Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy

We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS). Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy. In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background. The sensitivity of detection was much higher than that of brightfield microscopy. Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension. However, non-specific staining was also better visualized. The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions. A 25% reduction of the silver enhancement time was necessary for this purpose. However, these weaker labeling conditions also reduced the intensity of the specific staining. Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure. Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.     

Mechanical role of expiratory muscles during breathing in upright dogs

To examine the mechanical effects of the abdominal and triangularis sterni expiratory recruitment that occurs when anesthetized dogs are tilted head up, we measured both before and after cervical vagotomy the end-expiratory length of the costal and crural diaphragmatic segments and the end-expiratory lung volume (FRC) in eight spontaneously breathing animals during postural changes from supine (0 degree) to 80 degrees head up. Tilting the animals from 0 degree to 80 degrees head up in both conditions was associated with a gradual decrease in end-expiratory costal and crural diaphragmatic length and with a progressive increase in FRC. All these changes, however, were considerably larger (P less than 0.005 or less) postvagotomy when the expiratory muscles were no longer recruited with tilting. Alterations in the elastic properties of the lung could not account for the effects of vagotomy on the postural changes. We conclude therefore that 1) by contracting during expiration, the canine expiratory muscles minimize the shortening of the diaphragm and the increase in FRC that the action of gravity would otherwise introduce, and 2) the end-expiratory diaphragmatic length and FRC in upright dogs are thus actively determined. The present data also indicate that by relaxing at end expiration, the expiratory muscles make a substantial contribution to tidal volume in upright dogs; in the 80 degrees head-up posture, this contribution would amount to approximately 60% of tidal volume.     

Tricyclic Imipramine Modification of the Circadian Rhythms of Hypothalamic Serotonin, its Precursors and Acid Catabolite in Individually Housed Rats

Circadian rhythms of serotonin (5HT), its precursors tryptophan (TP) and 5-hydroxy-tryptophan (5HTP) and its acid catabolite 5-hydroxy-indoleacetic acid (5HIAA), were determined in the hypothalamus of control rats and rats which had been treated continuously with subcutaneous imipramine (10 mg/kg/day) for 2 weeks.

Rats were individually housed and entrained to LD12:12. Controls showed the 5HT and TP peaks in the light and dark periods respectively, as reported in the literature, but no inverted correlation (antiphase) between SHT and 5HIAA rhythms.

Imipramine significantly modified circadian rhythm characteristics: the 5HT acrophase was advanced, that of TP and 5HIAA was delayed. Imipramine also significantly increased hypothalamic SHT and TP concentrations.     

The role of morphometry in the cytology of well-differentiated hepatocarcinoma and cirrhosis with atypia

A morphometric study was performed on 200 nuclei per case in six well-differentiated hepatocarcinomas and in six cirrhoses with cytologic atypia, using samples obtained by fine needle aspiration (FNA) biopsy of the liver. The parameters measured were the nuclear area, the nuclear perimeter and the maximum nuclear diameter. The nuclei of well-differentiated hepatocarcinomas could be distinguished from those of cirrhoses on the basis of the larger size and greater anisonucleosis of the former. A statistical analysis (using a two-sided t-test) of the means of the parameters showed significant differences between the two diagnostic groups. These results suggest that morphometric analysis can help in the differential diagnosis between well-differentiated hepatocellular carcinoma and cirrhosis with cytologic atypia in FNA biopsy samples.     

Microspectrofluorimetric analysis of the formaldehyde-induced fluorophores of 5-hydroxytryptamine and dopamine in intrapulmonary neuroepithelial bodies after administration ofl-hydroxytryptophan andl-DOPA

Summary To demonstrate the intracellular store of 5-hydroxytryptamine and dopamine in pulmonary neuroepithelial bodies of the neonatal rabbit after treatment with the corresponding amino-acid precursorsl-5-hydroxytryptophan orl-3,4-dihydroxyphenylalanine, formaldehyde-induced flourescence in combination with microspectrofluorimetric analysis has been used. Emission spectra and excitation spectra in an extended wavelength range from 240 to 460 nm, the displacement of excitation peaks after exposure to hydrochloric acid vapour, and calculation of peak ratio values 410/260, 380/320, 320/260 for phenylethylamine fluorophores and 385/315 for indolylethylamine fluorphores were performed. Thus, the presence of 5-hydroxytryptamine without occurrence of 5-hydroxytryptophan was demonstrated in pulmonary neuroepithelial bodies after administration of the corresponding biological precursor, while dopamine combined with 5-hydroxytryptamine were clearly revealed after administration ofl-3,4-dihydroxyphenylalanine. The rate of photodecomposition always corroborated these findings.Dedicated to Prefessor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by grant nr. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium)     

