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N Groome

Publications and source records attributed to N Groome.

At least 37 records · Page 2Linked to original sources
PubMed1999

Prospectively measured levels of serum follicle-stimulating hormone, estradiol, and the dimeric inhibins during the menopausal transition in a population-based cohort of women.

The aims of this study were: 1) to describe, in relation to the date of final menses, the average hormone levels of women in the years before and after this date and to determine the extent to which these average levels were dependent on age and body mass index (BMI); and 2) to determine the degree of tracking in residual hormone levels [i.e., the extent to which individuals above (below) the mean for their age or time relative to final menstrual period (FMP) and BMI remain above (below) the mean as time progresses]. Serial levels of serum FSH, circulating estradiol (E2), and the dimeric inhibins (INH) A and B were measured annually in 150 women who experienced a natural menopause during 6 years of follow-up. Means of the log-transformed hormonal levels were analyzed as a double-logistic function of time relative to FMP, as well as age and BMI and correlations between repeated hormonal levels, were measured. Mean FSH levels started to increase from about 2 years before the FMP, increased most rapidly about 10 months before the FMP, and had virtually plateaued by 2 years after the FMP. FSH levels were, on average, 3% greater for each year of age and 2% lower for each kg/m2 of BMI. After adjusting for time relative to the FMP, logFSH showed modest tracking. Age-adjusted values of logFSH were moderately correlated across time, and much of this tracking was explained by the actual timing of a woman's FMP. Mean E2 levels started to decrease about 2 years before the FMP, decreased most rapidly around the time of the FMP, and had virtually plateaued by 2 years after the FMP. E2 levels were lower, on average, by about 9% per year of age, and residual values showed weak tracking. Levels of both INHA and INHB decreased, on average, in the years before the FMP and were undetectable (INHA, < 10 pg/mL; INHB, < 25 pg/mL) in the majority of women by the time of the FMP and in almost all women by 4 years post-FMP. Significant negative correlations between log serum FSH and log E2 (r = -0.73) and log INHA (r = -0.41) and log INHB (r = -0.36) were observed. It is concluded that substantial changes in reproductive hormone levels occur within 1-2 yr on each side of the FMP, that falling concentrations of E2 and the INH contribute to the rising concentrations of FSH, and that there is no single reliable hormonal marker of menopausal status for an individual woman.

Aging↗
PubMed1999

Control of early ovarian follicular development.

Early follicular growth refers to the development of an ovarian follicle from the primordial to early antral phase. In sheep and cows these phases of growth can be classified by the configuration of granulosal cells in the largest cross-section of the follicle as types 1 (primordial), 1a (transitory) 2 (primary), 3 and 4 (preantral) and 5 (early antral). Follicles classified as type 1 may be highly variable within each species with respect to number of granulosal cells and diameter of oocyte. Much of the variation in granulosal cell composition of type 1 follicles may occur at formation and this may account for the variability in granulosal cell composition throughout subsequent stages of growth. There appear to be important differences among species (for example sheep and cattle) in the number and function of granulosal cells relative to the diameter of the oocyte during the initiation of follicular growth. There is evidence that most, if not all, of the growth phases from types 1 to 5 are gonadotrophin-independent and that follicles develop in a hierarchical manner. In sheep, cows and pigs, numerous growth factor, growth factor receptor and gonadotrophin receptor mRNAs and peptides (for example c-kit, stem cell factor, GDF-9, beta B and beta A activin/inhibin subunit, alpha inhibin subunit, follistatin, FGF-2, EGF, EGF-R, TGF beta 1,2 and 3 FSH-R and LH-R) are expressed in a phase of growth (for example types 1-5)-specific and cell-specific manner. However, the roles of many of these factors remain to be determined.

Animals↗
PubMed1998

Apoptosis is induced in glioma cells by antisense oligonucleotides to protein kinase C alpha and is enhanced by cycloheximide.

