New insights into the effects of ethylene on ABA catabolism, sweetening and dormancy in stored potato tubers.

Continuous ethylene supplementation suppresses postharvest sprouting, but it can increase reducing sugars, limiting its use as an alternative to chlorpropham for processing potatoes. To elucidate the mechanisms involved, tubers were treated after curing with or without the ethylene binding inhibitor 1-methylcyclopropene (1-MCP at 1 µL L-1 for 24 h), and then stored in air or air supplemented with continuous ethylene (10 µL L-1). Across three consecutive seasons, changes in tuber physiology were assessed alongside transcriptomic and metabolomic analysis. Exogenous ethylene alone consistently induced a respiratory rise and the accumulation of undesirable reducing sugars. The transient respiratory peak was preceded by the strong upregulation of two genes encoding 1-aminocyclopropane-1-carboxylate oxidase (ACO), typical of wound and stress induced ethylene production. Profiles of parenchymatic tissue highlighted that ethylene triggered abscisic acid (ABA) catabolism, evidenced by a steep fall in ABA levels and a transient rise in the catabolite phaseic acid, accompanied by upregulation of transcripts encoding an ABA 8'-hydroxylase. Moreover, analysis of non-structural carbohydrate-related genes revealed that ethylene strongly downregulated the expression of the Kunitz-type invertase inhibitor, already known to be involved in cold-induced sweetening. All these ethylene-induced effects were negated by 1-MCP with one notable exception: 1-MCP enhanced the sprout suppressing effect of ethylene whilst preventing ethylene-induced sweetening. This study supports the conclusions that: i) tubers adapt to ethylene by regulating conserved pathways (e.g. ABA catabolism); ii) ethylene-induced sweetening acts independently from sprout suppression, and is similar to cold-induced sugar accumulation.

Fructans redistribution prior to sprouting in stored onion bulbs is a potential marker for dormancy break.

Continuous supply of high quality onion bulbs to meet year-round demand is dependent on maintaining dormancy and bulb quality during storage. Sprouting impacts negatively on the storage quality of onion bulbs. Ethylene supplementation has previously been revealed to inhibit sprout growth in stored onion bulbs. Fructans content, especially those at higher degree of polymerisation (DP), are reported to positively correlate with delayed sprouting. However, little is known about the impact of pre-harvest irrigation regimes on fructans accumulation and redistribution in relation to onion bulb dormancy and quality in store. Across two seasons, onion plants of cultivars 'Red Baron' and 'Sherpa' were subjected to full irrigation (FI) (100% replenishment of crop evapotranspiration) or deficit irrigation (DI) (50% of FI treatment) from bulb initiation to harvest. Bulbs were harvested at full maturity and stored at 1 °C for five months. Bulbs were treated with or without 1-MCP (1 µL L-1) for 24 h before storage under continuous ethylene supplementation (10 µL L-1) or air. DI had no effect on dormancy-break, sprout emergence, total fructans content and total sugar content. In contrast, ethylene delayed sprout emergence and suppressed sprout growth; added 1-MCP enhanced this effect. The concentration of DP3-8 fructans were higher in top and bottom sections compared to the baseplate. Before sprout emergence, fructans of DPs 7-8 were no longer present in the top and bottom wedges, while they accumulated in the baseplate; irrespective of pre- or postharvest treatments. This redistribution of fructans within the bulb suggested a transition in dormancy state and could be used as a predictive marker for sprouting in stored onion bulbs.

Bone marrow and periosteal skeletal stem/progenitor cells make distinct contributions to bone maintenance and repair.

A fundamental question in bone biology concerns the contributions of skeletal stem/progenitor cells (SSCs) in the bone marrow versus the periosteum to bone repair. We found that SSCs in adult bone marrow can be identified based on Leprcre and Adiponectin-cre/creER expression while SSCs in adult periosteum can be identified based on Gli1creERT2 expression. Under steady-state conditions, new bone arose primarily from bone marrow SSCs. After bone injuries, both SSC populations began proliferating but made very different contributions to bone repair. Drill injuries were primarily repaired by LepR+/Adiponectin+ bone marrow SSCs. Conversely, bicortical fractures were primarily repaired by Gli1+ periosteal SSCs, though LepR+/Adiponectin+ bone marrow cells transiently formed trabecular bone at the fracture site. Gli1+ periosteal cells also regenerated LepR+ bone marrow stromal cells that expressed hematopoietic niche factors at fracture sites. Different bone injuries are thus repaired by different SSCs, with periosteal cells regenerating bone and marrow stroma after non-stabilized fractures.

