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The alternative sigma factor E (σE) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σE-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σEmay indirectly participate in post-transcriptional regulation. In this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σEin Salmonella. Samples were analyzed from wild-type and isogenic rpoEmutant Salmonellacultivated in three different conditions: nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of the observed proteome was regulated by σEcombining all three conditions. In different growth conditions, σEaffected the expression of a broad spectrum of Salmonellaproteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σEand found that post-transcriptional regulation was responsible for a majority of changes observed in the σE-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfqmutant of Salmonellademonstrated that σE-mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.

ABSTRACTPeptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF)3RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is β-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP(which encodes acyl carrier protein). The MIC of (RFF)3RXB-AcpP was 2.5 μM (14 μg/ml) in Escherichia coliW3110. The rate of spontaneous resistance of E. colito (RFF)3RXB-AcpP was 4 × 10−7mutations/cell division. A spontaneous (RFF)3RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)3RXB-AcpP was 40 μM (224 μg/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpPwas identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)3RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)3RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmArestored susceptibility to (RFF)3RXB-AcpP. Deletion of sbmAcaused resistance to (RFF)3RXB-AcpP. We conclude that resistance to (RFF)3RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)3R-XB PPMOs may be transported across the plasma membrane by SbmA.

This paper reviews the progress that has been made in fabricating biomimetic bone structures using synthetic composite materials. The specification for long bone applications is developed and we identify the candidate materials for delivering cortical and cancellous bone properties and function. The role of composite materials is discussed together with the factors influencing fibre and matrix type. Challenges associated with moderating their performance in-vivo are discussed, relating to the properties of the starting materials and the dependence, for fibre reinforced systems, on interface quality. Fabrication routes for producing complex biomimetic structures are also reviewed and the state of current clinical developments is described along with the associated technical and regulatory issues.

Boron trifluoride catalyzed ɛ‐caprolactone polymerization in the presence of glycerol can produce poly(ɛ‐caprolactone) with a high weight‐average molecular weight and a broad molecular weight distribution. This article reports an investigation of the polymerization mechanism to determine the formation of these molecular weight features through a study of the polymerization kinetics and the molecular structure with NMR. The polymerization proceeds via an activated monomer mechanism, resulting in polymer molecules with hydroxyl chain ends. The broad molecular weight distribution can be attributed to the etherification reactions between hydroxyl chain ends. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 3900–3906, 2006

The impact properties of commingled E-glass-polypropylene composites with varying fibre architectures have been investigated. The fabric structures include balanced and unbalanced twill weaves and a three-dimensional woven fabric. Comparative data for glass mat thermoplastics are included and additional comparisons with crossply continuous filament tape laminates are also made. All the materials were processed into flat panels via non-isothermal compression moulding. First, voidage was correlated with processing parameters to produce well consolidated laminates. Mechanical tests included tensile, flexural, and interlaminar shear. Impact tests include Charpy and falling weight; the latter ranged from 15 J to full penetration and were followed by IR thermography and microscopy to determine the extent of damage areas. The results demonstrate the evolution of damage with increasing impact energies and the mode of impact damage propagation for the different fibre architectures.

A carbon fibre recycling process for scrap composites based on fluidised bed technology has been developed. This paper describes the recycling process and the characterisation methods used to analyse the quality of recycled fibre. They include: the measurement of fibre length distribution by image analysis; tensile properties by single fibre testing; and the examination of surface contamination and surface chemistry of fibre by SEM and XPS. Recycled fibres of up to 10 mm mean length were recovered and they retained ∼75% of their tensile strength, while the Young's modulus remained unchanged and the surface condition was similar to the virgin fibre.

This paper presents an overview of a method of predicting the permeability of textile preforms. The equations involved in the calculation of the pressure distribution and flow field resulting from the imposition of a pressure gradient between two opposed faces of a porous domain are outlined. The presentation is for a two-dimensional cross-section but the method applies to a full three-dimensional domain featuring porous tows and free channels defined between the tows. The mathematical treatment was kept as simple as possible in order to gain speed and improve productivity. Fast calculation methods allow the prediction of the variation of permeability over a preform, providing valuable information to the simulation of the filling. The method does take into account the heterogeneity of the preform, the architecture of the textiles, as well as the porosity and shape of the tows.