The protein kinase C (PKC) activity of human glioma cells correlates with their rate of proliferation. We report here that the down-regulation of the predominant PKC isoform of glioma cells, alpha, by antisense phosphorothioate oligonucleotides (AS PTO) significantly reduced the rate of proliferation of three human glioma cell lines. This reduction in growth rate was attributed to apoptosis, as assessed by terminal deoxynucleotidyl transferase (TdT) assay. Unexpectedly, when low concentrations of the protein synthesis inhibitor cycloheximide (CHX) were administered to A172 cells immediately prior to AS PTO treatment, a marked enhancement in the number of apoptotic cells was observed. These findings suggest that PKC alpha plays a pivotal role in the ability of gliomas to avoid apoptotic cell death.

Apoptosis↗
PubMed1997

Association of the common gamma-chain with the human IL-7 receptor is modulated by T cell activation.

IL-7 acts on both resting and activated T cells. The functional IL-7R is reported to consist of two subunits, the ligand binding IL-7R and the common gamma-chain (gamma c chain), where IL-7R:gamma c chain association is driven solely by ligand binding. However, we now demonstrate that in primary T cells this event is also controlled by cellular activation. We show that IL-7R:gamma c chain complexes are detected in activated, but not in resting, T cells despite similar levels of gamma c chain expression, implying that the gamma c chain is not associated with the IL-7R in unstimulated T cells. Furthermore, IL-7R:gamma c chain association correlates with the expression of JAK-3 in T cells, but not in transfected COS-7 cells. The finding that IL-7R:gamma c chain assembly is controlled at a level beyond that of receptor expression has important implications for the control of cytokine function in T cells.

Antibodies, Monoclonal↗
PubMed1997

Interleukin-1beta-induced expression of protein kinase C (PKC)-delta and epsilon in NIH 3T3 cells.

NIH 3T3 cells express the alpha, delta, epsilon and zeta isoenzymes of protein kinase C(PKC). Following stimulation of cells (24 h) with the pro-inflammatory cytokine, interleukin 1beta (IL-1beta), we observed, by Western blotting, a dose-dependent effect on the levels of PKC-epsilon and delta, but not on alpha or zeta. Moreover, time course analysis revealed that the isoenzymes, PKC-delta and epsilon were induced by IL-1beta after 7 h. Again, no change in PKC-alpha or zeta levels after IL-1beta treatment were detected. Incubation with selective PKC inhibitor peptides blocked the PKC-alpha, delta, epsilon and zeta antibodies binding to their respective isoenzyme bands. We also observed that the addition of the tumour-promoting phorbol ester, Phorbol 12-myristate 13-acetate (PMA), downregulated PKC-alpha, delta and epsilon by 7 h in NIH 3T3 cells. PMA did not affect constitutively produced PKC-zeta protein levels even after 24-h treatment. In summary, these results demonstrate that IL-1beta induces protein synthesis of the Ca2+-independent PKC-delta and epsilon isoforms in NIH 3T3 cells. The differences observed here between PKC isoenzymes in response to IL-1beta suggest that each isoenzyme may have a unique role in the signal transduction pathways of IL-1beta and that such isoenzyme may have a unique role in the signal transduction pathways of IL-1beta and that such selective expression may influence the action of agents which require PKC for signal transduction acting in concert with IL-1.

3T3 Cells↗
PubMed1997

Gonadotropic control of secretion of dimeric inhibins and activin A by human granulosa-luteal cells in vitro.

PURPOSE: It is well established that human granulosa cells and luteal cells express inhibin/activin subunit protein and secrete immunoreactive inhibin. The gonadotropic control of secretion of different molecular forms of inhibin and activin A by granulosa-luteal cells (G-LCs) was investigated using recently developed specific enzyme immunoassays (EIAs). METHODS: Granulosa-luteal cells obtained at IVF egg pickup were cultured in a serum-free medium at 37 degrees C in a water-saturated incubator with 5% CO2 for up to 5 days. Experiments with varying concentrations of human FSH, hLH, and hCG were carried out. RESULTS: FSH raised the secretion of inhibin A and pro-alpha C-containing inhibins after 24 and 48 hr in culture. Inhibin B was raised after 24 hr and activin A was raised after 48 hr of FSH treatment. LH treatment for 24 hr stimulated inhibin A, inhibin B, pro-alpha C, and activin A. hCG stimulated G-LC secretion of inhibin A after 48 hr and pro-alpha C after 24 hr. Paradoxically, inhibin B secretion was inhibited by 1 and 10 ng/ml hCG after 48 hr. Activin A was stimulated by hCG after 24 and 48 hr of incubation. G-LC secretion of estradiol and progesterone was also stimulated significantly by LH and hCG. CONCLUSIONS: Secretion of dimeric inhibins and activin A is controlled differentially by gonadotropins.

