The immunolocalization of ACTH and alpha MSH in human and rat pituitaries.

Human and rat pituitaries were investigated immunohistochemically for ACTH and alpha MSH activity by means of the enzyme-labeling technique. In rat pituitaries cells present in both the anterior and intermediate lobes were reactive with the anti-ACTH antibodies, the cells from the intermediate lobe were also reactive with anti-alpha MSH antibodies. In human pituitaries, ACTH-immunoreactivity was found in cells from the anterior lobe and cells invading the posterior lobe. In 5 out of 15 pituitaries ACTH-immunoreactive cells located at or invading the posterior lobe were also reactive with the anti-alpha MSH antibodies. It is concluded that the human pituitary cells that invade the posterior lobe represent a population which is at least immunohistochemically identical with the intermediate lobe cells of the rat. The ACTH-immunoreactivity of intermediate lobe cells may be explained by the presence of a corticotropin-like intermediate lobe peptide (CLIP) which has been suggested to be a prohormonal fragment of alpha MSH.     

Comparative immunoenzymatic localization of prolactin and growth hormone in human and rat pituitaries.

Twelve human and twelve rat pituitaries were stained by an immunohistochemical method using a rabbit anti-ovine prolactin serum, a rabbit anti-human growth hormone serum and a sheep anti-rabbit immunoglobulin serum conjugated with horseradish peroxidase. On the same pituitary section, growth hormone cells were stained brown by using 3-3'-diaminobenzidine as peroxidase substrate, and prolactin cells were stained purplish blue by using 4-chloro-1-naphtol. Growth hormone cells outnumbered prolactin cells, especially in human pituitaries where the proportion is at least 10:1. No cells containing both brown granules stained for growth hormone and blue granules stained for prolactin were found in any of the sections examined. In the fetal pituitaries, there was no apparent hypertrophy of the prolactin cells, although the circulating levels of the hromone are known to be as high in the fetus at term as in the mother and much higher than in nonpregnant women.     

Fluorochrome-coupled lectins reveal distinct cellular domains in human epidermis

The distribution of saccharide moieties in human interfollicular epidermis was studied with fluorochrome-coupled lectins. In frozen sections Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin I (RCAI), and wheat germ agglutinin (WGA) stained intensively both dermis and viable epidermal cell layers, whereas peanut agglutinin (PNA) bound only to living epidermal cell layers. Ulex europaeus agglutinin I (UEAI) bound to dermal endothelial cells and upper cell layers of the epidermis but left the basal cell layer unstained. Dolichos biflorus agglutinin (DBA) bound only to basal epidermal cells, whereas both soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) showed strong binding to the spinous and granular cell layers. On routinely processed paraffin sections, a distinctly different staining pattern was seen with many lectins, and to reveal the binding of some lectins a pretreatment with protease was required. All keratin-positive cells in human epidermal cell suspensions, obtained with the suction blister method, bound PNA, whereas only a fraction of the keratinocytes bound either DBA or UEAI. Such a difference in lectin binding pattern was also seen in epidermal cell cultures both immediately after attachment and in organized cell colonies. This suggests that in addition to basal cells, more differentiated epidermal cells from the spinous cell layer are also able to adhere and spread in culture conditions. Gel electrophoretic analysis of the lectin-binding glycoproteins in detergent extracts of metabolically labeled primary keratinocyte cultures revealed that the lectins recognized both distinct and shared glycoproteins. A much different lectin binding pattern was seen in embryonic human skin: fetal epidermis did not show any binding of DBA, whereas UEAI showed diffuse binding to all cell layers but gave a bright staining of dermal endothelial cells. This was in contrast to staining results obtained with a monoclonal cytokeratin antibody, which showed the presence of a distinct basal cell layer in fetal epidermis also. The results indicate that expression of saccharide moieties in human epidermal keratinocytes is related to the stage of cellular differentiation, different cell layers expressing different terminal saccharide moieties. The results also suggest that the emergence of a mature cell surface glycoconjugate pattern in human epidermis is preceded by the acquisition of cell layer-specific, differential keratin expression.     

