Choroideremia gene product affects trophoblast development and vascularization in mouse extra-embryonic tissues

Choroideremia (CHM) is a hereditary eye disease caused by mutations in the X-linked CHM gene. Disruption of the Chm gene in mice resulted in prenatal death of Chm-/Y males and Chm-/Chm+ females that had inherited the mutation from their mothers. Male chimeras and Chm+/Chm- females with paternal transmission of the mutation were viable and had photoreceptor degeneration reminiscent of human choroideremia. Here, we show that Chm-/Y males and Chm-/Chm+ females were retarded at e7.5 and died before e11.5 due to multiple defects of the extra-embryonic tissues. Mutant embryos exhibited deficiency of diploid trophoblasts associated with overabundance of giant cells. In yolk sac and placenta, severe defects in vasculogenesis were obvious. Chm-/Y males exhibited more pronounced phenotypes than Chm-/Chm+ females. The lethal genotypes could be rescued by tetraploid aggregation. Chm-/Chm+ females, but not Chm-/Y males, could also be rescued when their Chm+/Chm- mothers were mated with Mus spretus males. Backcross analysis suggested that the viability of interspecies hybrid Chm-/Chm+ females may be due to expression from the Chm allele on the M. spretus X-chromosome rather than a modifier effect. Our results demonstrate that Chm is essential for diploid trophoblast development and plays a role in the vascularization in placenta and yolk sac.     

Dehydroascorbate reductase affects leaf growth, development, and function

Ascorbic acid (Asc) is a major antioxidant in plants that detoxifies reactive oxygen species (ROS) and maintains photosynthetic function. Expression of dehydroascorbate reductase (DHAR), responsible for regenerating Asc from an oxidized state, regulates the cellular Asc redox state, which in turn affects cell responsiveness and tolerance to environmental ROS. Because of its role in Asc recycling, we examined whether DHAR is important for plant growth. Suppression of DHAR expression resulted in a preferential loss of chlorophyll a, a lower steady state of Rubisco as measured by the amount of the large subunit of Rubisco (RbcL), and a lower rate of CO(2) assimilation. As a consequence, a slower rate of leaf expansion and reduced foliar dry weight were observed. In addition, an accelerated rate of loss of chlorophyll, RbcL, light-harvesting complex II, and photosynthetic functioning was observed in mature leaves, resulting in premature leaf aging. Reduced growth rate as measured by plant height and leaf number was consistent with the DHAR-mediated reduction of photosynthetic function. Increasing DHAR expression maintained higher levels of chlorophyll, RbcL, light-harvesting complex II, and photosynthetic functioning, resulting in delayed leaf aging. The effect of DHAR expression on leaf aging inversely correlated with the level of lipid peroxidation, indicating that DHAR functions to protect against ROS-mediated damage. These observations support the conclusion that through its Asc recycling function, DHAR affects the level of foliar ROS and photosynthetic activity during leaf development and as a consequence, influences the rate of plant growth and leaf aging.     

GD3 expression in CHO-K1 cells increases growth rate,induces morphological changes,and affects cell-substrate interactions

We have generated a panel of CHO-K1 cell clones with different glycolipid compositions by stable transfection of appropriate glycosyltransferases and studied the morphological and growth phenotype of a clone stably expressing Sial-T2. Compared with the GM3 expressing parental cells, Sial-T2 transfectants show low expression of GM3 and neo expression of GD3 and GT3. These cells show about 60% reduction of the mean cell area, and about 2-fold increase of the mean colony area and growth rate. Cells over expressing Sial-T2 showed a flattened appearance, and with time in culture they detached from the substrate leaving adhered material that was GD3 immunoreactive. No apoptotic or proteome differences could be detected in the Sial-T2 transfectants. Thus, increased expression of GD3 and GT3 influence parameters of growth and social behavior of CHO-K1 cells. However, the molecular and cellular basis underlying these influences requires further investigation.     

Endometriosis as a neurovascular condition: estrous variations in innervation, vascularization, and growth factor content of ectopic endometrial cysts in the rat

Endometriosis is a poorly understood, estradiol-dependent condition associated with severe pelvic pains and defined by vascularized endometrial growths outside the uterus. Endometriosis is produced in cycling rats by autotransplanting pieces of uterus onto abdominal arteries where they develop into cysts. The surgery induces vaginal and abdominal muscle hyperalgesia, whose severity is greatest in proestrus and nearly absent in estrus. The cysts contain growth factors and cytokines and develop their own sympathetic and sensory C- and Adelta-fiber innervation. Here, we used quantitative immunostaining and protein array analyses to test the hypothesis that the innervation and growth factor/cytokine content of the cysts, but not uterine horn, contribute to proestrous-to-estrous changes in hyperalgesic severity. If so, these characteristics in the cysts, but not the uterine horn, should change with estrous stage. In cysts, the density of sympathetic (but not sensory) neurites and amounts of NGF and VEGF proteins (but not cytokines IL-1, IL-6, IL-10, or TNF-alpha) were greater in proestrus than estrus. These changes were accompanied by vascular changes. Both sympathetic and sensory fibers in both stages colabeled with TrkA, indicating that changes in NGF could act on both afferent and efferent fibers. In contrast with the cysts, no changes occurred in the uterine horn between proestrus and estrus. Together, these results suggest that coordinated proestrous-to-estrous changes in innervation and vascularization of the cysts contribute to similar changes in hyperalgesic severity. The findings also encourage consideration of endometriosis as a neurovascular condition.     