Activins↗
PubMed1997

The location of acidic fibroblast growth factor in the breast is dependent on the activity of proteases present in breast cancer tissue.

Acidic fibroblast growth factor (FGF1) and two of its receptors, FGFR1 and FGFR4, were localized in cryostat sections of normal, benign and malignant human breast tissue by immunohistochemistry. Without pretreatment, FGF1 staining was mainly seen in normal epithelial cells. However, polymerase chain reaction (PCR) analysis and immunoblotting of isolated normal epithelial and myoepithelial cells showed FGF1 mRNA and protein to be present in both cell types. Following incubation of frozen sections at 37 degrees C in phosphate-buffered saline, FGF1 staining was also revealed in myoepithelial cells and basement membrane adjacent to carcinoma cells. Treatment with protease inhibitors demonstrated that this effect was due to the activity of an endogenous protease. In contrast, FGF1 staining was found to be associated with the stroma adjacent to malignant cells only in the presence of protease inhibitors. FGFR1 and FGFR4 immunostaining was localized to both normal and malignant epithelial cells and to a lesser extent to myoepithelial cells. There was no difference in the staining intensity for the FGF receptors between normal and cancer samples. The change in location of FGF1 between normal and malignant tissues and the sensitivity of stored FGF1 to the action of endogenous proteases raises the possibility of both autocrine and paracrine roles for FGF1 in the normal and malignant human breast.

Animals↗
PubMed1997

Down-regulation of a novel form of fibroblast growth factor receptor 1 in human breast cancer.

Monoclonal antibodies against two epitopes of FGFR-1 have been used to investigate FGFR-1 expression in the normal and neoplastic human breast. Different forms are detected in the different cell types constituting the normal breast. Moreover, breast cancer cells lack one form of FGFR-1. Western blot analysis showed 115-kDa and 106-kDa forms of FGFR-1 within the human breast. The 115-kDa band corresponds to the beta form of FGFR-1, whereas the 106-kDa band is truncated at the carboxyl terminus. The 106-kDa form of FGFR-1 is the major form present in breast fibroblasts and myoepithelial cells, whereas epithelial cells contain equal amounts of the 115-kDa and 106-kDa forms. Breast cancer cells, however, appear to contain only the 115-kDa form of FGFR-1. This expression pattern is reflected in malignant and non-malignant tissue samples. Using reverse transcription polymerase chain reaction (RT-PCR) analysis, we have shown that the 106-kDa FGFR-1 isoform is not the previously described alpha 2 receptor that arises from a 25-base pair insertion in the second kinase domain. It is probable that the 106-kDa FGFR-1 has different signalling properties to the full-length receptor, having lost at least one tyrosine at amino acid 766, which is required for phospholipase C activation. This form of FGFR-1 appears to be lost in all breast cancer cells analysed and its absence may have a bearing on malignancy.

Amino Acid Sequence↗
PubMed1997

The biological and immunological characterization of inhibin A and B forms in human follicular fluid and plasma.

