Biomedical subjects

D Djakiew

Publications and source records attributed to D Djakiew.

50 records · Page 3Linked to original sources

PubMed1987

Polarized secretion of androgen-binding protein and transferrin by Sertoli cells grown in a bicameral culture system.

Sertoli cells in vivo are believed to secrete androgen-binding protein (ABP) as well as other proteins in a polarized manner, primarily into the adluminal (apical) compartment of the seminiferous tubules. It is now possible to examine polarized secretion by Sertoli cells in vitro by growing them in dual environment (bicameral) culture chambers such that there is a separation of the apical and basal compartments of the cells. Sertoli cells obtained from 17-day-old rats were grown in these chambers on Millipore filters coated with a reconstituted basement membrane matrix. Within 3 days a confluent epithelial sheet of Sertoli cells (30-40 microns in height) is established, and these cells are joined along their baso-lateral plasma membranes by typical Sertoli-Sertoli tight junctions. We have previously shown that this epithelial sheet of Sertoli cells establishes an electrical resistance, as well as a permeability barrier to small molecules, between the basal and apical compartments of the culture chamber. In the absence of Sertoli cells, proteins equilibrate within 8 h across the matrix-coated filter support. Between 12 and 48 h of culture the Sertoli cell monolayers secrete approximately 4- and 2-fold more ABP and transferrin (Tf), respectively, into the apical compartment than into the basal compartment when grown in serum-free defined medium. ABP secretion is diminished 10-fold by the removal of testosterone from the serum-free defined medium. In addition, the apical/basal ratio of ABP secretion decreases from 4.1 to 1.7 in the absence of testosterone. In contrast, neither the amount nor the direction of Tf secretion is altered by the removal of testosterone. These results demonstrate the bidirectional secretion of ABP and Tf by Sertoli cells grown in bicameral chambers. In addition, the polarity of secretion of these two proteins by Sertoli cells appears to be under differential regulation by testosterone.

Androgen-Binding Protein↗

PubMed1986

Micropuncture studies of receptor-mediated endocytosis of transferrin in the rat epididymis.

The concentration of transferrin in fluids collected by micropuncture techniques from the rete testis and zones 1A, 2, 5A, and 6B of the rat ductus epididymidis was 44, 527, 113, and 49 ng/microliter, respectively. When changes in fluid volume were taken into account, it was found that transferrin was concentrated by the efferent ducts, whereas the amount of endogenous luminal transferrin declined from the caput to the cauda epididymidis. Using transferrin-gold as an electron dense marker, we showed that the decline in the concentration of transferrin along the epididymis could be attributed to its cumulative receptor-mediated endocytosis by the lining principal cells. No significant difference in the net receptor-mediated endocytosis of transferrin-gold was found between the proximal caput and corpus epididymidis in vivo.

Animals↗

PubMed1985

Structural features of rat epididymal epithelial cells in vitro.

Epididymal epithelial fragments, free of stromal elements were isolated from mature rats using two sequential collagenase digestions. Within 24 h these attached efficiently to a variety of substrates including glass, plastic, placental collagen, type IV collagen and epididymal extracellular matrix material. Cells spreading away from the fragments rapidly assumed a flattened, overlapping, monolayer appearance typical of epithelial cells in culture. Cells still associated with the fragments or adjacent to them remained more polarized and more closely resembled epididymal principal cells in vivo than did cells that had migrated to the periphery of the monolayer. Apical microvilli characteristic of these cells in vivo were common during the first 4 days in culture but diminished in number and size thereafter. Cultured cells maintained many of the structural features characteristic of principal cells in vivo, including a well developed Golgi apparatus, coated pits and vesicles, and many multivesicular bodies. An extensive filamentous network, shown immunocytochemically to consist of keratin, was present in the cytoplasm of all cells but was more obvious in flattened cells at the periphery of the monolayer. Rhodamine phalloidin labelling of filamentous actin showed that concentrations of actin occurred corresponding to microvilli on the apical surface, in a continuous ring just below the apical surface, and also in stress fibres at the base of the cells. Cells isolated and cultured from the distal caput epididymidis possessed lobulated nuclei, in contrast to the round or oval nuclei found in cells cultured from the proximal caput epididymidis. Cells from the distal caput epididymidis were also characterized by the presence of many lipid droplets in their cytoplasm. Autofluorescent granules were observed in epithelial cells from both regions but were larger and more numerous in cells isolated from the distal caput epididymidis. Tritiated thymidine incorporation by the cells after 4 days in culture showed that cells adjacent to the parent epithelial fragment were dividing at a greater rate than cells that had migrated to the periphery of the monolayer.

