Biochemical responses of the ethylene-insensitive Never ripe tomato mutant subjected to cadmium and sodium stresses

In order to further address the known interaction between ethylene and components of the oxidative system, we have used the ethylene-insensitive Never ripe (Nr) tomato (Solanum lycopersicum L) mutant, which blocks ethylene responses. The mutant was compared to the control Micro-Tom (MT) cultivar subjected to two stressful situations: 100 mM NaCl and 0.5 mM CdCl(2). Leaf chlorophyll, lipid peroxidation and antioxidant enzyme activities in roots, leaves and fruits, and Na and Cd accumulation in tissues were determined. Although we verified a similar growth pattern and Na and Cd accumulation for MT and Nr, the mutant exhibited reduced leaf chlorophyll degradation following stress. In roots and leaves, the patterns of catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX), superoxide dismutase (SOD) enzyme activity as well as malondialdehyde (MDA) and hydrogen peroxide (H(2)O(2)) production under the stressful conditions tested were very similar between MT and Nr mutant. However, Nr fruits showed increased H(2)O(2) production, reduced and enhanced APX activity in NaCl and CdCl(2), respectively, and enhanced GPOX in NaCl. Moreover, through non-denaturing PAGE, a similar reduction of SOD I band intensity in both, control MT and Nr mutant, treated with NaCl was observed. In leaves and fruits, a similar SOD activity pattern was observed for all periods, genotypes and treatments. Overall the results indicate that the ethylene signaling associated with NR receptor can modulate the biochemical pathways of oxidative stress in a tissue dependent manner, and that this signaling may be different following Na and Cd exposure. (C) 2011 Elsevier B.V. All rights reserved.

Enhanced transpiration rate in the high pigment 1 tomato mutant and its physiological significance

Tomato high pigment (hp) mutants represent an interesting horticultural resource due to their enhanced accumulation of carotenoids, flavonoids and vitamin C. Since hp mutants are known for their exaggerated light responses, the molecules accumulated are likely to be antioxidants, recruited to deal with light and others stresses. Further phenotypes displayed by hp mutations are reduced growth and an apparent disturbance in water loss. Here, we examined the impact of the hp1 mutation and its near isogenic line cv Micro-Tom (MT) on stomatal conductance (gs), transpiration (E), CO(2) assimilation (A) and water use efficiency (WUE). Detached hp1 leaves lost water more rapidly than control leaves, but this behaviour was reversed by exogenous abscisic acid (ABA), indicating the ability of hp1 to respond to this hormone. Although attached hp1 leaves had enhanced gs, E and A compared to control leaves, genotypic differences were lost when water was withheld. Both instantaneous leaf-level WUE and long-term whole plant WUE did not differ between hp1 and MT. Our results indicate a link between exaggerated light response and water loss in hp1, which has important implications for the use of this mutant in both basic and horticultural research.

Increased class A scavenger receptor and glomerular lipid precede mesangial matrix expansion in the bGH mouse model

Objective: Elevated neutral lipid content and mRNA expression of class A scavenger receptor (SRA) have been found in the renal cortex of the bovine growth hormone (bGH) mouse model of progressive glomerulosclerosis (GS). We hypothesize that the increased expression of SRA precedes glomerular scarring in this model. Design: Real time RT-PCR and immunofluorescence were employed to measure SRA and collagen types I and IV in the bGH transgenic and control mice at 5 and 12 weeks (wk) of age to determine the chronology of change in SRA expression in relation to glomerular scarring. Alternative mechanisms for increasing glomerular lipid were assessed by measuring mRNA expression levels of low-density lipoprotein receptor (LDL-r), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), and ATP-binding cassette transporter A1 (ABCA1). In addition, the involvement of macrophages in early GS was assessed by CD68 mRNA expression in kidney cortex. Results: Both mRNA and protein levels of SRA were significantly increased in 5-wk bGH compared with control mice, whereas the expression of collagen I and IV was unaltered. Unchanged levels of LDL-r and HMGR mRNA indicate that neither regulated cholesterol uptake via LDL-r nor the cholesterol synthetic pathway played a role in the early lipid increase. The finding of increased ABCA1 expression was an indicator of excess intracellular lipid in the renal cortex of bGH mice at 5 wk. CD68 expression in bGH did not differ significantly from that of controls at 5 wk suggesting that cortical macrophage infiltration was not increased in bGH mice at this time point. Conclusion: An early increase in SRA mRNA and protein expression in the bGH kidney precedes glomerular scarring and is independent of macrophage influx. Published by Elsevier Ltd. on behalf of Growth Hormone Research Society.

