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Pretreatment of sections of fixed tissue with selective blocking reagents for indigenous thiol groups did not, it was found, impair the fluorescence subsequently obtainable with the acetic anhydridesalicylhydrazide-zinc technique (Stoward and Burns, 1967) for localizing C-terminal carboxyl groups of proteins. This suggests that thiol and S-acetyl groups do not participate in the complex reactions involved in the method. In further support of this suggestion it was found that artificially introduced thiol groups also do not take part. Sites containing either, glycogen or neutral periodate-reactive mucosubstances did not fluoresce in sections subjected to the technique. This indicates that O-acetyl groups, although probably formed, are not involved in the reactions giving rise to the fluorescence reaction in proteins.

Neurothelin/HT7, a transmembrane glycoprotein of the immunoglobulin superfamily, is a marker of blood–brain barrier (BBB)-forming endothelial cells. We have studied the expression of neurothelin in tumors grown on the chorioallantoic membrane (CAM) of chick embryos. We inoculated each 3–5×106 rat C6 glioma, rat 10AS pancreatic carcinoma, human A375 melanoma, and human mammary duct adenoma cells on the CAM of 10-day-old chick embryos. The tumors were harvested on day 17. All four tumor cell lines formed solid tumors which were supplied by vessels of CAM origin. Foci of bleeding were regularly observed within the tumors. All four tumors induced the expression of neurothelin/HT7 (but not of glucose transporter-1) in tumor endothelial cells, whereas expression in adjacent endothelial cells of normal CAM did not occur. Confocal laser scanning microscopy revealed that the pattern of neurothelin expression in tumor endothelial cells was different from that in normal central nervous system (CNS) endothelium, but the relative molecular weight of neurothelin, studied by western blot analysis, was the same in brain and in tumors. It has been shown that, with increasing malignancy, vessels of CNS tumors lose their morphological characteristics, and BBB markers such as the glucose transporter-1 are downregulated. Our results show that, in contrast, the BBB marker, neurothelin, is expressed de novo in tumor endothelial cells. Potential common functions of neurothelin in endothelial cells of the CNS and tumors are discussed.

Flow microfluorometric techniques have been applied to experiments concerned with the penetration of cells by lipid vesicles. The high sensitivity of laser flow systems enables to measure the weak fluorescence emitted by individual liposomes tagged with perylene, inserted into their multilamellar layer. The total fluorescence of cells which have incorporated such perylene-loaded liposomes could be measured and well separated from that of unbound liposomes. Significant differences in the incorporation rates of cationic and anionic liposomes were shown by means of time-course analyses of cellular fluorescence spectra. The advantages of the rapid data analysis by flow fluorescence techniques is discussed in comparison with conventional radio-isotopic methods.

The localization of proteoglycans in rat epiphyseal growth plate cartilage was investigated immunoelectron microscopically by the post-embedding method, using mouse monoclonal antibody (2-B-6) which specifically recognizes 4-sulphated chondroitin or dermatan sulphate after digestion of proteoglycans with chondroitinase ABC. Fixation with ruthenium hexamine trichloride (RHT) and embedding in LR White served to preserve chondrocytes in the expanded state and matrix proteoglycans were observed as a reticular network of filaments. Immunoelectron microscopy revealed gold labelling of the secondary antibodies for the demonstration of proteoglycans on these filamentous structures and in elements of the Golgi apparatus. Filaments associated with matrix vesicles were also labelled. After fixation in the presence of RHT, it was clearly demonstrated that cartilage matrix proteoglycans are retained approximately in their original spatial distribution and their antigenicity is well preserved.

As a part of a microfluorometric investigation of the nucleoproteins of nuclei whose chromatin displays varying degrees of condensation, a comparison was made of mouse small thymocyte and hepatocyte nuclei stained with the acidic dye, brilliant sulfaflavine, at pH 2.8. These estimates of total protein content were compared with measurements obtained in similarly stained nuclei after extraction either with 0.4 N H2SO4 to remove all histones or with 0.35 M NaCl to remove nucleoplasmic proteins and some loosely bound non-histone chromosomal proteins. Treatment with 5% TCA at 60°C was used to remove nucleic acids and to reverse the effects of formaldehyde fixation. In all instances, the fluorescence of 2c hepatocyte nuclei greatly exceeded that of similarly treated thymocyte nuclei. While extraction with 0.4 N H2SO4 resulted in reductions of as much as 75% of the total fluorescence of small thymocyte nuclei, the losses of fluorescence in 2c hepatocyte nuclei amounted to only 20–30%. Nevertheless, the absolute values of fluorescence lost in both types of nuclei were very similar. After extraction in 0.35 M NaCl, thymocyte nuclei displayed slightly greater fluorescence than control thymocyte nuclei, while the total fluorescence of hepatocyte nuclei declined. In hepatocyte nuclei extracted with TCA, with and without treatment with 0.35 M NaCl, two populations of diploid nuclei were apparent: one corresponding to parenchymal cell nuclei and the other comprised of non-parenchymal cell nuclei. Only single diploid populations were visible in acid-extracted material. The ratios of 4c∶2c, 8c∶4c, and 8c∶2c hepatocyte nuclei in control, acid-extracted, and NaCl-extracted groups were generally lower than the expected 2∶2∶4 values. These results indicate that total nuclear histones may be estimated microfluorometrically by computing the difference between acid-extracted and unextracted preparations treated in otherwise equivalent ways. In addition, despite very similar absolute losses of fluorescence after removal of histones in thymocyte and 2c hepatocyte nuclei, the proportion of total protein ascribable to histones is much greater in thymocyte nuclei than in 2c hepatocyte nuclei — or, conversely, the percentage of total protein attributable to non-histone proteins is much greater in 2c hepatocyte nuclei than in thymocyte nuclei.