The resistance of epithelia to vascularization: Proteinase and endothelial cell growth inhibitory activities in urinary bladder epithelium

The avascularity of epithelia may be attributed to the presence of an extractable, low-molecular-weight factor. This factor contains potent inhibitors of proteolytic enzymes, as well as a growth inhibitory activity directed against endothelial cells in vitro. It is extracted from the epithelium of bovine urinary bladders by 1 M NaCl. The extract is ultrafiltered through an Amicon XM-50 membrane, then concentrated and dialyzed into a 0.9% NaCl solution, using a UM-2 membrane. This ultrafiltrate, called the UM-2 retentate (UM-2R). contains approximately 6 μg protein/ g tissue. The UM-2R has a low content of uronic acid and is practically devoid of hydroxyproline. SDS-PAGE reveals that the UM-2R consists of six major proteins. The UM-2R contains a Trasylol-like proteinase inhibitor that expresses strong trypsin inhibitory activity. Comparisons between bladder and serum UM-2Rs and electrophoretic mobility assays indicate that this proteinase inhibitory activity is derived from the bladder epithelium and not from the serum. The UM-2R is cytotoxic to cultured endothelial cells. Cultures of other cell types (normal and neoplastic) are not affected. The bladder-derived proteinase and endothelial cell growth inhibitory activities may protect epithelia from vascular invasion.     

Homeotic genes and their role in development of wheat's morphological traits

The information concerning major families of plant homeotic genes, ways of their expression regulation and role in plant morphogenesis is outlined. Role of known homeotic genes in wheat development and growth habit establishment is presented. A supposed role of homeotic genes in major morphologic traits formation is discussed.     

Homeotic genes and their role in development of morphological traits in wheat

Information about main families of plant homeotic genes, ways of their activity regulation, and role in morphogenesis is presented. The role of known homeotic genes in wheat development and possible participation of homeotic genes in the development of main morphological traits of this species is discussed.     

Quantitative and morphological studies of retinal development in mice with trisomy 19

To investigate the development of the retinal layers, the eyes of mice with trisomy 19 have been examined by light microscopy between the 2nd and 15th postnatal day. The diameter of the eye, thickness of the entire retina and both relative thickness and nuclear density of each of the retinal layers have been measured and compared to those of chromosomally balanced control animals. Malformations of the eye, alterations of cell morphology or disturbed lamination can not be observed. Retinal differentiation of trisomy 19 mice is delayed by approximately two days. The development of all cellular constituents, i.e., of both neuroectodermal and mesenchymal origin, is retarded accordingly. The eyes of trisomy 19 mice are of reduced size. The relative thickness of each retinal layer follows a normal growth pattern; there is no indication for a selective impairment of the development of one particular layer. With the exception of the ganglion cell layer, nuclear densities of each retinal layer do not differ from those of control mice. The comparison of nuclear densities in the ganglion cell layer suggests that in trisomy 19 mice fewer postmitotic cells differentiate into mature retinal cells.     

Spatial covariance in resources affects photosynthetic rate and water potential,but not the growth of Glechoma longituba fragments

Glechom longituba is a widespread clonal plant and usually experiences diverse patchiness of its growing sites. The hypothesis was tested that spatial covariance in resources differentially affects the growth and physiology of G. longituba. Plants were exposed to two patchy habitats where above- and below-ground resources were either positively or negatively associated, and to four control habitats. When equal amounts of resources were given, G. longituba fragments from two patchy habitats had similar biomass, root weight ratio, leaf weight ratio, fluorescence yield, specific petiole length, and specific root length; fragments under reciprocal patchiness significantly increased photosynthetic rate and decreased leaf water potential. Biomass of clones grown in patchy habitats was equal or less than that of counterparts from the control habitats, not supporting the notion that clonal growth is more advantageous in patchy habitats than in uniform habitats. In addition, no evidence was detected for spatial division of labour because biomass allocation to roots and leaves was similar in patchy habitats compared with the control habitats.     