Prions are secreted in milk from clinically normal scrapie-exposed sheep.

The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrP(Sc) within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.

Detection of PrPsc in blood from sheep infected with the scrapie and bovine spongiform encephalopathy agents.

The role of blood in the iatrogenic transmission of transmissible spongiform encephalopathy (TSE) or prion disease has become an increasing concern since the reports of variant Creutzfeldt-Jakob disease (vCJD) transmission through blood transfusion from humans with subclinical infection. The development of highly sensitive rapid assays to screen for prion infection in blood is of high priority in order to facilitate the prevention of transmission via blood and blood products. In the present study we show that PrP(sc), a surrogate marker for TSE infection, can be detected in cells isolated from the blood from naturally and experimentally infected sheep by using a rapid ligand-based immunoassay. In sheep with clinical disease, PrP(sc) was detected in the blood of 55% of scrapie agent-infected animals (n = 80) and 71% of animals with bovine spongiform encephalopathy (n = 7). PrP(sc) was also detected several months before the onset of clinical signs in a subset of scrapie agent-infected sheep, followed from 3 months of age to clinical disease. This study confirms that PrP(sc) is associated with the cellular component of blood and can be detected in preclinical sheep by an immunoassay in the absence of in vitro or in vivo amplification.

1-Methylcyclopropene maintains postharvest quality in Norwegian apple fruit.

Norwegian fruit production is mostly destined for the local market and can suffer from poor-quality retention during storage. 1-Methylcyclopropene (1-MCP) is an inhibitor of ethylene perception used to maintain the physical and functional quality of pome fruit. Extensive work has been carried out on the effect of 1-MCP on apples, but not on cultivars grown in Norway. In this work, the potential of 1-MCP application (0.625 ml l -1 for 24 h at 0 ± 1℃) for ripening control of the apple cultivars 'Aroma', 'Red Gravenstein', and 'Summered' was studied during 1 and 1.5 months of cold storage; both scenarios were followed by five days of shelf life. The application of 1-MCP reduced softening by an average of 12% in 'Aroma', 'Red Gravenstein', and 'Summered' apples when cold stored for both 1 and 1.5 months as compared to control. External colour remained similar to initial values in 1-MCP fruit when compared to control apples, which presented a significant skin darkening. This indicated a delay in the ripening process. 1-MCP treatment did not affect total soluble solids content. 'Aroma' samples treated with 1-MCP showed a low sucrose hydrolysis, indicating a slower ripening process. This work confirms that 1-MCP postharvest treatment shows great potential for maintenance of apple cvs. in Norway during cold storage and shelf life.

Development of a disposable pyruvate biosensor to determine pungency in onions (Allium cepa L.).

A disposable prototype pyruvate biosensor was constructed using pyruvate oxidase immobilised on mediated meldolas blue electrodes to determine pungency in onions (Allium cepa L.). The optimum operating potential was +150 mV (versus Ag/AgCl). A strong correlation between the biosensor response and untreated onion juice of known pyruvate concentration 2-12 micromol/g fresh weight (FW) was demonstrated. The biosensor was able to differentiate between low and high pungency onions. The detection limit using 1 unit of pyruvate oxidase was 1-2 micromol/g FW. Optimum concentrations of co-factors TPP, FAD and MgSO4 comprising the enzyme cocktail were determined as being 0.04, 0.1 and 30 mM, respectively.

Generation of anti-human CD8 beta-specific antibodies using transfectants expressing mixed-species CD8 heterodimers.

The CD8 glycoprotein is a lymphocyte differentiation antigen comprised of two distinct polypeptide chains, alpha and beta, which have the capacity to form homodimeric (CD8 alpha/alpha) or heterodimeric (CD8 alpha/beta) cell surface complexes. The majority of monoclonal antibodies which recognize the human CD8 antigen react with the CD8 alpha chain, while a single mAb, referred to as T8/2T8-5H7 (or 2ST8-5H7), has been identified which binds to the CD8 alpha/beta heterodimer. In order to generate antibodies specific for CD8 beta, murine fibroblast transfectants were constructed which express the human CD8 beta chain in combination with either the human CD8 alpha chain or the murine CD8 alpha homologue, the Lyt-2 molecule. These transfectants were used to raise polyclonal heteroantisera in mice. Transfectants expressing human CD8 alpha/beta heterodimers induced moderate anti-CD8 alpha titers, but were weakly effective in generating anti-CD8 beta titers, despite high level cell surface expression of this protein. In contrast, transfectants expressing mixed-species CD8 heterodimers (murine CD8 alpha and human CD8 beta) induced high anti-CD8 beta titers in immunized mice. Following fusion of splenocytes from mice immunized with mixed-species CD8 transfectants, the mAb 5F2 was isolated which specifically recognizes the human CD8 beta chain. Unlike T8/2T8-5H7, the mAb 5F2 can bind the CD8 beta chain irrespective of its pairing partner, and can immunoprecipitate the CD8 beta protein from cells transfected with the CD8 beta gene in the absence of the human or mouse CD8 alpha gene product. Anti-CD8 beta antibodies should help elucidate the extent of biochemical heterogeneity of the CD8 beta protein, and will also be useful in defining the role of the CD8 beta protein in thymocyte and lymphocyte development, recognition and activation.

