Biomedical subjects

B A Keel

Publications and source records attributed to B A Keel.

At least 19 recordsLinked to original sources

PubMed1992

Purified human alpha fetoprotein inhibits growth factor-stimulated estradiol production by porcine granulosa cells in monolayer culture.

Purified alpha fetoprotein (AFP) synergizes with transforming growth factor alpha (TGF alpha) and insulin-like growth factor I (IGF-I) to enhance proliferation of porcine granulosa cells (pGC) in primary culture, suggesting a role for AFP in the modulation of growth factor-mediated cell growth. TGF alpha stimulates basal estrogen production by pGC and is in fact more potent than FSH in these cells. In this study, we investigated the effects of AFP on growth factor-stimulated estradiol (E2) production by pGC. Basal production of E2 was not altered by the addition of AFP. AFP dose-dependently inhibited TGF alpha-stimulated E2 production with statistically significant inhibition observed with 2.5 micrograms/ml. We have previously shown that the mitogenic effects of AFP are maximized with TGF alpha+IGF-I. E2 production was even more sensitive to AFP inhibition when the two growth factors were combined. Human serum albumin (HSA; 10 micrograms/ml) was without effect. AFP did not interfere with the E2 RIA, affect the uptake of or display specific in vitro binding of the androgen substrate. Furthermore, human AFP and HSA did not exhibit specific in vitro binding of E2, in contrast to purified rat AFP (positive control). These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit growth factor-stimulated E2 production by pGC in culture. Since AFP is known to increase TGF alpha+IGF-I mediated cell growth, these data suggest that AFP may be inhibiting the differentiated function (steroidogenesis) of pGC while enhancing the proliferation of these cells.

Amniotic Fluid↗

PubMed1991

Ovine luteinizing hormone. V. Significance of flow-through peaks observed during chromatofocusing as revealed by various methods of sample preparation and application.

In a previous study [Keel et al., Biol, Reprod., 36 (1987) 1102] the ovine luteinizing hormone (oLH) in pituitary extracts was chromatofocused on pH 10.5-7 gradients after equilibration in 25 mM triethylamine-HCl, pH 11.0, by gel permeation. Under these conditions, some immunoreactive oLH flowed through the columns unrestricted and this was interpreted to represent extremely basic isoforms. However, when selected flow-through peaks were re-chromatofocused, each was contaminated with other isoforms of oLH. In order to clarify this dilemma, various methods of sample preparation and application were systematically compared. Consistent with previous observations, variable amounts of the immunoreactive oLH in pituitary extracts equilibrated in triethylamine by gel permeation, dialysis, flow dialysis or ion-retardation chromatography eluted as flow-through peaks when chromatofocused. In contrast, when the ionic components in the pituitary homogenization buffer were removed by these methods as well as ultrafiltration and the proteins were applied to the resin in the elution buffer (1:45 Pharmalyte 8-10.5-HCl, pH 7.0), none of the immunoreactive oLH in pituitary extracts eluted as a flow-through peak. Thus, it appears that oLH eluting as a flow-through peak results from incomplete binding of the hormone to the chromatofocusing resin when applied in triethylamine.

Animals↗

PubMed1991

Chromatofocusing profile of purified human alpha-fetoprotein and albumin differs from those of crude samples: effect of protein concentration of the elution of the sample.

Chromatofocusing was utilized to characterize charge microheterogeneity of purified human alpha-fetoprotein (AFP) and human serum albumin (HSA). Crude cord blood samples yielded three isoforms: AFP-IA, IB, and II, with pIs 4.57 (52%), 4.27 (43%), and less than 4.00 (5%), respectively. In contrast, 10 micrograms of purified AFP or 250,000 cpm of 125I-AFP eluted entirely as isoform AFP-II. 125I-AFP focused in the presence of crude cord blood, amniotic fluid, adult male serum, or 25 mg purified HSA resulted in elution profiles similar to those of crude cord blood. Pure AFP focused along with 0.1, 1.0, 5.0, or 10 mg HSA showed a gradual shift from AFP-II to AFP-I. With greater than or equal to 5 mg HSA, isoform I was further resolve into AFP-IA and IB. Similarly, 250,000 cpm of 125I-HSA, which also eluted entirely as isoform II, showed a gradual shift to isoform I when increasing concentrations of unlabeled HSA were added. The resolution of isoform HSA-I in HSA-IA, IB, and IC was again improved with greater than or equal to 5 mg unlabeled HSA. When carrier proteins of varying pI values were chromatofocused along with purified AFP, it was observed that only those proteins with pIs in the range of AFP caused significant alteration in the relative distribution of AFP. We conclude that sample protein concentration and composition must be carefully considered when chromatofocusing is being used for purified samples and when the elution profiles of samples from different origins and varying protein concentrations are being compared.