Fibroblast growth factor receptor signaling affects development and function of dopamine neurons - inhibition results in a schizophrenia-like syndrome in transgenic mice

Developing and mature midbrain dopamine (DA) neurons express fibroblast growth factor (FGF) receptor-1 (FGFR1). To determine the role of FGFR1 signaling in the development of DA neurons, we generated transgenic mice expressing a dominant negative mutant [FGFR1(TK-)] from the catecholaminergic, neuron-specific tyrosine hydroxylase (TH) gene promoter. In homozygous th(tk-)/th(tk-) mice, significant reductions in the size of TH-immunoreactive neurons were found in the substantia nigra compacta (SNc) and the ventral tegmental area (VTA) at postnatal days 0 and 360. Newborn th(tk-)/th(tk-) mice had a reduced density of DA neurons in both SNc and VTA, and the changes in SNc were maintained into adulthood. The reduced density of DA transporter in the striatum further demonstrated an impaired development of the nigro-striatal DA system. Paradoxically, the th(tk-)/th(tk-) mice had increased levels of DA, homovanilic acid and 3-methoxytyramine in the striatum, indicative of excessive DA transmission. These structural and biochemical changes in DA neurons are similar to those reported in human patients with schizophrenia and, furthermore, these th(tk-)/th(tk-) mice displayed an impaired prepulse inhibition that was reversed by a DA receptor antagonist. Thus, this study establishes a new developmental model for a schizophrenia-like disorder in which the inhibition of FGF signaling leads to alterations in DA neurons and DA-mediated behavior.     

Ozone affects gas exchange, growth and reproductive development in Brassica campestris (Wisconsin fast plants)

Exposure to ozone (O(3)) may affect vegetative and reproductive development, although the consequences for yield depend on the effectiveness of the compensatory processes induced. This study examined the impact on reproductive development of exposing Brassica campestris (Wisconsin Fast Plants) to ozone during vegetative growth. Plants were exposed to 70 ppb ozone for 2 d during late vegetative growth or 10 d spanning most of the vegetative phase. Effects on gas exchange, vegetative growth, reproductive development and seed yield were determined. Impacts on gas exchange and foliar injury were related to pre-exposure stomatal conductance. Exposure for 2 d had no effect on growth or reproductive characteristics, whereas 10-d exposure reduced vegetative growth and reproductive site number on the terminal raceme. Mature seed number and weight per pod and per plant were unaffected because seed abortion was reduced. The observation that mature seed yield per plant was unaffected by exposure during the vegetative phase, despite adverse effects on physiological, vegetative and reproductive processes, shows that indeterminate species such as B. campestris possess sufficient compensatory flexibility to avoid reductions in seed production.     

Regulation of expansin gene expression affects growth and development in transgenic rice plants

To investigate the in vivo functions of expansins, we generated transgenic rice plants that express sense and antisense constructs of the expansin gene OsEXP4. In adult plants with constitutive OsEXP4 expression, 12% of overexpressors were taller and 88% were shorter than the average control plants, and most overexpressors developed at least two additional leaves. Antisense plants were shorter and flowered earlier than the average control plants. In transgenic plants with inducible OsEXP4 expression, we observed a close correlation between OsEXP4 protein levels and seedling growth. Coleoptile and mesocotyl length increased by up to 31 and 97%, respectively, in overexpressors, whereas in antisense seedlings, they decreased by up to 28 and 43%, respectively. The change in seedling growth resulted from corresponding changes in cell size, which in turn appeared to be a function of altered cell wall extensibility. Our results support the hypothesis that expansins are involved in enhancing growth by mediating cell wall loosening.     

Site-directed mutagenesis of the magB gene affects growth and development in Magnaporthe grisea

G protein signaling is commonly involved in regulating growth and differentiation of eukaryotic cells. We previously identified MAGB, encoding a Galpha subunit, from Magnaporthe grisea, and disruption of MAGB led to defects in a number of cellular responses, including appressorium formation, conidiation, sexual development, mycelial growth, and surface sensing. In this study, site-directed mutagenesis was used to further dissect the pleiotropic effects controlled by MAGB. Conversion of glycine 42 to arginine was predicted to abolish GTPase activity, which in turn would constitutively activate G protein signaling in magB(G42R). This dominant mutation caused autolysis of aged colonies, misscheduled melanization, reduction in both sexual and asexual reproduction, and reduced virulence. Furthermore, magB(G42R) mutants were able to produce appressoria on both hydrophobic and hydrophilic surfaces, although development on the hydrophilic surface was delayed. A second dominant mutation, magB(G203R) (glycine 203 converted to arginine), was expected to block dissociation of the Gbetagamma from the Galpha subunit, thus producing a constitutively inactive G protein complex. This mutation did not cause drastic phenotypic changes in the wild-type genetic background, other than increased sensitivity to repression of conidiation by osmotic stress. However, magB(G203R) is able to complement phenotypic defects in magB mutants. Comparative analyses of the phenotypical effects of different magB mutations are consistent with the involvement of the Gbetagamma subunit in the signaling pathways regulating cellular development in M. grisea.     

