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Biomedical subjects

A J Fleming

Publications and source records attributed to A J Fleming.

15 recordsLinked to original sources
PubMed2003

Viability of hydroxyethyl methacrylate-methyl methacrylate-microencapsulated PC12 cells after omental pouch implantation within agarose gels.

Hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA, 75 mol% HEMA). Microcapsules containing viable PC12 cells (as an allogeneic transplant model) were implanted into omental pouches in Wistar rats. Two different capsule preparations were tested, based on differences in polymer solutions during extrusion: 10% HEMA-MMA in TEG, and 9% HEMA-MMA in TEG with 30% poly(vinyl pyrrolidone) (PVP). The omental pouch proved to be an ideal transplant site in terms of implantation, recovery, and blood vessel proximity (nutrient supply). To minimize the fibrous overgrowth and damaged capsules previously seen on implantation of individual capsules, agarose gels were used to embed the capsules before implantation. Cells proliferated within the microcapsule-agarose device during the first 7 days of implantation, but overall cell viability declined over the 3-week period, when compared with similar capsules maintained in vitro. Nonetheless, approximately 50% of the initial encapsulated cells were still viable after 3 weeks in vivo. This approach to HEMA-MMA microcapsule implantation improved cell viability and capsule integrity after 3 weeks in vivo, compared with capsules implanted without agarose.

Animals↗
PubMed2001

Sucrose synthase expression pattern in young maize leaves: implications for phloem transport.

To study whether sucrose synthase (SuSy, EC 2.4.1.13) plays a role in phloem loading/unloading, we have directly assessed the distribution of SuSy transcripts and protein in young maize (Zea mays L.) leaves. Our data show that at the RNA and protein levels there is no expression of SuSy in phloem of sink tissue and that SuSy expression is specifically excluded from this tissue. These data, in conjunction with others in the literature, indicate that the localisation of SuSy to phloem may be limited to source tissue, whereas a non-vascular expression is linked with sink tissue activity.

Biological Transport↗
PubMed2001

Novel marker genes for early leaf development indicate spatial regulation of carbohydrate metabolism within the apical meristem.

To identify genes expressed at the earliest stages of leaf development, we have performed a differential display analysis using portions of meristems destined to form leaves. Our analysis led to the identification of five genes showing an asymmetric pattern of gene expression within the meristem associated with leaf formation. Surprisingly, three of these genes encoded enzymes involved in carbohydrate metabolism (ADPglucose pyrophosphorylase, sucrose synthase and an SNF1-like kinase). Furthermore, specific transcript patterns were responsive to specific sugar and hormonal treatments. The other two genes identified encoded a Phantastica-like myb transcription factor (associated with the acquisition of leaf dorsiventrality) and CYP85 (a cytochrome P450, which plays a pivotal role in brassinolide metabolism). These data, firstly, identify a novel set of marker genes for the analysis of the earliest stages of leaf formation. Secondly, the function of the proteins encoded by these genes and their expression patterns within the meristem indicate that carbohydrate metabolism is spatially regulated within a tissue involved in key developmental processes. Finally, our data provide the first indication of an asymmetry in gene expression related to hormone biosynthesis in the meristem.

Amino Acid Sequence↗
PubMed2000

Activation of neurokinin NK(2) receptors by tachykinin peptides causes contraction of uterus in pregnant women near term.

