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P Tuohimaa

Publications and source records attributed to P Tuohimaa.

At least 73 records · Page 4Linked to original sources
PubMed1994

Immunocytochemical localization of human 5 alpha-reductase 2 with polyclonal antibodies in androgen target and non-target human tissues.

We studied the tissue distribution and cellular localization of 5 alpha-reductase 2, the human prostatic isoenzyme, in different human tissues, both cryostat-sectioned and paraffin-embedded. Polyclonal antibodies raised in rabbits against a native peptide (C-terminal amino acids 229-254) or synthetic peptides (amino acids 234-245), either as carrier-conjugated linear peptides or multiple antigen peptides (MAP), were assayed for specificity and sensitivity with Western blotting and an ELISA system. One antibody showing monospecificity on Western blots and in ELISA was used for immunohistochemical detection of the respective antigen in tissues from male and female subjects. Positive cells were found (with decreasing intensity) in inner epithelial sheath of hair follicles, pyramidal cells of the cerebral cortex, hepatocytes and bile duct cells, prostate epithelial cells, seminal vesicle epithelial cells, endothelial cells of small vessels, fat cells, fibrocytes of genital and extragenital organs, and smooth muscle cells of prostate and seminal vesicles. Some variation in the immunoreactivity of testis and ovary tissue was seen with different antibodies. 5 alpha-Reductase 2 is obviously not restricted to androgen target organs in the male, but is present in a large number of cells and tissues in both males and females.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1993

Nuclear progesterone receptor is mainly heat shock protein 90-free in vivo.

Heat shock protein 90 (hsp90) is associated with many steroid receptors in tissue homogenates. It is widely accepted that hsp90 regulates the binding of the receptor to the corresponding gene regulatory element. However there is no unequivocal evidence that steroid receptor-hsp90 complexes are present in the intact cells. We demonstrate here the absence of progesterone receptor (PR)-hsp90 complexes in intact target cell nuclei, using immunohistochemical and biochemical methods to determine the location and composition of the nonliganded (aporeceptor) and liganded (holoreceptor) PR complexes. In the chicken oviduct cells, both apo- and holoreceptors were nuclear, while hsp90 was exclusively cytoplasmic. When expressed transiently in HeLa cells, hsp90 was detected in the cytoplasm and PR was detected in the nucleus. Their location or staining intensity was not affected when they were coexpressed in the same cells. To confirm that the sensitivity of the immunohistochemical detection of hsp90 and PR did not differ significantly, a chimeric hsp90-PR was transiently expressed in HeLa cells. Both hsp90 and PR antigens of the chimera were detected in nuclei with the same intensity. In homogenates of the same tissue samples that were used for immunohistochemistry, the PR was complexed with hsp90. Hsp90-PR complexes were formed in vitro when immature bursa of Fabricius, known to contain high levels of hsp90, was homogenized in the presence of hsp90-free aporeceptor, while holoreceptor did not associate with hsp90. Our data show that nuclear PR is not complexed with hsp90 in vivo and suggest that the 8S-PR may be an in vitro artifact generated during tissue processing.

Animals↗
PubMed1993

Immunohistochemical localization of the avian progesterone receptor and its candidate receptor binding factor (RBF-1).

