Dismantling Cell–Cell Contacts during Apoptosis Is Coupled to a Caspase-dependent Proteolytic Cleavage of β-Catenin

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis.

In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we provide demonstration for the mechanism that could contribute to the disassembly of cytoskeletal organization at cell–cell adhesion. In fact, β-catenin, a known regulator of cell–cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated β-catenin into a form which is similar in size to that observed in cells undergoing apoptosis. β-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting β-catenin product is unable to bind α-catenin that is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at cell–cell contacts could be dismantled during apoptosis. Our observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton occurring during cell death via cleavage of different regulators of the microfilament system.

     

Hepatitis B virus replication is cell cycle independent during liver regeneration in transgenic mice.

The content of hepatitis B virus (HBV) replicative forms and HBV core protein in the liver of HBV transgenic mice is transiently reduced during massive liver regeneration following partial hepatectomy while the steady-state content of viral RNA is unchanged. This antiviral effect is triggered by interferon and tumor necrosis factor that are induced in the liver following hepatectomy and either prevent the formation or accelerate the degradation of viral nucleocapsids in the cytoplasm of the hepatocyte. Despite massive hepatocellular turnover, this effect is independent of liver cell division, indicating that HBV replicates efficiently in resting and dividing hepatocytes.     

Interleukin-12 inhibits hepatitis B virus replication in transgenic mice.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that has the ability to induce gamma interferon (IFN-gamma) secretion by T and natural killer cells and to generate normal Th1 responses. These properties suggest that IL-12 may play an important role in the immune response to many viruses, including hepatitis B virus (HBV). Recently, we have shown that HBV-specific cytotoxic T lymphocytes inhibit HBV replication in the livers of transgenic mice by a noncytolytic process that is mediated in part by IFN-gamma. In the current study, we demonstrated that the same antiviral response can be initiated by recombinant murine IL-12 and we showed that the antiviral effect of IL-12 extends to extrahepatic sites such as the kidney. Southern blot analyses revealed the complete disappearance of HBV replicative intermediates from liver and kidney tissues at IL-12 doses that induce little or no inflammation in these tissues. In addition, immunohistochemical analysis demonstrated the disappearance of cytoplasmic hepatitis B core antigen from both tissues after IL-12 treatment, suggesting that IL-12 either prevents the assembly or triggers the degradation of the nucleocapsid particles within which HBV replication occurs. Importantly, we demonstrated that although IFN-gamma, tumor necrosis factor alpha, and IFN-alpha/beta mRNA are induced in the liver and kidney after IL-12 administration, the antiviral effect of IL-12 is mediated principally by its ability to induce IFN-gamma production in this model. These results suggest that IL-12, through its ability to induce IFN-gamma, probably plays an important role in the antiviral immune response to HBV during natural infection. Further, since relatively nontoxic doses of recombinant IL-12 profoundly inhibit HBV replication in the liver and extrahepatic sites in this model, IL-12 may have therapeutic value as an antiviral agent for the treatment of chronic HBV infection.     

Production of neosolaniol byFusarium tumidum

Extracts from autoclaved maize culture ofFusarium tumidum strain R-5823 were toxic towardsArtemia salina. Bioassay-guided fractionation of the organic extract led to the isolation of the toxic compound that was identified as the trichothecene toxin neosolaniol (NEOS) by¹H,¹³C nuclear magnetic resonance spectroscopy and low-resolution electronic impact mass spectrometry. The amount of NEOS produced by the strain R-5823 was 300 mg/kg maize culture. NEOS was also detected by HPLC in cultures of four out of seven additional strains ofF. tumidum andGibberella tumida with different origin, in amounts ranging from 1 to 311 mg/kg. This is the first report on the production of a trichothecene toxin byF. tumidum.     

Influence of mature and immature myeloid cells on lymphocyte reactivity in chronic myelocytic leukemia

Summary Earlier studies have shown anergy in chronic myeloid leukemia (CML), and it is known that myeloid cells influence lymphocyte responses. Therefore, lymphocytes from CML patients who had received no cytostatics for 2 weeks were stimulated in 89 tests with PHA and ConA. In 39 control tests, normal lymphocytes were used.Lymphocytes from CML patients were significantly less (p<0.05) markedly stimulated than normal ones. Lymphocytes from CML patients with more than 10×10⁹ white blood cells (WBC) per liter blood were inhibited to a greater degree than those from patients with a normal WBC count.When normal lymphocytes were stimulated with PHA in the presence of mononuclear cells from the blood of CML patients (mostly leukemic myelocytes), their response was significantly (p<0.05) inhibited. Inhibition with leukemic myelocytes was significantly (p<0.05) greater than that with mature granulocytes from CML patients. The latter did not seem to have an inhibitory effect.We suggest that patients with manifest CML are anergic to some extent because leukemic myelocytes have a suppressor effect.Visiting scientist and Anna Villa Rusconi Fellow, on secondment from Institute of Medical Pathology, University of Ferrara, Italy     

New pulsed field gradient NMR experiments for the detection of bound water in proteins

Summary New H₂O-selective homonuclear and heteronuclear 2D NMR experiments have been designed for the observation of protein hydration (PHOGSY, Protein Hydration Observed by Gradient Spectroscop Y). These experiments utilize selective excitation of the H₂O resonance and pulsed field gradients for coherence selection and efficient H₂O suppression. The method allows for a rapid and sensitive detection of H₂O molecules in labelled and unlabelled proteins. In addition it opens a way to measure the residence time of water bound to proteins. Its application to uniformly ¹⁵N-labelled FKBP-12 (FK-506 binding protein) is demonstrated.     

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.     

Serum-mediated regulation of amino acid transport in cultured chick embryo fibroblasts

Three different temperature sensitive mutants derived from the Syrian hamster cell line BHK 21 were found to have greatly reduced DNA synthesis at the non-permissive temperature. These mutants are distinct by complementation analysis and behave at the non-permissive temperature as cell cycle traverse defective mutants. Microfluorometric analysis of mutant populations arrested at the non-permissive temperature shows an accumulation of cells with G1 DNA content. Mutants ts 13 and ts HJ4 synchronized in G1 by serum or isoleucine deprivation and shifted to the non-permissive temperature at the time of release do not enter the S phase, while in the case of mutant ts 11 preincubation at the non-permissive temperature before release is required to completely prevent its entry into S. Ts 13 and ts 11 are able to traverse the S phase at the non-permissive temperature when synchronized at the boundary G1/S; in this case, preincubation of ts 11 at the non-permissive temperature before release does not affect the ability of these cells to perform DNA synthesis. On the other hand, ts HJ4 appears to traverse S only partially when tested under similar conditions. Temperature shift experiments of mutant populations at different times after isoleucine synchronization suggest that ts 13 and ts 11 are blocked at the non-permissive temperature in early G1, whereas ts HJ4 is probably affected near the initiation of DNA synthesis, or in some early S function.     

Apigenin 7-glucoside diacetates in ligulate flowers of Matricaria chamomilla

From ligulate flowers of Matricaria chamomilla was isolated a mixture of apigenin 7-O-β-glucoside diacetates, which was shown to be based on (2″, 3″)- and (3″, 4″)-diacetates.