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R M DePhilip

Publications and source records attributed to R M DePhilip.

At least 19 recordsLinked to original sources
PubMed1999

E-, P-, and N-cadherin are co-expressed in the nasopharyngeal carcinoma cell line TW-039.

The cadherin/catenin complex plays a key role in the initiation of cell-cell recognition, and adhesion, and the elaboration of structural and functional organization in multicellular tissues and organs. It is associated with tumor metastasis and also acts as an "invasion suppressor" of cancer cells. Nasopharyngeal carcinoma (NPC) is notorious for its highly metastatic nature. The expression of the E-cadherin/catenin complex is down-regulated in NPC tumor specimens. To obtain better insight into the intercellular adhesive property of NPC cells, we used immunofluorescence microscopy, immunoprecipitation, and immunoblot analysis to examine the expression of the classical cadherins and beta-catenin in a NPC cell line, TW-039. The results demonstrate a change in the distribution of E-cadherin from cytosolic flakes to cell-cell contacts with increasing time in culture. Between days 1 and 5 after plating, the detergent-insoluble fraction of E-cadherin increased from 20% to 37% of total E-cadherin, and that for P-cadherin increased from 33% to 40%. By contrast, the values for beta-catenin remained unchanged (26% and 25%). Both immunofluorescence and immunoblot studies suggested that P-cadherin may be involved in pioneer contact adhesion of TW-039 cells. Interestingly, E-, P-, and N-cadherin are co-expressed in this cell line. Immunoprecipitation studies also showed that other members of the cadherin family may be involved in the contact adhesion of TW-039 cells.

Blotting, Western↗
PubMed1998

Detection of steroidogenic acute regulatory protein (stAR) in mitochondria of cultured rat Sertoli cells incubated with follicle-stimulating hormone.

Previous work established biochemical homology between proteins named SCc1 and SCc2 in FSH-treated Sertoli cell cultures and a family of 30-kDa proteins, collectively referred to as Steroidogenic Acute Regulatory protein (StAR), in hormone-treated adrenal and gonadal cells. The purpose here was to establish the presence of StAR in FSH-treated Sertoli cell cultures using a StAR-specific antiserum. Immunofluorescence microscopy and immunoblot analysis were used to detect StAR in Sertoli cell cultures and in R2C rat Leydig tumor cell cultures that served as a positive control for StAR expression. FSH dramatically enhanced the appearance of StAR in mitochondria of Sertoli cells, whereas R2C cells expressed StAR constitutively. It has been proposed that StAR participates in steroidogenesis by facilitating the conversion of cholesterol to pregnenolone by the cytochrome P450 side-chain cleavage enzyme. FSH-treated Sertoli cells were shown here to lack detectable levels of side-chain cleavage enzyme protein and RNA, while R2C cells expressed both. We conclude that the appearance of StAR in Sertoli cell mitochondria is regulated by FSH, but that its function in Sertoli cells is not associated with the conversion of cholesterol to pregnenolone.

Animals↗
PubMed1996

Differential expression of placental (P)-cadherin in sertoli cells and peritubular myoid cells during postnatal development of the mouse testis.

BACKGROUND: In previous work, RNA transcripts for placental (P)-cadherin, a calcium-dependent cell adhesion molecule, were identified in the rat testis during the first 4 weeks of postnatal development. However, the cells in the testis responsible for P-cadherin expression have not yet been identified. METHODS: We used conventional epifluorescence microscopy to examine P-cadherin immunoreactivity in cryostat sections of mouse testis and scanning laser confocal microscopy to localize P-cadherin and beta-catenin in wholemount preparations of mouse seminiferous tubules. We used fluorescent phalloidin to identify actin filaments. RESULTS: Sertoli cells expressed P-cadherin on postnatal days 1, 3, and 8, but not on any day thereafter. In contrast, peritubular cells did not express P-cadherin on postnatal day 8 and continued expression in adulthood. beta-Catenin was localized near contact areas between peritubular cells on postnatal days 12 and 15. A mature pattern of actin filament organization in peritubular cells appeared on day 15 and coincided with the uniform appearance of P-cadherin and beta-catenin near areas of contact between adjacent peritubular cells. CONCLUSIONS: During postnatal development of the testis, the earlier expression of P-cadherin by Sertoli cells is replaced by the subsequent expression of P-cadherin by peritubular cells. The expression of P-cadherin in peritubular cells is correlated temporally with the expression of beta-catenin and the development of a mature network of actin filaments and is consistent with a role in intercellular adhesion and junction formation.