The aim of this study was firstly to elucidate whether the mammalian tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)-regulated contractility of myometrium obtained from near-term pregnant women, and secondly to investigate the receptor subtype(s) responsible. In the presence of peptidase inhibitors, i.e. thiorphan (3 micromol/l; endopeptidase 24.11 inhibitor), captopril (10 micromol/l; angiotensin converting enzyme inhibitor) and bestatin (10 micromol/l; aminopeptidase inhibitor); all three mammalian tachykinins elicited concentration-related contractions of isolated myometrial preparations. The rank order of agonist potency of the mammalian tachykinins in the presence of the peptidase inhibitors was NKA > SP = NKB, indicating that the contractile effects were mediated by activation of an NK(2) receptor. The NK(2) receptor-selective agonist, [Lys(5), MeLeu(9), Nle(10)]NKA(4-10), produced concentration-related contractile responses, while the respective NK(1) and NK(3) receptor-selective agonists, [Sar(9), Met(O(2))(11)]SP and [N-MePhe(7)]NKB, had no effect either in the absence or presence of the peptidase inhibitors. The NK(2) receptor-selective antagonist, SR48968, produced concentration-related rightward shift in the log concentration curve to [Lys(5), MeLeu(9), Nle(10)]NKA(4-10). This study shows that tachykinins elicit contractile effects on human myometrium obtained from pregnant women near term, and that these effects are mediated by an NK(2) receptor. An excitatory effect of the tachykinins on these preparations could indicate a physiological role for these peptides in enhancing contractility of the uterus in women at term.

Adult↗
PubMed2000

Fc-gamma receptor cross-linking by immune complexes induces matrix metalloproteinase-1 in U937 cells via mitogen-activated protein kinase.

Matrix metalloproteinase-1 (MMP-1) secreted by macrophages potentially contributes to plaque rupture. Because large quantities of immunoglobulin G and ICs (ICs), including low density lipoprotein-containing ICs (LDL-ICs), are present in atherosclerotic lesions, we examined the effect of LDL-ICs on macrophage MMP-1 expression. With the use of ICs prepared with human LDL and rabbit anti-LDL antiserum, our enzyme-linked immunosorbent assays and Northern blots showed that MMP-1 secretion and expression by U937 histiocytes were induced by LDL-ICs. Furthermore, our results showed that blocking of Fc-gamma receptor I and II (FcgammaRI and FcgammaRII) inhibited 70% and 55%, respectively, of the LDL-IC-induced secretion of MMP-1. Finally, our data showed that both PD98059, an inhibitor of the mitogen-activated protein kinase pathway, and Ro-31-2880, an inhibitor of protein kinase C, inhibited LDL-IC-stimulated MMP-1 secretion in a dose-dependent manner, with 96% and 95% inhibition, respectively, at the respective doses of 50 micromol/L and 80 nmol/L. In conclusion, this study demonstrated for the first time that LDL-ICs induce macrophage MMP-1 secretion by cocross-linking FcgammaRI and FcgammaRII and triggering a protein kinase C-dependent mitogen-activated protein kinase pathway. These results suggest that LDL-ICs and other ICs localized in atherosclerotic plaques may be potent stimulators for macrophage MMP-1 expression and may contribute to plaque rupture and acute coronary events.

Antibodies, Anti-Idiotypic↗
PubMed1996

Hormonal and environmental regulation of a plant PDR5-like ABC transporter.

The PDR5 gene from yeast encodes an ABC (ATP-binding cassette) transporter involved in the ATP-dependent efflux of a variety of structurally unrelated cytotoxic compounds. We report here on the cDNA cloning and characterization of a PDR5 homolog (TUR2) from a higher eukaryote, the aquatic plant Spirodela polyrrhiza. We show that TUR2 transcripts accumulate throughout the plant following treatment with the steroid-like hormone, abscisic acid, and that this induction can be repressed by the adenine-derived hormone, kinetin. Furthermore, TUR2 gene expression is induced by environmental stress treatments such as low temperature and high salt. These data indicate that PDR5 homologs are present in plants, that they may function during stress conditions in an analogous fashion to that described in yeast, and that the expression of such ABC transporters is subject to a complex hormonal and environmental regulation.

ATP-Binding Cassette Transporters↗
PubMed1996

Fluorescent imaging of GUS activity and RT-PCR analysis of gene expression in the shoot apical meristem.

