Mapping the conditions of spider silk proteins along the silk gland, and combining with molecular studies, reveals a pH controlled switch between lock and trigger forms, providing insights into spider silk formation.

The localization of carbonic anhydrase by histochemistry, of Na-K-ATPase by immunocytochemistry and of rod-shaped intramembranous particles by freeze-fracture electron microscopy, was determined in the collecting duct of rabbits. In the cortical collecting duct (CCD), rod-shaped particles, which are abundant in intercalated cells were observed in both the apical and basolateral membrane of all intercalated cells examined. In the outer stripe of the outer medullary collecting duct (OMCDo) a high density of rod-shaped particles was found only in the apical membrane of intercalated cells. All cells of the inner stripe of the outer medullary collecting duct (OMCDi) had rod-shaped particles in the apical membrane but not in the basolateral membrane. As the collecting duct entered the inner medulla the density of rod-shaped particles decreased until they were virtually absent in the terminal segment. Na-K-ATPase, localized to the basolateral membrane, was more abundant in principal cells than in intercalated cells in the CCD. In the OMCDo, staining was equal in principal and intercalated cells. All cells of the OMCDi and the inner medullary collecting duct (IMCD) stained for Na-K-ATPase. Carbonic anhydrase in the CCD was localized to the cell membranes and cytoplasm of intercalated cells. Principal cells did not stain for carbonic anhydrase. A similar pattern was seen in the OMCDo. In the outer region of the OMCDi most cells did not stain for carbonic anhydrase, whereas in the inner region the apical and lateral membranes of all cells stained for carbonic anhydrase. Weak cytoplasmic staining was occasionally seen. A similar pattern was seen in the initial half of the IMCD, while the terminal half of the IMCD did not stain. In this study, the localization of enzymes and rod-shaped intramembranous particles associated with Na+, K+, and H+ transport shows both segmental and cellular heterogeneity, and correlates with the known transport properties of tubule segments. The distribution of these enzymes and rod-shaped intramembranous particles is different in rabbits and rats, and may explain some of the functional differences between homologous segments in these species.

The rat kidney was studied by light and electron microscope after it was histochemically stained for carbonic anhydrase activity. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed intense activity at the brush border and the basolateral membranes. Cytoplasmic activity also was found. Straight proximal tubules had considerable enzyme activity at basolateral membranes but only low activity at the brush border and in the cytoplasm. In nephrons with long loops, the descending thin limb contained cytoplasmic enzyme activity, whereas the ascending thin limb was inactive. Thin limbs of short loops showed a varying enzyme pattern. In the thick limb of Henle's loop, most enzyme activity was found at the luminal cell border. Distal convoluted tubules showed enzyme activity only at basal infolded membranes. In the late distal tubule, intercalated cells appear among the "ordinary" distal cells, and they contained abundant cytoplasmic enzyme. Many highly active intercalated cells were found also in the cortical and outer medullary segments of the collecting duct. The chief cells in these segments also showed some cytoplasmic enzyme activity. In the inner medullary segment of the collecting duct, enzyme activity disappeared gradually, and the tip of the papilla lacked activity. Acetazolamide (10 microM) completely abolished visible staining, whereas Cl 13850 (10 microM), an inactive acetazolamide analogue, did not interfere with the staining.

Spider silk is made of unique proteins-spidroins-secreted and stored as a protein solution (dope) in specialized glands. The major ampullate gland, source of the dragline silk, is composed of a tail, a sac and an elongated duct. For this gland, several different types of epithelial cells and granules have been described, but it is largely unknown how they correlate with spidroin production. It is also not settled what parts of the large spidroins end up in the final silk, and it has been suggested that the N-terminal domain (NT) is lacking. Here we show that NT is present in the dope and throughout dragline silk fibers, including the skin layer, and that the major ampullate tail and sac consist of three different and sharply demarcated zones (A-C), each with a distinct epithelial cell type. Finally, we show that spidroins are produced in the A and B zone epithelia, while the C zone granules lack spidroins.

Eggshell thinning among wild birds has been an environmental concern for almost half a century. Although the mechanisms for contaminant-induced eggshell thinning are not fully understood, it is generally conceived to originate from exposure of the laying adult female. Here we show that eggshell thinning in the domestic hen is induced by embryonic exposure to the synthetic oestrogen ethynyloestradiol. Previously we reported that exposure of quail embryos to ethynyloestradiol caused histological changes and disrupted localization of carbonic anhydrase in the shell gland in the adult birds, implying a functional disturbance in the shell gland. The objective of this study was to examine whether in ovo exposure to ethynyloestradiol can affect eggshell formation and quality in the domestic hen. When examined at 32 weeks of age, hens exposed to ethynyloestradiol in ovo (20 ng/g egg) produced eggs with thinner eggshells and reduced strength (measured as resistance to deformation) compared with the controls. These changes remained 14 weeks later, confirming a persistent lesion. Ethynyloestradiol also caused a decrease in the number of shell gland capillaries and in the frequency of shell gland capillaries with carbonic anhydrase activity. These data suggested that a disrupted carbonic anhydrase expression was involved in the mechanism for the oestrogen-induced eggshell thinning found in this study. The results support our hypothesis that eggshell thinning in avian wildlife can result from a structural and functional malformation in the shell gland, induced by xeno-oestrogen exposure during embryonic development.