Chromatography↗

PubMed1991

Human alpha-fetoprotein purified from amniotic fluid enhances growth factor-mediated cell proliferation in vitro.

Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth-promoting activity of alpha-fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5-20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25-5.0 micrograms/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin-like growth factor (IGF-I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF-I alone. The response of pGC to the proliferative effects of AF-AFP and CB-AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly.

Amniotic Fluid↗

PubMed1991

Human mammary tumor cell proliferation: primary role of platelet-derived growth factor and possible synergism with human alpha-fetoprotein.

Human mammary medullary carcinoma cells (passages 16 to 21) were cultured for 2 days to allow for attachment, followed by 6 days of culture in either fetal calf serum, human cord blood, human amniotic fluid, or growth factors in the presence or absence of purified human alpha-fetoprotein (AFP). When growth factors were tested alone, only platelet-derived growth factor produced a significant increase in cell proliferation. Although up to 40% amniotic fluid had no effect on cell proliferation, human cord blood was two-fold more potent than fetal calf serum at similar concentrations. The addition of 10 ng/ml of platelet-derived growth factor increased the proliferative activity of human cord blood 1.5- to 2.5-fold. Ablation of endogenous AFP by affinity chromatography reduced the proliferative activity of cord blood by 75%. Similarly, the mitogenic activity of cord blood plus platelet-derived growth factor was reduced by 56% when AFP was removed. Purified AFP dose-dependently enhanced the proliferative activity of platelet-derived growth factor. This synergistic effect was specific for platelet-derived growth factor. We conclude that platelet-derived growth factor is a major growth factor controlling the proliferation of these tumor cells and that AFP may enhance growth factor proliferative activity and human mammary tumor growth.

Amniotic Fluid↗

PubMed1991

Synergistic action of purified alpha-fetoprotein and growth factors on the proliferation of porcine granulosa cells in monolayer culture.

alpha-Fetoprotein (AFP) is present in high levels in fetal fluids, certain neoplasias, and regenerating liver. Although AFP's physiological role remains an enigma, we have recently demonstrated mitogenic activity for AFP. Using a primary monolayer culture system, we have further investigated the proliferative activity of purified AFP. Porcine granulosa cells from small ovarian follicles were attached for 2 days in Ham's F-12-Dulbecco's Modified Eagle's Medium (1:1) and 5% fetal calf serum, followed by 6 days of culture in medium containing 0.25% plasma-derived serum plus 25 micrograms/ml low density lipoprotein with or without growth factors and/or purified human AFP. In this system AFP alone does not stimulate proliferation. However, when combined with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I; 10 ng/ml each), AFP (5 micrograms/ml) significantly (P less than 0.01) enhanced growth factor-mediated proliferation 4.5-fold over that of medium controls. Equivalent doses of purified human serum albumin or transferrin demonstrated no effect. The effects of AFP were dose dependent, with significant (P less than 0.05) enhancement of proliferation (2.7-fold over controls) observed with as little as 0.313 micrograms/ml AFP. Increased proliferation was noticed as early as 24 h after the addition of AFP and by 48 h AFP, EGF, and IGF-I had significantly (P less than 0.05) increased proliferation over that seen in medium controls, cells treated with EGF plus IGF-I, or cells treated with 10% fetal calf serum plus EGF, and this trend continued linearly over 5 days of culture. AFP (5 micrograms/ml) significantly increased the proliferative response observed with increasing doses of EGF, IGF-I, or EGF plus IGF-I, but did not appear to alter the dose-response curves. AFP dose-dependently (1.25-5 micrograms/ml) and significantly (P less than 0.05) increased proliferation of porcine granulosa cells in response to platelet-derived growth factor (PDGF) and EGF (25 and 10 ng/ml, respectively), but not to PDGF alone. In contrast, AFP produced no further proliferation of porcine thecal cells in response to PDGF plus EGF. Binding of EGF, IGF-I, or PDGF to purified AFP could not be demonstrated. These results demonstrate that physiological levels of AFP, although not mitogenic alone, can significantly enhance the mitogenic activity of EGF plus IGF-I/PDGF and may function to modulate growth factor-mediated cell proliferation during development and neoplasia.