In a previous study (see Ref.7), the molecular weight distribution of inhibin activity in fractionated human follicular fluid (hFF) and human male and female plasma/serum was determined by in vitro bioassay using ovine pituitary cells in culture and various specific inhibin A and inhibin alpha-subunit-directed immunoassays. It was shown, however, that the ovine in vitro bioassay detected inhibin B poorly. These findings are extended in the present study by the determination of the molecular weight profile of in vitro bioactivity using rat pituitary cells, which detects both inhibin A and B, a specific inhibin B enzyme-linked immunosorbent assay (ELISA), an RIA detecting the alpha N region of the alpha-subunit, an alpha-subunit ELISA (Pro-alpha C) directed to the inhibin forms containing the Pro sequence, and an alpha C subunit immunofluorometric assay that detects all inhibin forms. The profile in hFF of inhibin in vitro bioactivity, using rat pituitary cells in culture, significantly (P < 0.001) correlated with in vitro bioactivity using ovine pituitary cells (r = 0.85), inhibin A immunoactivity (r = 0.70), inhibin B immunoactivity (r = 0.89), and the combination of inhibin A + B immunoactivities (r = 0.93), with peaks of activity identified at 66K, 55K, 36K and 33K, consistent with presumed known mol wt forms of inhibin. Inhibin B profiles in fractionated serum from women stimulated with gonadotropins and male plasma consisted of two forms (66K and 36K), whereas inhibin A in female serum included, in addition, the 55K form. These findings indicated that higher molecular weight forms of inhibin B are present in biological samples, and their distribution differs from that of inhibin A, suggesting a differential processing of the precursor forms in the circulation. Pro-alpha C immunoactivity was identified in serum samples with prominent peaks at 36K and 29K (known Pro-alpha C subunit forms) and not with any high mol wt dimeric forms of inhibin. If this observation applies to a wider range of serum samples, the Pro-alpha C ELISA may provide an appropriate and specific assay for the measurement of free alpha-subunit. To compare immunoactivity levels between assays, the inhibins A, B, and Pro-alpha C standards were calibrated in terms of their alpha C subunit content, as determined by an alpha C subunit immunoassay, with the inhibin B standard containing 60% of the alpha C subunit content compared with either the inhibin A or Pro-alpha C standard. After adjustments of the various standards for this difference in alpha C subunit content, a comparison was undertaken of the combined levels of inhibins A, B, and Pro-alpha C immunoactivity across the hFF and serum chromatograms and compared with levels determined by the alpha-subunit-directed immunoassays. A high correlation (r = 0.59-0.96) was observed, indicating that the alpha-subunit immunoactivity in serum consists largely of a composite of presumed known molecular weight forms of inhibins A, B, and Pro-alpha C. It is concluded that: 1) inhibin in vitro bioactivity in hFF is largely attributed to the presence of 33-36K and 50-66K forms of inhibins A and B; and 2) inhibin alpha-subunit immunoactivity in hFF and serum is a composite of presumed known forms of inhibin A, inhibin B, and the alpha-subunit.

Animals↗
PubMed1997

Inhibin A and activin A in the first trimester of human pregnancy.

Recent studies show that high concentrations of inhibin A and activin A are present in the maternal serum throughout human pregnancy. The aim of this study was to determine whether the corpus luteum produces significant quantities of inhibin A and activin A during the first trimester of pregnancy. This prospective study examined two groups of women who had blood samples taken from 5-12 weeks gestation. One group consisted of 14 women with donor egg pregnancies (8 singletons and 6 multiples) who did not have corpora lutea, and the other group consisted 5 women with spontaneous pregnancies who had corpora lutea. Inhibin A and activin A were measured at weekly intervals using specific enzyme immunoassays. All pregnancies progressed to term, with healthy babies being delivered. Maternal serum concentrations of inhibin A significantly increased throughout the study period in the donor egg pregnancies (P < 0.001) and the control pregnancies (P < 0.001). Circulating concentrations of activin A also increased significantly in both the spontaneous and donor egg pregnancies (P < 0.001) during the study period. However, the concentrations of inhibin A and activin A in the first trimester of human pregnancy were not significantly different in the women with or without corpora lutea, suggesting a fetoplacental origin. Multiple donor egg pregnancies were found to have higher concentrations of inhibin A (P < 0.001) and activin A (P < 0.05) compared with singleton donor egg pregnancies, which also supports a placental source.

Activins↗
PubMed1997

Circulating concentrations of dimeric inhibin A and B in the male rhesus monkey (Macaca mulatta).