Aging↗
PubMed1996

Sex-dependent expression of placental (P)-cadherin during mouse gonadogenesis.

BACKGROUND: Placental (P)-cadherin is one of a family of cell adhesion molecules that participate in embryonic sorting and organogenesis. In previous work, P-cadherin was localized to Sertoli cells in the mouse testis as early as postnatal day 1. This early postnatal localization raised questions about when P-cadherin first appeared in the embryonic testis and whether P-cadherin was expressed differentially in the embryonic testis and ovary. METHODS: The localization of P-cadherin, epithelial (E)-cadherin, and Müllerian inhibiting substance was determined in frozen sections of mouse gonads between embryonic days 10.5 and 18 using indirect immunohistochemistry. Alkaline phosphatase reactivity was used to identify germ cells. RESULTS: The expression of P-cadherin was traced back to the indifferent stage of gonadogenesis where uniform distribution was observed in the indifferent gonad of both sexes. However, after sexual differentiation, the expression of P-cadherin in the testis was localized to Sertoli cells in the testicular cords, while its expression in the ovary fell below detectable levels. CONCLUSIONS: The localization of P-cadherin in the male and female indifferent gonad is similar and cannot be used to distinguish the future testis and ovary. The localization of P-cadherin in the testis after sexual differentiation suggests a role for P-cadherin in testicular cord formation. The common temporal pattern of P-cadherin and Müllerian inhibiting substance expression in Sertoli cells is consistent with a shared regulatory mechanism.

Aging↗
PubMed1995

A monoclonal antibody identifies vimentin filaments in Sertoli cells and in a subset of epithelial cells in the rat epididymis, urinary bladder, and prostate.

PURPOSE: We describe a monoclonal antibody (Mab B2) against vimentin filaments in rat Sertoli cells and in a subset of epithelial cells in the rat epididymis, prostate and urinary bladder. MATERIALS AND METHODS: The immunoreactivity of Mab B2 was examined on cryostat sections and immunoblots and compared with the reactivity of a previously characterized monoclonal antibody against vimentin. RESULTS: While both antibodies recognized vimentin in Sertoli cells, only Mab B2 recognized vimentin in urogenital tract epithelia. CONCLUSIONS: Vimentin is expressed in the urogenital tract, and differential reactivities of antibodies may be responsible for conflicting results reported elsewhere for vimentin expression.

Animals↗
PubMed1993

P-cadherin and E-cadherin are co-expressed in MDCK cells.

The Madin-Darby canine kidney (MDCK) cell line is a valuable model of epithelial cell function and the role of E (epithelial)-cadherin in MDCK cell activity is well-appreciated. Northern and immunoblot analyses were used here to demonstrate that, in addition to E-cadherin, MDCK cell cultures also express P(placental)-cadherin. Immunocytochemistry was then used to compare the localization and detergent extractability of P- and E-cadherin. While both cadherins were found at areas of cell-cell contact, the relative ease with which P-cadherin could be extracted suggests a weaker anchorage to the actin cytoskeleton than has been demonstrated for E-cadherin, and may correspond to less permanent cell-cell interactions than those attributed to E-cadherin. The co-expression of different cadherins by MDCK cells has not been fully explored and may influence interpretations of MDCK cell function that previously considered only the activity of E-cadherin.

Animals↗
PubMed1993

Expression of E-cadherin in immature rat and mouse testis and in rat Sertoli cell cultures.