The use of promoter-reporter gene constructs in transgenic plants is a powerful tool in the analysis of gene expression which can, however, be limited in the resolution of small structures, such as the apical meristem. This paper reports on a fluorescent imaging technique for the analysis of GUS reporter gene expression to cellular resolution in the apical meristem of tomato. Using this technique in combination with an RT-PCR analysis of RBS gene-specific transcript levels, it is shown that: 5' upstream sequences of RBCS genes are sufficient to mimic the pattern of transcripts revealed by in situ hybridisation (no expression in the apical meristem, high expression in the initiated leaf primordia); the genes RBCS2, RBCS3A and RBCS3B are transcriptionally activated upon primordium initiation with transcripts for RBCS1 and RBCS3C accumulating later in leaf development; and that RBCS promoter activity cannot be induced in the apical meristem by light, an environmental signal which elevates RBCS transcript level in other aerial parts of the plant. These data provide a detailed picture of the complex transcriptional events occurring on leaf initiation and the establishment of the photosynthetic machinery; they describe two complementary techniques which allow the analysis of such complex events at the tissue and cellular level; and they characterize an in vivo assay system which can be used to analyse the factors involved in the initiation and maintenance of gene expression patterns in the apical meristem.

Gene Expression Regulation, Plant↗
PubMed1996

Tissue-specific expression and promoter analysis of the tobacco Itp1 gene.

The Nicotiana tabacum Itp1 gene (Ntltp1) encodes a small basic protein that belongs to a class of putative lipid transfer proteins. These proteins transfer lipids between membranes in vitro, but their in vivo function remains hotly debated. This gene also serves as an important early marker for epidermis differentiation. We report here the analysis of the spatial and developmental activity of the Ntltp1 promoter, and we define a sequence element required for epidermis-specific expression. Transgenic plants were created containing 1346 bp of the Ntltp1 promoter fused upstream of the beta-glucuronidase (GUS) gene. In the mature aerial tissues, GUS activity was detected predominantly in the epidermis, whereas in younger aerial tissues, such as the shoot apical meristem and floral meristem, GUS expression was not restricted to the tunica layer. Unexpectedly, GUS activity was also detected in young roots particularly in the root epidermis. Furthermore, the Ntltp1 promoter displayed a tissue and developmental specific pattern of activity during germination. These results suggest that the Ntltp1 gene is highly expressed in regions of the plant that are vulnerable to pathogen attack and are thus consistent with the proposed function of lipid transfer proteins in plant defense. Deletions of the promoter from its 5' end revealed that the 148 bp preceding the translational start site are sufficient for epidermis-specific expression. Sequence comparison identified an eight-nucleotide palindromic sequence CTAGCTAG in the leader of Ntltp1, which is conserved in a number of other Itp genes. By gel retardation analysis, the presence of specific DNA-protein complexes in this region was demonstrated. The characterization of these factors may lead to the identification of factors that control early events in epidermis differentiation.

Base Sequence↗
PubMed1995

Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model.

The NeIF-4A10 gene belongs to a family of at least ten genes, all of which encode closely related isoforms of translation initiation factor 4A. The promoter region of NeIF-4A10 was sequenced, and four mRNA 5' ends were determined. Deletions containing 2750, 689 and 188 bp of untranscribed upstream DNA were fused to the GUS reporter gene and introduced into transgenic tobacco. The three constructs mediated GUS expression in all cells of the leaf, stem and shoot apical meristem. Control experiments using in situ hybridization and tissue printing indicated that the observed GUS expression matches the expression patterns of NeIF-4A mRNA and protein. This detailed analysis at the level of mRNA, protein and reporter gene expression shows that NeIF-4A10 is an ideal constitutively expressed control gene. We argue that inclusion of such a control gene in experiments dealing with specifically expressed genes is in many cases essential for the correct interpretation of observed expression patterns.

Base Sequence↗
PubMed1995

Organ-specific differential regulation of a promoter subfamily for the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit genes in tomato.

