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This article features a cyberspace discussion involving four communication scholars reflecting a variety of perspectives based on race, gender, age, regionality, and tenure within the academy. After reading each of the essays contained in this special issue, we engaged in an on-line discussion of our reactions to, perceptions of, and experiences with the issues raised by the collective voices contained in this volume. In short, this “finale piece” features diverse voices coming together to discuss the implications of issues raised throughout the special issue.

The primary goal of this study was to determine the extent to which religious frameworks inform lay public understandings of genes and disease. Contrary to existing research, there were minimal differences between racial groups. We did, however, observe two patterns in that data that are worthy of discussion. First, because participants were from the south, the finding that participants from both racial groups ascribe to a religious belief system to make sense of their lived experiences is not surprising. Rather, it appears to be reflective of the religious culture that is an integral part of the south and our identity as a nation. A second noteworthy finding is that while a significant number of participants believe that a relationship exists between health status, genes, and religious behaviors, they also recognize that positive health behaviors must also be adopted as a means for staving off disease. In some cases, however, there was a belief that health issues could dissolve or disappear as a result of certain religious behaviors such as prayer. © 2009 WileyLiss, Inc.

Background This study was performed to determine the variations in the branching pattern of the sphenopalatine artery medial to the crista ethmoidalis. Seventy-seven cadaver head sides that had been sectioned sagittally in the midline with their septum removed were used after injecting pink latex to highlight the arterial vessels.Methods The mucosa from the middle meatus from the level of the basal lamella was removed until the artery and its branches were seen and then was examined under the microscope to identify the position of the arterial branches.Results The sphenopalatine artery and its branches were identified in 75 specimens. Of these 75 specimens, 73 (97%) had 2 or more branches medial to the crista ethmoidalis, 49 (67%) had 3 or more branches, 26 (35%) had 4 or more branches, and 1 specimen had 10 branches. In two specimens the artery presented as a single trunk.Conclusion The sphenopalatine artery normally starts to branch lateral to the crista ethmoidalis and these branches vary widely. It is important that the surgeon who undertakes ligation or cautery of the artery is aware of these variations, otherwise they may overlook some of the branches. With an endoscopic approach, removal of the crista ethmoidalis helps visualize these branches.

A variety of explanations have been provided to elucidate the requirement of the large islet mass that is essential for a successful treatment of patients with type I diabetes by intrahepatic transplantation. The purpose of this study was to investigate islet cell survival under the effect of prolonged hypoxia and/or nutrient withdrawal, which mimics posttransplantation environment of transplanted islets in the liver. We studied the influence of 24 h of hypoxia (1% O2) in intact isolated human and rat islets as well as the effect of combined oxygen/nutrient deprivation in a mouse insulinoma cell line (MIN6). In intact human islets, 24 h of hypoxia led to central necrosis combined with apoptotic features such as nuclear pyknosis and DNA fragmentation. In the course of hypoxic treatment, ultrastructural analysis demonstrated a gradual transition from an apoptotic to a necrotic morphology particularly pronounced in central areas of large islets. In MIN6 cells, on the other hand, hypoxia led to a twofold (p < 0.01) increase in caspase-3 activity, an indicator of apoptosis, but not to necrosis, as determined by release of lactate dehydrogenase (LDH). Only in combination with nutrient/serum deprivation was a marked increase in LDH release observed (sixfold vs. control, p < 0.01). We therefore conclude that, similar to MIN6 cells, central necrosis in isolated hypoxic islets is the result of the combined effects of hypoxia and nutrient/serum deprivation, most likely due to limited diffusion. Provided that transplanted islets undergo a similar fate as shown in our in vitro study, future emphasis will require the development of strategies that protect the islet graft from early cell death and accelerate the revascularization process.