This report outlines the initial synthesis, degradation, and shortterm biocompatibility of sodium phosphate glasses, for use in the drawing of fibers and manufacture of biodegradable composites. Biocompatibility studies were performed using a macrophage cell line and primary human craniofacial osteoblasts. Sodium hydrogen phosphate and sodium dihydrogen phosphate glass synthesized for less than 1 h, resulted in a higher degradation rate than glass synthesized for 3 h or more 0.015 mg cm−2h−1. Glasses with high and low ratios of hydrogen phosphate to dihydrogen phosphate had very similar degradation rates. A condensation route for the formation of the glass should give rise to varying degradation rates with varying ratios of starting materials. It is suggested that the degradation rate of the glass is independent of the concentrations of the initial reagents and that ringopening polymerization, which reaches an equilibrium state, occurs. Biocompatibility studies suggest minimal macrophage activation low levels of peroxide and interleukin1β release and rounded morphology and high osteoblast biocompatibility. The ultimate aim of our studies is to produce a biocompatible soluble phosphate glass that can be drawn into fibers for incorporation into a polycaprolactone matrix for craniofacial bone repair. This report demonstrates the successful production of a soluble glass, which is biocompatible with regard to osteoblasts and macrophages. Recent data from our laboratory have demonstrated successful fiber drawing and production of a novel polycaprolactone. © 2001 Wiley Periodicals, Inc. J Biomed Mater Res 59: 481–489, 2002

In this paper, the shear properties of a number of woven and non-crimp (multiaxial warp knit) fabrics are analysed. These are shown to be related to the fibre architecture and, for non-crimp fabrics, the pattern and orientation of the stitching thread. A detailed geometric model has been developed for the deformation of woven fabrics during in plane shear, and this is used as a basis for a mechanical analysis to predict shear compliance. This model incorporates intertow friction, tow compaction, and in plane (membrane) tension. Finally an iterative model for fabric forming is described, based on fabric shear energy obtained either from the mechanical model or from experimental measurements. This is validated for a hemisphere and an automotive transmission tunnel and is shown to offer greater accuracy than the traditional geometric mapping, while associated computation times are at least an order of magnitude lower than those associated with non-linear finite element analysis.

This work is aimed at understanding and modelling the draping of engineered prepregs and reinforced thermoplastics through the development of constitutive models for use in numerical draping codes. Results from picture frame tests are presented, which indicate the behavioural response of these materials to shear deformation, when varying parameters such as temperature and shear rate. Observations are then presented illustrating how intraply shear deformation can be either heterogeneous or homogeneous on a mesoscopic scale, depending on the test's boundary conditions. Two distinct modes of deformation are identified and explained by consideration of simple shear kinematics within the ply. The mode of deformation is shown to influence dramatically the velocity field between crossovers in woven fabrics and a qualitative argument is presented to suggest that the contribution to the total deformation energy from crossovers is negligible. Implications of these different modes of deformation on the forming of three-dimensional components are analysed.

A geometric textile modelling package (modeller) is being developed, which forms the basis for a number of software tools predicting the processing properties of textile reinforcements and the final properties of composite parts. The modeller can provide interlacing patterns representing any textile reinforcement in a simple, universal format. In this paper the general concept of the modeller is presented and examples are provided for woven textiles and noncrimp textiles assembled by a warp knitted stitch. The modeller and the predictive tools are modular. The main advantage of this is that tools for the prediction ofthe reinforcement properties can accept any geometricdefinition, regardless of the textile type, provided that the definitions and models are expressed in the required format. The tools also have the capacity to assess properties such as permeability or behaviour in compaction and shear, for preforms containing textiles of different types.

Crush experiments have been performed on polyester and vinyl ester composite tubes. The preforms were made of random glass mat and the tubes were produced by resin transfer moulding.The effects ofdifferent processing parameters were investigated. Flat plaques were also produced under similar conditions in order to measure in plane properties of the composite material. The two main objectives of the study were to quantify the effect of industrial manufacturing conditions on the crush performance of composite structures and to correlate the performance to a number of in plane laminate properties. The manufacturing parameters considered are resin related:the mould temperature, post-cure time, and resin composition were varied according to a full factorial experimental plan. In addition to crush experiments, the tensile and compressive moduli and ultimate stresses were determined; the degree of conversion was also measured. The results demonstrate that while relationships between all in plane properties and the crush performance can be observed, the ultimate compressive stress is the most reliable indicator of this performance. The results also show clear advantages associated with the vinyl ester resin, and the many intricacies pertaining to the modelling of the effect of processing parameters on crush performance.

