Physical interaction of human T-cell leukemia virus type 1 Tax with cyclin-dependent kinase 4 stimulates the phosphorylation of retinoblastoma protein

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G(1) phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21(CIP). Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein.     

Two-way arginine transport in human endothelial cells: TNF-alpha stimulation is restricted to system y+

Humanumbilical vein endothelial cells transport arginine through twoNa⁺-independent systems. System y⁺L isinsensitive to N-ethylmaleimide (NEM), inhibited byL-leucine in the presence of Na⁺, and referableto the expression of SLC7A6/y⁺LAT2,SLC7A7/y⁺LAT1, and SLC3A2/4F2hc. System y⁺ isreferable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumornecrosis factor- (TNF-) and bacterial lipopolysaccharide induce atransient stimulation of arginine influx and efflux through systemy⁺. Increased expression of SLC7A2/CAT2B is detectable from3 h of treatment, while SLC7A1 expression is inhibited at latertimes of incubation. System y⁺L activity and expressionremain unaltered. Nitric oxide synthase type 2 mRNA is not detected inthe absence or presence of TNF-, while the latter condition lowersnitric oxide synthase type 3 expression at the mRNA and the proteinlevel. Nitrite accumulation is comparable in cytokine-treated andcontrol cells up to 48 h of treatment. It is concluded thatmodulation of endothelial arginine transport by TNF- orlipopolysaccharide occurs exclusively through changes in CAT2B and CAT1expression and is dissociated from stimulation of nitric oxide production.

     

The Zygosaccharomyces bailii antifungal virus toxin zygocin: cloning and expression in a heterologous fungal host

Zygocin, a monomeric protein toxin secreted by a virus-infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double-stranded (ds)RNA killer virus (ZbV-M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N-terminal amino acid sequence was determined, and a full-length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238-amino-acid preprotoxin precursor (pptox) consisting of a hydrophobic N-terminal secretion signal, followed by a potentially N-glycosylated pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N-terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix-assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post-translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin-sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.     

The viral killer system in yeast: from molecular biology to application

Since the initial discovery of the yeast killer system almost 40 years ago, intensive studies have substantially strengthened our knowledge in many areas of biology and provided deeper insights into basic aspects of eukaryotic cell biology as well as into virus-host cell interactions and general yeast virology. Analysis of killer toxin structure, synthesis and secretion has fostered understanding of essential cellular mechanisms such as post-translational prepro-protein processing in the secretory pathway. Furthermore, investigation of the receptor-mediated mode of toxin action proved to be an effective means for dissecting the molecular structure and in vivo assembly of yeast and fungal cell walls, providing important insights relevant to combating infections by human pathogenic yeasts. Besides their general importance in understanding eukaryotic cell biology, killer yeasts, killer toxins and killer viruses are also becoming increasingly interesting with respect to possible applications in biomedicine and gene technology. This review will try to address all these aspects.     

Validation data for periprosthetic bone remodelling theories

Periprosthetic adaptive bone remodelling after total hip arthroplasty (THA) has been frequently simulated in computer models, combining bone remodelling theory with finite element analysis. Unfortunately, there still subsist a lack of clinical data, which are necessary for validation of these simulation results. Therefore, the objective of the current project is to collect prospective volumetric bone density data with a clinical computerized tomography study in seven patients after THA. A retrospective study 12 years after implantation in 11 patients was added. A data set of about 100 000 bone voxels for each femur was collected. In all prospective cases, the predominant change is seen during the first year. The average density reduction in the horizontal slices was between 50 and 150 Hounsfield units (HU) (approx. 10%; p<0.001) after 2 years. Loss of density is particularly strong distal of the minor trochanter and decreases from proximal to distal.

For the 12 years retrospective study, the contralateral femur provided the control. Similar trends comparable to the prospective 2-year follow-up CT density values were seen in most cases with density reductions of up to 400 HU (30%). However, in one of these cases there was no difference between the operated and the control density.

As far as we are aware, this is the first collection of fully prospective 3D validation data in vivo for periprosthetic adaptive bone remodelling theories. The data are also unique as they are suitable for direct patient-specific 3D finite element meshing and individual weight-related loading.     

ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

In eukaryotic cells, secretion is achieved by vesicular transport. Fusion of such vesicles with the correct target compartment relies on SNARE proteins on both vesicle (v-SNARE) and the target membranes (t-SNARE). At present it is not clear how v-SNAREs are incorporated into transport vesicles. Here, we show that binding of ADP-ribosylation factor (ARF)-GTPase-activating protein (GAP) to ER-Golgi v-SNAREs is an essential step for recruitment of Arf1p and coatomer, proteins that together form the COPI coat. ARF-GAP acts catalytically to recruit COPI components. Inclusion of v-SNAREs into COPI vesicles could be mediated by direct interaction with the coat. The mechanisms by which v-SNAREs interact with COPI and COPII coat proteins seem to be different and may play a key role in determining specificity in vesicle budding.     

