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.

     

Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.     

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.     

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.     

Linear oligopeptides: 65′. Conformational analysis of the N-protected aromatic α-amino acid N-tert-butyloxycarbonyl-l-phenylalanine by X-ray diffraction and infrared absorption

The X-ray diffraction and i.r. absorption conformational analysis of $\text{N}\text{-tert-butyloxycarbonyl-}\text{l}\text{-phenylalanine}$ has showed the absence of intramolecularly hydrogen-bonded peptide conformations in the solid state. The molecules are held together in rows of ‘cyclic dimer’ motifs through intermolecular NH … OC (acid) and OH … OC {urethane} hydrogen bonds, the secondary amide-like group of the urethane moiety being in the unusual cis conformation, whereas the carboxylic acid group in the common syn conformation. The two molecules in the unit cell present a centrosymmetric set of ?, ψ¹, and ψ² values. In polar solvents solvated species largely predominate. In saturated hydrocarbon solution non-associated and associated (mostly involving the carboxylic acid CO as the proton acceptor) species simultaneously occur. The extent of association decreases with dilution. The amount of intramolecularly hydrogen-bonded oxy-C₇ and C₅ forms if any, should be extremely small. The type of association at saturation seems to differ from that found in the crystalline compound obtained by precipitation with saturated aliphatic hydrocarbons (from a diethyl ether solution).