The purpose of this study was to determine the relative concentrations of inhibin A and B in peripheral serum of the adult male rhesus monkey and to examine the testicular contribution to these circulating forms of inhibin. In addition, inhibin B concentrations were also determined in peripheral sera of neonatal and juvenile males and in spermatic vein blood of adults. Immunoradiometric assays specific for the measurement of inhibin A and B were used. These assays also provided an opportunity to reexamine the physiological significance of a replacement infusion of recombinant human (rh)-inhibin A previously employed to study the role of this hormone in regulating FSH secretion in the monkey. In intact adults, the mean (+/-SE) serum concentration of inhibin B was 1008 +/- 184 pg/mL. In contrast, circulating inhibin A concentrations were very low (< 46 pg/mL). Inhibin B was consistently detected in neonatal monkey serum (275 +/- 57 pg/mL), and concentrations of this inhibin dimer increased throughout postnatal development, reaching maximum values in adulthood. Circulating inhibin A concentrations in neonatal and juvenile monkeys were undetectable (< 7 pg/mL). Both forms of inhibin were generally undetectable in castrate sera. The ratio of inhibin B concentrations in testicular venous blood to those in the peripheral circulation was 1.4:1. These findings indicate that, in the male monkey, inhibin B is the principal form of circulating dimeric inhibin, and that this hormone is derived exclusively from the testis. The elevated levels of circulating inhibin B in the juvenile male monkey suggest that, during this phase of development, testicular inhibin B secretion is relatively gonadotropin independent. Additionally, we found that the concentration of circulating inhibin A in castrate animals that had earlier received an iv infusion of rh-inhibin A (832 ng/h/kg BW) was 9881 +/- 2135 pg/mL, indicating that this mode of inhibin replacement may not have been entirely physiological.

Animals↗
PubMed1996

Encephalitogenic epitopes of myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein for experimental allergic encephalomyelitis induction in Biozzi ABH (H-2Ag7) mice share an amino acid motif.

Myelin basic protein (MBP) and synthetic MBP peptides were screened for their ability to induce experimental allergic encephalomyelitis in Biozzi ABH (H-2Ag7) mice. In contrast to the failure of native MBP to induce experimental allergic encephalomyelitis, the use of overlapping MBP peptides revealed epitopes within MBP 12-26 and MBP 21-35, which induced mild disease. In comparison with disease induced by spinal cord homogenate or peptides of myelin oligodendrocyte glycoprotein (MOG) or proteolipid protein (PLP), the low incidence indicates that, at least in ABH mice, MBP is a minor encephalitogen. However, the data suggest the presence of a peptide core between MBP 21-26 (HARHGF), which contains similar elements to the previously defined encephalitogenic MOG 1-22 and PLP 56-70 peptides. The fine specificity of these epitopes was further investigated using frame-shifted peptides, which indicated cores between MOG 9-15 (GYPIRAL) and PLP 62-68 (NVIHAFQ). Based on these pathogenic peptides, a putative H-2Ag7 binding motif is suggested that contains a series of hydrophobic, basic, small, and large hydrophobic residues within a 6 to 7 amino acid core. The core and particular importance of these four residues in PLP 56-70 was confirmed in vitro using amino acid substitution studies. These findings support many of the predictions made by computer modeling of peptide:H-2Ag7 interactions. This may have relevance in the design of strategies in the treatment of experimental autoimmune diseases in animals that express this haplotype.

Amino Acid Sequence↗
PubMed1996

Identification of a mitogen-activated protein kinase site in human myelin basic protein in situ.

Ultrastructural localization of a specific phosphorylated isomer of myelin basic protein (MBP) has been achieved with a monoclonal antibody specific for human MBP sequence, 89-105, in which Thr98 was phosphorylated. Cryosections of human brain white matter revealed that gold particles were found localized almost exclusively to the major dense line demonstrating that threonine 98 in the sequence Thr-Pro-Arg-Thr-Pro-Pro-Pro, a mitogen-activated protein kinase-specific site, was phosphorylated in vivo. In two cases of multiple sclerosis, the density of gold particles in myelin was reduced by about 30%, in one case by 42%, and by 80% in a fourth case. However, gold labelling was seen in areas of demyelination suggesting that the phosphorylated threonyl peptide was protected from degradation.

Amino Acid Sequence↗
PubMed1996

Characterization of inhibin immunoreactivity in post-menopausal women with ovarian tumours.