Aligned fibre glass reinforced thermoplastic composites manufactured from commingled glass and polypropylene offer potential for structural crashworthy parts moulded in high volumes. In side-impact beam applications, their low densities capacitate more occupant-friendly beam geometries. This paper describes a simple implicit finite element damage model for commingled glass reinforced thermoplastic composites. The model is implemented as a user subroutine within ABAQUS/Standard, and is based on the well-known Chang and Lessard approach for thermosets. The material model includes various modes of failure and mode-dependent, non-linear post failure behaviour. The post failure behaviour requirements range from a slight reduction in modulus to a rapid drop-off in load carrying capability.An implicit analysis with the model is used to predict the behaviour of a thermoplastic side impact beam design. The results show good agreement with experimental testing to FMVSS 214.

Rlk/Txk is a T-cell-specific member of the Btk/Tec family of tyrosine kinases, whereas SLP-76 is a lymphoid adaptor that is essential for pre-TcR and mature TcR signaling. Although Rlk deficient T-cells show partial defects in T-cell proliferation, Rlk can complement ITK-/- cells with multiple defects in TcR initiated early events and interleukin (IL)-2 production. A key question is the nature of the target of Rlk responsible for bridging the TcR with the activation of IL-2 transcription. In this study, we identify a pathway in which Rlk phosphorylates SLP-76 leading to the phosphorylation of PLCgamma1, activation of ERKs, and the synergistic up-regulation of TcR-driven IL-2 NFAT/AP-1 transcription. Rlk phosphorylated the N-terminal region of SLP-76, a region that has been previously shown to serve as a target for ZAP-70. Loss of N-terminal YESP/YEPP sites of SLP-76 or the Rlk kinase activity attenuated cooperativity between Rlk and SLP-76. These observations support a model where the TcR can utilize Rlk (as well as ZAP-70) in the phosphorylation of key sites in SLP-76 leading to the up-regulation of Th1 preferred cytokine IL-2.

T-cell activation involves the participation of protein-tyrosine kinases p56(lck) and ZAP-70/SYK as well as lymphoid proteins such as SLP-76 and FYB/SLAP. FYB/SLAP has the hallmarks of an adaptor protein that binds to the SH2 domains of the Src kinase FYN-T and SLP-76. Whereas two forms of FYB at 120 and 130 kDa have been identified biochemically, a cDNA encoding only the lower molecular weight isoform has been cloned (termed FYB-120 or SLAP-130). In this study, we report the isolation of an alternative isoform of FYB with a molecular mass of 130 kDa (FYB-130) that has the same structure as FYB-120 except for an insertion of 46 amino acids toward the carboxyl-terminal region of the protein. FYB-120 and FYB-130 share an ability to bind to the SH2 domains of FYN-T and SLP-76, to act as substrates for p59(FYN-T), and to be expressed in the cytoplasm and nucleus of T-cells. Differences were noted between the isoforms in the efficiency of binding to SLP-76 and in the preferential expression of FYB-130 in mature T-cells. When co-expressed together with FYN-T and SLP-76, FYB-130 caused a significant increase in anti-CD3-driven NF-AT transcription. Finally, fluorescence in situ hybridization analysis localized the FYB gene to human chromosome 5 at position p13.1. FYB-130 therefore represents a novel variant of FYB protein that can up-regulate T-cell receptor-driven interleukin 2 production in mature T-cells.

Protein-tyrosine kinases p56(Lck), SYK, and ZAP-70 and downstream adaptors LAT and SLP-76 have been implicated as essential components in T-cell activation. Another lymphoid-specific adaptor FYB/SLAP has also been identified as a predominant binding partner of SLP-76 and the Src kinase FYN-T, although its role in the activation process has been unclear. In this study, we demonstrate that FYN-T selectively phosphorylates FYB providing a template for the recruitment of FYN-T and SLP-76 SH2 domain binding. This interaction is unusual in its distinct cytoplasmic localization and its long term stable kinetics of phosphorylation. Furthermore, we demonstrate for the first time that the co-expression of all three components of the FYN-T-FYB-SLP-76 matrix can synergistically up-regulate T-cell receptor-driven interleukin 2 transcription activity. These findings document the existence of a T-cell receptor-regulated FYN-T-FYB pathway that interfaces with the adaptor SLP-76 and up-regulates lymphokine production in T-cells.

Predictive calculations of the physical properties of fibrous preforms and composite parts require an appropriate description of the preform geometry. Most internal dimensions of a preform are set during its manufacturing through mechanical interactions occurring between the tows and threads. However, the global shape and the interlacing patterns of the constituent textiles are determined independently. In this paper, a formal procedure for the description of the interlacing patterns is proposed. This procedure, which is based on the individual textile manufacturing processes, is general in the textiles considered and in the possible applications. The interlacing patterns are expressed by a series of vectors that follow a universal set of criteria and are generated from the values taken by the processing parameters. Defining examples are given for three-dimensional woven textiles and three-dimensional tubular braided textiles, and geometrical applications are also presented. Further examples for warp-knitted textiles and multiple-layer stacks will be given in a subsequent paper, together with examples of physical applications.