Photoprotective effect of Pothomorphe umbellata on UVB radiation-induced biomarkers involved in carcinogenesis of hairless mouse epidermis

In this study we assessed the protective effect of topical application of Pothomorphe umbellata extract on ultraviolet B (UVB)-induced skin lesion parameters in hairless mouse epidermis. A single dose of UVB irradiation (0.23 kJ/m(2)) resulted in a significant decrease in thymine dimer-positive cells and apoptotic sunburn cells, with an increase in p53 and proliferating cell nuclear antigen-positive cells in the epidermis. After 5 weeks (total dose 13.17 kJ/m(2)) and 15 weeks (total dose 55.51 kJ/m(2)) of irradiation, P. umbellata treatment inhibited the hyperplasic response and induced an increase in p53-positive cells. These findings suggest that P. umbellata extract affords protection against UVB-induced skin lesions.

Transformation of external sensilla to chordotonal sensilla in the cut mutant of Drosophila assessed by single-cell marking in the embryo and larva

The cut gene of Drosophila melanogaster is an identity selector gene that establishes the program of development and differentiation of external sense organs. Mutations in the cut gene cause a transformation of the external sense organs into chordotonal organs, originally assessed by the use of immunostaining methods [Bodmer et al. (1987): Cell, 51:293-307]. Because of evidence that axonal projections of the transformed neurons within the central nervous system are not completely switched in cut mutants, the transformation of the four cells making up a sense organ was reassessed using single-cell staining with fluorescent dye and differential interface contrast (DIC) microscopy of the embryo and larva. The results provide strong evidence that all cells of the sense organs are completely transformed, exhibiting the morphologies and organelles characteristic of chordotonal sense organs. A comparison of the structures of external sense organs and chordotonal organs indicates that a number of the differences could be due to the degree of development of common structures, and that cut or downstream genes modulate effector genes that are normally utilized in both receptor types. The possible derivation of insect chordotonal and external sense organs from a receptor type found in crustaceans is discussed in the light of arthropod phylogenetics and the molecular genetics of sense organ development. (C) 1997 Wiley-Liss, Inc.

Th2-type CD4(+) cells neither enhance nor suppress antitumor CTL activity in a mouse tumor model

Many cervical cancers express the E7 protein of human papillomavirus 16 as a tumor-specific Ag (TSA). To establish the role of E7-specific T cell help in CD8(+) CTL-mediated tumor regression, C57BL/6J mice were immunized with E7 protein or with a peptide (GF001) comprising a minimal CTL epitope of E7, together with different adjuvants, Immunized mice were challenged with an E7-expressing tumor cell line, EL4.E7. Growth of EL4.E7 was reduced following immunization with E7 and Quil-A (an adjuvant that induced a Th1-type response to E7) or with GF001 and Quil-A, Depletion of CD8(+) cells, but not CD4(+) cells, from an immunized animal abrogated protection, confirming that E7-specific CTL are necessary and sufficient for TSA-specific protection in this model. Immunization with E7 and Algammulin (an alum-based adjuvant) induced a Th2-like response and provided; no tumor protection. To investigate whether a Th2 T helper response to E7 could prevent the development of an E7-specific CTL-mediated protection, mice were simultaneously immunized with E7/Algammulin and GF001/Quil-A or, alternatively, were immunized with GF011/Quil-A 8 wk after immunization with E7/Algammulin, Tumor protection was observed in each case. We conclude that an established Th2 response to a TSA does not prevent the development of TSA-specific tumor protective CTL.

A gene (Cargl) that regulates tissue resistance to Candida albicans maps to chromosome 14 of the mouse

Tissue susceptibility and resistance to infection with the yeast Candida albicans is genetically regulated. Analysis of the strain distribution pattern of the C. albicans resistance gene (Carg1) and additional gene and DNA segment markers in the AKXL recombinant inbred (RI) set showed that 13/15 RI strains were concordant for Carg1, Tcra and Rib1. Therefore, Carg1 is probably located within a 17 cM segment of chromosome 14, within approximately 4 cM of the other two genes. (C) 1998 Academic Press.