An avian oviduct nuclear matrix protein in the 6-10 kDa size range has been implicated to function in the cell-free nuclear binding of the avian oviduct progesterone receptor (PR). This protein, termed the receptor binding factor-1 (RBF-1), has been purified and partially characterized [Schuchard et al.: Biochemistry 30:4535-4542, 1991]. This paper describes the immunohistochemical co-localization of the RBF-1 and PR in the avian oviduct cell nuclei and rat reproductive cell nuclei using antibodies directed specifically against the RBF-1 and activated PR. In the undifferentiated oviduct, the immunoreactivities for both PR and RBF-1 were co-localized in the nuclei of only epithelial cells, but not the stromal cells or smooth muscle cells. In the partially differentiated oviduct of estrogen treated chicks, the immunoreactivity co-localized in the nuclei of not only epithelial but also glandular and stromal cells. Staining for the PR, but not RBF-1, was detected in the smooth muscle cells. The intensity of the PR but not the RBF-1 staining was markedly down-regulated in these cells at 2 and 6 h after treatment of the animals with progesterone (P). However, the band patterns for RBF-1 in the Western blots did show qualitative changes which may reflect P-induced posttranslational modifications which alter the epitope on the RBF-1. Interestingly, immunohistochemical analysis of several reproductive tissues of the rat showed that certain cell types in the uterus, ovary, and prostate displayed strong positive nuclear staining for an RBF-1-like antigen(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Vitamin D3 (soltriol) nuclear receptors in abdominal scent gland and skin of Siberian hamster (Phodopus sungorus) localized by autoradiography and immunohistochemistry.

In vivo autoradiography with [3H]1,25-dihydroxycholecalciferol (vitamin D, soltriol) and immunostaining with antibodies to vitamin D receptor were applied to identify specific binding sites in the abdominal scent gland of male Siberian hamster (Phodopus sungorus). Nuclear concentration of radiolabeled hormone and receptor antibodies was observed in the corresponding cell types including basal cells of sebaceous glands, cells of the outer hair sheaths and hair bulbs, and also keratinocytes in the epidermis. Cells of the hair dermal papillae and fibroblasts of the dermis did not show nuclear labeling. There was good correspondence between the autoradiographic and immunohistochemical data. The results indicate the presence of receptors for vitamin D-soltriol and suggest a seasonal regulation of scent gland marking activities by this steroid hormone of sunlight in cooperation with the sex steroid testosterone.

Animals↗
PubMed1993

Immunohistochemical demonstration of androgen receptors in human salivary glands.

Androgen receptors were demonstrated in human salivary glands by immunohistochemistry using polyclonal antibodies. Fresh, clinically healthy salivary gland samples (two from minor, seven from parotid and eight from submandibular glands) of both sexes were used. Frozen tissue sections were incubated with the antibody against human androgen receptor and visualized by an indirect immunoperoxidase technique. Androgen receptors could be detected in all salivary tissues studied. Positive staining was confined to nuclei of almost all acinar cells as well as to the majority of nuclei in ductal cells. Very few of the nuclei of connective tissue and endothelial cells stained positively. The presence of androgen receptors in human salivary glands suggests possible direct effects of androgens on these tissues.

Adult↗
PubMed1993

Functional properties of isolated stroma and epithelium from rat ventral prostate during androgen deprivation and estrogen treatment.

To identify the functional activities of prostatic stroma under different hormonal conditions, isolated stroma and epithelium from rat ventral prostate (RVP, intact or one week castrated or estrogen-treated), were studied in metabolic labeling experiments. Using a semiquantitative stereological procedure, the relative proportion of the epithelial and stromal compartment was determined in situ. In addition, the distribution of the androgen receptor was visualized by in situ hybridization and by immunocytochemistry. In castrated animals protein biosynthesis of the stroma and epithelium exceeded the control value by a factor 7 and 5, respectively. In estrogen-treated animals protein biosynthesis was reduced, reaching only between one tenth and one fifth of the control value. The amount of stroma obtained from these animals was very low. These results were confirmed by stereological findings and indicate a differential regulation of prostatic stroma and epithelium after estrogen challenge and androgen deprivation. Estrogen receptor was induced in epithelium and stroma in estrogenized animals whereas the androgen receptor was reduced in experimental specimens. During estrogenization the biosynthetic activity of both stroma and epithelium is depressed, while estrogen responsivity of the epithelium in terms of estrogen receptor expression is increased. Androgen withdrawal results in active transformation of the gland through increased stromal biosynthetic activity and epithelial regression.

