A novel two-chain proteinase inhibitor generated by circularization of a multidomain precursor protein

Female reproductive tissues of the ornamental tobacco amass high levels of serine proteinase inhibitors (PIs) for protection against pests and pathogens. These PIs are produced from a precursor protein composed of six repeats each with a protease reactive site. Here we show that proteolytic processing of the precursor generates five single-chain PIs and a remarkable two-chain inhibitor formed by disulfide-bond Linkage of Nand C-terminal peptide fragments. Surprisingly, PI precursors adopt this circular structure regardless of the number of inhibitor domains, suggesting this bracelet-like conformation is characteristic of the widespread potato inhibitor II (Pot II) protein family.

Single mode Lamb waves in composite laminated plates generated by piezoelectric transducers

The technique of permanently attaching piezoelectric transducers to structural surfaces has demonstrated great potential for quantitative non-destructive evaluation and smart materials design. For thin structural members such as composite laminated plates, it has been well recognized that guided Lamb wave techniques can provide a very sensitive and effective means for large area interrogation. However, since in these applications multiple wave modes are generally generated and the individual modes are usually dispersive, the received signals are very complex and difficult to interpret. An attractive way to deal with this problem has recently been introduced by applying piezoceramic transducer arrays or interdigital transducer (IDT) technologies. In this paper, the acoustic wave field in composite laminated plates excited by piezoceramic transducer arrays or IDT is investigated. Based on dynamic piezoelectricity theory, a discrete layer theory and a multiple integral transform method, an analytical-numerical approach is developed to evaluate the input impedance characteristics of the transducer and the surface velocity response of the plate. The method enables the quantitative evaluation of the influence of the electrical characteristics of the excitation circuit, the geometric and piezoelectric properties of the transducer array, and the mechanical and geometrical features of the laminate. Numerical results are presented to validate the developed method and show the ability of single wave mode selection and isolation. The results show that the interaction between individual elements of the piezoelectric array has a significant influence on the performance of the IDT, and these effects can not be neglected even in the case of low frequency excitation. It is also demonstrated that adding backing materials to the transducer elements can be used to improve the excitability of specific wave modes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen

Up-regulation of receptor-ligand pairs during interaction of an MHC-presented epitope on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In this study, we used replication deficient adenoviruses to introduce a model tumor-associated Ag (the E7 oncoprotein of human papillomavirus 16) and the T cell costimulatory molecule 4-IBBL into murine DCs, and monitored the ability of these recombinant DO to elicit E7-directed T cell responses following immunization. Splenocytes from mice immunized with DCs expressing E7 alone elicited E7-directed effector and memory CTL responses. Coexpression of 4-1BBL in these E7-expressing DO increased effector and memory CTL responses when they were used for immunization. 4-1BBL expression up-regulated CD80 and CD86 second signaling molecules in DO. We also report an additive effect of 4-IBBL and receptor activator of NF-kappaB/receptor activator of NF-kappaB ligand coexpression in E7-transduced DC inummogens on E7-directed effector and memory CTL responses and on MHC class II and CD80/86 expression in DCs. Additionally, expression of 4-1BBL in E7-transduced DCs reduced nonspecific T cell activation characteristic of adenovirus vector-associated immunization. The results have generic implications for improved or tumor Ag-expressing DC vaccines by incorporation of exogenous 4-1BBL. There are also specific implications for an improved DC-based vaccine for human papillomavirus 16-associated cervical carcinoma.

Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B(RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8(+) T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-gamma-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.

Molecular cloning reveals that the p160 myb-binding protein is a novel, predominantly nucleolar protein which may play a role in transactivation by Myb

We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alpha beta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes, Here we describe a DNA plasmid encoding a polyepitope or polytope protein, which contained multiple contiguous minimal murine CTL epitopes, Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models, CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help, The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.

Proliferation in monocyte-derived dendritic cell cultures is caused by progenitor cells capable of myeloid differentiation

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [H-3]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor-alpha led to maturation of the DC, but did not inhibit proliferation. Ki67(+) cells were observed in cytospins of these DC, and by double staining were CD3(-)CD19(-)CD11c(-)CD40(-) and myeloperoxidase(+), suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34(+)CD33(-)CD14(-) progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes. (C) 1998 by The American Society of Hematology.

