FISH-mapping of LEP and SLC26A2 genes in sheep, goat and cattle R-banded chromosomes: comparison between bovine, ovine and caprine chromosome 4 (BTA4/OAR4/CHI4) and human chromosome 7 (HSA7).

Sheep (OAR), goat (CHI) and cattle (BTA) R-banded chromosome preparations, obtained from synchronized cell cultures, were used to FISH-map leptin (LEP) and solute carrier family 26 member 2 (SLC26A2) genes on single chromosome bands. LEP maps on OAR4q32 and CHI4q32, being the first assignment of this gene to these two species. SLC26A2 maps on BTA7q24, OAR5q24 and CHI7q24. This gene, too, was assigned for the fist time to both sheep and goat chromosomes, while it was more precisely localized on a single chromosome band in cattle. Improved cytogenetic maps of BTA4/OAR4/CHI4 were constructed and compared with HSA7 revealing five main conserved segments and complex chromosome rearrangements, including a centromere repositioning, differentiating HSA7 and BTA4/OAR4/CHI4.

Susceptibility to ozone-induced airway inflammation is associated with decreased levels of surfactant protein D.

BACKGROUND: Ozone (O3), a common air pollutant, induces exacerbation of asthma and chronic obstructive pulmonary disease. Pulmonary surfactant protein (SP)-D modulates immune and inflammatory responses in the lung. We have shown previously that SP-D plays a protective role in a mouse model of allergic airway inflammation. Here we studied the role and regulation of SP-D in O3-induced inflammatory changes in the lung. METHODS: To evaluate the effects of O3 exposure in mouse strains with genetically different expression levels of SP-D we exposed Balb/c, C57BL/6 and SP-D knockout mice to O3 or air. BAL cellular and cytokine content and SP-D levels were evaluated and compared between the different strains. The kinetics of SP-D production and inflammatory parameters were studied at 0, 2, 6, 12, 24, 48, and 72 hrs after O3 exposure. The effect of IL-6, an O3-inducible cytokine, on the expression of SP-D was investigated in vitro using a primary alveolar type II cell culture. RESULTS: Ozone-exposed Balb/c mice demonstrated significantly enhanced acute inflammatory changes including recruitment of inflammatory cells and release of KC and IL-12p70 when compared with age- and sex-matched C57BL/6 mice. On the other hand, C57BL/6 mice had significantly higher levels of SP-D and released more IL-10 and IL-6. Increase in SP-D production coincided with the resolution of inflammatory changes. Mice deficient in SP-D had significantly higher numbers of inflammatory cells when compared to controls supporting the notion that SP-D has an anti-inflammatory function in our model of O3 exposure. IL-6, which was highly up-regulated in O3 exposed mice, was capable of inducing the expression of SP-D in vitro in a dose dependent manner. CONCLUSION: Our data suggest that IL-6 contributes to the up-regulation of SP-D after acute O3 exposure and elevation of SP-D in the lung is associated with the resolution of inflammation. Absence or low levels of SP-D predispose to enhanced inflammatory changes following acute oxidative stress.

Development and activity-dependent expression of neuronal marker proteins in organotypic cultures of rat visual cortex.

We are interested in activity-dependent mechanisms which govern the structural and functional maturation of neurons in the visual cortex. We have asked whether the expression of neuronal markers microtubule-associated proteins tau, MAP-2, synaptophysin (p38), and the growth-associated protein GAP-43 are dependent on cortical afferents or spontaneous activity. As a model system we have employed organotypic monocultures of rat visual cortex (OTCs, isolated from subcortical structures) in comparison with visual cortex in vivo (innervated by thalamic and other afferents) at different postnatal ages. We know from previous work that the OTCs, like the cortex in vivo, display a high rate of spontaneously generated action potentials. Therefore, as a third objective, we have analysed OTCs grown as monocultures under chronic blockade of spontaneous action potentials. Protein expression was detected by protein blots and/or immunohistochemistry. The proteins examined in this study are expressed in OTCs, even when grown under activity blockade. However, the pattern of expression differs from the cortex in vivo. Tau is expressed much weaker in OTCs than in cortex in vivo. The expression of the major band of about 50 kDa increases over time in vivo and in OTCs. Smaller isoforms of tau are dramatically downregulated, and larger (adult) isoforms do not appear within 35 days in vitro (DIV). Under activity blockade the expression of tau reaches a maximum by 21 DIV and decreases dramatically, so that the protein is hardly detectable by 47 DIV. MAP-2-immunoreactive proteins are localized in somata and dendrites, but also persist in axons. The expression in OTCs of p38 and GAP-43 correlates well with the expression observed in vivo. Synaptophysin (p38) occurs with a similar time course and amount in OTCs as in cortex in vivo. Synaptic boutons appear in all layers, and specialized terminal elements have been observed. Activity blockade slightly affects the p38 expression, although the late postnatal decline in p38 immunoreactivity observed on protein blots from cortex in vivo and in normal OTCs appears more accentuated in activity-blocked OTCs. The GAP-43 expression is prominent from birth onwards in vivo and in OTCs. However, in normal OTCs GAP-43 is not declining as it is in vivo, although it is downregulated in activity-blocked OTCs. As a major finding we report that neuronal markers which are normally expressed in immature neurons and axons during the period of differentiation and structural plasticity are continuously expressed in OTCs, suggesting that a monocultured cortex retains the ability for growth and structural changes longer than the cortex in vivo.

Oligodendrocytes differentiate in organotypic cultures of rat visual cortex and myelinate efferent axons.

We have investigated the presence and function of glia cells, especially of oligodendrocytes (OL) in organotypic cultures of rat visual cortex grown for 1-6 weeks in vitro. OL identified by strong Galactocerebroside-immunoreactivity (GalC-ir) displayed rather small somata and elaborately ramified processes. They were most concentrated in layers VIa and VIb and the remnant of the white matter. Silver staining revealed long descending or oblique processes in layers V and VI, which were often arranged in patches, and horizontal processes in the white matter. Proximal processes of OL cell bodies were connected to these long processes. DiI-labeling revealed very similar patches of processes, termed OL domains. They were identified as membraneous sheaths formed by processes of single OL around axons passing the OL domain. Confocal microscopy revealed single axons running through the membrane sheaths. We compared the molecular differentiation of glial cells in cultures to the in vivo situation with protein blots and immunohistochemistry for glial cell marker molecules. In homogenates of visual cortex in vivo, protein blots revealed the increase in expression by OL of myelin basic protein (MBP) during the fourth postnatal week. The astrocytic marker glial fibrillary acidic protein (GFAP), blotted as a control, increased over time in vivo, beginning at P14, indicating the differentiation of astrocytes. In homogenates of organotypic cortex cultures, the times course of expression of GFAP was very similar: it increased dramatically during the first 10 DIV, and remained fairly constant in older cultures.(ABSTRACT TRUNCATED AT 250 WORDS)