BACKGROUND AND OBJECTIVE: We have previously reported elevated serum immunoreactive inhibin (INH) levels in patients with ovarian malignancies, particularly granulosa cell and mucinous tumours. The present study was designed to compare INH measurements using a heterologous radioimmunoassay with cross-reactivity for inhibin alpha-subunit derived peptides with measurements obtained using a new ELISA specific for dimeric inhibin-A. It was hypothesized that granulosa cell tumours may secrete significant quantities of inhibin-A whereas mucinous tumours were unlikely to do so because of the lack of a relation between INH and FSH measurements in the latter group. DESIGN: Serum samples obtained from women found to have ovarian cancer were assayed using the heterologous radioimmunoassay (the Monash assay) and using an ELISA specific for dimeric inhibin (the Groome assay) and the results were compared. PATIENTS: Samples for assay were available from 69 normal post-menopausal control women, 12 patients with mucinous tumours of the ovary, 26 with serous tumours, 7 with granulosa cell tumours and 8 with various other ovarian tumours. Patients were post-menopausal or had been subjected to bilateral oophorectomy at the time these samples were collected. MEASUREMENTS: The Monash and Groome assays were carried out as described previously. The upper limit of normal for post-menopausal women in the Monash assay was 122 U/l and for the Groome assay was calculated to be 32 ng/l. RESULTS: Among the 69 normal subjects, 4 were found to have elevated inhibin levels using the Monash RIA (133-190 U/l) and 4 were found to have elevated levels in the Groome ELISA (45.5-55.3 ng/l). Among 12 patients with mucinous tumours, 10 (83%) had elevated inhibin levels using the Monash assay but only 3 (25%) had elevated levels with the Groome assay (P < 0.005). Among 26 with serous tumours, 15 (58%) had elevated levels in the Monash assay but only 1 (4%) in the Groome assay (P < 0.001). Among 7 samples from patients with granulosa cell tumours, 100% were elevated in the Monash assay and 71% in the Groome assay (NS, non-significant). In a miscellaneous group of tumours all 8 had elevated levels in the Monash assay and 2 in the Groome assay (P < 0.001). CONCLUSIONS: It was concluded that in normal postmenopausal subjects, INH is generally undetectable or present at low levels, consistent with the loss of ovarian function. The majority of granulosa cell tumours appear to secrete significant amounts of dimeric inhibin-A, whereas mucinous tumours secrete predominantly other forms of INH, presumably related to the alpha-subunit. Serous tumours may also secrete inhibin-related peptides but not dimeric inhibin-A. The nature of the inhibin related peptides produced by epithelial ovarian cancers remains to be characterized.

Adenocarcinoma, Mucinous↗
PubMed1996

Inhibin A and inhibin B are inversely correlated to follicle-stimulating hormone, yet are discordant during the follicular phase of the rat estrous cycle, and inhibin A is expressed in a sexually dimorphic manner.

Inhibin A and inhibin B are related dimeric protein hormones and endocrine regulators of the reproductive axis. Specifically, inhibin inhibits FSH secretion from the anterior pituitary. The inhibins are synthesized by the gonads and are themselves modulated by FSH. Although the activity of these ligands has been well characterized, the circulating concentrations of dimeric inhibin A and dimeric inhibin B have not previously been reported for the rat. Our group examined the serum concentration of inhibin A and inhibin B in normally cycling female rats, male rats, and in gonadectomized animals. Both inhibin isoforms are detected in intact female rat serum. Interestingly, inhibin B, but not inhibin A, is detected in intact male rat serum. Neither inhibin isoform is detected in long-term castrate female or male rats. In normally cycling female rats, inhibin A was low on the morning of metestrus and rose steadily to a peak on proestrus. In contrast, inhibin B was elevated on the mornings of metestrus, diestrus, and proestrus. Both ligands persisted in the serum until proestrus evening. Serum inhibins then declined beginning at 2100 h (inhibin A) or 1800 h (inhibin B) on proestrus, and the concentrations reached a nadir on the morning of estrus (0600 h). The nadir coincided with the peak of the secondary FSH surge. Both inhibins rebounded later on the morning of estrus. The results of this study demonstrate that dimeric, ovarian-derived inhibin A and inhibin B circulate in the female rat. The inverse relationship of the inhibins during the secondary FSH surge is consistent with the hypothesis that they participate in the regulation of reproductive cyclicity. The differing patterns of inhibin A and inhibin B during the period of follicular development on metestrus and diestrus suggest different follicle sources or regulation of these molecules during this period. We further demonstrate that inhibin B is the dominant form of FSH regulating protein in the male rat.

