ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27^Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF^Skp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF^Skp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.     

Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Photoreduction of autooxidized albumin-heme hybrid in saline solution: revival of its O(2)-binding ability.

Recombinant human serum albumin (rHSA) incorporating 2-[8-[N-(2-methylimidazolyl)]octanoyloxymethyl]-5,10,15,20-tetrakis(alpha,alpha,alpha,alpha-o-pivalamido)phenylporphinatoiron(II)s (Fe(II)Ps) [rHSA-Fe(II)P] is a synthetic hemoprotein which can bind and release O(2) reversibly under physiological conditions (saline solution [NaCl]: 150 mM, pH 7.3) as do hemoglobin and myoglobin. However, the central ferrous ions of Fe(II)Ps are slowly oxidized to O(2)-inactive ferric forms. Based on the UV-vis. absorption spectroscopy, the majority of the autooxidized Fe(III)Ps in albumin are determined to be six-coordinate high-spin complexes with a proximal imidazole and a chloride anion, which show ligand-to-metal charge transfer (LMCT) absorption at 330 nm. Interestingly, photoirradiation of this LMCT band under an argon atmosphere led to reduction of the central ferric iron of Fe(III)P, allowing the revival of the O(2)-binding ability. The ratio of the photoreduction reached a maximum of 83%, which is probably due to the partial dissociation of the axial imidazole. The same photoirradiation under a CO atmosphere provides the corresponding carbonyl rHSA-Fe(II)P. Laser flash photolysis experiments revealed that the reduction was completed within 100 ns. The quantum yields (Phi) of these photoreductions were approximately 0.01.     

Autoradiographic demonstration of gastrin binding sites in rat gastric mucosa

The location of 125I-iodotyrosyl gastrin I binding sites in rat gastric mucosa was studied. Peptide specificity was demonstrated by competitive binding studies through the addition of a large dose of cold human gastrin I or cholecystokinin-octapeptide. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and dry-mounting the emulsion film by means of the wire-loop method to prevent loss of the labeled substance. Specific binding sites for gastrin were found on parietal and chief cells, whereas few binding sites were seen on the surface mucous or mucous neck cells. Binding sites on the parietal cells were dispersed in the cytoplasm, while those on the chief cells were found near the basal plasma membrane.     

Evolution of genes for allelic and isotypic forms of immunoglobulin kappa chains and of the genes for T-cell receptor beta chains in rabbits

New insights into the evolution of the families of genes encoding immunoglobulins and T-cell receptors of rabbits (Oryctolagus cuniculus) have come from molecular genetic studies. In contrast to human and mouse, rabbits were shown to have two genes for the constant region of immunoglobulin light chains (C kappa 1 and C kappa 2 isotypes) and complex allelic variants of K1 (allotypes). Although K1 allotype protein sequences differed at up to 41% of the amino acid positions, 3' untranslated, 5', and 3' flanking regions were conserved, and in the coding regions 78-80% of the codons with differences had replacement changes. Proportions of silent changes and changes in noncoding regions were comparable. Thus, in spite of their markedly different protein sequences, the K1b4, b5, and b9 allotypes appeared to be products of allelic genes. Molecular genetic analyses suggested that they may have undergone rapid divergence after an ancestral K2-like gene duplicated. Some rabbits were found to have two similar T-cell receptor C beta genes as do humans and many strains of mice, but others appeared to have three different C beta. In addition, we found allotypic forms of C beta. Some of the C beta allotypic differences occurred at positions where analogous C kappa allotypic differences were found. We also found V beta in mouse and human that were more similar to rabbit V beta than closely linked rabbit genes were to each other. This contrasts with rabbit immunoglobulin VH gene sequences that reflect concerted evolution. The data suggested that T-cell receptor V beta genes duplicated prior to mammalian radiation.     

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.     

Isolation and Characterization of an Enterobacter cloacae Strain That Reduces Hexavalent Chromium under Anaerobic Conditions

An Enterobacter cloacae strain (HO1) capable of reducing hexavalent chromium (chromate) was isolated from activated sludge. This bacterium was resistant to chromate under both aerobic and anaerobic conditions. Only the anaerobic culture of the E. cloacae isolate showed chromate reduction. In the anaerobic culture, yellow turned white with chromate and the turbidity increased as the reduction proceeded, suggesting that insoluble chromium hydroxide was formed. E. cloacae is likely to utilize toxic chromate as an electron acceptor anaerobically because (i) the anaerobic growth of E. cloacae HO1 accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurred more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. The chromate reduction in E. cloacae HO1 was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C).     

Mutagenesis of human granulocyte colony stimulating factor

To define the structure-function relationship, we have made a number of mutants of human granulocyte colony-stimulating factor (hG-CSF) by in vitro mutagenesis. The results indicate that most of the mutations located in the internal and C-terminal regions of the molecule abolished the activity, whereas the mutants without N-terminal 4, 5, 7, or 11 amino acids retained the activity. N-terminal amino acids were also altered by cassette mutagenesis using a synthetic oligonucleotide mixture. Among them, KW2228, in which Thr-1, Leu-3, Gly-4, Pro-5 and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg and Ser, showed more potent granulopoietic activity than that of intact hG-CSF both in vitro and in vivo.     

Alterations in cardiac membrane Ca2+ transport during oxidative stress

Although cardiac dysfunction due to ischemia-reperfusion injury is considered to involve oxygen free radicals, the exact manner by which this oxidative stress affects the myocardium is not clear. As the occurrence of intracellular Ca²⁺ overload has been shown to play a critical role in the genesis of cellular damage due to ischemia-reperfusion, this study was undertaken to examine whether oxygen free radicals are involved in altering the sarcolemmal Ca²⁺-transport activities due to reperfusion injury. When isolated rat hearts were made globally ischemic for 30 min and then reperfused for 5 min, the Ca²⁺ -pump and Na⁺-Ca²⁺ exchange activities were depressed in the purified sarcolemmal fraction; these alterations were prevented when a free radical scavenger enzymes (superoxide dismutase plus catalase) were added to the reperfusion medium. Both the Ca²⁺- pump and Na⁺- Ca²⁺ exchange activities in control heart sarcolemmal preparations were depressed by activated oxygen-generating systems containing xanthine plus xanthine oxidase and H₂O₂; these changes were prevented by the inclusion of superoxide dismutase and catalase in the incubation medium. These results support the view that oxidative stress during ischemia-reperfusion may contribute towards the occurrence of intracellular Ca²⁺ overload and subsequent cell damage by depressing the sarcolemmal mechanisms governing the efflux of Ca²⁺ from the cardiac cell.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-α, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-α mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-α protein by DLD-1 and CoLo320DM cells was confirmed by TGF-α specific monoclonal antibody binding assay. The spontaneous³H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-α monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-α produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.