Distribution and levels of cellular retinol- and cellular retinoic acid-binding protein in various types of rat testis cells

The distribution and levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) were measured in rat testicular peritubular and Sertoli cells and in isolated rat pachytene spermatocytes and spermatids. Two Sertoli cell preparations, one containing some germ cells and another that had been osmotically shocked to destroy germ cells, were examined. CRBP and CRABP levels were measured by specific and sensitive radioimmunoassays. Testicular peritubular cell cytosol preparations were found to contain high levels of CRBP (1.48 +/- 0.87 microgram CRBP/mg protein) but CRABP could not be detected. The mean CRBP level in Sertoli cell preparations that contained some germ cells was 0.93 +/- 0.24 microgram CRBP/mg protein; this value was similar to the level of 1.11 +/- 0.20 microgram CRBP/mg protein measured for Sertoli cells free of germ cells. The level of CRABP found in Sertoli cell preparations containing germ cells (0.81 +/- 0.32 microgram CRABP/mg protein) was approximately five times greater than was observed in Sertoli cells free of germ cells (0.16 +/- 0.03 microgram CRABP/mg protein). CRBP and CRABP levels in cultured Sertoli cells were not affected by time in culture for up to five days of culture. Pachytene spermatocytes and spermatids were very enriched in CRABP (0.72 +/- 0.26 microgram CRABP/mg protein for spermatocytes and 0.65 +/- 0.21 microgram CRABP/ml protein for spermatids). A search for a high molecular weight retinol-binding protein did not demonstrate the existence of such a protein in Sertoli cell-conditioned medium. In summary, these studies provide quantitative information about the distribution of the cellular retinoid-binding proteins in the cell types that compose the rat testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of beta-endorphin-(1-9) from human and rat pituitaries

Using a radioimmunoassay specific for the carboxyl terminus of beta-endorphin-(1-9) large amounts of beta-endorphin-(1-9)-immunoreactive material was detected in the human pituitary. The major peak of immunoreactivity was purified and characterized by fast atom bombardment-mass spectrometry and Edman degradation sequencing as authentic beta-endorphin-(1-9). In the rat pituitary the highest concentration of beta-endorphin-(1-9) immunoreactivity was in the posterior neurointermediate lobe. This material was identified as N-acetyl beta-endorphin-(1-9) by multiple radioimmunoassays, gel chromatography, and reversed-phase high-performance liquid chromatography. Control experiments determined that beta-endorphin-(1-9) was not formed postmortem or during the extraction procedure. These studies suggest that single lysine residues, similar to single arginine residues, are potential sites of posttranslational processing.     

Widespread cellular distribution of aldehyde oxidase in human tissues found by immunohistochemistry staining

Aldehyde oxidase (EC 1.2.3.1) is a xenobiotic metabolizing enzyme that catalyzes a variety of organic aldehydes and N-heterocyclic compounds. However, its precise pathophysiological function in humans, other than its xenobiotic metabolism, remains unknown. In order to gain a better understanding of the role of this enzyme, it is important to know its exact localization in human tissues. In this study, we investigated the distribution of aldehyde oxidase at the cellular level in a variety of human tissues by immunohistochemistry. The enzyme was found to be widespread in respiratory, digestive, urogenital, and endocrine tissues, though we also observed a cell-specific localization in the various tissues studied. In the respiratory system, it was particularly abundant in epithelial cells from the trachea and bronchium, as well as alveolar cells. In the digestive system, aldehyde oxidase was observed in surface epithelia of the small and large intestines, in addition to hepatic cells. Furthermore, the proximal, distal, and collecting tubules of the kidney were immunostained with various intensities, while glomerulus tissues were not. In epididymus and prostate tissues, staining was observed in the ductuli epididymidis and glandular epithelia. Moreover, the adrenal gland, cortex, and notably the zona reticularis, showed strong immunostaining. This prevalent tissue distribution of aldehyde oxidase in humans suggests some additional pathophysiological functions besides xenobiotic metabolism. Accordingly, some possible roles are discussed.     

Indomethacin and gonadotrophin release from rat pituitaries.

The influence of low (5 mcM) and high (200 mcM) concentrations of indomethacin on gonadotropin release from rat pituitaries was studied in vitro. Low concentrations significantly (p less than .05) reduced the release of luteinizing hormone (LH) in comparison with controls, whereas high concentrations significantly (p less than .05) increased the rate of release. The release of follicle stimulating hormone (FSH) was not affected. When pituitary tissue was stimulated by LH-releasing hormone (LH-RH), the high concentration of indomethacin significantly (p less than .05) increased LH release and produced a nearly significant (p less than .01) increase in FSH. The low concentration was without effect. The effect of the addition of prostaglandins (PGs) alone and in combination with indomethacin was also investigated. PGE-1 significantly (p less than .05) increased the release of LH. However, there was no significant (p greater than .1) interaction between the 2 drugs. The effects on the release of FSH were similar. The addition of PGE-2 or PGF-2alpha slightly increased the release of LH and FSH (p greater than .1). It is suggested that high concentrations of indomethacin probably do not inhibit PG synthesis, but may inhibit cyclic nucleotide phosphodiesterase.     