Androgens↗
PubMed1993

Change in location and processing of inhibin alpha-subunit precursors during sexual maturation of the Djungarian hamster testis.

Immunohistochemical location and immunoblot of inhibin alpha-subunit peptides were analyzed in the testis of the Djungarian hamster from days 0-31 of postnatal development using a specific antibody. An intense immunoreaction was observed in the centrally located T1 prespermatogonia at day 0. The staining intensity decreased gradually in the spermatogonia when they make contact with the basal lamina at days 8-10. At days 13 and 15 there is no staining. Thereafter the immunoreactivity in Sertoli cells as well as in A spermatogonia gradually increased, being highest in sexually mature animals. The intensity of alpha-subunit staining in the seminiferous tubules was stage specific, being strongest at stages III and IV. Immunoblot analysis of testis homogenates with the anti-INH alpha 1-32 antibody showed several bands: 88K, 80K, and 43K in immature hamster testis (0-, 2-, 6-, 8-, or 10-day-old). In the adult hamster (31-day-old) 88K, 80K, 28K, and 20K bands were seen, but no 43K band. Dimeric inhibin was not detected. The 43-44K band most likely corresponds to the pro-alpha N alpha C, the 28K band to intermediate forms between alpha N alpha C and alpha C (alpha I alpha C), and the 20K band to mature alpha-subunit (alpha C). The shift from the immature pattern to mature occurs at about 20 days of age. Freezing of the samples was deleterious to alpha C, since it could be detected only in freshly homogenized samples. The results suggest that prespermatogonia produce predominantly monomeric alpha-subunit precursor pro-alpha N alpha C, whereas the mature Sertoli cells as well as A spermatogonia contain mainly monomeric alpha I alpha C. The alpha-inhibin precursors may act as auto-/paracrine regulators of spermatogenesis. Our results suggest that different alpha-subunit precursors, pro-alpha N alpha C and alpha I alpha C, might be involved in the differentiation and maintenance of spermatogenesis, respectively. The posttranslational processing of alpha-subunit precursors seems to play an important role in the physiology of reproduction.

Aging↗
PubMed1993

Expression of epithelial phenotype is enhanced by v-Ha-ras in rat endometrial cells immortalized by SV40 T antigen.

To study the interplay of steroid hormones and oncogenes in the control of endometrial cell proliferation and differentiation we have generated cell lines derived from rat endometrium by expressing the immortalizing oncogenes adeno E1A or SV40 large T antigen. These lines are positive for mesenchymal markers and contain very few characteristic epithelial proteins. Cell lines expressing a temperature-sensitive mutant of SV40 T antigen exhibit a temperature-dependent morphology and growth behavior, but do not manifest an epithelial phenotype at the non-permissive temperature. Cell lines additionally infected with retroviral vectors carrying the v-Ha-ras oncogene (p21rasArg-12) no longer express collagen type III and recover part of their epithelial potential by expressing cytokeratins and/or cadherin E. Some of these cells also express characteristic decidual marker proteins such as desmin, whereas others express glandular epithelial markers such as uteroglobin. Uteroglobin mRNA levels in these cells are increased by glucocorticoids. The parental temperature-sensitive cells do not contain progesterone receptor but become positive for progesterone receptor at the permissive temperature after infection with the v-Ha-ras-expressing retrovirus. Our results indicate that there is a fluent transition and overlapping between mesenchymal, glandular epithelial and decidual phenotypes of endometrial cells, suggesting that these three cell types are derived from the same stem/precursor cells. The v-Ha-ras oncogene product appears to act on the differentiation pathway at an early step prior to the distinction between decidual and glandular epithelial lineage.

Animals↗
PubMed1992

Arguments against the prostatic origin of the R-3327 Dunning H tumor.

