Progressive Age-Related Changes Similar to Age-Related Macular Degeneration in a Transgenic Mouse Model

Age-related macular degeneration (AMD) is the major cause of blindness in the developed world. its pathomechanism is unknown and its late onset, complex genetics and strong environmental components have all hampered investigations. Here we demonstrate the development of an animal model for AMD that reproduces features associated with geographic atrophy, a transgenic mouse line (mcd/mcd) expressing a mutated form of cathepsin D that is enzymatically inactive thus impairing processing of phagocytosed photoreceptor outer segments in the retinal pigment epithelial (RPE) cells. Pigmentary changes indicating RPE cell atrophy and a decreased response to flash electroretinograms were observed in 11- to 12-month-old mcd/mcd mice. Histological studies showed RPE cell proliferation, photoreceptor degeneration, shortening of photoreceptor outer segments, and accumulation of immunoreactive photoreceptor breakdown products in the RPE cells. An accelerated photoreceptor cell death was detected in 12-month-old mcd/mcd mice. Transmission electron microscopy demonstrated presence of basal laminar and linear deposits that are considered to be the hallmarks of AMD. Small hard drusen associated with human age-related maculopathy were absent in the mcd/mcd mouse model at the ages analyzed. in summary, this model presents several features of AMD, thus providing a valuable tool for investigating the underlying biological processes and pathomechanism of AMD.

Electrolyte transport in the mouse trachea: No evidence for a contribution of luminal K+ conductance

Recent studies on frog skin acini have challenged the question whether Cl- secretion or Na+ absorption in the airways is driven by luminal K+ channels in series to a basolateral K+ conductance. We examined the possible role of luminal K+ channels in electrolyte transport in mouse trachea in Ussing-chamber experiments. Tracheas of both normal and CFTR (-/-) mice showed a dominant amiloride-sensitive Na+ absorption under both, control conditions and after cAMP-dependent stimulation. The lumen-negative transepithelial voltage was enhanced after application of IBMX and forskolin and Cl- secretion was activated. Electrolyte secretion induced by IBMX and forskolin was inhibited by luminal glibenclamide and the blocker of basolateral Na(+)2Cl(-)K(+) cotransporter azosemide. Similarly, the compound 29313, a blocker of basolateral KCNQ1/KCNE3 K+ channels effectively blocked Cl- secretion when applied to either the luminal or basolateral side of the epithelium. RT-PCR analysis suggested expression of additional K+ channels in tracheal epithelial cells such as Slo1 and Kir6.2. However, we did not detect any functional evidence for expression of luminal K+ channels in mouse airways, using luminal 29313, clotrimazole and Ba2+ or different K+ channel toxins such as charybdotoxin, apamin and alpha-dendrotoxin. Thus, the present study demonstrates Cl- secretion in mouse airways, which depends on basolateral Na(+)2Cl(-)K(+) cotransport and luminal CFTR and non-CFTR Cl- channels. Cl- secretion is maintained by the activity of basolateral K+ channels, while no clear evidence was found for the presence of a luminal K+ conductance.

Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities

Increasingly, cystic fibrosis (CF) is regarded as an inflammatory disorder where the response of the lung to Pseudomonas aeruginosa is exaggerated as a consequence of processes mediated by the product of the CF gene, CFTR. Of importance to any gene-replacement strategy for treatment of CF is the identification of the cell type(s) within the lung milieu that need to be corrected and an indication whether this is sufficient to restore a normal inflammatory response and bacterial clearance. We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages. Following chronic pulmonary infection with P. aeruginosa, CF mice with epithelial-expressed but not macrophage-specific CFTR showed an improvement in pathogen clearance and inflammatory markers compared with control CF animals. Additionally, these data indicate the general role for epithelial cell-mediated events in the response of the lung to bacterial pathogens and the importance of CFTR in mediating these processes.

Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V-te) and amiloride-sensitive short-circuit current (Isc-Amil) in mouse trachea. Exposure to apical ATP or UTP (each 100 mumol/l) caused a large initial increase in lumen negative V-te and I-sc corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of Isc-Amil that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 mumol/l) or the Ca2+ ionophore ionomycin (1 mumol/l). Removal of extracellular Cl- or exposure to 200 muM DIDS reduced UTP-mediated inhibition of Isc-Amil Substantially. The phospholipase inhibitor U73122 (10 mumol/l) and pertussis toxin (PTX 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of Isc-Amil. Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.

Regulation of the mouse Nas1 promoter by vitamin D and thyroid hormone

The renal sodium-sulfate cotransporter, NaSi-1, a protein implicated to control serum sulfate levels, has been shown to be regulated in vivo by 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) and tri-iodothyronine (T-3). Recently, we cloned the mouse NaSi-1 gene (Nas1) and in the present study identified a 1,25-(OH)(2)D-3- and T-3-responsive element located within the Nas1 promoter. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3-responsive element (IR0 T3RE) at -436 to -425 which conferred 1,25(OH)(2)D-3 and T3 responsiveness, respectively. In summary, we have identified responsive elements that mediate the enhanced transcription of Nas1 by 1,25-(OH)(2)D-3 and T-3, and these mechanisms may provide important clues to the physiological control of sulfate homeostasis.

The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel

In renal collecting ducts, a vasopressin-induced cAMP increase results in the phosphorylation of aquaporin-2 (AQP2) water channels at Ser-256 and its redistribution from intracellular vesicles to the apical membrane. Hormones that activate protein kinase C (PKC) proteins counteract this process. To determine the role of the putative kinase sites in the trafficking and hormonal regulation of human AQP2, three putative casein kinase II (Ser-148, Ser-229, Thr-244), one PKC (Ser-231), and one protein kinase A (Ser-256) site were altered to mimic a constitutively non-phosphorylated/phosphorylated state and were expressed in Madin-Darby canine kidney cells. Except for Ser-256 mutants, seven correctly folded AQP2 kinase mutants trafficked as wild-type AQP2 to the apical membrane via forskolin-sensitive intracellular vesicles. With or without forskolin, AQP2-Ser-256A was localized in intracellular vesicles, whereas AQP2-S256D was localized in the apical membrane. Phorbol 12-myristate 13-acetate-induced PKC activation following forskolin treatment resulted in vesicular distribution of all AQP2 kinase mutants, while all were still phosphorylated at Ser-256. Our data indicate that in collecting duct cells, AQP2 trafficking to vasopressin-sensitive vesicles is phosphorylation-independent, that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and that PMA-induced PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state.

Complementary and layered expression of Ephs and Ephrins in developing mouse inner ear

The distributions of the Eph-class receptors EphA4 and EphB 1, and their ligands ephrin-A2, ephrin-B1, and ephrin-B2, were analysed by immunostaining in the mouse inner ear. Complementary patterns of EphA4 and its potential ligand ephrin-A2 were found, with ephrin-A2 in many of the structures lining the cochlear duct and within the cochlear nerve cells, and EphA4 in the deeper structures underlying the cochlear duct and in the cells lining the nerve pathway. EphB1 and its potential ligands ephrin-B1 and ephrin-B2 showed a segregated layered expression in the lateral wall of the cochlear duct (the external sulcus), which together with EphA4 expressed in the area, form a four-layered structure with an alternating pattern of receptors and ligands in the different layers. This arrangement gives the potential for different bidirectional Eph-mediated interactions between each of the layers. The results suggest that the Eph system in the cochlea may have a role in maintaining cell segregation during phases of cochlear development. (C) 2002 Wiley-Liss, Inc.