Cadherins are Ca(2+)-dependent cell adhesion molecules that play essential roles in organogenesis. Northern blot analysis was used to determine the levels of epithelial (E)-, neural (N)-, and placental (P)-cadherin expression in developing rat testes from 1-, 2-, 3-, and 4-wk-old rats. Highest expression of all three cadherins occurred during the first two postnatal weeks, prior to the establishment of the blood-testis barrier. Transcripts of all three cadherins were also detected in RNA isolated from Sertoli cells cultured from 3-wk-old rat testes. Immunoblot analysis demonstrated levels of E- and P-cadherin protein in the testis and Sertoli cells that corresponded to the levels of their respective RNA transcripts. Immunohistochemical reactivity of E-cadherin in 8-day-old mouse testis was associated with germ cells situated basally in the seminiferous tubules. This report establishes the expression of E-cadherin in the testis and the simultaneous expression of E-, N-, and P-cadherin in both testis and Sertoli cell cultures.

Animals↗
PubMed1993

An autoradiographic study of calcium phosphate ceramic bone implants in turkeys.

Sintered, porous cylinders of beta-tricalcium phosphate ceramic (TCP) and hydroxyapatite (HAP) labeled with 45Ca were implanted into turkey ulnae for up to six months. Histologic examination of ground sections revealed extensive bone remodeling in the defect regions and resorption of both TCP and HAP. Autoradiograms suggest time- and composition-dependent events. Initial release of calcium ions was observed from both ceramics into adjacent new bone. Diffusion of calcium from TCP, but not HAP, extended into original bone at later periods. At six months, however, calcium from HAP was located intracellularly and within channels of distribution in hard tissue, away from the implant site. These data suggest that physical processes dominate initial HAP resorption while biologic (cellular) mechanisms become more significant later.

Animals↗
PubMed1992

Follicle-stimulating hormone-regulated Sertoli cell proteins SCc1 and SCc2 are phosphorylated and mitochondrially associated.

Sertoli cell intracellular protein 1 (SCc1) and 2 (SCc2) are polypeptides found in rat Sertoli cell cultures incubated with either FSH or (Bu)2cAMP. They were first identified in [35S]methionine-labeled Sertoli cell lysates using two-dimensional gel electrophoresis. Here we extend these observations by showing that SCc1 and SCc2 are present in rat seminiferous tubules, ovaries, and granulosa cells incubated with either FSH or (Bu)2cAMP and in testicular peritubular cells incubated with (Bu)2cAMP. Peritubular cells do not, however, respond to FSH with the production of SCc1 and SCc2. Peptide mapping with N-chlorosuccinimide revealed that SCc1 and SCc2 have similar cleavage patterns, suggesting a common primary amino acid sequence that is modified posttranslationally. Metabolic labeling with [32P]orthophosphate provided direct evidence that SCc1 and SCc2 are phosphoproteins. A shift in mobility of SCc1 and SCc2 toward the basic region of the gel to positions designated SCc1' and SCc2' occurred when cell lysates were treated with alkaline phosphatase before electrophoresis, providing additional evidence that SCc1 and SCc2 are phosphoproteins. SCc1 and SCc2 are also shown to be mitochondrially-associated in the Sertoli cell. Peptide maps of SCc1, SCc2, SCc1', and SCc2' obtained by treatment with alpha-chymotrypsin, are identical to proteolytic maps of proteins pp30', p30, and pp30 from adrenocortical cells. SCc1, SCc2, SCc1', and SCc2' are homologous with regard to their regulated expression, electrophoretic mobility, and mitochondrial localization to the adrenal proteins pp30' and pp30 as well as a series of 30 kilodalton proteins from MA-10 Leydig tumor cells. Both the adrenal cell proteins and the Leydig tumor cell proteins are thought to participate in cholesterol transport to the inner mitochondrial membrane, providing substrate for the cholesterol side-chain cleavage enzyme complex, an activity which the Sertoli cell does not perform, suggesting that alternative functions must be sought for SCc1 and SCc2 in Sertoli cells.