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm and ectoderm) of the organism, including the germline. To study early stages of development, as well as to investigate the impact of a gene knock-out in vitro, ES cells are differentiated into three-dimensional structures called embryoid bodies, because of their ability to mimick post-implantation embryonic tissues. This review summarises the work on ES cell differentiation into haematopoietic and vascular cells, neuronal and glial cells, myocytes, and adipocytes, using this in vitro model of early embryogenesis. We also present the potential of this method to analyse the impact of genetic alterations in vitro

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm and ectoderm) of the organism, including the germline. To study early stages of development, as well as to investigate the impact of a gene knock‐out in vitro, ES cells are differentiated into three‐dimensional structures called embryoid bodies, because of their ability to mimick post‐implantation embryonic tissues. This review summarises the work on ES cell differentiation into haematopoietic and vascular cells, neuronal and glial cells, myocytes, and adipocytes, using this in vitro model of early embryogenesis. We also present the potential of this method to analyse the impact of genetic alterations in vitro

Background:Retinoids are candidate differentiation-inducing agents for glial tumors. Differentiation therapy is an attractive approach to cancers that resist surgery, irradiation and chemotherapy. Methods:We examined the effects of retinoids on proliferation, morphology and sensitivity to apoptosis in human malignant glioma and neuroblastoma cell lines. Results:In contrast to neuroblastoma cells, retinoids are devoid of acute cytotoxic effects and have only moderate antiproliferative effects on human glioma cell lines upon long-term exposure at high concentrations. Electron microscopy fails to reveal features of differentiation or apoptosis in retinoid-treated glioma cells and untransformed rat astrocytes. Retinoids do not modulate CD95 or CD95L expression or susceptibility to CD95-mediated apoptosis and fail to act in synergy with interferon (IFN)-α or IFN-γ or cancer chemotherapy drugs to promote growth inhibition or apoptosis. Conclusion:Glioma cell lines are refractory to the induction of differentiation or apoptosis by retinoids.

Borrelia burgdorferi (Bb) is the tick-borne etiologic agent of Lyme borreliosis, which has many aspects of autoimmune diseases. Bb is unable to recycle synthesized membrane lipids and lipoproteins. Consequently, a large amount of liposome-like vesicle (Bb-blebs) is shed from the outer bacterial membrane. The influence of Bb-blebs on the cellular immune response is not yet known. As a Bb-blebs model, we established standardized Bb-liposomes, produced from freshly extracted lipids and lipoproteins of live Bb. Bb-liposomes were incorporated via nonendocytotic mechanisms by different human cell types, namely dendritic cells (DC), lymphocytes, and fibroblasts, as visualized by immunofluorescence and transmission electron microscopy. Bb-liposomes were localized in the cytosol and in the nucleus of the cells. With this in mind, we generated in vitro Bb-specific T-cells from nonadherant peripheral blood mononuclear cells by use of Bb-liposomes loaded autologous DC. More than 95% of those T-cells were CD8+ and they killed autologous Bb-liposome-loaded T-cell blasts. These results suggest that Bb-blebs may be responsible for the autoimmune-like appearance of Lyme disease.

The spirochaetal bacteria Borrelia burgdorferi (Bb) is the tick-borne causative agent of lyme disease. The major membrane immunogens of Bb are outer surface proteins. The lipid component of these lipoproteins is relevant for the immunogenicity of Bb-lipoproteins. To characterize the antigenic properties, the native lipid component of lipoproteins was isolated and the detailed molecular structure was analyzed. The molecular structure of the lipoprotein–lipid component turned out to be S(propane-2′,-3′diol)-3-thio-2-aminopropanic acid (S-glyceryl-cysteine) with one ester-linked fatty acid, one acetyl group, and one N-terminal amide-bound fatty acid. Fatty acid analysis of the lipid component indicated a heterogeneous composition comprising C16:0, C18:0, C18:1, C18:2, and C 20:0. The antigenicity was tested with in vitro bioassays using human blood-derived dendritic cells (DCs) as antigen-presenting cells and autologous Bb-specific T-cells. We found that human DCs present the lipid component of Bb-lipoproteins via MHC class II inducing an antigen-specific T-cell immune response in vitro.