Temporal synergism of neurotransmitters (serotonin and dopamine) affects testicular development in mice

The temporal phase relation of circadian oscillations is reported to regulate reproduction in many seasonally breeding avian and mammalian species, but its role in the reproductive regulation of continuous breeders is not yet known. Hence in the present study, six experimental groups of 3-week-old male Parkes strain mice, Mus musculus, were injected with 5-hydroxytryptophan (5-HTP, serotonin precursor) and l-dihydroxyphenylalanine (l-DOPA, dopamine precursor) at intervals of 0, 4, 8, 12, 16 or 20 hr (5 mg/100 g body weight per day for 13 days). Control mice received two daily injections of normal saline. When observed 24 days post-treatment, 8-hr mice exhibited low body weight and suppression of gonadal activity (spermatogenesis, sperm count/motility/viability and plasma testosterone concentration), while body weight and degree of gonadal development were higher in the 12-hr mice as compared to the controls. It is concluded that normal somatic and gonadal growth of pre-puberal mice may be suppressed with an 8-hr phase relation of circadian serotonergic and dopaminergic oscillations. On the other hand, a 12-hr phase relation accelerated the rate of gonadal maturation, while other relations led to more or less similar gonadal development as in the control mice. This study suggests the importance of circadian organization as a function of specific temporal phase relations of neural oscillations in the maturation of gonads. Although the exact mechanism still needs to be investigated, this seems to be mediated via effects on the neuroendocrine axis.     

goosecoid expression represses Brachyury in embryonic stem cells and affects craniofacial development in chimeric mice

The homeobox gene goosecoid, originally identified in Xenopus, is expressed in the organizer or its equivalent during gastrulation in the frog, chick, zebrafish and mouse. To investigate the role of goosecoid in mouse development, we have generated embryonic stem cells that stably overexpress the murine homolog of goosecoid. These cells show a repression of the gastrulation-associated gene Brachyury. Interestingly, repression of Brachyury is conserved between Xenopus and mouse despite the lack of conservation of the Brachyury promoter. Further characterization of the goosecoid-overexpressing ES cells revealed that they maintain the expression of stage-specific embryonic antigen-1, and teratomas derived from goosecoid-overexpressing cells show the presence of cell types derived from all three germ layers. Some highly chimeric mice derived from goosecoid-overexpressing cells displayed skull defects. These observations suggest that goosecoid may play a role in specification of anterior mesendodermal fates and specifically in mouse craniofacial development.     

Physiological and morphological characterisation of Limonium species in their natural habitats: Insights into their abiotic stress responses

Background and aims

Morphological and biochemical traits of four halophytes of the genus Limonium were analysed in plants sampled from salt marshes in SE Spain. This work aimed to explore the mechanism(s) behind the adaptation of these species to stressful habitats, with particular emphasis on responses to drought.

Methods

Plants of each species together with soil samples were collected in summer, which is the most stressful season in the Mediterranean. Soil parameters and plant morphological traits were determined, and the levels of several biochemical stress markers in plants were measured using spectrophotometric assays. A multivariate analysis was performed to correlate soil and plant data.

Results

Morphological characteristics regarding the underground system topology and several biochemical traits (higher foliar Ca²⁺, sucrose and glucose, and lower proline, glycine-betaine and fructose) clearly separate L. santapolense individuals from plants of the other three species.

Conclusions

Drought tolerance of L. santapolense in the field is mostly dependent on morphological adaptations: when growing in an arid location, plants of this species develop long taproots that can extract water from the deep, moist layers of the soil.

     

Haemopoietic growth factors: their role in cell development and their clinical use

Mature blood cells are derived from haemopoietic stem cells which grow and proliferate to give rise to progenitor cells more restricted in their proliferation and differentiation capacity. These in turn give rise to cells belonging to any of the haemopoietic lineages. The haemopoietic growth factors interleukin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony stimulating factor, macrophage colony-stimulating factor and erythropoietin act on haemopoietic cells to promote cell survival, proliferation, differentiation and maturation, as well as many functions of the mature cells. These factors, now purified to homogeneity and molecularly cloned have recently become available. This has facilitated studies of their roles in cell production, and the range of target cells sensitive to them in vitro and in vivo in several species. The latter experimental data led to the first clinical trials where these factors have been used successfully in several clinical settings: erythropoietin to correct the anaemia of renal disease; granulocyte and granulocyte-macrophage colony-stimulating factors to accelerate haemopoietic regeneration after chemotherapy and bone marrow transplantation, and in other situations where increase in the numbers of white cells and stimulation of their function were required. The results to date allow optimism; the clinical use of growth factors not only in haematology and oncology, but in wider fields of medicine may well constitute a major breakthrough in the near future.