The Dunning tumor, originally described as a carcinoma of the rat dorsal prostate, has for long been used as an experimental model of prostatic cancer. We have recently presented a number of morphological findings that are incompatible with the prostatic origin of the H-subline of the Dunning tumor. In this paper, biochemical and immunohistochemical markers of rat prostate and mammary gland are studied in the R-3327 Dunning H tumor. Pieces of the H tumor were inoculated in male or lactating female rats. The electrophoretic protein pattern of Dunning tumor extracts was more similar to that of the mammary gland than the dorsolateral prostate. Proteins selectively appearing after metabolic labeling in Dunning tumors grown in lactating rats corresponded to labeled proteins in mammary glands from the same animals. Secretory proteins typical of the lateral prostate (SVS II) and dorsal prostate (transglutaminase) could not be detected immunohistochemically in the Dunning tumor. Western blot studies of tumor extracts and slot blot analysis of RNA preparations from the tumor confirmed the absence of SVS II and prostate specific transglutaminase from the Dunning tumor. On the other hand, the presence of mammary gland proteins such as milk fat globule membrane proteins, lactoperoxidase and lactalbumin were detected in the Dunning tumor by immunohistochemistry and Western blotting, but were absent from the dorsolateral prostate. Transferrin-mRNA, expressed in the male urogenital tract and also in the liver and other tissues, was detected in the mammary gland and Dunning tumor, but not in the dorsolateral prostate. The absence of mammary gland secretory beta-casein in the Dunning tumor was related to the elevated Ha-ras oncogene expression in the tumor, previously reported to suppress casein expression. The findings clearly demonstrate that the prostate cannot be the origin of the Dunning tumor, presently being used in prostatic cancer research. The designation prostatic adenocarcinoma for this tumor is therefore invalid. Furthermore, the data support our view that mammary gland might be the origin of the Dunning tumor, although the derivation from the bulbourethral or the parotid glands cannot strictly be excluded.

Adenocarcinoma↗
PubMed1992

Immunohistochemical detection of androgen receptors in human oral mucosa.

Oral mucosal biopsies from 11 healthy volunteers, 7 women and 4 men, were analysed for the localization of androgen, oestrogen and progesterone receptors. The samples were dissected as quickly as possible and immediately frozen in liquid nitrogen to be stored at -70 degrees C. Only androgen receptors could be detected by the methods used. These were mainly located in the nuclei of basal epithelial cells, and to some extent in the nuclei of fibroblasts and endothelial cells. Failure in the localization of oestrogen and progesterone receptors might have been due either to low numbers of receptors or to the insensitivity of antibodies used. The method is thus suitable only for detecting androgen receptors.

Adult↗
PubMed1992

Inducibility of the avidin gene by progesterone is suppressed during estrogen-induced cytodifferentiation.

We have studied epithelial differentiation of the chick oviduct as induced by diethylstilbestrol (DES) and 17 beta-estradiol (E2). The proportion of goblet cells in the oviduct was slightly higher after E2 than after DES treatment. Also avidin induction by progesterone was stronger following DES than E2 priming. In the estrogen pretreated oviduct epithelium, avidin expression was induced by progesterone in the surface epithelial cells, protodifferentiated gland cells and tubular gland cells, but not in goblet cells. During prolonged estrogen treatment, however, the inducibility of avidin by progesterone ceased in tubular gland cells but not in surface epithelial cells. The estrogen action on the expression of avidin could be explained by estrogen-induced terminal differentiation of the epithelial gland cells or by a direct effect of estrogen on the progesterone action, for instance interaction of estrogen receptor and progesterone receptor in the regulation of transcription.

Animals↗
PubMed1992

Characterization of human 1,25-dihydroxyvitamin D3 receptor anti-peptide antibodies.

