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Biomedical subjects

R E Canfield

Publications and source records attributed to R E Canfield.

At least 19 recordsLinked to original sources
PubMed1999

Efficacy of superovulation and intrauterine insemination in the treatment of infertility. National Cooperative Reproductive Medicine Network.

BACKGROUND: Induction of superovulation with gonadotropins and intrauterine insemination are frequently used to treat infertility. We conducted a large, randomized, controlled clinical trial of these treatments. METHODS: We studied 932 couples in which the woman had no identifiable infertility factor and the man had motile sperm. The couples were randomly assigned to receive intracervical insemination, intrauterine insemination, superovulation and intracervical insemination, or superovulation and intrauterine insemination. Treatment continued for four cycles unless pregnancy was achieved. RESULTS: The 231 couples in the group treated with superovulation and intrauterine insemination had a higher rate of pregnancy (33 percent) than the 234 couples in the intrauterine-insemination group (18 percent), the 234 couples in the group treated with superovulation and intracervical insemination (19 percent), or the 233 couples in the intracervical-insemination group (10 percent). Stratified, discrete-time Cox proportional-hazards analysis showed that the couples in the group treated with superovulation and intrauterine insemination were 3.2 times as likely to become pregnant as those in the intracervical-insemination group (95 percent confidence interval, 2.0 to 5.3) and 1.7 times as likely as those in the intrauterine-insemination group (95 percent confidence interval, 1.2 to 2.6). The couples in the intrauterine-insemination group and in the group treated with superovulation and intracervical insemination were nearly twice as likely to conceive as those in the intracervical-insemination group. CONCLUSIONS: Among infertile couples, treatment with induction of superovulation and intrauterine insemination is three times as likely to result in pregnancy as is intracervical insemination and twice as likely to result in pregnancy as is treatment with either superovulation and intracervical insemination or intrauterine insemination alone.

Abortion, Spontaneous↗
PubMed1998

Measuring human chorionic gonadotropin in the absence of implantation with use of highly sensitive urinary assays for intact beta-core and free beta epitopes.

OBJECTIVE: To determine if human chorionic gonadotropin (hCG) can be absorbed from the uterine cavity in the absence of an embryo. DESIGN: Prospective study. SETTING: University-based assisted reproduction program. PATIENT(S): Eight functionally agonadal patients (age range, 33-46 years) who were taking hormone replacement therapy so that they could receive donated oocytes. INTERVENTION(S): Intrauterine instillation of 50 microL of hCG (10,000 IU) during a mock cycle before an attempt at oocyte donation. MAIN OUTCOME MEASURE(S): Spot urine measurements of different hCG epitopes (intact beta, beta-core, and free beta) at timed intervals (12, 20, 44, and 68 hours after instillation). RESULT(S): All hCG epitopes were detected in the urine at the first sampling interval, and levels decreased in subsequent sampling intervals. Measurement of the serum hCG level confirmed that systemic absorption had occurred and that the urine measurements were not a result of specimen contamination through the cervix. CONCLUSION(S): hCG may be systemically absorbed into the blood through the uterine cavity, even in the absence of implantation, and its metabolites may be measured with use of highly sensitive urinary assays.

Adult↗
PubMed1998

The expression of the urinary forms of human luteinizing hormone beta fragment in various populations as assessed by a specific immunoradiometric assay.

Human gonadotrophins undergo metabolic transformations which result in the presence of several smaller, structurally and immunologically related forms of gonadotrophins in the urine. For luteinizing hormone (LH), a beta core fragment (LHbeta cf) has been isolated from the pituitary and characterized. The corresponding urinary fragment is inferred from mass spectral and immunochemical analysis of chromatographically separated urinary forms. Physicochemical characteristics, primarily mass spectral and chromatographic, indicate that the pituitary and urinary forms of LHbeta cf have a different structure, probably in the carbohydrate moieties. This communication characterizes the expression of LHbeta cf in the urine of both reproductive and post-reproductive age women and in men, employing assays highly specific for the pituitary form of the fragment. It was found that LHbeta cf is the predominant LH associated molecular form in the urine during peri-ovulatory period, peaking 1-3 days later than intact LH and reaching a concentration of approximately 600 fmol/mg creatinine, 7-fold higher than either LH or LH free beta subunit. Corresponding concentrations of human chorionic gonadotrophin (HCG) beta cf were <1% that of LHbeta cf. LHbeta cf cross-reaction with some LH or LHbeta monoclonal antibodies may well interfere with the accurate estimation of the day of the LH surge when urinary tests are utilized.

Adolescent↗
PubMed1996

Three-dimensional structures of gonadotropins.