The unquestionable aims for a less invasive operations are less morbidity, less discomfort, and a reduced hospital stay through an operation which proves equally durable to the conventional approach. Such an operation must be carried out without further risk to the patient or increased difficulty for the surgeon. Whilst most definitions of less invasive coronary surgery include the phrase without cardiopulmonary bypass, this is clearly not yet possible in valve surgery. In valve surgery, the definition of less invasive relates only to the size of incision and rate of recovery. As a result of the discussions during the Heart Lab International Workshop on video-assisted heart surgery in Zürich, October 22-25, 1998, the following conclusions emerged. The partial upper sternotomy with J- or L- shaped extension to the right is the preferred approach for minimally invasive aortic valve surgery. Other methods which sacrify the internal thoracic arteries, open pleural cavities or predispose to long hernia are less satisfactory. A detailed description of the technique proposed is given and its indications and contraindications are discussed.

β-lapachone and camptothecin are structurally unrelated agents thought to inhibit topoisomerase-I activity through distinct mechanisms. We find that β-lapachone is much more potent than camptothecin in inducing acute cytotoxic effects on human malignant glioma cells. Acute cytotoxicity induced by both drugs is apoptotic by electron microscopy, but not blocked by inhibitors of RNA or protein synthesis and not associated with changes in the expression of bcl-2, bax, p53, p21 or GADD45 proteins. In contrast, prolonged exposure of glioma cells to both drugs for 72 hr results in growth inhibition and apoptosis, with EC50 values around 1 μM. None of 7 glioma cell lines tested were resistant to either drug. LN-229 cells which have partial p53-wild-type activity show enhanced expression of p53, p21 and bax protein, whereas bcl-2 levels decrease, after exposure to camptothecin. In contrast, β-lapachone increases bax protein expression in the absence of p53 activation. T98G cells are mutant for p53. In these cells, p53 levels do not change and p21 is not induced. bax accumulation in T98G cells is induced by both drugs, with bcl-2 levels unaltered. Surprisingly, ectopic expression of murine bcl-2 fails to abrogate the toxicity of either drug. Camptothecin, but not β-lapachone, sensitizes human malignant glioma cells to apoptosis induced by the cytotoxic cytokines, tumor necrosis factor-α and CD95 ligand. Thus, both drugs have potent anti-glioma activity that may be mediated by enhanced bax expression but is not inhibited by ectopic bcl-2 expression. Camptothecin-like agents are particularly promising for immunochemotherapy of malignant glioma using cytotoxic drugs and CD95 ligand. Int. J. Cancer 73:707–714, 1997. © 1997 Wiley-Liss, Inc.

A model explaining the mechanism of initiation of differentiation is presented. It is based on the finding that sporulation in B. subtilis can be induced by the same nutrient deficiencies that also induce the synthesis of highly phosphorylated nucleotides. Two of these nucleotides are synthesized by membrane vesicles. Synthesis can be inhibited by the same metabolites of glucose that also inhibit sporulation. It is concluded, therefore, that the plasma membrane synthesizes unusual nucleotides in response to nutrient deficiencies. By several as yet unknown steps, these nucleotides then cuase changes in the metabolism of the organism leading to the formation of spores. Both structure and mechanism of synthesis of adenosine hexaphosphate, pppAppp, have been elucidated by use of ATP analogues.