Errors in lamina growth of primary olfactory axons in the rat and mouse olfactory bulb

In the adult olfactory nerve pathway of rodents, each primary olfactory axon forms a terminal arbor in a single glomerulus in the olfactory bulb. During development, axons are believed to project directly to and terminate precisely within a glomerulus without any exuberant growth or mistargeting. To gain insight into mechanisms underlying this process, the trajectories of primary olfactory axons during glomerular formation were studied in the neonatal period. Histochemical staining of mouse olfactory bulb sections with the lectin Dolichos biflorus-agglutinin revealed that many olfactory axons overshoot the glomerular layer and course into the deeper laminae of the bulb in the early postnatal period. Single primary olfactory axons were anterogradely labelled either with the lipophilic carbocyanine dye, 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), or with horseradish peroxidase (HRP) by localized microinjections into the nerve fiber layer of the rat olfactory bulb. Five distinct trajectories of primary olfactory axons were observed in DLI-labelled preparations at postnatal day 1.5 (P1.5). Axons either coursed directly to and terminated specifically within a glomerulus, branched before terminating in a glomerulus, bypassed glomeruli and entered the underlying external plexiform layer, passed through the glomerular layer with side branches into glomeruli, or branched into more than one glomerulus. HRP-labelled axon arbors from eight postnatal ages were reconstructed by camera lucida and were used to determine arbor length, arbor area, and arbor branch number. Whereas primary olfactory axons display errors in laminar targeting in the mammalian olfactory bulb, axon arbors typically achieve their adult morphology without exuberant growth. Many olfactory axons appear not to recognize appropriate cues to terminate within the glomerular layer during the early postnatal period. However, primary olfactory axons exhibit precise targeting in the glomerular layer after P5.5, indicating temporal differences in either the presence of guidance cues or the ability of axons to respond to these cues. (C) 1999 Wiley-Liss, Inc.

Differentiation of the mononuclear phagocyte system during mouse embryogenesis: The role of transcription factor PU.1

During mouse embryogenesis, macrophage-like cells arise first in the yolk sac and are produced subsequently in the liver. The onset of liver hematopoiesis is associated with the transition from primitive to definitive erythrocyte production. This report addresses the hypothesis that a similar transition in phenotype occurs in myelopoiesis. We have used whole mount in situ hybridization to detect macrophage-specific genes expressed during mouse development. The mouse c-fms mRNA, encoding the receptor for macrophage colony-stimulating factor (CSF-1), was expressed on phagocytic cells in the yolk sac and throughout the embryo before the onset of liver hematopoiesis, Similar cells were detected using the mannose receptor, the complement receptor (CR3), or the Microphthalmia transcription factor (MITF) as mRNA markers. By contrast, other markers including the F4/80 antigen, the macrophage scavenger receptor, the S-100 proteins, S100A8 and S100A9, and the secretory product lysozyme appeared later in development and appeared restricted to only a subset of c-fms-positive cells. Two-color immunolabeling on disaggregated cells confirmed that CR3 and c-fms proteins are expressed on the same cells. Among the genes appearing later in development was the macrophage-restricted transcription factor, PU.1, which has been shown to be required for normal adult myelopoiesis. Mice with null mutations in PU.1 had normal numbers of c-fms-positive phagocytes at 11.5dpc. PU.1(-/-) embryonic stem cells were able to give rise to macrophagelike cells after cultivation in vitro. The results support previous evidence that yolk sac-derived fetal phagocytes are functionally distinct from those arising in the liver and develop via a different pathway. (C) 1999 by The American Society of Hematology.

A null mutation in the inflammation-associated S100 protein S100A8 causes early resorption of the mouse embryo

S100A8 (also known as CP10 or MRP8) was the first member of the S100 family of calcium-binding proteins shown to be chemotactic for myeloid cells. The gene is expressed together with its dimerization partner S100A9 during myelopoiesis in the fetal liver and in adult bone marrow as well as in mature granulocytes. In this paper we show that S100A8 mRNA is expressed without S100A9 mRNA between 6.5 and 8.5 days postcoitum within fetal cells infiltrating the deciduum in the vicinity of the ectoplacental cone. Targeted disruption of the S100A8 gene caused rapid and synchronous embryo resorption by day 9.5 of development in 100% of homozygous null embryos. Until this point there was no evidence of developmental delay in S100A8(-/-) embryos and decidualization was normal. The results of PCR genotyping around 7.5-8.5 days postcoitum suggest that the null embryos are infiltrated with maternal cells before overt signs of resorption. This work is the first evidence for nonredundant function of a member of the S100 gene family and implies a role in prevention of maternal rejection of the implanting embryo. The S100A8 null provides a new model for studying fetal-maternal interactions during implantation.