The surge of LH that induces ovulation in mammals showing spontaneous ovulation is precipitated by the positive feedback of increasing oestrogens from the developing follicles in the ovary. In eutherians, exogenous oestrogens can mimic this effect by eliciting an LH surge in females, but not usually in males. The absence of a positive LH response in eutherian males is either due to an acute suppression by the secretory products of the testes during adulthood or the permanent disabling of the system by testosterone during early development. This phenomenon is examined in tammar wallabies, Macropus eugenii. The results show that the oestradiol–LH positive feedback response is sexually dimorphic in this marsupial. A surge in plasma LH occurred between 15 and 28 h after injection of 2.5 μg oestradiol benzoate kg−1in 13 of 16 intact females and 4 of 4 ovariectomized females, but in none of 11 intact males. Five females each implanted with a 100 mg testosterone pellet 3 months earlier failed to produce an LH surge. Four males castrated in adulthood and three adult males castrated before puberty also failed to show an LH surge. However, three males castrated 24–26 days after birth showed an unambiguous LH surge when challenged with oestradiol benzoate during adulthood. Thus, in tammar wallabies, the ability to generate an LH surge to oestradiol is a sexually dimorphic response that is suppressed in the male by the organizational effects of the testes in early life and presumably supplemented by an inhibitory effect of circulating testosterone in adulthood.

The CD4 (T4) antigen is a cell-surface glycoprotein that is expressed predominantly on the surface of helper T cells and has been implicated in the regulation of T-cell activation and in the associative recognition of class II antigens of the major histocompatibility complex. In addition, the CD4 antigen appears to serve as a receptor for the human immunodeficiency virus (HIV). An important question has been whether the CD4 receptor is linked to an intracellular mediator that could regulate the activation of the CD4+ subset. In this paper, we provide preliminary evidence that the CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (PTK) of 55-60 kDa, which is expressed specifically in T cells. The PTK is the human analogue of the murine pp56LSTRA (pp56lck) and has significant homology with c-src, c-yes, and other members of the src family. The identification of the PTK associated with CD4 receptor was made by use of an antiserum to a synthetic peptide that was deduced from the DNA sequence of PTK. Two-dimensional nonequilibrium pH gradient gel electrophoresis/NaDodSO4/PAGE revealed the kinase to focus as a heterogeneous collection of spots in the pH range of 4.0-5.0. Furthermore, in vitro phosphorylation revealed the phosphorylation of two additional polypeptides at 40 and 80 kDa, in addition to the autophosphorylation of the PTK at 55-60 kDa. The potential importance of the association between the CD4 receptor and the PTK of T cells is discussed in relation to T-cell activation and HIV infectivity.

The resin temperature distributions in a heated mould during the impregnation stage of resin transfer moulding have been investigated in this work. Effects such as the fibre content, the filling time and the mould and preform temperatures have been evaluated. It was shown that the fibre content and the filling time effects on the resin temperature distributions are less significant than the mould and preform temperatures. It was also shown that approximately 70% of the resin temperature increase is obtained within a flow path length of 12.5 cm. These trends led to the development of an empirical relation used to evaluate the influence on fill times of non-isothermal effects during filling.

The T-cell antigen CD28 provides a costimulatory signal that is required for T-cell proliferation. T-cell receptor zeta/CD3 engagement without CD28 ligation leads to a state of nonresponsiveness/anergy, thereby implicating CD28 in the control of peripheral tolerance to foreign antigens or tumors. A key unresolved question has concerned the mechanism by which CD28 generates intracellular signals. Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase with Src-homology 2 (SH2) domain(s) that binds to the platelet-derived growth factor receptor (PDGF-R), an interaction that is essential for signaling by growth factor. In this study, we demonstrate that CD28 binds to PI 3-kinase by means of a Y(P)MXM motif within its cytoplasmic tail. CD28-associated PI 3-kinase was detected by lipid kinase and HPLC analysis as well as by reconstitution experiments with baculoviral-expressed p85 subunit of PI 3-kinase. CD28 bound directly to the p85 subunit without the need for the associated p110 subunit. Site-directed mutagenesis and peptide competition analysis using Y(P)-MXM-containing peptides showed that PI 3-kinase bound to a Y(P)MXM motif within the CD28 cytoplasmic tail (residues 191-194). Mutation of the Y191 within the motif resulted in a complete loss of binding, while mutation of M194 caused partial loss of binding. Binding analysis showed that the CD28 Y(P)-MXM motif bound to the p85 C- and N-terminal SH2 domains with an affinity comparable to that observed for PDGF-R and insulin receptor substrate 1. In terms of signaling, CD28 ligation induced a dramatic increase in the recruitment and association of PI 3-kinase with the receptor. CD28 is likely to use PI 3-kinase as the second signal leading to T-cell proliferation, an event with implications for anergy and peripheral T-cell tolerance.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Purpose:To assess the frequency of retinopathy and vision loss in patients with insulin-dependent diabetes mellitus and their relations to potentially modifiable risk factors.