Effector-target conjugates, formed by coincubation of lymphokine-activated killer (LAK) cells with either K562 or Daudi cells, were separated from single cells by Percoll sedimentation. The occurrence of various CD molecules (CD3, CD56, CD57, CD16, γ/δ-TCR) was compared in both fractions. Only LAK cells expressing the γ/δ T cell receptor (TCR) were found in a significantly increased percentage in fractions containing conjugates indicating that γ/δ-TCR+ LAK cells were preferably bound to target cells at the time of separation. In order to determine whether γ/δ-TCR+ LAK cells also show a preferred killing activity against the targets, cultures enriched with or depleted of γ/δ-TCR+ cells were established. Against K562 cells, γ/δ-TCR+-enriched cultures showed a greatly reduced killing activity compared to LAK bulk cultures or cultures depleted of γ/δ-TCR+ cells. Using Daudi cells as targets the enriched fraction revealed a slightly increased killing activity compared to bulk cultures or depleted fractions. Preincubation of γ/δ-TCR+ LAK cells with anti-γ/δ or anti-CD3 mAb resulted in a distinct increase of the killing activity against K562 cells, but in only a slightly enhanced activity against Daudi cells. It is postulated that γ/δ-TCR+ LAK cells use the same adhesion mechanism for both targets but that only Daudi cells express a specific ligand for the γ/δ-TCR. Occupation of the γ/δ-TCR/CD3 complex by mAb, however, seems to substitute for the absent epitope on K562 cells by eliciting stimulatory signals in γ/δ-TCR+ LAK cells which, in combination with the binding stimulus, trigger cytolytic activity.

Cytotoxic T lymphocytes mediate lysis of target cells by various mechanisms, including exocytosis of lytic proteins (perforin, granzymes) and receptor-ligand binding of Fas/APO molecules. Death of target cells is characterized by either necrosis or apoptosis, depending on the killing mechanism used and on the metabolism of the target cell itself.

Energy systems combine the sources of energy, the means for converting these sources to usable forms, the distribution devices and procedures, the using community and the ways it employs energy, and the surrounding natural and economic environment. Thus, energy systems are socioeconomic systems whose analysis includes various sciences with different limitations to scientific findings. While their physical behavior may be modeled on the computer with sufficient precision at least on a local or regional level, the parameters necessary for economic modeling are subject to large principle uncertainties. Qualitative parameters may enter the analysis via model restrictions or exogenous assumptions, which inevitably involve personal views. Solving the global energy problem is an urgent task that cannot be postponed until a provable scientific solution is found, especially since such a solution may never emerge. In this situation, systems analysis based on energy models may support the necessary decisions by enhancing the understanding of energy systems. However, such models will not lead to correct or validated quantitative results, but to reasonable qualitative suggestions.

The planning of energy supply systems is getting more and more complex and involves increasing amounts of data. In the past, these data have been stored on the computer in simple text files, which were accessible via text editors. At present, powerful database management systems (DBMS) provide mature tools for handling data. However, these instruments are not yet widely used in the energy sector. Therefore, sample solutions for two standard problems of energy plannmg are presented, namely, the management of time series and of technology data. In addition, the database structure of the energy-optimization model ecco is discussed in detail.

Dynamic substitution processes between energy-conversion techniques are very important phenomena for the restructuring of energy systems. Using the method of synergetics, we develop a probabilistic model framework for individual decisions and their effects on the macroscopic configuration of energy supply systems. The derived probability transition rates are associated with socioeconomic parameters that are the basis for individual decisions. We state preliminary results of a case study for the room heating of private households in the former West Germany, which are based on a small data sample and a nonrepresentative poll. We find that the number of heating systems of one type, which are already installed, their ease of use, and the cost are the most important parameters for the selection of a new heating system. Using the estimated parameters, we simulate the diffusion of a new type of heating system, which is regarded as better than all the others, into the energy supply system. Its market share will reach 11-18% after 35 years.

Low density lipoprotein (LDL), at concentrations high enough for receptor binding but not high enough to saturate the receptor, induces activation of phosphatidylinositol (PtdIns) turnover in a variety of cell types with various biological functions. Using both biochemical and electron microscopic studies, we have shown that blood platelets take up and degrade LDL in a manner reminiscent of phagocytic cell types. The activation of both PtdIns turnover and LDL metabolism is inhibited by high density lipoprotein. Thus, LDL at hormonal concentrations causes general cellular activation. Since all cell types studied responded to LDL with increased PtdIns turnover and uptake of LDL cholesterol, the PtdIns cycle may also be involved in the cellular regulation of LDL cholesterol metabolism.