Actins↗

PubMed1984

Receptor-mediated endocytosis of alpha 2-macroglobulin and transferrin in rat caput epididymal epithelial cells in vitro.

The endocytic activity of epithelial cells from the rat epididymis in vitro has been examined by following the uptake of tracer compounds conjugated to proteins. Transferrin-gold and alpha 2-macroglobulin-gold were taken up initially in coated pits, internalized and sequestered into tubular-vesicular structures, multivesicular bodies and, in the case of alpha 2-macroglobulin, into lysosomes. Uptake could be prevented by an excess of unlabeled protein. Studies using 125I-alpha 2-macroglobulin and 125I-transferrin also showed that the uptake of these proteins was specific and could be displaced with increasing amounts of unlabeled protein. In addition, binding of 125I-transferrin to cells was saturable at 4 degrees C. These studies indicate that transferrin and alpha 2-macroglobulin are taken up by receptor-mediated endocytosis. In contrast, a fluid phase marker, bovine serum albumin-gold (BSA-gold), was initially taken up predominantly in uncoated caveolae rather than coated pits, and could not be displaced with excess BSA. By virtue of their charge, polycationized ferritin and unlabeled colloidal gold were taken up and internalized by adsorptive endocytosis, a pathway which is similar to fluid phase endocytosis. The uptake and internalization of alpha 2-macroglobulin and transferrin differed in a number of respects. Uptake and internalization of alpha 2-macroglobulin but not of transferrin was dependent on extracellular calcium. Only alpha 2-macroglobulin was transferred into lysosomes, whereas transferrin was recycled to the cell surface. Although the proton ionophore, monensin, and the transglutaminase inhibitor, dansylcadaverine, did not stop uptake and internalization of either alpha 2-macroglobulin or transferrin, they did prevent the transfer of alpha 2-macroglobulin to lysosomes.

Adsorption↗

PubMed1983

Sperm maturation, fluid transport, and secretion and absorption of protein in the epididymis of the echidna, Tachyglossus aculeatus.

Extratesticular sperm maturation in the echidna mainly occurs in the initial segment of the ductus epididymidis. The process involves the development of motility, migration and loss of the cytoplasmic droplet, a decrease in permeability to Congo red and the formation of sperm bundles. The spermatozoa are supported in the bundles by a matrix of electron-dense material; the bundles are very motile is undiluted samples of luminal fluids. Micropuncture studies of anaesthetized echidnas showed that the ductuli efferentes absorb 74% of the fluid and 46% of the soluble protein that enters them. The initial segment of the ductus epididymidis absorbs 83% of the fluid which enters it, and its secretions increase the concentration of protein in luminal fluid by 107%. Denatured, linear-gradient polyacrylamide gel electrophoresis of micropuncture samples showed that 1 protein (apparent M4 = 100 500) which is not present in blood plasma is present in rete testis fluid, and a glycoprotein which is present in rete testis fluid (apparent Mr = 78500) is absorbed by the ductuli efferentes. Six proteins which are not present in blood plasma are secreted into the initial segment of the ductus epididymidis; 5 are glycoproteins (apparent Mr = 48500, 39000, 32000, 20500 and 19000) and one (apparent Mr = 82500) is not. The most prominent electrophoresis bands corresponded to the glycoproteins with apparent molecular weights of 48500, 20500 and 19000.

Animals↗

PubMed1982

Ultrastructure of the ductus epididymidis of the echidna, Tachyglossus aculeatus.

