E7 oncoprotein of human papillomavirus type 16 expressed constitutively in the epidermis has no effect on E7-specific B- or Th-repertoires or on the immune response induced or sustained after immunization with E7 protein

A line of FVB (H-2(q)) mice transgenic for the E6/E7 open reading frames of Human Papillomavirus type 16 driven from the alpha-A crystallin promoter expresses E7 mRNA in lens and skin epithelium. E7 protein is detectable in adult skin, coinciding with the development or inflammatory skin disease, which progresses to papillomata and squamous carcinomata in some mice. By examining the outcome of parenteral immunization with E7 protein, we sought to determine whether endogenous expression of E7 in skin had induced a preexisting immune outcome, i.e., specific immunity or tolerance, or whether the mice remain naive (''ignorant'') to E7. Our data show that the antibody response to defined E7 B-epitopes, the proliferative response to Th epitopes, and the delayed-type hypersensitivity (DTH) response to whole E7 did not differ between groups or young and old E6/E7 transgenic mice (likely having different degrees of lifetime exposure to E7 protein) or between E6/E7-transgenic and nontransgenic parental strain control mice. Although an E7-specific CTL response could not be induced in the H-2(q) background of these mice, incorporation of a D-b allele into the genome allowed comparison of D-b-restricted CTL responses in E6/E7 transgenic and nontransgenic mice. Experiments indicated that the E7-immunization-induced CTL response did not differ significantly between E6/E7 transgenic and nontransgenic mice. We interpret these results to indicate that in spite of expression of E7 protein in adult skin, E6/E7 transgenic mice remain immunologically naive (ignorant) of E7 epitopes presented by immunization. (C) 1997 Academic Press.

Functional growth hormone (GH) receptors and GH are expressed by preimplantation mouse embryos: A role for GN in early embryogenesis?

The results of this study challenge the widely held view that growth hormone (GH) acts only during the postnatal period. RNA phenotyping shows transcripts for the GH receptor and GH-binding protein in mouse preimplantation embryos of all stages from fertilized eggs (day 1) to blastocysts (day 4). An antibody specific to the cytoplasmic region of the GH receptor revealed receptor protein expression, first in two-cell embryos, the stage of activation of the embryonic genome (day 2), and in all subsequent stages, In cleavage-stage embryos this immunoreactivity was localized mainly to the nucleus, but clear evidence of membrane labeling was apparent in blastocysts. GH receptor immunoreactivity was also observed in cumulus cells associated with unfertilized oocytes but not in the unfertilized oocytes. The blastocyst receptor was demonstrated to be functional, exhibiting the classic bell-shaped dose-response curves for GH stimulation of both 3-O-methyl glucose transport and protein synthesis. Maximal stimulation of 40-50% was seen for both responses at less than 1 ng/ml recombinant GH, suggesting a role for maternal GK. However mRNA transcripts for GH were also detected from the morula stage (day 3) by using reverse transcription-PCR, and GH immunoreactivity was seen in blastocysts. These observations raise the possibility of a paracrine/autocrine GH loop regulating embryonic development in its earliest stages.

Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we describe the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution. (C) 1997 Elsevier Science B.V.

A Mox homeobox gene in the gastropod mollusc Haliotis rufescens is differentially expressed during larval morphogenesis and metamorphosis

We have isolated a homeobox-containing cDNA from the gastropod mollusc Haliotis rufescens that is most similar to members of the Mox homeobox gene class, The derived Haliotis homeodomain sequence is 85% identical to mouse and frog Mox-2 homeodomains and 88.9% identical to the partial cnidarian cnox5-Hm homeodomain. Quantitative reverse transcription-polymerase chain reaction analysis of mRNA accumulation reveals that this gene, called HruMox, is expressed in the larva, but not in the early embryo, Transcripts are most prevalent during larval morphogenesis from trochophore to veliger. There are also transient increases in transcript prevalence 1 and 3 days after the intitiation of metamorphosis from veliger to juvenile. The identification of a molluscan Mox homeobox gene that is more closely related to vertebrate genes than other protostome (e.g. Drosophila) genes suggests the Mox class of homeobox genes may consist of several different families that have been conserved through evolution, (C) 1997 Federation of European Biochemical Societies.