Animals↗

PubMed1991

A simple two-step purification of human and monkey alpha fetoprotein from amniotic fluid and serum.

We developed a two-step purification system to characterize alpha fetoprotein (AFP) in early gestation amniotic fluid and late gestation fetal serum or cord blood from monkey and human. It involves only two chromatographic steps, allows preparative purification using up to 12 ml of starting sample, can purify up to 350 micrograms of AFP at one time, and can be used to purify both fetal serum or amniotic fluid AFP from two different species. This procedure will allow detailed biochemical analysis of purified AFP from different stages of fetal development.

Amniotic Fluid↗

PubMed1990

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development.

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.

Amniotic Fluid↗

PubMed1990

A microcomputer data base system for an in vitro fertilization clinic programmed in BASIC.

A computer program was developed for the IBM personal computer to be used for in vitro fertilization and gamete intrafallopian transfer clinics. This program, written in BASIC, allows input, editing, updating, sorting, and printing of patient data. Statistical functions permit summation of patient data based on various combinations of user-defined treatment cycles, diagnoses, and protocols, thus making possible comparison of pregnancy and other patient data between and among various treatment groups and diagnoses. The statistical information can be continually updated and revised when new data become available on patients (such as confirmation of pregnancy by ultrasound or live births) and at the end of each cycle. The formats used are useful in assimilating individual clinic data for various surveys and other reporting requirements. The program can be easily modified by anyone with minimal training in the BASIC programming language.

Ambulatory Care Facilities↗

PubMed1990

The charge characterization of native and deglycosylated thyrotropin.

Chromatofocusing was used to characterize the charge microheterogeneity of crude pituitary, highly purified native bovine (b) and deglycosylated (dg) thyrotropin (TSH) preparations. Greater than 90% of crude pituitary TSH and native bTSH-I-1 bound to concanavalin-A (conA) columns while dgbTSH was excluded from the column. Extracts of ovine (o) pituitaries contained at least nine species (isohormones) of immunoreactive TSH when chromatofocused on pH 7.5-4 gradients. Highly purified native bTSH-I-1 displayed a similar elution profile. In contrast, dgbTSH eluted as a single homogeneous species with an apparent pI greater than 7.5. When subjected to chromatofocusing on a pH 10.5-7 gradient, 68% of crude pituitary oTSH and 96% of native bTSH-I-1 was bound to the column but could be eluted with NaCl indicating acidic species, while at least three peaks of dgbTSH could be resolved having apparent pI's of 9.12, 9.03 and 8.98. These data suggest that although removal of the carbohydrate moieties markedly alters the isohormone pattern of TSH, chemical deglycosylation does not completely eliminate the charge microheterogeneity of bTSH.

Animals↗

PubMed1990

Human alpha fetoprotein enhances epidermal growth factor proliferative activity upon porcine granulosa cells in monolayer culture.

Alpha fetoprotein (AFP) is present at high concentrations in fetal fluids, certain neoplasias, and regenerating liver. Its physiological function remains largely unknown. Using a primary monolayer culture system, we investigated the proliferative activity of human (h) cord blood (CB) and highly purified AFP. hAFP, purified from hCB by Cibacron blue and immunoaffinity chromatography was homogeneous on SDS-PAGE and silver stain. Porcine granulosa cells from ovarian small follicles were cultured (25,000/cm2) for 2 days in medium (Ham's F-12:DMEM, 1:1) + 5% fetal calf serum (FCS) to facilitate attachment, followed by 6 days in medium containing: FCS, hCB or h amniotic fluid (1-20%)+/- EGF (10 ng/ml); or 0.25% plasma-derived serum (PDS) containing human low density lipoprotein (LDL, 25 ug/ml), +/- AFP (0.05-5 ug), and +/- EGF and IGF-I (10 ng/ml). In this system, single growth factors do not stimulate proliferation, a characteristic also exhibited by AFP. When combined with EGF, however, AFP dose-dependently increased proliferation to levels equal to that obtained with 10% FCS (2.3-fold increase vs PDS/LDL controls). When combined with EGF+IGF-I, AFP again dose-dependently increased proliferation to levels equal to that obtained with 10% FCS+EGF (6.7-fold increase vs controls). Purified human albumin used in place of AFP was not effective. TGF-a but not PDGF could replace the proliferative activity of EGF. These results suggest that AFP at physiological levels, although not itself mitogenic, can enhance the mitogenic activity of EGF and TGF-a and may function to modulate growth factor-mediated proliferation during development and neoplasia.