ABSTRACTThe increasing use of nucleic acid-based therapeutics has created a need for new methods of determining tissue distribution and levels. Radiolabel methods may not always be appropriate because nucleic acids are easily degraded. Quantitation using the polymerase chain reaction (PCR) has the advantage that only continuous stretches of DNA will be amplified. In situhybridization allows detection of specific sequences in histological preparations. We have used quantitative PCR and in situhybridization techniques to study the pharmacokinetics and distribution of PGagPol (a potential anti-HTV plasmid vaccine) in rabbits. Samples were obtained 4 hr, 24 hr, 7 days, and 28 days after intramuscular injection of 100 μμg or 400 μμgof plasmid. A simplified procedure for collecting and processing tissues for PCR that minimizes the risk of contamination was developed. Using PCR, plasmid was found principally in the skin and muscle of the injection site and in blood plasma. At 4 hr after dosing with 400 μμg, the plasmid was detected at the injection site with mean copy numbers of 106(in muscle) and 4 ×× 104(in skin) per microgram of tissue. Plasmid copy number declined rapidly in muscle during the first 24 hr and was undetectable at 7 and 28 days after injection. The decline was slower in the skin, and the plasmid was still detectable at 28 days. With in situhybridization, plasmid was detected in muscle, mainly in the perimysium and to a lesser degree in the endomysium and within the muscle fibers. These data indicate that quantitative PCR and in situhybridization are sensitive methods for examining tissue distribution of DNA used for gene therapy.

Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex.

CD80 (B7-1) and CD86 (B7-2) ligation of CD28 provide co-stimulatory signals required for optimal lymphokine production in response to TCR zeta-CD3 ligation. CD28 binds to several intracellular proteins including phosphatidylinositol 3-kinase (Pl3-kinase), the tyrosine kinase ITK and the growth factor receptor-bound protein/Son of Sevenless (GRB-2/SOS) complex. Previously, we showed that TCR zeta-CD3 and CD28 co-stimulation required Pl3-kinase binding to the pYMNM motif of the cytoplasmic domain of the co-receptor. In this study, we have investigated whether CD28-associated Pl3-kinase is required for CD80 and CD86 co-stimulation, as well as in co-signaling that involves different primary signals (i.e. TCR zeta-CD3 versus phorbol ester/lonomycin). In the presence of anti-CD3, ligation of CD28 by both CD80 and CD86 was found to induce Pl3-kinase recruitment and IL-2 production. Furthermore, mutations at Y-191 and M-194 within the pYMNM motif blocked the ability of both ligands to induce IL-2. CD80 and CD86 therefore share a common signaling pathway leading to IL-2 production. By contrast, CD28 mediated co-stimulation involving receptor ligation plus phorbol ester/lonomycin induced IL-2 independent of Pl3-kinase binding to CD28. These data indicate that TCR zeta-CD3-dependent CD80 and CD86 co-signaling requires Pl3-kinase binding to the CD28pYMNM motif, while phorbol ester and lonomycin can bypass this requirement in CD28 co-stimulation.

Present and future requirements for preforms in the automotive and aerospace industries are identified with respect to quality, processing, mechanical properties and economic manufacture for RTM and SRIM processes. The status of current preform technology is reviewed including textile, non-woven, continuous and chopped fiber based methods. Automotive applications are considered with an emphasis on structural parts. Key areas for development are identified and progress towards these presented as an integrated design method comprising of surface modeling, deformation analysis, flow considerations and high speed fiber placement.