Novel gene containing multiple epidermal growth factor-like motifs transiently expressed in the papillae of the ascidian tadpole larvae

We have investigated molecular mechanisms of the embryonic development of an ascidian, a primitive chordate which shares features of both invertebrates and vertebrates, with a view to identifying genes involved in development and metamorphosis, We isolated 12 partial cDNA sequences which were expressed in a stage-specific manner using differential display, We report here the isolation of a full-length cDNA sequence for one of these genes which was specifically expressed during the tailbud and larval stages of ascidian development, This cDNA, 1213 bp in length, is predicted to encode a protein of 337 amino acids containing four epidermal growth factor (EGF)-like repeats and three novel cysteine-rich repeats, Characterization of its spatial expression pattern by in situ hybridisation in late tailbud and larval embryos demonstrated strong expression localised throughout the papillae and anteriormost trunk and weaker expression in the epidermis of the remainder of the embryo, As recent evidence indicates that the signal for metamorphosis originates in the anterior trunk region, these results suggest that this gene may have a role in signalling the initiation of metamorphosis. (C) 1997 Wiley-Liss, Inc.

Functional CD40 ligand is expressed by T cells in rheumatoid arthritis

CD40 ligand (CD40-L), a member of the tumor necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. Interactions between CD40-L and CD40 induce B cell immunoglobulin production as well as monocyte activation and dendritic cell differentiation. Since these features characterize rheumatoid arthritis (RA), the expression and function of CD40-L in RA was examined. Freshly isolated RA peripheral blood (PB) and synovial fluid (SF)T cells expressed CD40-L mRNA as well as low level cell surface CD40-L. An additional subset of CD4+ RA SF T cells upregulated cell surface CD40-L expression within 15 min of in vitro activation even in the presence of cycloheximide, but soluble CD40-L was not found in SF. CD40-L expressed by RA T cells was functional, since RA PB and SF T cells but not normal PB T cells stimulated CD40-L-dependent B cell immunoglobulin production and dendritic cell IL-12 expression in the absence of prolonged in vitro T cell activation. In view of the diverse proinflammatory effects of CD40-L, this molecule is likely to play a central role in the perpetuation of rheumatoid synovitis. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.

Emotions at multiple levels: An integration

Weiss and Isen have provided many supportive comments about the multi-level perspective, but also found limitations. Isen noted the importance of integrating affect, cognition, and motivation. Weiss commented similarly that the model lacked an integrating “thread.” He suggested that, to be truly multilevel, each level should constrain processes at other levels, and also provide guidance for the development of new concepts. Weiss also noted that the focus on biological processes was a strength of the model. I respond by suggesting that these very biological processes may constitute the “missing” thread. To illustrate this, I discuss some of the recent research on emotions in organizational settings, and argue that biology both constrains and guides theory at each level of the model. Based on this proposition, I revisit each of the five levels in the model, to demonstrate how this integration can be accomplished in this fashion. Finally, I address two additional points: aggregation bias, and the possibility of extending the model to include higher levels of industry and region.

Emotions in organizations: A multi-level perspective

Recent years have seen an upsurge of interest in the study of emotions in organizations. Research, however, has been hampered by the ephemeral nature of emotions and a lack of an integrated multi-level model. This article therefore presents a five-level model of emotions in organizations. At the lowest level is within-person variation, defined in terms of affective events theory. Levels of the model then proceed through individual, dyadic relationship, group, and organization-wide perspectives. The article also outlines the neurophysiological processes that underlie the experience, perception, and communication of emotion; it concludes with a discussion of implications for research and practice.

Customer emotions in service failure and recovery encounters

Emotions play a significant role in the workplace, and considerable attention has been given to the study of employee emotions. Customers also play a central function in organizations, but much less is known about customer emotions. This chapter reviews the growing literature on customer emotions in employee–customer interfaces with a focus on service failure and recovery encounters, where emotions are heightened. It highlights emerging themes and key findings, addresses the measurement, modeling, and management of customer emotions, and identifies future research streams. Attention is given to emotional contagion, relationships between affective and cognitive processes, customer anger, customer rage, and individual differences.