Adrenal Cortex↗
PubMed1987

Germ cell binding to rat Sertoli cells in vitro.

The interaction between male germ cells and Sertoli cells was studied in vitro by co-incubation experiments using isolated rat germ cells and primary cultures of Sertoli cells made germ cell-free by the differential sensitivity of germ cells to hypotonic shock. The germ cell/Sertoli cell interaction was examined morphologically with phase-contrast and scanning electron microscopy and then quantified by measuring radioactivity bound to Sertoli cell cultures after co-incubation with added [3H]leucine-labeled germ cells. Germ cell binding to Sertoli cell cultures was the result of specific adhesion between these two cell types, and several features of this specific adhesion were observed. First, germ cells adhered to Sertoli cell cultures under conditions during which spleen cells and red blood cells did not. Second, germ cells had a greater affinity for Sertoli cell cultures than they had for cultures of testicular peritubular cells or cerebellar astrocytes. Third, germ cells fixed with paraformaldehyde adhered to live Sertoli cultures while similarly fixed spleen cells adhered less tightly. Neither live nor paraformaldehyde-fixed germ cells adhered to fixed Sertoli cell cultures. Fourth, germ cell binding to Sertoli cell cultures was not immediate but increased steadily and approached a maximum at 4 h of co-incubation. Saturation of germ cell binding to Sertoli cell cultures occurred when more than 4200 germ cells were added per mm2 of Sertoli cell culture surface. Finally, germ cell binding to Sertoli cell cultures was eliminated when co-incubation was performed on ice. Based on these observations, we concluded that germ cell adhesion to Sertoli cells was specific, temperature-dependent, and required a viable Sertoli cell but not necessarily a viable germ cell. These results have important implications for understanding the complex interaction between Sertoli cells and germ cells within the seminiferous tubule and in the design of future experiments probing details of this interaction.

Animals↗
PubMed1986

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions.

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates. Our observations should facilitate efforts to achieve a differentiated functional state of Sertoli and peritubular cells in culture as well as to select secretory proteins for assessing their possible biological role in testicular function.

Animals↗
PubMed1986

Temporal appearance and cyclic behavior of Sertoli cell-specific secretory proteins during the development of the rat seminiferous tubule.

Electrophoretic and morphologic methods have been used to study the time course of [35S] methionine-labeled proteins accumulated in the incubation medium of rat fetal testes and seminiferous cords/tubules during their development. We have found that Sertoli cell-specific secretory proteins S70, S45 and S35 became progressively prominent as premeiotic, meiotic and postmeiotic spermatogenic events were established in the seminiferous tubules. In the sexually mature rat, S70, S45 and S35 were expressed in a spermatogenic stage-dependent manner. While S70, S45 and S35 were present in Stage VII-VIII, S45 and S35 were observed in Stages X and XIV. Neither S70, S45 nor S35 were detected in Stage IV. A relevant group of high molecular weight proteins, previously reported as characteristic products of cultured peritubular cells, accumulated in the incubation medium of seminiferous cords from postnatal Day 0-15 rats. This group of high molecular weight proteins appears when peritubular cells are proliferative and are engaged in the organization of the seminiferous tubular wall. A low molecular weight protein, designated T35, was also detected. T35 was prominent in the medium of incubated fetal testes and seminiferous cords of postnatal rats 0- to 5-days-old and disappeared gradually thereafter. A set of proteins (designated SP1 and SP2) previously ascribed to both cultured Sertoli and peritubular cells, were recognized during the early postnatal stages of seminiferous tubular development. SP1 and SP2 displayed age-dependent fluctuations in their [35S] methionine labeling. The timing of appearance of S70, S45, and S35 indicates both age- and spermatogenic stage-related activity that, in the future, may prove to be functionally significant in the spermatogenic process.

Animals↗
PubMed1983

Isolation and culture of rat seminal vesicle epithelial cells. The use of the secretory protein SVS IV as a functional probe.