It is concluded that the ductus epididymidis of the echidna is divided into only two structurally distinct segments which are each homogeneous along their length. The initial segment in the echidna is structurally very similar to the initial segment proper in the epididymis of all other mammals which have been studied, whereas the terminal segment is structurally quite different from the terminal segment in other mammals. The terminal segment in the echidna is involved in considerable apocrine secretion of highly membranous material. The secretions gradually degenerate and occupy a large proportion of the lumen of the duct.

Animals↗

PubMed1981

Structural differentiation of the male genital ducts of the echidna (Tachyglossus aculeatus).

Seven ductuli efferentes radiate from the testis of the echidna and join the ductus epididymidis either directly or after joining one of their neighbours. They are pigmented brown and appear to be structurally and functionally similar to the ductuli efferentes of therian mammals. The epididymis is anatomically differentiated into a large head and small tail which appear to be, respectively, larger and smaller than the similar regions of the epididymis of scrotal mammals; they also contain, respectively, larger and smaller proportions of the animals' extragonadal spermatozoa. Only the head of the epididymis is adjacent to the testis: the tail and the ductus deferens are distal to the testis. The ductus epididymis is also histologically differentiated into two segments (initial and terminal segments) which correspond precisely with the anatomical differentiation. The initial segment is structurally similar to the initial segment of the epididymis of scrotal mammals (i.e. tall epithelium with long stereocilia, a farily homogeneous supranuclear cytoplasma containing Golgi apparatus and a low concentration of spermatozoa in the lumen). The terminal segment has adaptations of the duct (as in scrotal mammals) for the storage of spermatozoa such as a wide lumen containing a high concentration of spermatozoa, low epithelium and thick layers of periductal muscle. However, it is peculiar in that the duct epithelium is thrown into folds and it is involved in considerable apocrine secretion.

Animals↗

PubMedDate not supplied

Receptor-mediated endocytosis of alpha 2-macroglobulin by principal cells in the proximal caput epididymidis in vivo.

Micropuncture techniques were used to study receptor-mediated endocytosis of alpha 2-macroglobulin bound to colloidal gold (alpha 2M-gold) by principal cells in the proximal caput epididymidis of control and efferent duct-ligated rats. The pathway of receptor-mediated endocytosis of alpha 2-macroglobulin-gold in vivo was similar to that which occurs in vitro. Alpha 2-macroglobulin-gold was taken up and internalized in coated pits and coated vesicles and was localized sequentially in uncoated vesicles (endosomes), tubular-vesicular structures, multivesicular bodies, and lysosomes. However, a 100-fold excess of alpha 2-macroglobulin did not displace the uptake of alpha 2-macroglobulin-gold in principal cells from control rats. In contrast, uptake of alpha 2-macroglobulin-gold by principal cells from efferent duct-ligated rats was six-fold greater than in control rats, and could be displaced to control levels by a 100-fold excess of alpha 2-macroglobulin. It is suggested that the inability of a 100-fold excess of alpha 2-macroglobulin to displace uptake of alpha 2-macroglobulin-gold in control rats was due to the normal saturation of apparent alpha 2-macroglobulin receptors on principal cells. The effect of efferent duct ligation was to remove the high levels of endogenous alpha 2-macroglobulin, which depleted the receptors of alpha 2-macroglobulin, thereby allowing a higher uptake of alpha 2-macroglobulin-gold in the efferent duct-ligated rats.

Animals↗

PubMedDate not supplied

Growth and characterization of polarized monolayers of epididymal epithelial cells and Sertoli cells in dual environment culture chambers.

Epididymal epithelial cells isolated from mature rats and Sertoli cells isolated from 10-day-old rats were cultured in serum-free defined media on extracellular matrix impregnated filters maintained in dual environment culture chambers. Epididymal epithelial cells had a polarized appearance only when plated at high density (greater than 1 X 10(6) cells/cm2). Confluent monolayers of these cells formed a permeability barrier to inulin. Sertoli cells were columnar and highly polarized when grown on extracellular matrix-impregnated filters, cuboidal when grown on filters alone, and squamous when grown on plastic. Confluent polarized monolayers of these cells excluded the electron-dense tracer lanthanum nitrate by way of basal-tight junctions. Therefore, polarized monolayers of epididymal epithelial cells and Sertoli cells can be obtained by growing the cells at high density on extracellular matrix-impregnated permeable supports. By maintaining the monolayers in specially constructed culture chambers, the cells can develop a permeability barrier, and are able to achieve the separation of apical from basal compartments so important for their function in vivo.