Animals↗

PubMed1990

Characterization of the acidic forms of ovine pituitary luteinizing hormone.

The naturally occurring acidic forms of ovine pituitary LH were examined by chromatofocusing. Immunoreactive oLH eluted as six variants (elutions pHs of greater than 7.4, 6.83, 6.59, 6.23, 5.5-4.0, and less than 4.0, designated as "basic forms" and variants II-VI, respectively). In rams, the percentage in each variant averaged 46, 9, 28, 10, 5 and 2, respectively, of the total LH recovered. oLH from non-implanted wethers or wethers implanted with dihydrotestosterone, E2 or dihydrotestosterone + E2 eluted as six similar variants but differences were noted in the distribution of oLH among the variants. Compared to rams, pituitary extracts from non-implanted and dihydrotestosterone-implanted wethers contained significantly higher percentages of oLH eluting as basic forms. In contrast, E2- or dihydrotestosterone-implanted wethers exhibited significantly higher percentages of oLH in the acidic variants. Each of these variants was demonstrated to contain biologically active oLH using an in vitro bioassay. Furthermore, the basic forms of oLH were the most abundant biologically active forms present in pituitary extracts. These results suggest that in addition to the seven basic forms of oLH previously described, there are at least five naturally occurring acidic forms of oLH in pituitary extracts which are biologically active, and the distribution of pituitary oLH among its isohormones, including the acidic forms, appears to be modified by steroids.

Analysis of Variance↗

PubMed1989

Semen analysis data from fresh and cryopreserved donor ejaculates: comparison of cryoprotectants and pregnancy rates.

Patients (155) were selected at random for fresh or cryopreserved semen and inseminated on the predicted day of ovulation. Semen analysis was performed using a microcomputerized multiple-exposure photography system. Frozen semen was used with either glycerol or TEST-yolk (TEST-buffered 20% egg yolk with 10% glycerol) as the cryoprotectant. Cryopreservation resulted in significant decreases in all semen parameters measured. Of these, velocity appeared to be the least effected. TEST-yolk provided significantly more protection against a reduction in velocity compared with glycerol. A total of 18, 17, and 27 patients conceived using fresh, glycerol, or TEST-yolk-preserved semen, respectively. For these same groups, a cumulative pregnancy rate of 52.9%, 27.1%, and 68.5%, respectively, was observed (not significant). The total number of motile sperm per insemination used for fresh artificial inseminations resulting in conception (132.4 X 10(6] was significantly greater than the number used for successful glycerol- and TEST-yolk-preserved semen (approximately 24 X 10(6]. These results demonstrate that although the number of motile sperm of cryopreserved ejaculates are dramatically reduced compared with the fresh counterparts, if a minimum criteria for ejaculate quality is established, the use of cryopreserved semen can offer a viable, effective, and relatively safe alternative to artificial insemination by donor with fresh semen.

Cell Survival↗

PubMed1989

A microcomputer data base system for maternal serum alpha fetoprotein screening programmed in BASIC.

A computer program was developed for the IBM PC computer to be used for maternal serum alpha fetoprotein screening (MSAFP). This program, written in BASIC, 1) maintains a data base for patients tested, 2) allows input and storage of MSAFP results, 3) calculates the gestational age dependent result in multiple of the median (MOM), 4) makes an individualized interpretation of the result based on maternal age, weight and diabetes, 5) makes an appropriate recommendation for subsequent action based on the result, and 6) prints a report containing the above information. In addition, the program will print a daily log of patients tested, provides a follow-up sheet to assist in tracking abnormal results to term, converts the data files into files which can be easily intergrated into more powerful data base programs, and generates a monthly statement for billing purposes. The program can be easily modified by someone with minimal training in the BASIC programming language.

Female↗

PubMed1988

Correlation of human sperm motility characteristics with an in vitro cervical mucus penetration test.