Dynamic spatiotemporal expression patterns of neurocan and phosphacan indicate diverse roles in the developing and adult mouse olfactory system

The chondroitin sulfate proteoglycans neurocan and phosphacan are believed to modulate neurite outgrowth by binding to cell adhesion molecules, tenascin, and the differentiation factors heparin-binding growth-associated molecule and amphoterin. To assess the role of these chondroitin sulfate proteoglycans in the olfactory system, we describe here their expression patterns during both embryonic and postnatal development in the mouse. Immunoreactivity for neurocan was first detected in primary olfactory neurons at embryonic day 11.5 (E11.5). Neurocan was expressed by primary olfactory axons as they extended toward the rostral pole of the telencephalon as well as by their arbors in glomeruli after they contacted the olfactory bulb. The role of neurocan was examined by growing olfactory neurons on an extracellular matrix substrate containing neurocan or on extracellular matrix in the presence of soluble neurocan. In both cases, neurocan strongly promoted neurite outgrowth. These results suggest that neurocan supports the growth of primary olfactory axons through the extracellular matrix as they project to the olfactory bulb during development. Phosphacan, unlike neurocan, was present within the mesenchyme surrounding the E11.5 and E12.5 nasal cavity. This expression decreased at E13.5, concomitant with a transient appearance of phosphacan in nerve fascicles. Within the embryonic olfactory bulb, phosphacan was localised to the external and internal plexiform layers. However, during early postnatal development phosphacan was concentrated in the glomerular layer. These results suggest that phosphacan may play a role in delineating the pathway of growing olfactory axons as well as defining the laminar organization of the bulb. Together, the spatiotemporal expression patterns of neurocan and phosphacan indicate that these chondroitin sulfate proteoglycans have diverse in situ roles, which are dependent on context-specific interactions with extracellular and cell adhesion molecules within the developing olfactory nerve pathway. (C) 2000 Wiley-Liss, Inc.

Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Specific Expression by a Keratin 6a Minigene

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta -galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.

Mutant GABA(A) receptor gamma 2-subunit in childhood absence epilepsy and febrile seizures

Epilepsies affect at least 2% of the population at some time in life, and many forms have genetic determinants(1,2). We have found a mutation in a gene encoding a GABA, receptor subunit in a large family with epilepsy. The two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS), There is a recognized genetic: relationship between FS and CAE, yet the two syndromes have different ages of onset, and the physiology of absences and convulsions is distinct. This suggests the mutation has age-dependent effects on different neuronal networks that influence the expression of these clinically distinct, but genetically related, epilepsy phenotypes. We found that the mutation in GABRG2 (encoding the gamma2-subunit) abolished in vitro sensitivity to diazepam, raising the possibility that endozepines do in fact exist and have a physiological role in preventing seizures.

Myb-binding protein 1a augments AhR-dependent gene expression

We have studied the mechanism by which an acidic domain (amino acids 515-583) of the aromatic hydrocarbon receptor (AhR) transactivates a target gene. Studies with glutathione S-transferase fusion proteins demonstrate that the wild-type acidic domain associates in vitro with Myb-binding protein la, whereas a mutant domain (F542A, 1569A) does not. AhR-defective cells reconstituted with an AhR containing the wild-type acidic domain exhibit normal AhR function; however, cells reconstituted with an AhR containing the mutant acidic domain do not function normally. Transient transfection of Myb-binding protein la into mouse hepatoma cells is associated with augmentation of AhR-dependent gene expression. Such augmentation does not occur when Myb-binding protein la is transfected into AhR-defective cells that have been reconstituted with an AhR that lacks the acidic domain. We infer that 1) Myb-binding protein la associates with AhR, thereby enhancing transactivation, and 2) the presence of AhR's acidic domain is both necessary and sufficient for Myb-binding protein la to increase AhR-dependent gene expression.

Low-affinity neurotrophin receptor with targeted mutation of exon 3 is capable of mediating the death of axotomized neurons

1. In vivo studies have shown that the low-affinity 75 kDa neurotrophin receptor (p75NTR) is involved in axotomy-induced cell death of sensory and motor neurons. To further examine the importance of p75NTR in mediating neuronal death in vivo , we examined the effect of axotomy in the p75NTR-knockout mouse, which has a disrupted ligand-binding domain. 2. The extent of sensory and motor neuron loss in the p75NTR-knockout mouse following axotomy was not significantly different to that in wild-type mice. This suggests that disruption of the ligand-binding domain is insufficient to block the cell death process in axotomized neurons. 3. Immunohistochemical studies showed that axotomized neurons continue to express this mutant receptor with its intracellular death-signalling moiety intact. 4. Treatment with antisense oligonucleotides targeted against p75NTR resulted in significant reduction in the loss of axotomized neurons in the knockout mouse. 5. These data suggest that the intracellular domain of p75NTR is essential for death-signalling and that p75NTR can signal apoptosis, despite a disrupted ligand-binding domain.