Cytotoxic and proliferative allospecific T-cell clones contain perforin and mediate anti-CD3-induced cytotoxicity.

Some in vitro-generated allospecific T-cell clones can kill target cells bearing specific antigen, whereas others can only proliferate in response to that antigen. The mechanism of target lysis by clones that exhibit antigen-specific cytotoxicity is thought to involve the exocytosis of lytic granules, which contain the pore-forming protein perforin. Here, CD4+, CD8+, and CD4-8- T-cell clones, positive for CD3 and the alpha/beta T-cell receptor, were tested for their ability to lyse the mouse-anti-human CD3 hybridoma OKT3; this hybridoma has been shown to trigger the cytolytic mechanism in cytotoxic T cells regardless of their clonal specificity. We found that all in vitro-generated allospecific T-cell clones can efficiently lyse the OKT3 targets whether or not they can kill alloantigen-bearing lymphoblastoid B-cell line targets. Furthermore, all tested clones contained perforin. The OKT3 hybridoma was not lysed by perforin-negative, CD3+ leukemic T-cell lines or by CD3- NK clones. Thus, the presence of perforin in T-cell clones correlated with their ability to lyse OKT3 targets, but not with their ability to lyse alloantigen-bearing targets. These results demonstrate that T-cell clones that are nonlytic when activated by specific antigen nevertheless contain a complete lytic mechanism and also support the proposed central role in perforin in that mechanism.

Detection of disease-specific PrP in the distal ileum of cattle exposed orally to the agent of bovine spongiform encephalopathy.

The immunohistochemical localisation of the disease-specific protein, PrP(Sc), was examined in the distal ileum of cattle up to 40 months after they had been exposed orally to the agent of bovine spongiform encephalopathy (BSE), in the intestines and mesenteric lymph nodes of an additional group of cattle, killed six months after a similar exposure, and in the distal ileum of naturally occurring clinical cases of BSE. PrP(Sc) was detected, mainly in macrophages, in a small proportion of the follicles of Peyer's patches in the distal ileum of the experimentally exposed cattle throughout much of the course of the disease. The observations are in agreement with the infectivity data derived from mouse bioassays of the distal ileum. At the later stages of the disease, the proportion of immunostained follicles increased as the total number of follicles decreased with age. In the additional experimental group of cattle, PrP(Sc) was confined to the Peyer's patches in the distal ileum. No immunostaining was detected in the lymphoid tissue of the distal ileum of naturally occurring clinical cases of BSE. In some of the clinically affected experimentally induced and naturally occurring cases of BSE there was sparse immunostaining of the neurons of the distal ileal myenteric plexus.

The monoclonal antibody, UCHL1, recognizes a 180,000 MW component of the human leucocyte-common antigen, CD45.

The leucocyte-common antigen (L-CA or CD45) is a family of high molecular weight glycoproteins, ranging from 180,000 to 220,000 MW that are expressed only on cells of lymphoid and myeloid origin. CD45 monoclonal antibodies (mAbs) recognize epitopes present on all polypeptides of the family, while other mAbs, termed CD45R, recognize determinants found only on the 220,000 MW and 200,000 MW polypeptides. In contrast the mAb UCHL1 recognizes a 180,000 MW antigen. UCHL1-coupled Sepharose beads were used to absorb antigen from lysates of cell lines. CD45 mAbs bound to this immobilized antigen. Antigen immobilized with CD45 mAb-coupled Sepharose beads bound UCHL1. Antigen purified by absorption and elution from the MOLT-4 cell line with CD45 mAb-coupled beads yielded molecules of 180,000 and 190,000 MW. Reprecipitation of the eluted antigen with UCHL1 resulted in a 180,000 MW band only. In a reciprocal experiment, CD45 mAb reprecipitated a 180,000 MW molecule from purified UCHL1 antigen. UCHL1 and the CD45R mAb 2H4 showed a mutually exclusive pattern of reactivity with human T- and B-cell lines, but co-expression of the antigens was seen on two myeloid and one erythroleukaemic cell line. In contrast, epitopes recognized by other putative CD45R mAbs were co-expressed with UCHL1 both on myeloid, erythroid and many T- and B-cell lines. We conclude that UCHL1 recognizes an epitope present only on the 180,000 MW polypeptide of CD45. Expression of this antigen is essentially reciprocal to the epitope detected by the CD45R mAb 2H4.

