Human growth hormone presented by K14hGH-transgenic skin grafts induces a strong immune response but no graft rejection

Although immune responses leading to rejection of transplantable tumours have been well studied, requirements for epithelial tumour rejection are unclear. Here, we use human growth hormone (hGH) expressed in epithelial cells (skin keratinocytes) as a model neo-self antigen to investigate the consequences of antigen presentation from epithelial cells. Mice transgenic for hGH driven from the keratin 14 promoter express hGH in skin keratinocytes. This hGH-transgenic skin is not rejected by syngeneic non-transgenic recipients, although an antibody response to hGH develops in grafted animals. Systemic immunization of graft recipients with hGH peptides, or local administration of stimulatory anti-CD40 antibody, induces temporary macroscopic graft inflammation, and an obvious dermal infiltrate of inflammatory cells, but not graft rejection. These results suggest that a neo-self antigen expressed in somatic cells in skin can induce an immune response that can be enhanced further by induction of specific immunity systemically or non-specific immunity locally. However, immune responses do not always lead to rejection, despite induction of local inflammatory changes. Therefore, in vitro immune responses and in vivo delayed type hypersensitivity are not surrogate markers for immune responses effective against epithelial cells expressing neoantigens.

GLUT4 overexpression or deficiency in adipocytes of transgenic mice alters the composition of GLUT4 vesicles and the subcellular localization of GLUT4 and insulin-responsive aminopeptidase

The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2GLUT4- Tg) mice were studied. GLUT4 amount was reduced by 80 - 95% in aP2-GLUT4-/- adipocytes and increased similar to10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase ( IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2GLUT4- Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.

Transgenic mice that express Cre recombinase in osteoclasts

To study the physiological control of osteoclasts, the bone resorbing cells, we generated transgenic mice carrying the Cre recombinase gene driven by either the tartrate-resistant acid phosphatase (TRAP) or cathepsin K (Ctsk) promoters. TRAP-Cre and Ctsk-Cre transgenic mouse lines were characterized by breeding with LacZ ROSA 26 (R26R) reporter mice and immunohistochemistry for Cre recombinase. The Cre transgene was functional in all lines, with Cre-mediated recombination occurring primarily in the long bones, vertebrae, ribs, and calvaria. Histological analyses of the bones demonstrated that functional Cre protein was present in 1) osteoclasts (Ctsk-Cre); 2) osteoclasts, columnar proliferating, and hypertrophic chondrocytes (TRAP-Cre line 4); and 3) round proliferating chondrocytes (TRAP-Cre line 3). In conclusion, we generated transgenic mouse lines that will enable the deletion of floxed target genes in osteoclasts, which will be valuable tools for studying the regulation of osteoclast function. (C) 2004 Wiley-Liss, Inc.

Influence of growth hormone on the craniofacial complex of transgenic mice

Growth hormone (GH) secretion affects bone and cartilage physiology. This study investigated the effect of GH on the size of the craniofacial structures and their angular relationship. Three different models of mice with a genetically altered GH axis were used: GH excess (giant), dwarf GH antagonist (dwarf-Ant), and dwarf GH receptor knockout (dwarf-KO) mice. Each model was compared with the corresponding wild type (Wt). Five craniofacial distances were analysed: craniofacial length, upper face height, mandibular anterior height, mandibular ramus length, and mandibular corpus length. In addition, upper and lower incisor lengths and four angular relationships, nasal bone with cranial base, maxillary plane with cranial base, mandibular plane with cranial base, and the angle of the mandible, were determined. Data were analysed by one-way ANOVA. Craniofacial length, upper face height and mandibular corpus length were significantly increased in the giant mice and significantly reduced in the dwarf mice. Mandibular anterior height and mandibular ramus length were significantly affected in the dwarf-KO mice but not in the giant mice. The length of both the upper and lower incisors was significantly increased and reduced in the giant and dwarf-KO mice, respectively. In addition, the angle of the mandible was significantly increased in the giant mice and significantly reduced in the dwarf mice. It is concluded that GH plays a major role in the growth and development of the craniofacial complex by directly and indirectly modulating the size and the angular relationships of the craniofacial structures, including the incisor teeth.

Do squash players accurately report use of appropriate protective eyewear?