Two-dimensional polyacrylamide gel analyses of immunoprecipitates of HLA-D region antigens prepared from [35S]methionine-labeled B lymphoblastoid cells revealed a number of invariant polypeptides (Ii and theta) that co-precipitate with the alpha and beta polypeptides of the class II (Ia) antigens. The invariant polypeptides comprised at least three Ii spots of Mr = 31,000 (Ii1-Ii3) and a series of six theta spots of Mr = 34,000 (theta 1-theta 6). The structural inter-relationships of these polypeptides have been investigated. Tryptic peptide fingerprints showed that Ii and theta have closely related amino acid sequences. In contrast, the fingerprints of the HLA-DR alpha and beta polypeptides clearly differed from those of theta and Ii as well as from each other. Analyses of immunoprecipitates prepared from cells cultured in the presence of tunicamycin revealed the presence of two N-linked oligosaccharides on each invariant polypeptide and suggested that the more acidic theta polypeptides (theta 1 and theta 2) differed from the other invariant polypeptides by the presence of sialic acid on one or both N-linked oligosaccharides. Removal of sialic acid by neuraminidase simplified the pattern of theta spots into three distinct Ii-related polypeptides. Endo-beta-N-acetylglycosaminidase H digestion indicated that the individual theta polypeptides represent stages in carbohydrate processing whereby Ii with two N-linked immature oligosaccharides are converted initially to theta 6-theta 3 with one immature and one complex, but nonsialylated, oligosaccharide and finally to theta 2-theta 1 with two complex oligosaccharides. Digestion of the theta polypeptides with N-acetylgalactosamine oligosaccharidase indicated that the theta spots are also derived by O-glycosylation from the Ii polypeptides. This assignment is supported by results obtained using monensin to block glycosylation within the Golgi. At least three spots persisted after complete removal of the N- and O-linked oligosaccharides, suggesting the presence of a family of invariant polypeptides differing in amino acid sequence.

AbstractLiquid composite molding techniques offer potential for cost and weight reduction in the transport industries. One of the limiting factors in the growth of new applications is the de velopment of short cycle times without the need for investment in costly reactive processing equipment. The status of liquid molding technology and applications is reviewed and new processing techniques for rapid cycling are summarized.

This paper describes an investigation of the substantial electrochemical potentials which have been found to arise if dissimilar metals are immersed in an electrolyte of polyester resin while it is curing. Sharp initial changes in the e.m.f. indicate the moment of arrival of resin at the electrodes, and the subsequent magnitude can be used to indicate the state of cure. The e.m.f. was found to be influenced by the addition of calcium carbonate filler powder to the base resin, as well as by the choice of electrode metals. Aluminium and copper were selected for practical applications, and tested under transfer moulding conditions. A prototype instrument suitable for incorporating in a mould to indicate the completion of filling and the state of cure in a resin transfer moulding process is described. The results will also have application to other thermoset moulding processes.