Immunofluorescence staining using human autoantibodies is a simple and reliable method for investigation of meiotic and post-meiotic cells. Patients suffering from autoimmune diseases often produce circulating autoantibodies to antigens of germ cells and Sertoli cells. Four hundred human autoimmune sera were screened by indirect immunofluorescence on mouse seminiferous tubule cells. Autoantibodies of several specificities were found: one group reacted with organelles of meiotic prophase spermatocytes or spermatozoa. Included in this group were autoantibodies to synaptonemal complexes, sex vesicle, acrosome, and sperm tail. A second group of autoantibodies was found to stain different spermatogenic cell types uniformly, such as round spermatids or Sertoli cells.

Growth cones and neurites of chicken dorsal root ganglia neurons cultured on laminin, Ng-CAM, or axonin-1 exhibit substratum-dependent morphology and growth patterns which are accompanied by distinctive distributions of axonin-1 and Ng-CAM in the growth cone membrane. On either Ng-CAM or axonin-1 substratum, both Ng-CAM and axonin-1 were depleted from some areas of the apical growth cone membrane. In contrast, on laminin, both axonin-1 and Ng-CAM remained randomly distributed. Removal of axonin-1 from growth cones resulted in a blockage of neurite outgrowth on both Ng-CAM and axonin-1 substrata, indicating that in these neurons axonin-1 cooperates with Ng-CAM in the activation of axon growth. Based on these results possible molecular models for cooperation between axonin-1 and Ng-CAM on the growth cone are discussed.

The structure of the Escherichia colichaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiaewith a destabilizing Gly→Ala mutation and RTEM β-lactamase from Escherichia coliwith two Cys→Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPγS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.

To test our model on the mechanism of initiation of differentiation in Bacillus subtilis, we tested early blocked (stage 0) sporulation mutants for their ability to synthesize highly phosphorylated nucleotides. We also isolated early blocked asporogenous mutants with the aid of the intercalating drug tilorone. Among all mutants tested we found that the spo0F-bearing strain was unable to synthesize adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp. A revertant of this mutant regained the ability to both sporulate and synthesize pppAppp. Ribosomes of the asporogenous mutant isolated at T2 (2 hr after the end of logarithmic growth) of sporulation, in contrast to the wild type, do not synthesize adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp, or adenosine 3'(2')-diphosphate 5'-triphosphate, pppApp, but synthesize guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp. This behavior is characteristic of ribosomes from vegetative, not sporulating, cells. Ribosomes from the sporogenous revertant behave like those of the wild type. The results suggest that the spo0F mutation may be a mutation in the structural gene for pppAppp synthetase. The inability to synthesize pppAppp in this strain also prevents the formation of "sporulation-specific ribosomes," i.e., ribosomes that synthetize ppApp and pppApp. The present experiments suggest that the nucleotide pppAppp participates in the initiation of sporulation by triggering a sequencies of events required for the production of heat-resistant spores.

Bacillus subtiliscontains an enzyme that synthesizes the highly phosphorylated nucleotide adenosine 5′,3′(2′)-bis(triphosphate), abbreviated p3Ap3. This enzyme can be demonstrated to be present in the cytoplasmic membrane of B. subtilisat all stages of growth and development. During vegetative growth its enzymatic activity is inhibited by phosphorylated metabolites of energy-rich carbon sources, as was shown with the aid of conditional sporulation mutants. The finding that a temperature-sensitive sporulation mutant with a mutation in the spoOFgene, an early sporulation gene, shows a temperature-sensitive activity of p3Ap3-synthetase indicates that p3Ap3plays an important role in sporulation, probably initiation of sporulation.