Cellular and molecular markers of anteroposterior and dorsoventral organisation in the vitellogenic follicles of adult Sarcophaga bullata (Diptera) and dorsoventral orientation of follicles in the ovary

Summary Polar organisation in the follicles of adult Sarcophaga bullata is reflected in the nurse cell-oocyte axis and in the orientation of the two polar cell pairs in the follicular epithelium. The internal organisation of the nurse cell chamber contributes to polarity but not to dorsoventral asymmetry. Dorsoventral asymmetry is correlated with the eccentric position of the germinal vesicle and the orientation of the polar cell pairs; no other follicle cell specialisations are seen. In an ovary, follicles are preferentially orientated with the dorsal side to the centre of the ovary. Cytoskeletal and some haemolymph proteins are molecular markers of polarity. Thus, in pre-vitellogenic stages, tubulin immunoreactivity is higher in the oocyte than in the nurse cells, actin immunoreactivity is the same over the cystocytes and larval serum proteins are restricted to the poles. During vitellogenesis, both actin and tubulin become more concentrated in the nurse cells and larval serum protein 1 accumulated in the polar cells during border cell migration when yolk polypeptides also accumulate in the oocyte. At the end of vitellogenesis a lipophorin is taken up by the oocyte. No molecular marker of dorsoventral asymmetry was identified.     

Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes

The synthesis and intracellular distribution of actin were studied in isolated dictyate and metaphase II mouse oocytes by (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of newly synthetized oocyte protein and (2) cytochemical F-actin labeling by fluorescent phalloidin. Unpermeabilized, fully grown oocytes bound phalloidin intensely at the level of the zona pellucida (ZP), such ZP-associated actin representing a significant portion of total actin found in these cells. In contrast, phalloidin binding to ZP was very low in growing oocytes and was undetectable in ovulated, metaphase II eggs. When ZP-associated actin of fully grown oocytes was removed by prolongedly exposing oocytes to α-chymotrypsin, the amount of newly synthesized actin displayed by cumulus-enclosed oocytes was reduced to a level comparable to that shown by oocytes isolated from granulosa cells. We demonstrate that ZP-associated actin belongs to granulosa cell processes that remain within the ZP as a consequence of oocyte isolation procedures. We conclude that actin synthesis of mouse oocytes is not regulated by granulosa cells.     

Interaction of opioid peptides and other drugs with multiple kappa receptors in rat and human brain. Evidence for species differences.

Previous experiments resolved four kappa binding sites in guinea pig brain termed kappa 1a, kappa 1b, and kappa 2b. The present study was undertaken to examine the occurrence of kappa receptor subtypes in rat and human brain. [3H]U69,593 and [3H]bremazocine were used to label kappa 1 and kappa 2 binding sites, respectively, present in brain membranes depleted of mu and delta binding sites by pretreatment with the irreversible ligands, BIT and FIT. Low levels of [3H]U69,593 binding precluded a detailed quantitative study of kappa 1 binding sites in these species. Quantitative examination of [3H]bremazocine binding resolved two kappa 2 binding sites in both rat and human brain whose ligand selectivity patterns differed from that of the guinea pig. These observations suggest that there may be considerable variation in the ligand recognition site of kappa receptor subtypes among mammalian species.     

Bovine pancreatic trypsin inhibitor and homologous polypeptide inhibitors in nephron cells.

Bovine pancreatic trypsin inhibitor (BPTI, aprotinin) is a fifty-eight amino acid polypeptide, which is present together with related molecular isoforms in various bovine organs. In the present study these protease inhibitors were isolated from bovine kidney by affinity chromatography on immobilized trypsin and a subsequent FPLC step. Due to their electrophoretic, structural, and inhibitory properties, the inhibitors were strictly similar to the polypeptides identified previously in other bovine organs. Immunohistochemical experiments showed a widespread localization of these polypeptides in nephron epithelial cells (proximal and distal tubules, loop of Henle, collecting tubules).