Different efficiency of heat shock proteins (HSP) to activate human monocytes and dendritic cells: superiority of HSP60

One essential immunoregulatory function of heat shock protein (HSP) is activation of the innate immune system. We investigated the activation of human monocytes and monocyte-derived dendritic cells (DC) by recombinant human HSP60, human inducible HSP72, and preparations of human gp96 and HSP70 under stringent conditions, in the absence of serum and with highly purified monocytes. HSP60 induced human DC maturation and activated human DC to secrete proinflammatory cytokines. HSP72 induced DC maturation to a lesser extent, but activated human monocytes and immature DC as efficiently as HSP60 to release proinflammatory cytokines. The independence of the effects of HSP60 and HSP72 from endotoxin or another copurifying bacterial component was shown by the resistance of these effects to polymyxin B, their sensitivity to heat treatment, the inactivity of endotoxin controls at concentrations up to 100-fold above the endotoxin contents of the HSP, and the inactivity of a recombinant control protein. Preparations of HSP70, which consisted mainly of the constitutively expressed HSP73, induced only marginal cytokine release from monocytes. The gp96 preparations did not have significant effects on human monocytes and monocyte-derived DC, indicating that these human APC populations were not susceptible to gp96 signaling under the stringent conditions applied in this study. The biological activities of gp96 and HSP70 preparations were confirmed by their peptide binding activity. These findings show that HSP can differ considerably in the capacity to activate monocyte-derived APC under certain conditions and underline the potential of HSP60 and HSP72 as activation signals for the innate immune system.     

Characterization of the peptide-binding specificity of Mamu-B*17 and identification of Mamu-B*17-restricted epitopes derived from simian immunodeficiency virus proteins

The SIV-infected rhesus macaque is an excellent model to examine candidate AIDS virus vaccines. These vaccines should elicit strong CD8(+) responses. Previous definition of the peptide-binding motif and optimal peptides for Mamu-A*01 has created a demand for Mamu-A*01-positive animals. We have now studied a second MHC class I molecule, Mamu-B*17, that is present in 12% of captive-bred Indian rhesus macaques. The peptide-binding specificity of the Mamu-B*17 molecule was characterized using single substitution analogs of two Mamu-B*17-binding peptides and libraries of naturally occurring sequences of viral or bacterial origin. Mamu-B*17 uses position 2 and the C terminus of its peptide ligands as dominant anchor residues. The C terminus was found to have a very narrow specificity for the bulky aromatic residue W, with other aromatic residues (F and Y) being only occasionally tolerated. Position 2 is associated with a broad chemical specificity, readily accommodating basic (H and R), bulky hydrophobic (F and M), and small aliphatic (A) residues. Using this motif, we identified 50 peptides derived from SIV(mac)239 that bound Mamu-B*17 with an affinity of 500 nM or better. ELISPOT and intracellular cytokine-staining assays showed that 16 of these peptides were antigenic. We have, therefore, doubled the number of MHC class I molecules for which SIV-derived binding peptides have been characterized. This allows for the quantitation of immune responses through tetramers and analysis of CD8(+) function by intracellular cytokine-staining assays and ELISPOT. Furthermore, it is an important step toward the design of a multiepitope vaccine for SIV and HIV.     

Transformation of mutagenic aromatic amines into non-mutagenic species by alkyl substituents. Part II: alkylation far away from the amino function

Alkyl and trifluoromethyl derivatives of 4-aminobiphenyl (1) (4ABP) and 2-aminofluorene (7) (2AF) were synthesised and assayed for mutagenicity using Salmonella typhimurium tester strains TA98 and TA100 with and without the addition of S9 mix. Modification of 1 was achieved by attachment of alkyl groups (methyl, ethyl, iso-propyl, n-butyl, tert-butyl) and a trifluoromethyl group (CF(3)) in the 4'-position, the 3'-position (Me, CF(3)) and the 3'-, 5'-positions (DiMe, DiCF(3)). Compound 7 was modified by introduction of alkyl groups (methyl, tert-butyl, adamantyl) and a trifluoromethyl group (CF(3)) in the 7-position. The derivatives of 1 and 7 show for groups with growing steric demand decreased mutagenic activity. The bulkiest groups (CF(3), tert-butyl and adamantyl) induce the strongest effects on the mutagenicity. It was even possible to eliminate the mutagenicity of 1 and 7 by introduction of such substituents. In the last part of the work, we compared the experimental mutagenicities with calculated values derived from QSAR correlations. Our findings show that the predictions for aromatic amines with bulky substituents were generally too high. The strongest deviations were observed in the case of the CF(3)-, tert-butyl- and the adamantyl-group. Only the parent compounds and derivatives with small alkyl groups were predicted well. These investigations show that "large" substituents have an influence on the mutagenicity caused by their steric demand. To predict the correct mutagenicities of such compounds, it is necessary to introduce steric parameters in the respective QSAR equations which will be done in a forthcoming paper.