Purpose:: Forty-one months after we performed bilateral implantation of in vitro endothelialized femoropopliteal bypass grafts in a 69-year-old patient, we obtained a central graft segment for histologic and ultrastructural investigation. Methods:: Before implantation the grafts were confluently lined with autologous first passage mass cultures of pure cephalic vein endothelial cells. The precoating of the expanded polytetrafluoroethylene prosthesis was done with fibrinolytically inhibited fibrin glue. Reoperation became necessary because of symptomatic unilateral atherosclerotic lesions located in the center of one of the two in vitro lined grafts. A 21 cm long graft segment was removed and replaced by a new in vitro endothelialized expanded polytetrafluoroethylene graft. Results:: On scanning electron microscopy a confluently covering mature endothelium was found throughout the whole length of the removed prosthesis. The endothelial identity was confirmed by a positive immunohistochemical CD 34, von Willebrand factorstaining, and the ultrastructural demonstration of Weibel Pallade bodies. The endothelium rested on a collagen IV positive basement membrane. Histologic cross sections revealed uniformly developed subintimal tissue of 1.21 +/- 0.19 mm thickness, which was separated from the intima by a distinct internal elastic membrane. The cells of this cell-rich matrix stained strongly positive for actin. Ultrastructurally, this matrix was dominated by highly contractile myofibroblasts loaded with peripherally located well-developed actin fillaments. A number of these cells also showed signs of secretory cells with a distinct endoplasmic reticulum and a Golgi complex. In areas of atherosclerotic lesions the subendothelial matrix was partially exposed, and the internal elastic membrane had to a certain extent disintegrated. Only in these areas KP-1 and MG-Ml positive foamy macrophages and CD 34 positive capillaries were found. The myofibroblasts of this diseased part of the subintimal tissue contained large lipid vacuoles. Conclusions:: We conclude that the confluent in vitro lining of synthetic vascular grafts with pure autologous endothelial cells facilitates graft healing, which may result in a hybrid structure with features of a native vessel.

Cytoplasmic granules of cytolytic T lymphocytes contain at least six related serine esterases (granzymes) that are released together with perforin, a pore-forming protein related to complement component C9, during target-cell lysis. Polyclonal antibodies were used to isolate a large number of cDNA clones from an expression library derived from cytolytic-T-cell mRNA. Three distinct full-length cDNA clones coding for granzymes D, E, and F were identified by restriction site mapping and nucleotide sequencing. The three deduced amino acid sequences are highly similar to one another (between 72% and 90% amino acid identities) and to the sequences of granzymes B and C, cathepsin G, and rat mast-cell proteases I and II (between 43% and 57% amino acid identities). Cysteine residues capable of forming intramolecular disulfide bonds are conserved, as are the catalytic-site residues characteristic of serine proteases. Comparison of the cDNA-derived protein sequences with the amino termini of the isolated granzymes provides evidence that they are stored in a fully processed, activated form after removal of the signal peptide and two additional residues (propeptide) at the amino terminus. Immunoelectron microscopic studies demonstrated that granzymes D, E, and F are present in the same morphologically distinct cytoplasmic granules in which perforin has been found previously.

The ultrastructure of blood platelets was related to platelet function and secretion products before, during, and after cardiopulmonary bypass. Circulating platelets from 15 patients undergoing aorta-coronary bypass operations were investigated at ten predetermined points of time by scanning and transmission electron microscopy. Simultaneously, platelet adenosine triphosphate, diphosphate, and serotonin, as well as plasma levels of platelet factor 4, β-thromboglobulin, serotonin, thromboxane B2, lactic dehydrogenase, and free hemoglobin were measured. Moreover, platelet responsiveness toward adenosine diphosphate and collagen was determined by optical aggregometry. By scanning electron microscopy, the number of unactivated platelets dropped from 96% ± 4% to 54% ± 19% (p < 0.05) 8 minutes after the onset of bypass. Simultaneously, the percentage of “shape changed” platelets significantly increased. No major release reaction was detected at this time. After the initial activation, platelet morphology began to recover although the bypass continued. During the late period of bypass, a highly significant correlation between increasing plasma levels of α-granule compounds (platelet factor 4 and β-thromboglobulin) and lysis parameters (lactic dehydrogenase and free hemoglobin) was found. However, transmission electron microscopic analysis of the arterial filter and scanning electron microscopic findings of circulating platelets indicated that the release products in plasma were due not only to platelet lysis but also to a limited extent to secondary aggregation. In an inverse and probably causative manner, platelet morphology recovered, whereas the sensitivity of platelets to adenosine diphosphate and collagen decreased toward the end of bypass.