Glen St J. Barclay. A Very Small Insurance Policy. The Politics of Australian Involvement in Vietnam. 1954-1967. St Lucia: University of Queensland Press, 1988. 199pp. No price given.Robert J. Cooksey. Review of Australia's Defence Exports and Defence Industry. Report to the Minister for Defence. Canberra: Australian Government Publishing Service, 1986. x + 569pp. No price given.Ernest McNamara, Robin Ward, Desmond Ball, J. O. Langtry and Richard Q. Agnew. Australia's Defence Resources: a compendium of data. Sydney. Pergamon Press Australia, 1986. 186pp. $ 16.Jacob Bercovitch. Social Conflicts and Third Parties: Strategies of Conflict Resolution. Boulder. Westview Press, 1984. xv + 163pp. $US23.50.Norman P. Barry. The New Right. London, New York and Sydney. Croom Helm, 1987. 205pp. $87.95.Douglas E. Ashford. The Emergence of the Welfare States. Oxford: Basil Blackwell, 1986. x+352pp. $87.50.J.B. Ghartey. Crisis Accountability and Development in the Third World. The Case of Africa. Aldershot: Avebury, 1987. x + 170pp. $34.50.Jan-Erik Lane and Svante O. Ersonn. Politics and Society in Western Europe. London: Sage Publications, 1987. x + 370pp. £20.00 (cloth), £7.50 (paper).John Toye. Dilemmas of Development: Reflections on the Counter-Revolution in Development Theory and Policy. Oxford: Basil Black-well, 1987. ix+ 177pp. $29.95.Eva Kolinsky. Opposition in Western Europe. London and Sydney: Croom and Helm, 1987. 400pp. £29.95.Allen Lynch. The Soviet Study of International Relations. Cambridge: Cambridge University Press, 1987. xii+ 197pp. $90.00.Uri Ra'anan, Francis Fukuyama, Mark Falcoff, Sam C. Sarkesian and Richard H. Shultz, Jr.. Third World Marxist-Leninist Regimes: Strengths, Vulnerabilities, and U.S. Policy. Washington, D.C: Institute for Foreign Policy Analysis, Inc., Pergamon-Brassey's, 1985. xv + 130pp. No price given.Helene Carrere d'Encausse. Big Brother: The Soviet Union and Soviet Europe. Translated by George Holoch. New York and London: Holmes and Meier, 1987. xii+332pp. $39.50 (cloth), $US24.50 (paper).Ferenc Feher, Agnes Heller and Gyorgy Markus. Dictatorship Over Needs: An Analysis of Soviet Societies. Oxford: Basil Blackwell, 1984. xii+312pp. $62.00 (cloth), $ 18.95 (paper).Bruno Rizzi. The Bureaucratization of the World. The USSR: Bureaucratic Collectivism. London and New York: Tavistock Publications, 1985. viii +111 pp. No price given.Stephen Fortescue. The Communist Party and Soviet Science. London: Centre for Russian and East European Studies, University of Birmingham/Macmillan, 1986. x+ 234pp. $77.00.Colin Mackerras and Nick Knight (eds). Marxism In Asia. London & Sydney. Croom Helm, 1985. 297pp. £22.50.Harry Harding. China's Second Revolution: Reform After Mao, Washington, D.C.: The Brookings Institution, 1987. xx + 369pp. $US32.95 (cloth), $US12.95 (paper).Jim Masselos (ed.). Struggling and Ruling: The Indian National Congress 1885-1985. New Delhi: Sterling Publishers Private Limited, 1987. 224pp. Rs. 150.00.Lawrence B. Krause, Koh Ai Tee and Lee (Tsao) Yuan. The Singapore Economy Reconsidered. Singapore: The Institute of Southeast Asian Studies, 1987. 230pp. $S38.50 (cloth) $19.90 (paper).Raj Vasil. Governing Singapore: Interviews with the new leaders. Singapore and Kuala Lumpur: Times Books International, 1980 (Revised Edition 1988). 247pp. $S28.50.Martin Stuart-Fox. Laos: Politics, Economics and Society. London: Francis Pinter, Boulder. Lynne Rienner Publishers Inc., 1986. xxiv +220pp. £22.50 (cloth), £7.95 (paper).United Nations Centre on Transnational Corporations. Transnational Corporations and the Electronics Industries oj ASEAN Economics. New York: ESCAP/UNCTC Joint Unit on Transnational Corporations, 1987. v+49pp. $7.50.Stewart Firth. Nuclear Playground. Sydney: Allen and Unwin, 1987. xii +176pp. $14.95.Lars Schoultz. National Security and United States Policy towards Latin America. Princeton, New Jersey: Princeton University Press, 1987. 337pp. $US42.50 (cloth), $US12.95 (paper).Rose J. Spalding (ed.). The Political Economy of Revolutionary Nicaragua. Sydney. Allen and Unwin, 1987. 255pp. $87.50 (cloth), $34.95 (paper).Abby L. Bloom (ed.). Primary Health Care. Australian Council for Overseas Aid, Development Dossier No. 20. Canberra: ACFOA, 1987.iv+80pp.$5.00.

The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex.

Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human.

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

The co-stimulatory antigen CD28 has been shown to bind to several intracellular proteins including phosphatidylinositol 3-kinase, growth factor receptor-bound protein 2 (Grb2), and ITK. Paradoxically, Grb2 and phosphatidylinositol 3-kinase binding has been mapped to a similar pYMNM motif within the CD28 cytoplasmic tail. Given the importance of CD28 co-signaling to T cell function, questions exist regarding the mechanism by which Grb2 binds to CD28, and whether the interaction plays a role in co-stimulation. To biochemically characterize Grb2/CD28 binding, we initially utilized glutathione S-transferase-Grb2 fusion proteins carrying inactivating mutations within the SH2 and SH3 domains of Grb2, and assessed their ability to bind to CD28. In vitro binding experiments indicated that the Grb2 SH2 domain is critical for the association, while the SH3 domain plays an additional role in facilitating optimal binding. Enhanced binding via the SH3 domains was not observed when the C-terminal PXXP motif within CD28 was disrupted, thereby indicating that both SH2 and SH3 domains contribute to CD28 binding. Mutations that alter Grb2 binding were found to block the CD28-dependent interleukin-2 production. Further, tyrosine phosphorylation of Vav and the costimulation-dependent activation of Jun N-terminal kinase was blocked in cells defective in CD28/Grb2 binding. These results provide evidence for an alternate CD28-mediated signaling process involving Grb2 binding to the co-receptor.