Animals↗
PubMed1996

Biological and immunological characterization of inhibin forms in human plasma.

A number of immunoassay methods have been developed recently to detect specifically the bioactive alpha-beta A subunit inhibin dimer (inhibin A) in human plasma. However, the specificity of these assays in terms of their ability to detect the range of inhibin forms found in plasma and their relationship to bioactivity have not been investigated. Inhibin was fractionated from human follicular fluid (hFF) and serum/plasma from women stimulated with gonadotropins (IVF serum), and from postmenopausal and male plasma, using a combined immunoaffinity/preparative SDS-PAGE procedure. The molecular weight profile of inhibin was established by inhibin in vitro bioassay, three alpha-beta A subunit specific immunoassays, and three alpha subunit-directed immunoassays that detect the alpha subunit as well as inhibin A and B forms. In hFF inhibin forms of 33, 36, 55 and 66K were detected by in vitro bioassay and by most immunoassays except for 33 k inhibin, which was nondetectable by one alpha-beta A ELISA. The alpha subunit-directed assays also detected activity in the 29-31K region, in some assays in considerably high levels. In IVF serum in vitro bioactivity and immunoactivities were detected between 27 and 100K with the alpha-beta A assays failing to detect all bioactive forms. Alpha subunit-directed assays gave similar immunoactive profiles. Neither in vitro bioassay nor alpha-beta A assays detected activity in post-menopausal plasma or male plasma, while alpha subunit-directed assays showed peaks predominantly at 36 k, although at low levels. It is concluded that dimeric inhibin A specific assays detected bioactive inhibin forms in hFF and to a lesser extent in IVF serum. Alpha subunit-directed assays correlated poorly with in vitro bioassay in hFF because of the high alpha subunit levels in this sample. The higher correlation between these assays in IVF serum suggested that there was little free alpha subunit. The 36K form in male plasma may be free alpha subunit or inhibin B.

Animals↗
PubMed1996

Reduced daunomycin accumulation in drug-sensitive and multidrug-resistant human carcinoma KB cells following phorbol ester treatment: a potential role for protein kinase C in reducing drug influx.

We have investigated the role of protein kinase C (PKC) in the multidrug resistance (MDR) phenotype of human KB carcinoma cell lines. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced daunomycin accumulation in both drug-sensitive KB-3-1 and MDR KB-C1 cells in a time-dependent manner. The inactive phorbol ester 4 alpha TPA did not reduce daunomycin accumulation, and the PKC inhibitor, Ro 31-8220, reversed the TPA effect. TPA had no effect on daunomycin efflux and did not induce Pgp expression in KB-3-1 cells or alter Pgp levels in KB-C1 cells. Linear, short-term daunomycin accumulation was reduced by pretreatment with TPA, an effect that could be reversed by Ro 31-8220. The effects of TPA on PKC subspecies localisation and downregulation were also examined. TPA initially induced translocation of PKCs alpha and delta, and to a lesser extent, PKC epsilon to the membrane fraction; 8 h after TPA treatment, differential effects on downregulation of PKCs alpha and delta were observed between cell lines, although PKC epsilon was not reduced in either cell line. We therefore propose that the TPA-induced reduction in daunomycin accumulation in KB cells is due to a PKC-mediated process, which is maintained after depletion of certain PKC subspecies or is due to activation of downregulation insensitive PKC subspecies. These results suggest that PKC may regulate drug resistance by reducing drug influx in a Pgp-independent manner in KB cells. This may represent a mechanism of drug-resistance independent of, or in addition, to, Pgp-mediated drug efflux.

ATP Binding Cassette Transporter, Subfamily B, Mem↗