Heterogeneity in olfactory neurons in mouse revealed by differential expression of glycoconjugates

Cell surface glycoconjugates have been implicated in the growth and guidance of subpopulations of primary olfactory axons. While subpopulations of primary olfactory neurons have been identified by differential expression of carbohydrates in the rat there are few reports of similar subpopulations in the mouse. We have examined the spatiotemporal expression pattern of glycoconjugates recognized by the lectin from Wisteria floribunda (WFA) in the mouse olfactory system. In the developing olfactory neuroepithelium lining the nasal cavity, WFA stained a subpopulation of primary olfactory neurons and the fascicles of axons projecting to the target tissue, the olfactory bulb. Within the developing olfactory bulb, WFA stained the synaptic neuropil of the glomerular and external plexiform layers. In adults, strong expression of WFA ligands was observed in second-order olfactory neurons as well as in neurons in several higher order olfactory processing centres in the brain. Similar, although distinct, staining of neurons in the olfactory pathway was detected with Dolichos biflorus agglutinin. These results demonstrate that unique subpopulations of olfactory neurons are chemically coded by the expression of glycoconjugates. The conserved expression of these carbohydrates across species suggests they play an important role in the functional organization of this region of the nervous system.

The interaction of water flow and nutrients on aquatic plant growth

A long-term experiment was conducted to compare the effects of flowing and still water on growth, and the relationship between water flow and nutrients, in Aponogeton elongatus, a submerged aquatic macrophyte. A. elongatus plants were grown for 23 weeks with three levels of nutrition (0, 0.5 and 1g Osmocote Plus(R) fertiliser pot(-1)) in aquaria containing stirred or unstirred water. Fertilized plants grew much better than non-fertilized. The highest fertilizer level produced 29% wider leaves and 58% higher total dry weight in stirred water. Stirred water increased leaf area by 40% and tuber size by 81%, but only with the highest level of nutrition. These results suggest that this plant depends on its roots for mineral uptake, rather than from the open water, and the major limitation to growth in still water is the supply of dissolved inorganic carbon. It was the combined effects of nutrient availability and stirring that produced the strongest response in plant growth, morphology and composition. This study provides some explanation for the observations of others that these plants grow best in creeks or river systems with permanently flowing water.

Dry matter production and allocation in Eucalyptus cloeziana and Eucalyptus argophloia seedlings in response to soil water deficits

Effects of soil water availability on seedling growth, dry matter production and allocation were determined for Gympie ( humid coastal) and Hungry Hills ( dry inland) provenances of Eucalyptus cloeziana F. Muell. and for E. argophloia Blakely ( dry inland) species. Seven-month-old seedlings were subjected to well-watered (100% field capacity, FC), moderate (70% FC) and severe (50% FC) soil water regimes in a glasshouse environment for 14 wk. There were significant differences in seedling growth, biomass production and allocation patterns between species. E. argophloia produced twice as much biomass at 100% FC, and more than three times as much at 70% and 50% FC than did either E. cloeziana provenance. Although the humid provenance of E. cloeziana had a greater leaf area at 100% FC conditions than did the dry provenance, total biomass production did not differ significantly. Both E. cloeziana provenances were highly sensitive to water deficits. E. argophloia allocated 10% more biomass to roots than did E. cloeziana. Allometric analyses indicated that relative biomass allocation patterns were significantly affected by genotype but not by soil water availability. These results have implications for taxon selection for cultivation in humid and subhumid regions.

Leaf water relations of Eucalyptus cloeziana and Eucalyptus argophloia in response to water deficit

Leaf water relations responses to limited water supply were determined in 7-month-old plants of a dry inland provenance of Eucalyptus argophloia Blakely and in a humid coastal provenance (Gympie) and a dry inland provenance (Hungry Hills) of Eucalyptus cloeziana F. Muell. Each provenance of E. cloeziana exhibited a lower relative water content at the turgor loss point, a lower apoplastic water content, a smaller ratio of dry mass to turgid mass and a lower bulk modulus of elasticity than the single provenance of E. argophloia. Osmotic potential at full turgor and water potential at the turgor loss point were significantly lower in E. argophloia and the inland provenance of E. cloeziana than in the coastal provenance of E. cloeziana. There was limited osmotic adjustment in response to soil drying in E. cloeziana, but not in E. argophloia. Between-species differences in water relations parameters were larger than those between the E. cloeziana provenances. Both E. cloeziana provenances maintained turgor under moderate water stress through a combination of osmotic and elastic adjustments. Eucalyptus argophloia had more rigid cell walls and reached lower water potentials with less reduction in relative water content than either of the E. cloeziana provenances, thereby enabling it to extract water from dryer soils.

Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis

This study examined the nature of the infiltrating cells in Porphyromonas gingivalis-induced lesions and immunoglobulins in the serum samples of BALB/c (H-2(d)), C57BL6 (H-2(b)), DBA/2J (H-2(d)) and CBA/CaH (H-2(k)) mice. Mice were immunized intraperitoneally with P. gingivalis outer membrane antigens or sham-immunized with phosphate-buffered saline followed by subcutaneous challenge with live organisms 1 week after the final immunization. The resulting skin abscesses were excised 7 days later, cryostat sections cut and an immunoperoxidase method used to detect the presence of CD4(+) and CD8(+) T-cell subsets, CD14(+) macrophages and CD19(+) B cells. Peroxidase positive neutrophils and IgG1- and IgG2a-producing plasma cells were also identified. Anti P. gingivalis IgG1 and IgG2a subclass antibodies were determined in serum obtained by cardiac puncture. Very few CD8(+) T cells and CD19(+) B cells were found in any of the lesions. The percentages of CD4(+) cells, CD14(+) cells and neutrophils were similar in lesions of immunized BALB/c and C57BL6 mice, with a trend towards a higher percentage of CD14(+) cells in sham-immunized mice. The percentage of CD14(+) cells was higher than that of CD4(+) cells in immunized compared with sham-immunized DBA/2J mice. The percentages of CD4(+) and CD14(+) cells predominated in immunized CBA/CaH mice and CD4(+) cells in sham-immunized CBA/CaH mice. The percentage of neutrophils in immunized CBA/CaH mice was significantly lower than that of CD14(+) cells and CD4(+) cells in sham-immunized mice. IgG1(+) plasma cells were more dominant than IgG2a(+) cells in immunized BALB/c, C57BL6 and DBA/2J mice, whereas IgG2a(+) plasma cells were more obvious in sham-immunized mice. IgG2a(+) plasma cells were predominant in immunized and sham-immunized CBA/CaH mice. In the serum, specific anti-P. gingivalis IgG2a antibody levels (Th1 response) were higher than IgG1 levels (Th2 response) in sham-immunized CBA/CaH and DBA/2J mice. In immunized BALB/c mice, IgG2a levels were lower than IgG1 levels, while IgG2a levels were higher in immunized C57BL6 mice. In conclusion, this study has shown differences in the proportion of infiltrating leukocytes and in the subclasses of immunoglobulin produced locally and systemically in response to P. gingivalis in different strains of mice, suggesting a degree of genetic control over the response to P. gingivalis.

Horizontal rotating cylinder - A compact apparatus for studying the effect of water wetting on carbon dioxide corrosion of mild steel

Water wetting is a crucial issue in carbon dioxide (CO.) corrosion of multiphase flow pipelines made from mild steel. This study demonstrates the use of a novel benchtop apparatus, a horizontal rotating cylinder, to study the effect of water wetting on CO2 corrosion of mild steel in two-phase flow. The setup is similar to a standard rotating cylinder except for its horizontal orientation and the presence of two phases-typically water and oil. The apparatus has been tested by using mass-transfer measurements and CO2 corrosion measurements in single-phase water flow. CO2 corrosion measurements were subsequently performed using a water/hexane mixture with water cuts varying between 5% and 50%. While the metal surface was primarily hydrophilic under stagnant. conditions, a variety of dynamic water wetting situations was encountered as the water cut and fluid velocity were altered. Threshold velocities were identified at various water cuts when the surface became oil-wet and corrosion stopped.

Assessing childrens competence in computer interactions: Preliminary reliability and validity of the test of mouse proficiency

The Test of Mouse Proficiency (TOMP) was developed to assist occupational therapists and education professionals assess computer mouse competency skills in children from preschool to upper primary (elementary) school age. The preliminary reliability and validity of TOMP are reported in this paper. Methods used to examine the internal consistency, test-retest reliability, and criterion- and construct-related validity of the test are elaborated. In the continuing process of test refinement, these preliminary studies support to varying degrees the reliability and validity of TOMP. Recommendations for further validation of the assessment are discussed along with indications for potential clinical application.