Self-report surveys are a common method of collecting data on protective equipment use in sport. The aim of this study was to assess the validity of self-reported use of appropriate protective eyewear by squash players. Surveys of squash players' appropriate protective eyewear behaviours were conducted over two consecutive years (2002 and 2003) at randomly-selected squash venues in Melbourne, Australia. Over the two years, 1219 adult players were surveyed (response rate of 92%). Trained observers also recorded the actual on-court appropriate protective eyewear behaviours of all players during the survey sessions. Eyewear use rates calculated from both data sources were compared. The self-reported appropriate protective eyewear use rate (9.4%; 95% CI 7.8, 11.0) was significantly higher (1.6 times more) than the observed rate (5.9%; 95%CI 4.6, 7.2). This suggests that players may over-report their use of appropriate protective equipment, though some may have incorrectly classified their eyewear as being appropriate or suitably protective. Studies that rely only on self-report data on protective equipment use need to take into account that this could lead to biased estimates.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

The effectiveness of a squash eyewear promotion strategy

Objective: To evaluate the protective eyewear promotion ( PEP) project, which was a comprehensive educational strategy to increase the use of appropriate protective eyewear by squash players. Methods: An ecological study design was used. Four squash venues in one playing association were randomly chosen to receive PEP and four in another association maintained usual practice and hence formed a control group. The primary evaluation measurements were surveys of cross sectional samples of players carried out before and after the intervention. The surveys investigated players' knowledge, behaviours, and attitudes associated with the use of protective eyewear. The survey carried out after the intervention also determined players' exposure to PEP. Univariate and multivariate analyses were undertaken to describe differences at PEP venues from pre- to post-intervention and to compare these with the control venues. Results: The PEP players had 2.4 times the odds (95% confidence interval, 1.3 to 4.2) of wearing appropriate eyewear compared with control group players post-intervention, relative to the groups' preintervention baselines. Components of PEP, such as stickers and posters and the availability and prominent positioning of the project eyewear, were found to contribute to players adopting favourable eyewear behaviours. Conclusions: Components of the PEP intervention were shown to be effective. The true success will be the sustainability and dissemination of the project, favourable eyewear behaviours, and evidence of the prevention of eye injuries long into the future.

Unprotected eyes in squash: not seeing the risk of injury

The use of appropriate eyewear in squash can protect the eyes against injury. However, few adult squash players adequately protect their eyes against potential severe injuries. We describe the characteristics of non-users of protective eyewear and examine predictors of appropriate eyewear use. Self-report surveys of adult players were conducted in metropolitan Melbourne, Australia. Information on players' knowledge, behaviours and attitudes associated with protective eyewear use was collected, in addition to player demographic data. Appropriate eyewear was defined as Standards-approved polycarbonate lens eyewear. The majority 92.2% of players did not adequately protect their eyes while playing squash. Significant predictors of reported eyewear use were: previous eye injury; playing squash on average more than 2 hr per wk; having played for more than 20 y; and having more favourable attitudes towards eye safety in squash. The significant predictors of appropriate eyewear use were: being female; previous eye injury; playing squash on average more than 2 hr per week; and having more favourable attitudes towards eye safety in squash. Understanding the characteristics of both users and non-users of appropriate eye protection in squash is essential for informing future prevention strategies.

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Analysis of distinct tartrate-resistant acid phosphatase promoter regions in transgenic mice