Quantification of the cellular proliferation on freshly dispersed cells from rat anterior pituitaries after in vivo and in vitro labelling with bromodeoxyuridine.

Summary The labelling index i.e., the proportion of cells in S phase of the cell cycle, has been calculated in cytospin preparations of rat anterior pituitary cells after labelling eitherin vivo orin vitro with the thymidine analogue bromodeoxyuridine (BrdU). The aims of this work were (1) to check whether enzymatic digestion interferes with the incorporation of BrdU into S phase cells and/or whether it has any deletereous effect on the immunohistochemical detection of cells that have already incorporated BrdU, and (2) to check the viability of simultaneous staining for BrdU and markers for the different types of pituitary cells in the cytospins. No statistical difference was found between the labelling index afterin vivo orin vitro labelling with BrdU. Identification of doubly-immunostained cells was straightforward and up to 40% of BrdU-labelled cells were immunopositive for pituitary hormones. It is suggested that cytospin preparations from biopsy samples may be used to study cellular proliferation without exposing the patient to the hazardous effects of BrdU infusion and without the interference of cell culture methods.     

Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Polη, Polι and Polκ, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Polη has three PCNA-interacting protein (PIP) boxes (PIP1, 2, 3) that contribute differentially to two distinct functions, stimulation of DNA synthesis and promotion of PCNA ubiquitination. The latter function is strongly associated with formation of nuclear Polη foci, which co-localize with PCNA. We also show that Polκ has two functionally distinct PIP boxes, like Polη, whereas Polι has a single PIP box involved in stimulation of DNA synthesis. All three polymerases were additionally stimulated by mono-ubiquitinated PCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Polη exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases.     

Monoclonal antibody LICR-LON-E36 recognizes gonadotrophs in female rat pituitaries

The distribution of the antigen localized by monoclonal antibody LICR-LON-E36 has been studied by means of light and electron microscopical immunocytochemical procedures on resin-embedded pituitaries from male and female rats. Using both immunoperoxidase and immunogold labeling techniques, the localization of LICR-LON-E36 has been compared with that obtained with antibodies against the beta subunit of rat luteinizing hormone (beta LH) and human follicle stimulating hormone (beta FSH), porcine adrenocroticotropic hormone (ACTH) and rat S-100. LICR-LON-E36 is localized in a portion of beta LH-containing cells in female rat pituitaries and where present both antigens are localized within the same storage granules. LICR-LON-E36 is rarely detectable within beta LH-containing cells of male rat pituitaries that also stain positively with anti-beta FSH antibodies. ACTH and S-100 were localized within different cell populations.     

Mating types in cellular slime molds

We have discovered mating types in three species of cellular slime mold. One of these species is $\text{Dictyostelium}\text{discoideum}$, hitherto the subject of extensive biochemical investigations, and now amenable to genetic studies.     

Two immunologically distinct human DNA polymerase alpha-primase subpopulations are involved in cellular DNA replication

Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase alpha-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G1, whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G2. Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2'-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork.     

Double-labeling immunogold electron-microscopic study of hormonal colocalization in nontumorous and adenomatous rat pituitaries

According to the one cell, one hormone theory, the pituitary gland is composed of 5 cell types which secrete 6 hormones. Recent investigations indicate that this theory must be modified, as there are some bihormonal cells containing 2 hormones, i.e., mammosomatotrophs prolactin-growth hormone (PRL-GH). This study was undertaken in order to elucidate whether other adenohypophysial cells are capable of producing 2 hormones and to demonstrate the presence of cells coexpressing PRL-GH, PRL-thyrotropin (TSH), or TSH-GH. Sixteen nontumorous and 16 adenomatous male and female Sprague-Dawley and Long Evans rat pituitaries were removed immediately after the animals were killed and processed for transmission electron microscopy and the immunogold double-labeling technique. Coexpression of PRL-GH, PRL-TSH, and TSH-GH was found in both nontumorous and adenomatous pituitaries. Double labeling was present not only in the same cell cytoplasm but also in the same secretory granules. The question of whether these double-labeled cells represent different cell populations, transitional cell types, or precursor cells requires further investigation.