Cytotoxic T lymphocytes mediate lysis of target cells by various mechanisms, including exocytosis of lytic proteins (perforin, granzymes) and receptor-ligand binding of Fas/APO molecules. Death of target cells is characterized by either necrosis or apoptosis, depending on the killing mechanism used and on the metabolism of the target cell itself.

The phosphatase inhibitors okadaic acid and calyculin A were used to examine the role of phosphorylation processes in T cell apoptosis induced by interleukin-2 (IL-2) deprivation or transforming growth factor-β₂(TGF-β₂. Okadaic acid and calyculin A inhibited IL-2-driven T cell proliferation and induced apoptosis at concentrations known to inhibit protein phosphatase 1. High concentrations of both agents caused toxic changes of prominent cellular swelling and dilatation of rough endoplasmic reticular profiles. When the T cells were induced to undergo apoptosis by IL-2 deprivation, okadaic acid and calyculin A delayed loss of membrane integrity, nucleosomal size DNA fragmentation, and loss of bcl-2 mRNA. However, T cells deprived of IL-2 in the presence of okadaic acid or calyculin A revealed DNA breaks by in situ DNA end labeling and apoptotic morphology by electron microscopy and failed to show enhanced survival after reexposure to IL-2. Although TGF-β-mediated signaling is thought to involve the dephosphorylation of specific substrates, okadaic acid and calyculin A not only failed to inhibit, but actually augmented, TGF-β₂-induced inhibition of T cell proliferation and induction of apoptosis. Exposure to either TGF-β₂ or the phosphatase inhibitors prevented phosphorylation of the retinoblastoma protein RB. In summary, okadaic acid and calyculin A: (i) induce T cell apoptosis in the presence of IL-2, (ii) allow us to distinguish essential from epiphenomenal features of T cell apoptosis after IL-2 deprivation, and (iii) cooperate with TGF-β₂ in inducing growth arrest and apoptosis of murine T cells via intracellular cascades that converge in the prevention of RB phosphorylation. Copyright 1995, 1999 Academic Press

We have investigated virus-lymphocyte interactions by using cloned subpopulations of interleukin-2-dependent effector lymphocytes maintained in vitro. Cloned lines of H-2-restricted hapten- or virus-specific cytotoxic T lymphocytes (CTL) and alloantigen-specific CTL were resistant to productive infection by vesicular stomatitis virus (VSV). In contrast, cloned lines of natural killer (NK) cells were readily and persistently infected by VSV, a virus which is normally highly cytolytic. VSV-infected NK cells continued to proliferate, express viral surface antigen, and produce infectious virus. Furthermore, persistently infected NK cells showed no marked alteration of normal cellular morphology and continued to lyse NK-sensitive target cells albeit at a slightly but significantly reduced level. The persistence of VSV in NK cells did not appear to be caused by the generation of temperature-sensitive viral mutants, defective interfering particles, or interferon. Consequently, studies comparing the intracellular synthesis and maturation of VSV proteins in infected NK and mouse L cells were conducted. In contrast to L cells, in which host cell protein synthesis was essentially totally inhibited by infection, the infection of NK cells caused no marked diminution in the synthesis of host cell proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of viral proteins from infected cells showed that the maturation rate and size of VSV surface G glycoprotein were comparable in L cells and NK cells. Nucleocapsid (N) protein synthesis also appeared to be unaffected in NK cells. In contrast, the viral proteins NS and M appeared to be selectively degraded in NK cell extracts. Mixing experiments suggested that a protease in NK cells was responsible for the selective breakdown of VSV NS protein. Finally, VSV-infected NK cells were resistant to lysis by virus-specific CTL, suggesting that persistently infected NK cells may harbor virus and avoid cell-mediated immune destruction in an immunocompetent host.