Rabbit and chicken antibodies were raised against two peptides synthesized according to the structure of human 1,25-dihydroxyvitamin D3 receptor (hVDR): rabbit alpha hVDR-103 against the N-terminal amino acids 5-18 and alpha hVDR-104 against the amino acids 172-186 in the hinge region and chicken alpha hVDR-cab11 against the amino acids 172-186, respectively. The specificity of the antibodies was tested by peptide saturation, SDS-PAGE immunoblotting, gel shift assay and sucrose gradient centrifugation. Immunoblotting of a soluble extract (cytosol) from osteosarcoma cell line MG-63 showed a single band with an M(r) of about 48,000 and human intestine cytosol a broad band (50-63,000) for both antibodies. The antibodies recognized activated (3.2S) hVDR by shifting the centrifugation sedimentation profile to 5-6S. The antibodies showed nuclear immunostaining of unoccupied VDR in human osteosarcoma cells MG-63, U2-Os and SaOs-2. The immunoreaction could be saturated with the corresponding synthetic peptide. In immunoblot alpha hVDR-103 reacted with human and rat VDR, whereas alpha hVDR-104 recognized human VDR only. Similarly in immunohistochemistry, alpha hVDR-103 showed staining with hVDR and rVDR, whereas alpha hVDR-104 reacted only with hVDR. All antibodies recognized the native hVDR as verified with sucrose gradient centrifugation or immunoprecipitation but only alpha hVDR-103 and alpha hVDR-cab11 in gel shift assay of hVDR associated with the vitamin D-responsive element of human osteocalcin gene promoter.

Amino Acid Sequence↗
PubMed1992

In situ hybridization of ovalbumin mRNA in the chick oviduct reveals target cell specificity for estrogen and progesterone.

An in situ hybridization method using paraffin-embedded sections was used to characterize the chicken oviduct cells synthesizing ovalbumin mRNA due to the action of estrogen and progesterone. The cytodifferentiation of the oviduct cells was induced by 17 beta-estradiol administration to newly hatched female chicks. To avoid possible effect of estrogen on the action of progesterone the chicks were withdrawn from the estrogen by six days withdrawal period without hormone treatment. Ovalbumin mRNA was not synthesized after a period of estrogen withdrawal. Administration of estrogen induced ovalbumin mRNA in the tubular gland cells. Administration of progesterone induced the expression of ovalbumin mRNA in the surface epithelial cells. It was also found that progesterone induced mucus producing goblet cells in the surface epithelium. Estrogen did not have an effect on the mucus production, which suggests that progesterone could induce the terminal differentiation of the goblet cells. We conclude that the expression of ovalbumin in the surface epithelial cells and in the tubular gland cells is specific for progesterone and estrogen, respectively.

Animals↗
PubMed1992

Subcellular location of androgen receptor in rat prostate, seminal vesicle and human osteosarcoma MG-63 cells.

Location of the androgen receptor (AR) before and after dihydrotestosterone (DHT) administration was studied in 6 castrated and 2 normal male rats, as well as in MG-63 human osteosarcoma cell culture. Two days after castration, rats were injected with DHT and sacrificed 0, 6 and 24 h later. Cryosections of ventral prostate and seminal vesicle were stained with a polyclonal anti-AR antibody. Cultured MG-63 cells were also stained similarly. The intensity of immunoreaction was measured semiquantitatively by computer-assisted image analysis. In both normal and castrated rats, a positive reaction was seen mainly in the nuclei of epithelial cells and stromal cells of the prostate and seminal vesicle, as well as in those of smooth muscle cells of the seminal vesicle. AR immunoreactivity was up-regulated by DHT, it decreased clearly in both organs after castration. Nuclear AR and its up-regulation by androgen were also seen in MG-63 cells. At the immunoelectron microscopy, silver enhanced gold particles were predominantly found in the heterochromatin of cell nuclei. Treatment with DHT caused a decondensation of the heterochromatin and AR was more dispersed. Thus, AR appears to be nuclear independently of the ligand.

Animals↗
PubMed1992

Induction of avidin messenger ribonucleic acid in the chick oviduct by progesterone and other steroids.