Semen analysis was performed on 226 ejaculates by an integrated microcomputerized system employing the multiple-exposure photography (MEP) method. Mucus penetration tests were performed in vitro using commercial preparations of bovine cervical mucus. A highly significant (P less than 0.001) correlation between mucus penetration distance and sperm count (r = 0.582), motility (r = 0.357), velocity (r = 0.569), motile density (r = 0.582), motility index (r = 0.467), and morphology (r = 0.383) was observed. Increased percentages of immature germ cells (r = -0.318) and bent-tailed sperm (r = -0.221) were the most strongly correlated with mucus penetration. Approximately 10% to 15% of patients with otherwise normal semen parameters displayed poor penetration of mucus. Conversely, 5% to 40% of patients with abnormal semen parameters displayed excellent penetration of the mucus. Motile density and velocity demonstrated the strongest relationship with the outcome of the mucus penetration test. These results suggest that a significant subpopulation of patients can be identified as having inadequate (or adequate) penetration of mucus with otherwise normal (or abnormal) motility characteristics.

Animals↗

PubMed1988

Effects of aging on biologically active pituitary LH in rats.

The effects of aging in the male rat on serum testosterone and LH and on the amounts of immunoreactive and biologically active pituitary LH were studied. Animals were killed at three, six, 12 and 24 months of age and serum and pituitary extracts prepared. Serum testosterone was significantly reduced by six months of age and remained at this low level throughout the study. The serum testosterone circadian rhythm was severely blunted in old animals. Serum LH was not effected by aging. Pituitary immunoreactive LH content was significantly decreased by 24 months of age, whereas the biological to immunological (B/I) ratio was not altered. However, the absolute amount of biologically active pituitary LH declined steadily with age from six months. These age-related effects were not caused by a gross redistribution of pituitary LH charge isohormones. These results suggest that the hypostimulated testis in the aging male rat may be related to a reduced amount of biologically active LH available for secretion.

Aging↗

PubMed1988

Variation in the biologic and biochemical characteristics of human menopausal gonadotropin.

Lot differences in the biopotency of human menopausal gonadotropin (hMG) were evaluated and the potential biochemical basis was investigated. The in vivo biopotency of hMG was assessed by a unique bioassay that evaluates the number of ova shed in the cyclic hamster in response to hMG administration. Significant variation in hMG lots was observed using this assay. When subjected to chromatofocusing, hMG displayed five immunoreactive follicle-stimulating hormone (FSH) isohormones and nine luteinizing hormone (LH) isohormones. The relative distribution of FSH, but not LH isohormones, was slightly but significantly different between the lots tested. These data indicate that significant differences exist in the ability of commercially available hMG to stimulate follicular development and ovulation. The biochemical basis for these differences in in vivo biopotency remains to be elucidated.

Animals↗

PubMed1987

Charge microheterogeneity of ovine follicle-stimulating hormone in rams and steroid-treated wethers.

Chromatofocusing was utilized to separate the isohormones of ovine follicle-stimulating hormone (oFSH) in pituitary extracts from rams, wethers, and wethers receiving Silastic implants of dihydrotestosterone (DHT), 17 beta-estradiol (E2), or both DHT and E2. Pituitary extracts were prepared by homogenization and centrifugation at 100,000 x g. Extracts of ram pituitaries yielded at least nine species (isohormones) of immunoreactive oFSH having apparent isoelectric points (pIs) of greater than 7.40, 6.74, 6.52, 5.76, 5.20, 4.74, 4.44, 4.10, and less than 4.00. These isohormones were designated by the letters A-H and Z, respectively. Eighty-four percent of the immunoreactive ram oFSH recovered from the chromatofocusing column was very acidic in nature, having apparent pIs less than 5.30. The majority of the immunoreactive oFSH was focused in an area different from that of most of the immunoreactive ovine luteinizing hormone (oLH) and ovine thyroid-stimulating hormone (oTSH). Pituitary extracts from control and steroid-treated wethers also contained these nine oFSH isohormones, but significant differences were noted in the relative distribution among the isohormones. Castration resulted in a 4-fold increase in isohormone B and a concomitant reduction in isohormones E and H. DHT administration returned these levels to the values observed in the ram, whereas E2 administration produced a significant 2-fold increase in the most acidic form (isohormone Z). The implant combination produced isohormone profiles comparable to that of E2 alone. Neuraminidase treatment in vitro of both crude pituitary extracts or highly purified iodinated oFSH abolished the most acidic form and caused a marked shift in the isohormone pattern to more basic species. These results demonstrate that 1) at least nine isohormones of oFSH can be separated reproducibly from the male ovine pituitary by chromatofocusing, 2) the majority of FSH in the pituitary exists in acidic form, 3) castration and steroid administration alter the distribution of oFSH in the pituitary among its isohormones, and 4) at least a portion of oFSH-charge microheterogeneity appears to be due to the presence of sialic acid residues on the molecule.

Animals↗