The tomato (Lycopersicon esculentum) gene family for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCS) has been investigated to determine the role of promoter regions and DNA-protein interactions in the differential organ-specific transcription of individual genes. Transgenic plants expressing RBCS-promoter-beta-glucuronidase fusion genes have confirmed that promoter fragments ranging from 0.6 to 3.0 kb of the RBCS1, RBCS2, and RBCS3A genes were sufficient to confer the temporal, organ-specific, and differential expression pattern observed for the endogenous genes. The individual temporal and organ-specific beta-glucuronidase enzyme activities closely reflect the qualitative and quantitative transcription activities of the respective RBCS genes, including the strongly reduced activity of RBCS3A (L.A. Wanner, W. Gruissem [1991] Plant Cell 3: 1289-1303). In particular, tissue-specific activity of all three promoters is similar in developing fruit, with high activity in the locular tissue and extremely reduced activity in the pericarp. This specific pattern of gene activity was further substantiated by in situ analysis of RBCS mRNA levels. Together, the data suggest an interesting correlation between RBCS gene activity and sink strength in different fruit tissues. DNA-protein interaction studies have revealed a novel fruit-specific DNA-binding protein called FBF that specifically interacts with a sequence element directly upstream of the G-box in the RBCS3A promoter. FBF binding thus correlates with the reduced activity of this promoter in developing tomato fruit, rendering it a candidate for a fruit-specific negative regulator of transcription in tomato.

Base Sequence↗
PubMed1993

A plant gene with homology to D-myo-inositol-3-phosphate synthase is rapidly and spatially up-regulated during an abscisic-acid-induced morphogenic response in Spirodela polyrrhiza.

The induction of turions (dormant vegetative buds) by the plant growth regulator abscisic acid (ABA) in the aquatic angiosperm Spirodela polyrrhiza is one of the few examples of a plant hormone triggering a morphogenic response. In order to aid our understanding of the molecular basis of this phenomenon a cDNA library has been prepared from ABA-induced S. polyrrhiza and by differential screening a number of early ABA-up-regulated genes have been isolated. This paper reports the characterization of one such cDNA (tur1) from S. polyrrhiza which codes for a protein highly homologous to yeast D-myo-inositol-3-phosphate synthase (EC.5.5.1.4), the catalyst for the first committed step in inositol biosynthesis. This is the first reported cloning of a plant gene involved in inositol production. It is shown by Northern analysis and in situ hybridization that ABA induces a rapid preferential up-regulation of this gene and that this is localized to the stolon tissue. The stolon connects the developing turion to the node of the mother frond (the meristematic region involved in the production of turions which arise as a morphogenic response to this hormone). This increase in tur1 transcript level is accompanied by a rise in the activity of D-myo-inositol-3-phosphate synthase. The possible role of inositol in ABA-induced events during turion formation is discussed.

Abscisic Acid↗
PubMed1993

The patterns of gene expression in the tomato shoot apical meristem.

In this paper, we describe the synthesis of a cDNA library from the vegetative shoot apical meristem and the analysis of clones selected from it. Using in situ hybridization, we characterized the patterns of expression of these genes in the tomato shoot apical meristem, as well as the patterns obtained from other sources. The results from the analysis of 15 cDNAs indicated the following six main patterns of gene expression in the shoot apical meristem: overall expression, zero expression, expression limited to the epidermis, expression excluded from the epidermis, punctate expression, and expression elevated in the flanks of the meristem. The patterns observed and the nature and number of the genes showing these patterns necessitate a reinterpretation of the models of meristem structure and function. In particular, the data suggest a compartmentation within the shoot apical meristem based on the spatial expression of particular subsets of genes. This paper also reports on the specific and precise criteria essential for the correct identification of meristem-specific gene expression. The data give new insight into the molecular organization of the shoot apical meristem and provide the framework for a detailed dissection of the factors controlling this organization.

Amino Acid Sequence↗
PubMed1992

Expression pattern of a tobacco lipid transfer protein gene within the shoot apex.

A genomic clone coding for a putative lipid transfer protein was isolated from Nicotiana tabacum. The gene is expressed in all aerial organs of the tobacco plant, with expression being highest in the upper part of the plant and declining towards the base (i.e. with increasing age). In the shoot apex transcript levels are highest in the LI layer, lower in the underlying cell layers, and are again higher in the submeristem region. This pattern is markedly different from those previously described for genes expressed within the shoot apex.

Amino Acid Sequence↗