ALBERT

Notes: Summary Calmodulin is a regulator of several calcium-dependent cellular processes. It has been suggested that it plays a role in the mechanism of secretion. Employing an indirect immunoperoxidase technique at the light microscope level, this study demonstrates the presence of calmodulin in several exocytotic cells (mast cells, thyroid follicular cells, neurohypophyseal neurosecretory terminals, pancreaticβ-cells and pancreatic acinus cells) in rat and man. The positive staining reaction for calmodulin was granular and at least in the case of rat mast cells it appeared to be associated with the granule membrane.

Notes: Summary Electron microscopic studies of sodium transporting epithelia from frog skin, sheep choroid plexus, rabbit gallbladder and small intestine, and rat kidney revealed the presence of a complex intracellular system of tubulo-cisternal endoplasmic reticulum which appeared to connect apical (luminal) and baso-lateral cell surfaces. The system was present in the tight epithelium of frog skin but was most abundant in leaky epithelia with low transepithelial resistance and isotonic transport. The basic structural features of the system and its relationship with some associated components are described. Our result, coupled with preliminary physiological studies, indicate that developmental and seasonal (hormone-induced) changes in the configuration of the tubulo-cisternal endoplasmic reticulum may be closely correlated with specific changes in epithelial permeability. The findings are discussed in the light of the hypothesis that epithelia possess two sodium transporting systems: One based on pump sites in the plasma membrane producing a hypertonic transportate and another located in the membranes of the tubulo-cisternal endoplasmic reticulum which, due to its extensive surface, would be well suited for producing an isotonic transportate.

Notes: Summary When an isolated frog skin (Rana temporaria) is exposed to a hydrostatic pressure difference between inside and outside bathing solutions (inside pressure higher than outside) of 20–50 cm of H2O and if under these conditions the skin is short-circuited electrically, small “vacuoles” appear light-microscopically in the outermost living cell layer in the epithelium. The number of such “vacuoles” shows a linear dependency on the rate of active sodium transport as measured by the short-circuit current. Electron-microscopically, the “vacuoles” are interpreted as previously undescribed organelles, the “scalloped sacs” which are about 0.5 μ in diameter, with a wrinkled surface and bounded by a unit membrane. This organelle is in intimate contact with sacs and tubules of smooth endoplasmic reticulum. The observed increase in the number of scalloped sacs usually is accompanied by a significant expansion of the whole system of endoplasmic reticulum. Some of the “vacuoles” seen light-microscopically must indeed be expanded cisternae of endoplasmic reticulum. The findings are discussed in light of the possibility that the scalloped sacs and the endoplasmic reticulum may be involved in active transport of sodium ions.

Notes: Abstract A technique is described for enhancing the reaction product of the staining reaction for iron in paraffin-embedded tissue from central nervous system (CNS). After amplification of the Prussian Blue staining reaction with 3,3′-diaminobenzidine (DAB), the reaction product was further intensified using a stepwise treatment with silver methenamine, gold chloride and uranyl nitrate (post-DAB treatment). Following the Prussian Blue-DAB staining reaction, iron was seen only in glial cells and choroid plexus epithelial cells, whereas the post-DAB treatment revealed that neurons and endothelial cells of the brain capillaries were also positively stained. The post-DAB treatment resulted additionally in an increased intensity of the reaction product within choroid plexus epithelial cells compared to that obtained in sections subjected only to the Prussian Blue-DAB reaction. The reliability of the method was evaluated using liver sections as positive controls. Furthermore the higher sensitivity of the method was assessed using nitrocellulose filters containing serially diluted iron-saturated transferrin. The post-DAB method is simple and can easily be applied to formalin- or glutaraldehyde fixed, paraffin-embedded nervous and non-nervous tissue.