Use of representational difference analysis for the identification of mdm2 oncogene amplification in diethylstilbestrol-induced murine uterine adenocarcinomas.

Exposure in utero to the synthetic estrogen diethylstilbestrol (DES) is associated with the subsequent development of reproductive-tract malignancies in female offspring. To search for the genetic targets of DES, representational difference analysis was used to compare genomic DNA from DES-associated mouse uterine adenocarcinoma cells with genomic DNA from normal CD-1 mouse tissue. Several difference clones were obtained, all of which recognized rearranged and amplified sequences in tumor compared with normal DNA. One of these difference fragments mapped to a region of mouse chromosome 10 that includes the mdm2 oncogene. Amplification and overexpression of mdm2 was found in all three early-passage cell lines established from independent DES-associated cancers. These findings demonstrate the potential power of representational difference analysis in cancer research and suggest a genetic mechanism for DES-induced carcinogenesis.

1,25-dihydroxyvitamin D3 and development of tuberculosis in cattle.

This report describes the presence and activity of 1,25-dihydroxyvitamin D3 (1,25-D3) in experimental bovine tuberculosis. Animals that went on to develop tuberculous lesions exhibited a rapid transient increase in serum 1,25-D3 within the first 2 weeks following infection with Mycobacterium bovis. 1,25-D3-positive mononuclear cells were later identified in all tuberculous granulomas by immunohistochemical staining of postmortem lymph node tissue. These results suggest a role for 1,25-D3 both at the onset of infection and in the development of the granuloma in these infected animals. Using a monoclonal antibody to the vitamin D receptor (VDR) as a VDR agonist, we confirmed that activation of the vitamin D pathway profoundly depresses antigen-specific, but not mitogenic, bovine peripheral blood T-cell responses (proliferation and gamma interferon production). Investigation of the mechanism of this suppression showed that the VDR antibody modified the expression of CD80 by accessory cells, such that a significant positive correlation between T-cell proliferation and accessory cell CD80 emerged.

Immune response to murine cell lines of glial origin transplanted into the central nervous system of adult mice.

Temperature-sensitive simian virus 40 (SV40) T antigen-transformed central nervous system (CNS)-derived murine cell lines were used to analyse the host response to transplantation in the mouse adult brain. The cell lines were shown to be susceptible to immune recognition in vitro by cytotoxic effector cells indicating that tissue-specific privilege was not in operation. Histological examination at time points post-implantation showed characteristic responses similar to those seen during graft rejection. Astrocytosis and up-regulation of major histocompatibility complex (MHC) class I and MHC class II activation of resident microglia and recruitment of macrophages were observed in both allogeneic and syngeneic hosts 10 days post-implantation suggesting a trauma-induced response. However, the response in allogeneic hosts was more widespread and evident when the syngeneic responses had returned to normal levels. Evidence of T-cell infiltration was also more pronounced in the allogeneic hosts. Despite quite extensive host reactions to these cellular grafts at early time-points the implants appeared to survive in the host CNS long after the responses had abated and could be detected at the maximum time-point studied of 40 days.

Differential expression and regulation of the human CD8 alpha and CD8 beta chains.