Animals↗

PubMedDate not supplied

Transferrin-mediated transcellular transport of 59Fe across confluent epithelial sheets of Sertoli cells grown in bicameral cell culture chambers.

The transferrin-mediated transcellular transport of 59Fe across confluent epithelial sheets of Sertoli cells grown on Millipore filters was investigated. These filters had been impregnated with reconstituted basement membrane and suspended in bicameral (two houses) culture chambers. After five days of culture, Sertoli cells from 10-day-old rats formed basally-located tight junctional complexes. Concomitantly, there was an increase in electrical resistance and the epithelial sheet became impermeable to lanthanum nitrate. The rate of passage of [3H]inulin across the epithelial sheet was considerably less than passage across a filter alone, a filter impregnated with reconstituted basement membrane or an epithelial sheet pretreated with 2 mM EGTA. We conclude from these permeability studies that the tight junctional complexes between Sertoli cells formed an effective transepithelial permeability barrier. Following addition of human serum [59Fe]transferrin to media bathing the basal cytoplasm of the cells, rat testicular [59Fe]transferrin was immunoprecipitated from apical media overlying the Sertoli cells. Cross-reactivity of the rabbit anti-rat transferrin antibody with human serum transferrin was less than 0.001%. Substitution of the primary antibody with normal rabbit serum reduced the amount of immunoprecipitable rat testicular [59Fe]transferrin to 20% of normal levels. Prior fixation of the Sertoli cell epithelial sheet in 2.5% glutaraldehyde, addition of a 100-fold excess of holotransferrin to the basal media, and incubation of the Sertoli cell epithelial sheet at 4 C all reduced the immunoprecipitable rat testicular [59Fe]transferrin in apical media to levels below that for the non-specific binding of the primary antibody. From these studies we conclude that 59Fe is shuttled across Sertoli cells by two different forms of transferrin. Serum transferrin delivers the 59Fe to the basal cytoplasm of the Sertoli cells. The 59Fe dissociates from the serum transferrin, is delivered to testicular transferrin, and is subsequently secreted from the apical surface of the epithelial sheet of Sertoli cells as testicular [59Fe]transferrin.

Cell Membrane Permeability↗

PubMedDate not supplied

Epidermal growth factor modulates the expression of vascular endothelial growth factor in the human prostate.

The growth and dissemination of tumors in the body has been associated with angiogenesis. Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates endothelial cell growth and enhances vascular permeability. VEGF exerts its action by binding to specific cell surface receptors. Three receptors, VEGFR-1 (flt-1), VEGFR-2 (flk-1), and VEGFR-3 (flt-4) have been identified. Very little information on the coordinated expression of VEGF and its receptors in normal prostate, benign prostatic hyperplasia (BPH), and prostate carcinoma is available. Therefore, we examined the immunohistochemical localization of VEGF and its receptors in tissues derived from normal human prostate, BPH, and prostatic carcinoma. Immunostaining for VEGF was absent in the normal prostate. Epithelium lining the glands of prostate derived from patients with BPH exhibited strong immunostaining. The intensity of staining was relatively less in prostate carcinoma. It is interesting that VEGFR-1 and VEGFR-3 were strongly expressed in both stromal and epithelial tissues in normal prostate, BPH, and carcinoma. In comparison, VEGFR-2 was not localized to normal prostate and its expression in the stroma of BPH and epithelium of carcinoma was very weak. Because progression of prostate cancer is accompanied by altered expression of epidermal growth factor (EGF) and its receptor (EGFR) in malignant cells, we investigated the effect of EGF on VEGF gene expression by Northern blot analysis in 2 human prostate cancer cell lines that express EGFR. EGF greatly enhanced the expression of VEGF messenger RNA in DU145 and PC3 cell lines in a dose-dependent manner. The EGF induction of VEGF gene expression suggests a mechanism by which angiogenesis could be accelerated in BPH and prostate carcinoma.