Notes: Abstract Tissue distribution and developmental expression of fetuin were studied in the sheep fetus from embryonic day (E) 30 to adult (gestational period is 150 days). The presence of fetuin was demonstrated immunocytochemically using anti-fetuin antibodies; in situ hybridisation using short anti-sense oligonucleotide probes labelled with digoxigenin was used to study the ability of the developing tissue to synthesise fetuin, and reverse transcription-polymerase chain reaction (RT-PCR) was used to estimate the level of fetuin mRNA in selected tissues. Tissue distribution of fetuin was widespread in the younger fetuses (E30 to E40). The most prominent presence due to in situ synthesis was demonstrated in the liver, central nervous system (CNS) including anterior horn cells, dorsal root ganglia and in skeletal muscle cells. Other developing tissues and organs that showed evidence of fetuin synthesis and presence of the protein included mesenchyme, kidney, adrenal, developing bone, gut, lung and heart. In the immature liver (E30–40) there was a strong signal for fetuin mRNA in hepatocytes and also in numerous haemopoietic cells; the proportion of these latter cells that was positive for fetuin mRNA increased between E30 and E40. Only some hepatocytes and a proportion of the haemopoietic stem cells were immunoreactive for fetuin itself at E30–40; immunoreactive hepatocytes were more frequently observed in the more mature outer regions of the developing liver. Lung and gut contained scattered fetuin-positive epithelial cells, especially at E30; a weak fetuin mRNA signal could be detected above background in many of these cells up to E40, but not at E60–E115 or in the adult. Particularly at E30 to E40, mesenchymal tissue both within organs such as the gut and lung and around forming bone and skeletal muscle contained cells that were positive for fetuin mRNA. Mesenchyme at these ages was also very strongly stained for fetuin protein, much of which may reflect fetuin in tissue extracellular spaces and be derived from the high concentration in plasma. By E80 fetuin mRNA was mainly present in the liver and the CNS; staining of the muscle tissue was becoming less pronounced. However in developing bone tissue, staining of chondrocytes for fetuin mRNA was still prominent in older (E80) fetuses; there was also fetuin protein staining of chondrocytes at the growing surfaces of bones and in bone marrow at this age. In the adult, weak immunocytochemical staining for fetuin itself was present in hepatocytes, but the mRNA signal was barely above the threshold limit of detection. Other tissues in the adult were generally negative for both fetuin mRNA and fetuin, except that fetuin could generally be detected immunocytochemically in precipitated plasma within vessels in many tissues and in their interstitial spaces. The highest levels of fetuin mRNA, as demonstrated by RT-PCR, were detected in E40 and E60 liver followed by E40 muscle. The very low level of fetuin mRNA in adult liver, evident from in situ hybridisation, was confirmed by RT-PCR (about 0.1% of that at E60). These results show that in many tissues in which fetuin could be demonstrated immunocytochemically, its presence is likely to be due to synthesis in situ. However in some instances (e.g. gut and mesenchymal tissue) fetuin probably originates predominantly by uptake from plasma or extracellular fluid. The functional significance of the presence of fetuin in different tissues during their development is considered.

Notes: Summary The specificity of Alcian blue staining has been investigated on a material comprising the adenohypophysis from 26 human foetuses. Two distinct types of alcianophilic cells were observed. The first cell type appears about 28 mm CRL forming small groups at the epithelial-mesenchymal junction and beneath the anlage of the fibrous capsule. Besides its alcianophilic property the cell shows a pronounced maltase-resistant PAS-positivity. The alcianophilia is due to carboxylgroups — probably of proteins as no sialic acid could be detected. By the performic acid — Alcian blue method the cell seems to show a high content of SH and S-S groups. The second type of cell appears about 70 mm CRL and is mostly located singly. It shows a pronounced alcianophilia — probably due to sulphate groups and to some extent to carboxyl groups. The role of the alcianophilic properties of the cells in typing definite cells of the foetal adenohypophysis was discussed. — Finally the pitfalls caused by different fixatives and by the use of the general polychrome staining methods for the typing of pituitary parenchymal cells were discussed.