The c-fps/fes proto-oncogene encodes a 92-kDa protein-tyrosine kinase that is expressed at high levels in macrophages. We have previously shown that overexpression of c-fps/fes in a CSF-1-dependent macrophage cell line (BAC1.2F5) partially released these cells from their factor dependence and that this correlated with the tyrosine phosphorylation of a subset of proteins in a tissue-specific manner. We have now identified one of the macrophage substrates of Fes as the crk-associated substrate (Cas) and a second substrate as a 130-kDa protein that has been previously described as a T cell activation-dependent substrate and is unrelated to Cas. Both of these proteins, which have optimal consensus sequences for phosphorylation by Fes, were tightly associated with this kinase through its SH2 domain, suggesting that they were direct substrates of Fes. Remarkably, when the Fes SH2 domain was used as an affinity reagent to identify potential substrates of endogenous Fes in control BAC1.2F5 cells, the phosphotyrosyl proteins that were recognized were the same as those that were specifically phosphorylated when Fes was overexpressed in the same cells. We conclude that the substrates we identified may be structurally related or identical to the physiological targets of this kinase in macrophages. The known functions of Cas and p130 suggest that Fes kinase may play a role in signaling triggered by cell adhesion and cell-cell interactions during immune responses of macrophages.

CTLA-4 is a T cell antigen that is structurally related to CD28 and serves as a high affinity ligand for the B cell antigen B7-1/2. Unlike CD28, the function of CTLA-4 is unclear, although reports have implicated the antigen in the costimulation of T cells. Recently, phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated in the costimulatory function of CD28 by virtue of its ability to bind to a pYMNM motif within the cytoplasmic tail of the antigen. In this study, we show that CTLA-4 can also associate with PI 3-kinase as detected by lipid kinase analysis and immunoblotting with anti-p85 antiserum. High pressure liquid chromatographic separation of deacylated lipids showed the presence of a peak corresponding to PI-3-P. Anti-CTLA-4 ligation of the receptor induced a significant increase in the levels of precipitable PI 3-kinase activity. Peptide binding studies revealed that the NH2- and COOH-terminal SH2 domains of p85 bind the CTLA-4 cytoplasmic pYVKM motif with an affinity (ID50: 0.6 and 0.04 microM), that is similar to CD28. CTLA-4 binding to PI 3-kinase provides further evidence that CTLA-4 is not an inert counterreceptor, but rather is coupled to an intracellular signaling molecule with the capacity to regulate cell growth.

The T4 (CD4) subset of T lymphocytes has been subdivided into two major subsets, a suppressor/inducer subset (T4+,2H4+) and a helper subset (T4+,2H4-) on the basis of the differential expression of the L-C/T200 (CD45) antigens. The 2H4 antigen itself comprises at least three distinct polypeptides at 125,200, and 220 X 10(3) Mr, of which the 200 and 220 X 10(3) Mr polypeptides constitute the highest Mr isoforms of a pool of five distinct L-C/T200 antigens. The T4+,2H4+ subset expresses at least four of these isoforms at 180, 190, 200, and 220 X 10(3) on the cell surface, while the T4+,2H4- subset expresses only the 180 and 190 X 10(3) Mr forms. Pulse-chase analysis and endoglycosidase treatment revealed that the 125 X 10(3) Mr chain of the 2H4 antigen is nonglycosylated, while the 200 and 220 X 10(3) polypeptides are structurally related and derived by N- and O-linked glycosylation from two nascent subunits at 150 and 160 X 10(3) Mr. The function of the T4+,2H4+ subset could be blocked only by an antibody reactive with the L-C/T200 isoforms enriched with O-linked oligosaccharides at 200 and 220 X 10(3) Mr.

Whether the expression of higher molecular weight isoforms of the T-200 complex represents different lineages of T cells and/or a sequential stage of the differential pathway of T cells has been unclear. Understanding T cell expression of higher molecular weight isoforms of the T-200 complex (CD45R) may be important because of their association with regulation of immune responses. By direct single cell cloning, we observed a number of long-term T cell clones that expressed CD45RA (2H4). CD45RA expression could be further regulated by ionomycin or the cytokines IL-1 and IL-6, but not IL-2, IL-4, or IFN-gamma. These results indicate that CD45RA expression may define T cell lineages of activated T cells partially controlled by the cytokines IL-1 and IL-6. Further, these results may associate regulatory actions of IL-1 and IL-6 with their ability to increase CD45RA expression in subpopulations of human T cells.