Carcinoma↗

PubMedDate not supplied

The neurotrophin receptor p75NTR is a tumor suppressor in human prostate cancer.

The loss of tumor suppressor gene function contributes to the transformation of human prostate epithelial cells to a malignant pathology. Previous studies have demonstrated a loss of protein expression of the neurotrophin receptor, the p75NTR, from the pathological human prostate. Through cell cycle analysis, we demonstrate that a dose-dependent increase in the expression of p75NTR protein induces increased quiescence of a series of prostate tumor cells in vitro. When the same series of tumor cells were injected into the flanks of SCID mice, the growth of prostate tumors was suppressed in proportion to increased p75NTR expression. Tumor analysis showed that a dose-dependent increase in p75NTR expression was associated with a dose-dependent increase in the proportion of apoptotic cells, as determined by TUNEL assay, and a dose-dependent decrease in the proportion of cells committed to proliferation, as determined by proliferating cell nuclear antigen assay. These results demonstrate that the neurotrophin receptor p75NTR is a tumor suppressor of prostate growth by enhancing cell cycle quiescence, reducing proliferation and increasing apoptosis of the tumor cells.

Animals↗

PubMedDate not supplied

Role of nerve growth factor-like protein in the paracrine regulation of prostate growth.

Prostate cancer is the most commonly diagnosed form of malignant neoplasia in men. Considerable evidence has accumulated suggesting that paracrine interactions between stromal cells and epithelial cells mediate, in part, the growth and development of the prostate. A nerve growth factor-like protein secreted by stromal cells has been implicated in the paracrine regulation of prostate epithelial tumor cell growth in vitro. This prostate-derived nerve growth factor-like protein differs from the known members of the neurotrophin family of proteins, and may represent a prostate-specific form of this family of gene products. Furthermore, corresponding nerve growth factor receptors have been localized to the epithelial cells of the human prostate in vivo, consistent with a role of the receptors and the adjacent nerve growth factor-like protein secreted by stromal cells, in the paracrine regulation of prostate growth and neoplasia.

Humans↗

PubMedDate not supplied

Anti-proliferative effect of the kinase inhibitor K252a on human prostatic carcinoma cell lines.

Members of the K252 family of kinase inhibitors have been demonstrated to inhibit a number of neurotrophin-mediated cellular responses, and to preferentially inhibit the activity of neurotrophin receptors. In this study, we examined the effects of K252a and K252b on the growth of human prostate carcinoma cells, whose growth is, in part, mediated by a prostatic nerve growth factor(NGF)-like protein(s). K252a inhibited [3H]thymidine incorporation by three androgen-independent prostate tumor cell lines (TSU-pr1, DU-145, and PC-3), under basal growth conditions, and in response to growth stimulation by human prostatic stromal (hPS) cell proteins and serum. K252b, which does not readily penetrate cell membranes, had no significant effect on [3H]thymidine incorporation by the prostate tumor cell lines. The decrease in [3H]thymidine incorporation by the cell lines in response to K252a did not appear to be the result of K252a cytotoxicity at concentrations as high as 100 nM, as measured by the Trypan blue assay for cell viability. Treatment of cells for 25 hours with 100 nM K252a resulted in accumulation of TSU-pr1, DU-145, and PC-3 cells in G0/G1, concurrent with a substantial decrease in cells synthesizing DNA. Treatment of androgen-responsive LNCaP prostatic carcinoma cells for 25 hours with 100 nM K252a also resulted in a significant decrease in DNA synthesis. Human recombinant NGF-mediated phosphorylation of a 140-kDa Trk NGF receptor in the TSU-pr1 cell line was inhibited by treatment with 100 nM K252a. Hence, K252a inhibition of Trk phosphorylation most probably contributed, in part, to the inhibition of prostate tumor cell growth in vitro. These results suggest that the mechanism of K252a action may be useful in the design of potential therapies for prostate cancer treatment.

Antineoplastic Agents↗