syntheses are catalyzed by different polymerases. (iii) A modification of the current eukaryotic bypass model is necessary to account for the accurate bypass synthesis in human cells.     

Organization of extracellular matrix components during differentiation of adipocytes in long-term culture

Summary Scanning electron microscopy (SEM) observation showed that fully differentiated spherical adipocytes were embraced by a network of collagens and fibroblastic preadipocytes. The properties of both the collagen networks and the preadipocytes allow the adipocytes to be interconnected, forming a fat-cell cluster, which can anchor to the bottom of a culture dish. In this network structure, collagen fibrils and fibrillar bundles were closely arranged and stratified. We found that immunostained collagens appeared to form extracellular network structures, which can be observed by SEM. The extracellular network of fibronectin was the first to develop among the extracellular matrix (ECM) components, though it became degraded with the progress of adipocyte differentiation. The type I collagen network was the last to develop and remained well organized through the late stage of adipocyte differentiation. The extracellular networks of type III, V, and VI collagen developed by the mid-stage and remained in the late stage of adipocyte differentiation. The network structures of type IV collagen and laminin became degraded during the differentiation process and localized at the surface of spherical cells. In addition to these basement membrane components, types III, V, and VI collagens also showed pericellular spherical staining patterns. These results demonstrated that the constitution and distribution of the ECM are altered during adipocyte differentiation, suggesting that the organization of each ECM component into a suitable structure is a requirement for the differentiation and maintenance of unilocular adipocytes.     

Differences in accumulation of elements in human cardiac valves

To elucidate changes of human cardiac valves with aging, the authors determined age-related changes of element contents in the four human cardiac valves by inductively coupled plasma-atomic emission spectrometry and attempted to examine the relationships in the element contents among the four cardiac valves. The subjects consisted of 10 men and 15 women, ranging in age from 65 to 102 yr. The accumulation of calcium and phosphorus was the highest in the aortic valve, and decreased in the order mitral, pulmonary, and tricuspid valves. The contents of calcium and phosphorus in the aortic valves corresponded to about 12 and 19 times the amounts of those in the tricuspid valves, in which the contents were very low. The contents of calcium and phosphorus in the aortic valves were about 2.5-fold the amounts of those in the mitral valves. An examination was attempted to determine whether or not there were relationships in element contents among the four cardiac valves. As for the aortic and mitral valves, there were no relationships in the contents of calcium and phosphorus between them, but there were relationships in the contents of sulfur and magnesium between them. Three out of 24 cases contained high contents of calcium and phosphorus in both the mitral and aortic valves, whereas 16 out of 24 cases contained high contents of calcium and phosphorus in the aortic valves alone, without the high contents in the mitral valves. Likewise, there were no relationships in the element contents, such as calcium, phosphorus, sulfur, and magnesium, between the mitral and pulmonary valves or between the mitral and tricuspid valves. It is suggested that the accumulation of calcium and phosphorus in the cardiac valve occurs independent of the other cardiac valves.     

Regulation of reactive oxygen species by Atm is essential for proper response to DNA double-strand breaks in lymphocytes

The ataxia telangiectasia-mutated (ATM) gene plays a pivotal role in the maintenance of genomic stability. Although it has been recently shown that antioxidative agents inhibited lymphomagenesis in Atm(-/-) mice, the mechanisms remain unclear. In this study, we intensively investigated the roles of reactive oxygen species (ROS) in phenotypes of Atm(-/-) mice. Reduction of ROS by the antioxidant N-acetyl-l-cysteine (NAC) prevented the emergence of senescent phenotypes in Atm(-/-) mouse embryonic fibroblasts, hypersensitivity to total body irradiation, and thymic lymphomagenesis in Atm(-/-) mice. To understand the mechanisms for prevention of lymphomagenesis, we analyzed development of pretumor lymphocytes in Atm(-/-) mice. Impairment of Ig class switch recombination seen in Atm(-/-) mice was mitigated by NAC, indicating that ROS elevation leads to abnormal response to programmed double-strand breaks in vivo. Significantly, in vivo administration of NAC to Atm(-/-) mice restored normal T cell development and inhibited aberrant V(D)J recombination. We conclude that Atm-mediated ROS regulation is essential for proper DNA recombination, preventing immunodeficiency, and lymphomagenesis.     

C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells

Glutamate decarboxylase (GAD) converts L-glutamate to gamma-aminobutyric acid (GABA), which is a non-protein amino acid present in all organisms. Plant GADs carry a C-terminal extension that binds to Ca(2+)/calmodulin (CaM) to modulate enzyme activity. However, rice possesses two distinct types of GAD, OsGAD1 and OsGAD2. Although they both have a C-terminal extension, the former peptide contains an authentic CaM-binding domain (CaMBD), which is common to dicotyledonous plants, while the latter does not. Therefore, the role of the C-terminal extension in functional expression of OsGAD2 was investigated. An in vitro enzyme assay using recombinant OsGAD2 proteins revealed low activity in the presence or absence of Ca(2+)/CaM. However, a truncated version of GAD2 (OsGAD2DeltaC) had over 40-fold higher activity than wild-type GAD at physiological pH. These two DNA constructs were introduced simultaneously into rice calli via Agrobacterium to establish transgenic cell lines. Free amino acids were isolated from several lines for each construct to determine GABA content. Calli overexpressing OsGAD2 and OsGAD2DeltaC had about 6-fold and 100-fold the GABA content of wild-type calli, respectively. Regenerated OsGAD2DeltaC rice plants had aberrant phenotypes such as dwarfism, etiolated leaves, and sterility. These data suggest that the C-terminal extension of OsGAD2 plays a role as a strong autoinhibitory domain, and that truncation of this domain causes the enzyme to act constitutively, with higher activity both in vitro and in vivo.     

Multiple amino acid substitutions in hemagglutinin are necessary for wild-type measles virus to acquire the ability to use receptor CD46 efficiently

Measles virus (MV) possesses two envelope glycoproteins, namely, the receptor-binding hemagglutinin (H) and fusion proteins. Wild-type MV strains isolated in B-lymphoid cell lines use signaling lymphocyte activation molecule (SLAM), but not CD46, as a cellular receptor, whereas MV vaccine strains of the Edmonston lineage use both SLAM and CD46 as receptors. Studies have shown that the residue at position 481 of the H protein is critical in determining the use of CD46 as a receptor. However, the wild-type IC-B strain with a single N481Y substitution in the H protein utilizes CD46 rather inefficiently. In this study, a number of chimeric and mutant H proteins, and recombinant viruses harboring them, were generated to determine which residues of the Edmonston H protein are responsible for its efficient use of CD46. Our results show that three substitutions (N390I and E492G plus N416D or T446S), in addition to N481Y, are necessary for the IC-B H protein to use CD46 efficiently as a receptor. The N390I, N416D, and T446S substitutions are present in the H proteins of all strains of the Edmonston lineage, whereas the E492G substitution is found only in the H protein of the Edmonston tag strain generated from cDNAs. The T484N substitution, found in some of the Edmonston-lineage strains, resulted in a similar effect on the use of CD46 to that caused by the E492G substitution. Thus, multiple residues in the H protein that have not previously been implicated have important roles in the interaction with CD46.