A method for the isolation and culture of seminal vesicle epithelial cells obtained from control and androgen-primed sexually-immature, uncastrated rats is described. This method allows the establishment of monolayer cultures from aggregates of seminal vesicle epithelial cells isolated after trypsin and collagenase digestion. Phase contrast and transmission electron microscopic methods demonstrate that cell aggregates, after attaching to the substrate, establish within 48 h a colony-like, epithelial-like growth pattern. Immunofluorescent localization studies of SVS IV, an androgen-dependent secretory protein purified from rat seminal vesicle secretion, show that cultured seminal vesicle epithelial cells are immunoreactive. An electrophoretic analysis of [35S]methionine-labeled secretory proteins immunoprecipitated with rabbit anti-SVS IV serum demonstrate that, whereas SVS IV is newly-synthesized and accumulated in the medium of cultured seminal vesicle cells established from androgen primed rats, cultured cells from control rats appear to synthesize and accumulate SVS IV in a precursor form. Results of this work show that seminal vesicle epithelial cells in culture not only retain several structural features representative of the tissue but also serve as a potential system for the study of androgen action.

Animals↗
PubMed1982

Hormonal regulation of protein synthesis, secretion, and phosphorylation in cultured rat Sertoli cells.

The accumulation of two polypeptides, SCm1 and SCm2, in the medium of Sertoli cell cultures is enhanced by follicle-stimulating hormone (FSH) but is unaffected by either the cAMP analog, N6,O2'-dibutyrl cAMP or luteinizing hormone. The assigned molecular weights of SCm1 and SCm2 differ from those of androgen-binding protein subunits or any other previously identified Sertoli cell secretory product. Incubation of Sertoli cell cultures with either FSH or N6,O2'-dibutyryl cAMP also stimulates the incorporation of [35S]methionine into two intracellular polypeptides, SCc1 and SCc2. In addition, the phosphorylation of three intracellular polypeptides, SCc3, SCc4, and SCc5, is intensified when Sertoli cell cultures are treated with either FSH or N6,O2'-dibutyryl cAMP. Based on these results and on previous work, we conclude that (i) SCm1 and SCm2 may, like androgen-binding protein, be secreted by Sertoli cells and function extracellularly while SCc1 and SCc2 are involved in FSH-dependent intracellular activity; (ii) SCc3, SCc4, and SCc5 are possible substrates for FSH-stimulated, cAMP-dependent protein kinase activity; and (iii) SCc5 is an isoelectric variant of vimentin-type intermediate filament protein presumably involved in FSH- and N6,O2'-dibutyryl cAMP-induced Sertoli cell shape changes.

Animals↗
PubMed1981

Lodish model and regulation of ribosomal protein synthesis by insulin-deficient chick embryo fibroblasts.

The production of ribosomal proteins in chick embryo fibroblasts that have been deprived of insulin is depressed in a much greater degree than that of most or all other cell proteins. Previous observations ruled out explanations for the preferential decrease in ribosomal protein formation that depend upon a selective destruction of ribosomal protein messages or a regulatory role for nascent ribosomal ribonucleic acid. The proposition has now been examined that ribosomal protein messenger ribonucleic acids (mRNAs) in the hormone-deficient chick embryo cells have a lower affinity for a limiting, early acting component of the initiating machinery than do most other cell messages and, in consequence, suffer from a translational disadvantage. The approach that was used depends upon the findings of Lodish and others that all mRNAs are not initiated with equal ease, that inhibitors of elongation favor the initiation of low-affinity mRNAs, and that agents that dampen an early step in initiation discriminate against the low-affinity messages. The idea was tested by comparing the effects of various inhibitors on the rates of synthesis of total cell protein and individual nonribosomal proteins, on the one hand, with those of individual ribosomal proteins, on the other. The results fit the Lodish model and are consistent with the conclusions that ribosomal protein mRNAs are more poorly initiated in the resulting fibroblasts than are most or all other cell messages and that this condition is largely or entirely responsible for the low rate of ribosomal protein formation.

Animals↗