The tartrate-resistant acid phosphatase (TRAP) is present in multiple tissues, including kidney, liver, lung, spleen, and bone. Recent study of (TRAP) gene expression has provided evidence for distinct promoters within the (TRAP) gene, suggesting that the gene has alternative, tissue-preferred mRNA transcripts. Examination of endogenous (TRAP) exon 1B and 1C mRNA transcripts revealed tissue-preferred transcript abundance with increased exon 1B transcripts detected in liver and kidney and increased exon 1C transcripts detected in bone and spleen. In this investigation, we have made transgenic mice that express a marker gene driven by two candidate promoters, designated BC and C, within the (TRAP) gene. The BC and C promoters are 2.2 and 1.6 kb, respectively, measured from the translation initiation site. Evaluation of BC transgenic lines demonstrated robust expression in multiple tissues. In contrast, significant transgene expression was not detected in C transgenic lines. Evaluation of transgene mRNAs in BC transgenic lines revealed that virtually all expression was in the form of B transcripts, suggesting that the tissue-preferred pattern of endogenous (TRAP) was not replicated in the BC transgenic line. Likewise, osteoclastogenic cultures from BC, but not C, transgenic bone marrow cells expressed the transgene following receptor activator of NFkappaB ligand/macrophage colony-stimulating factor stimulation. In conclusion, when compared with the 2.2-kb BC portion of the (TRAP) promoter region, the 1.6-kb C portion does not account for significant gene expression in vivo or in vitro; production of the bone- and spleen-preferred (TRAP) C transcript must depend on regulatory elements outside of the 2.2-kb promoter. As the majority of currently investigated transcription factors that influence transcriptional regulation of osteoclast gene expression bind within the 1.6-kb C portion of the (TRAP) promoter, it is likely that transcription binding sites outside of the 2.2-kb region will have profound effects on regulation of the gene in vivo and in vitro.

Possible incompatibility between transgenic cottons and parasitoids

We investigated the rates of egg and larval parasitism in transgenic and non-transgenic, conventional cottons. Sentinel eggs and larvae of the cotton bollworm, CBW, Helicoverpa armigera Hubner, were released and collected at regular intervals across the cotton growing season, and the relationship between parasitism and different cotton cultivars determined. Egg and larval parasitism were significantly lower in the transgenic cottons than in the non-transgenic conventional cottons. The egg parasitoid recovered was Trichogramma confusum Viggiani and the predominant larval parasitoids were Campoletis chlorideae Uchida and Meteorus pulchriconis (Wesmael). Our studies indicate a potential negative interaction between transgenic cottons and parasitoids of CBW but need to be interpreted with caution because no within-year replication was used and treatments were not spatially randomised across years.

Fate of hairpin transcript components during RNA silencing and its suppression in transgenic virus-resistant tobacco

Transgenic tobacco plants, carrying a Potato virus Y (PVY)-NIa hairpin sequence separated by a unique unrelated spacer sequence were specifically silenced and highly resistant to PVY infection. In such plants neither PVY-NIa nor spacer transgene transcripts were detectable by specific quantitative real time reverse transcriptase PCR (RT-qPCR) assays of similar relative efficiencies developed for direct comparative analysis. However, small interfering RNAs (siRNAs) specific for the PVY sequence of the transgene and none specific for the LNYV spacer sequence were detected. Following infection with Cucumber mosaic virus (CMV), which suppresses dsRNA-induced RNA silencing, transcript levels of PVY-NIa as well as spacer sequence increased manifold with the same time course. The cellular abundance of the single-stranded (ss) spacer sequence was consistently higher than that of PVY dsRNA in all cases. The results show that during RNA silencing and its suppression of a hairpin transcript in transgenic tobacco, the ssRNA spacer sequence is affected differently than the dsRNA. In PVY-silenced plants. the spacer is efficiently degraded by a mechanism not involving the accumulation of siRNAs, while following suppression of RNA silencing by CMV, the spacer appears protected from degradation. Crown Copyright (c) 2006 Published by Elsevier B.V. All rights reserved.

Strategies to obtain stable transgenic plants from non-embryogenic lines: complementation of the nn(1) mutation of the NORK gene in Medicago sativa MN1008

Strategies to introduce genes into non-embryogenic plants for complementation of a mutation are described and tested on tetraploid alfalfa (Medicago sativa). Genes conditioning embryogenic potential, a mutant phenotype, and a gene to complement the mutation can be combined using several different crossing and selection steps. In the successful strategy used here, the M. sativa genotype MnNC-1008(NN) carrying the recessive non-nodulating mutant allele nn(1) was crossed with the highly embryogenic alfalfa line Regen S and embryogenic hybrid individuals were identified from the F1 progeny. After transformation of these hybrids with the wild-type gene (NORK), an F2 generation segregating for the mutation and transgene were produced. Plants homozygous for the mutant allele and carrying the wild-type NORK transgene could form root nodules after inoculation with Sinorhizobium meliloti demonstrating successful complementation of the nn(1) mutation.