The CD8 glycoprotein is expressed by thymocytes, mature T cells and natural killer (NK) cells and has been implicated in the recognition of monomorphic determinants on major histocompatibility complex (MHC) Class I antigens, and in signal transduction during the course of T-cell activation. Both human and rodent CD8 antigens are comprised of two distinct polypeptide chains, alpha and beta. The majority of monoclonal antibodies (mAb) reactive with the human CD8 antigen bind the CD8 alpha chain, while a single mAb, T8/2T8-5H7, has been identified which binds to the CD8 alpha/beta heterodimer. While the two chains of CD8 have been presumed to be coordinately expressed in normal T cells, this is not always the case. Northern blot analysis of a panel of T-cell leukemias and normal cells demonstrate that CD8 alpha and CD8 beta are not invariably co-transcribed and phenotypic analysis of fresh and interleukin 2 (IL-2) expanded peripheral blood mononuclear cells (PBMC) confirm that the CD8 alpha and CD8 beta chains are differentially expressed at the cell surface. Four distinct subpopulations of CD8+ cells have been identified based on the expression of CD8 alpha/alpha or CD8 alpha/beta complexes: (1) T-cell receptor (TcR) alpha beta+ T cells which are CD8 alpha+/beta+; (2) TcR alpha beta+ T cells which are CD8 alpha+/beta-; (3) TcR gamma delta+ T cells which are CD8 alpha+/beta- and (4) natural killer (NK) cells which are CD8 alpha+/beta-. We also demonstrate the down-regulation of the CD8 alpha/beta heterodimers from the surface of a CD8+ T-cell clone following treatment with phorbol myristate acetate (PMA) while CD8 alpha/alpha homodimers remain on the cell surface. This observation demonstrates that a) a CD8+ T-cell clone can express both CD8 alpha/alpha homodimers and CD8 alpha/beta heterodimers and b) these two complexes do not have identical biological properties. Together, these data suggest that CD8 alpha/alpha and CD8 alpha/beta dimers may not subserve identical functions. The differential contribution of these two CD8 complexes should be considered in models of T-cell-mediated cytotoxicity and T-cell activation.

Estrogen upregulation of BRCA1 expression with no effect on localization.

Alterations in the expression of the breast and ovarian cancer susceptibility gene BRCA1 may contribute to the development of mammary and ovarian neoplasia. The sex-steroid estrogen modulates cell proliferation of normal and neoplastic breast and ovarian epithelial cells, but the role of estrogen regulation on the expression of BRCA1 remains to be defined. In this study, estrogen-regulated BRCA1 expression was examined in breast and ovarian cancer cells. Estrogen stimulated the proliferation of estrogen receptor (ER)-positive breast MCF-7, C7-MCF-7, and ovarian BG-1 cells as well as the expression of the estrogen-inducible pS2 gene. This was concomitant with upregulation of BRCA1 mRNA (2.5- to 5.0-fold) and a 3- to 10-fold induction of BRCA1 protein (230 kDa). Cell fractionation studies localized the BRCA1 protein to the nucleus in both unstimulated and estrogen-stimulated cells. The antiestrogen ICI-182780 inhibited estrogen-induced cell proliferation, BRCA1 mRNA induction, and BRCA1 protein expression in ER-positive cells. Conversely, estrogen did not influence expression of BRCA1 in HBL-100 cells that lacked the estrogen receptor, although the constitutive levels of BRCA1 mRNA (but not protein) in these cells were 5- to 30-fold higher than in other breast and ovarian cancer cells. Secretion of the BRCA1 protein into the cell medium did not account for the discrepancy between the mRNA and protein levels in HBL-100 cells. Proliferation of HBL-100 cells was not affected by either estrogen or ICI-182780. Taken together, these data support a role for the steroid estrogen and the involvement of the estrogen receptor pathway in the modulation of expression of BRCA1. We therefore propose that stimulation of cell proliferation may be a prerequisite for upregulation of BRCA1 in breast and ovarian cancer cells.

Mutational analysis of the p21/WAF1/CIP1/SDI1 coding region in human tumor cell lines.

p21/WAF1/CIP1/SDI1 is an important cell-cycle mediator with tumor suppressor gene capabilities, and its inactivation could potentially lead to tumor progression. Because tumor suppressor genes are commonly inactivated by somatic and germline mutations, we analyzed a variety of human tumor cell lines for p21 mutations. We used single-strand conformational analysis and direct sequencing to identify possible mutations in the p21 coding region. Two base-alterations were observed in 41 immortalized human tumor cell lines. A previously reported polymorphism that results in a serine-to-arginine amino-acid substitution at codon 31 was found in 24% (10 of 41) of the tumor cell lines but was also found in 10% (six of 62) of normal parental DNAs tested and 7% (three of 43) of normal DNAs from patients with primary endometrial tumors. Another nucleotide substitution found at codon 80 resulted in the replacement of threonine with methionine. Codon 80 changes were found in 7% (three of 41) of the tumor cell lines (all endometrial) and in 2% (one of 62) of the normal parental DNAs. This change was not found in any of the primary endometrial tumors examined. The biological activity of these base changes was analyzed by using in vitro cyclin-dependent kinase 2-cyclin A kinase assays and calcium phosphate transfections. We observed that wild-type p21 and the p21 variants had similar growth-inhibitory abilities. Thus, our results suggest that mutation of the p21 gene is not prevalent in human tumor cell lines and is not a probable mechanism of inactivation of this gene.