Avidin gene expression was analyzed using an avidin immunoassay and RNA hybridization analysis. To ascertain whether the induction of the avidin gene by progesterone remains specific also during secondary restimulation with diethylstilbestrol, chicks were given different steroid hormones or hormone combinations. Progesterone-specific induction of avidin protein and messenger RNA (mRNA) was 15- to 30-fold over the control even after secondary restimulation with diethylstilbestrol. A functional difference between the progesterone response element and glucocorticoid response element was suggested, since dexamethasone alone did not induce avidin in vivo. In spite of progesterone specificity, a combination of progesterone with other steroids nevertheless generated a synergistic increase in the amount of avidin mRNA. This may indicate that binding of progesterone receptor to the progesterone response element may be important to alter the functional activity of other hormone response elements present on the avidin gene. The time response curve of the avidin mRNA induction by progesterone was also determined. Avidin mRNA was detectable 8 h after progesterone induction, and its amount was maximal after 16-24 h. This would indicate that the avidin gene belongs in the so-called late responder genes, which also include chicken ovalbumin, ovomucoid, and lysozyme genes.

Animals↗
PubMed1992

Inhibin alpha- and beta A-subunit immunoreactivity in the chicken embryo during morphogenesis.

Antibodies against synthetic peptides selected from the amino acid sequences of human inhibin alpha- and beta A-subunits were used to examine the distribution of inhibin subunit immunoreactivity in chicken embryos during the first week of development. Inhibin alpha-subunit immunoreactivity was localized in skeletal and smooth muscle myoblasts as well as developing cardiac muscle cells. In somites, immunostaining was seen exclusively in myotomes. The appearance of alpha-subunit immunoreactivity was correlated with myogenic differentiation; immunoreactivity was not seen in non-differentiated mesenchymal cells or in terminally differentiated adult muscle cells. In cardiac muscle, some immunopositive myocytes were seen also in the adult. In the adult heart, the Purkinje fibers were strongly immunoreactive, suggesting a possible role of the immunoreactive protein in the impulse-conducting function of these specialized cells. Inhibin alpha-subunit immunoreactivity was also seen in the visceral and parietal cells of the Bowman's capsule in both mesonephric and metanephric kidneys. In addition to mesodermal derivatives, alpha-subunit immunoreactivity was localized in neuroepithelial cells and axons in the developing central nervous system. Immunoblotting with anti-alpha(1-32) revealed two protein bands with M(r) values of 50,000 and 32,000 in cytosol samples of whole embryos under nonreducing conditions. In reduced samples an approximately 14,000 M(r) protein species was detected. Inhibin beta A-subunit immunoreactivity was detected only in chondrocytes, suggesting that the immunoreactive protein might represent a chicken homologue of the various cartilage and bone morphogenetic proteins expressed in mammals.

Animals↗
PubMed1991

Sensitive immunometric assays for secretory peroxidase and myeloperoxidase in human saliva.

We have developed specific immunoassays for secretory peroxidase (SP) and for myeloperoxidase (MP) (polymorphonuclear leukocyte-derived peroxidase) in human saliva. Antibodies against SP and MP were produced using bovine milk lactoperoxidase (LP) and human MP as the immunogens, respectively. The methods developed are non-isotopic immunometric assays using biotinylated antibodies and avidin-enzyme conjugate. The detection limit was 0.1 ng/ml and the performance time less than 3 h for both assays. The determination ranges were 0.5-100 ng LP (SP)/ml and 0.5-200 ng MP/ml with intra- and interassay CVs of 4.3% and 15.6% for SP and 3.7% and 10.8% for MP, respectively. The mean analytical recoveries were 108.9% (SP) and 91.5% (MP). These assays correlated well (r = 0.849-0.871) with the colorimetric assays based on the oxidation of thiocyanate or chloride by peroxidases. However, compared to the colorimetric methods the new immunometric assays are much more sensitive and specific for salivary SP and MP. The assays are also more rapid since extensive dialysis to remove endogenous thiocyanate is not required.

Adult↗