Molecular motion in miscible polymer blends .1. Motion in blends of PEO and PVPh studied by solid-state C-13 T-1 rho measurements

Changes in molecular motion in blends of PEO-PVPh have been studied using measurements of C-13 T-1 rho relaxation times. C-13 T-1 rho relaxation has been confirmed as arising from spin-lattice interactions by observation of the variation in T-1 rho with rf field strength and temperature. In the pure homopolymers a minimum in T-1 rho is observed at ca. 50 K above the glass transition temperatures detected by DSC. After blending, the temperature of the minimum in T-1 rho for PEO increased, while that for PVPh decreased, however, the minima, which correspond to the temperatures where the average correlation times for reorientation are close to 3.1 mu s, are separated by 45 K (in a 45% PEO-PVPh blend). These phenomena are explained in terms of the local nature of T-1 rho measurements. The motions of the individual homopolymer chains are only partially coupled in the blend. A short T-1 rho has been observed for protonated aromatic carbons, and assigned to phenyl rings undergoing large-angle oscillatory motion, The effects of blending, and temperature, on the proportion of rings undergoing oscillatory motion are analyzed.

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xylp, and similar to 0.03 mol acetyl per mol of glucosyl residue. Treatment with alpha- and beta-D-galactosidases showed that the majority of the side-chains were either single Galp-alpha-(1 --> residues or the disaccharide Galp-beta-(1 --> 2)-Galp-alpha-(1 --> linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1 --> 4)-beta-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating --> 4)-D-Manp-beta-(1 --> and --> Lt)-D-Glcp-beta-(1 --> branched at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-beta-(1 --> 4)-[D-Galp-beta-(1 --> 2)-D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> 4)-D-Glcp-beta-(I --> 4)-[D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> (27%), and most of the other oligosaccharides produced in significant quantities were based on this structure. (C) 1997 Elsevier Science Ltd.

Post-transcriptional regulation of the GLI1 oncogene by the expression of alternative 5 ' untranslated regions

The oncogene GLI1 is involved in the formation of basal cell carcinoma and other tumor types as a result of the aberrant signaling of the Sonic hedgehog-Patched pathway. In this study, we have identified alternative GLI1 transcripts that differ in their 5' untranslated regions (UTRs) and are generated by exon skipping. These are denoted (alpha -UTR, beta -UTR, and gamma -UTR according to the number of noncoding exons possessed (three, two, and one, respectively). The alpha- and beta -UTR forms represent the major Gli1 transcripts expressed in mouse tissues, whereas the gamma -UTR is present at relatively low levels but is markedly induced in mouse skin treated with 12-O-tetradecanoylphorbol 13-acetate, Transcripts corresponding to the murine beta and gamma forms were identified in human tissues, but significantly, only the gamma -UTR form was present in basal cell carcinomas and in proliferating cultures of a keratinocyte cell line. Flow cytometry analysis determined that the gamma -UTR variant expresses a heterologous reporter gene 14-23-fold higher than the alpha -UTR and 5-13-fold higher than the beta -UTR in a variety of cell types. Because expression of the gamma -UTR variant correlates with proliferation, consistent with a role for GLI1 in growth promotion, up-regulation of GLI1 expression through skipping of 5' noncoding exons may be an important tumorigenic mechanism.

Heat of desorption measured by means of electrical heat excitation

A new method of measuring heat of desorption is proposed in this Letter. The principle of the method is to measure the amount of mass released when a controlled amount of energy is supplied directly to a solid adsorbent. This is in contrast to conventional methods such as microcalorimetry, where heat released upon adsorption is measured. In this method, a quantified heat supply is generated by passing a de current through a carbon pellet, which is equilibrated with a gas phase confined in a closed vessel. As a consequence of the heating, the particle temperature is increased, resulting in partial desorption of adsorbed molecules. The variations of pellet temperature and the system pressure with respect to time are used to determine the heat of desorption as a function of loading.