Most secreted proteins are modified post-translationally with the addition of carbohydrate. It has been difficult to use crystallography to solve the structures of these proteins due to the inherent heterogeneity of the carbohydrate. The structure of the chemically deglycosylated form (hydrogen fluoride treated) of human chorionic gonadotropin (hCG) has been solved through crystallographic techniques. Unfortunately this form of hCG is not biologically active, and exhibits immunochemical differences from native hormone. In addition, subunit interactions appear altered after chemical deglycosylation as indicated by the increased thermal stability of the HF-treated hormone. The Asn 52 glycan on the alpha-subunit of hCG has been identified as being required for biological activity, it is, therefore, of physiological importance to determine the structure of the hormone with its carbohydrate intact. Also, it has not been possible to obtain crystals of the individual glycosylated subunits of hCG. Therefore an alternative method to solve the structure of the biologically active form of the hormone in solution as well as its separated subunits is necessary. Structural information utilizing NMR techniques can be obtained from native hCG subunits in solution if they can be uniformly labeled with 13C and 15N isotopes. We have developed a universal nonradioactive isotope, labeling medium enriched in 13C and 15N which can be used to express uniformly labeled hCG from Chinese hamster ovary cells suitable for solving the structure of the individual subunits and ultimately that of the native, biologically active hormone. The isotopically labeled recombinant hCG and its purified subunits are essentially identical to urinary hCG on comparison by biochemical, immunochemical, biological activity and the ability of the isolated subunits to recombine to form a biologically active dimer. Mass spectrometric analysis and preliminary structural NMR data indicate that the labeling is uniform and there is greater than 90% incorporation, sufficient for complete structural determination studies. This labeled growth medium represents a technological advance which will enable the rapid solution of the structures of the other glycoprotein hormones, as well as other glycoproteins which have proven unsuitable for crystallographic study.

Animals↗
PubMed1996

Expression of human chorionic gonadotropin uniformly labeled with NMR isotopes in Chinese hamster ovary cells: an advance toward rapid determination of glycoprotein structures.

Most secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein backbone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with 13C and 15N isotopes. We report the first such uniform labeling of a glycoprotein using a universal 13C- and 15N-labeling medium to express 13C, 15N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The 13C, 15N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.

Animals↗
PubMed1995

The expression, characterization, and crystallization of wild-type and selenomethionyl human chorionic gonadotropin.

Although the glycoprotein hormone hCG was crystallized over 4 yr ago, it is only now that three-dimensional structural information is available. This manuscript reports the method for successful production of modified expressed hormone, the characteristics of the crystallized protein, and unexpected observations during the crystallization process. Two different routes of solution to the structure of hCG were followed. The first was based on the traditional method of heavy atom isomorphous replacement, and the second was the more novel method of expressing the protein with selenomethionine substituting for methionine and applying multiwavelength anomalous diffraction analysis. Selenomethionyl hCG was employed to successfully grow the crystals used for the solution of the structure of hCG after partial deglycosylation by hydrogen fluoride (HF) treatment. The selenomethionyl hCG proved to be more hydrophobic than the expressed form of native hCG. Furthermore, expressed forms of hCG that were deglycosylated by HF proved to be more intact and less susceptible to peptide bond cleavages during the crystallization process than the urinary form of HF-treated hCG studied previously. It was found that addition of reducing agent during the crystallization period was necessary for the growth of crystals of HF-treated selenomethionyl hCG suitable for diffraction studies. Growth of crystals of HF-treated expressed hCG were accelerated by the addition of dithiothreitol, but would successfully grow without reductant. HPLC analysis of the HF-treated hormones before and during the crystallization process was used to identify alterations in the molecules, including oxidation and aggregation, both of which may affect the growth of crystals.

Animals↗
PubMed1994

The development of assays for the detection of fibrin(ogen)olysis based on COOH-terminal A alpha chain epitopes.

The COOH-terminal two-thirds of the fibrinogen A alpha chain is a substrate for both factor XIIIa and plasmin and is, therefore, a source of structural markers for the clinical detection of fibrin(ogen)olysis. Monoclonal antibodies (MoAbs) that bind to epitopes within this region (F-102, A alpha 563-578; F-103, A alpha 259-276) have been applied towards the development of two sensitive and specific enzyme-linked immunosorbent assays (ELISAs). The first assay, a capture (F-102)-tag (F-103) ELISA, measures plasma fibrinogen molecules whose A alpha chains are intact. The second assay, a solution phase competitive ELISA based on MoAb F-102, quantifies circulating COOH-terminal A alpha chain degradation products (A alpha FDPs), among the earliest peptides released from fibrinogen during plasmin-mediated fragment X formation. This assay features a novel preliminary plasma absorption step on concanavalin A to recover A alpha FDPs (if present in the sample) in a milieu free of immunologically cross-reactive fibrinogen. Both ELISAs use highly purified fibrinogen as the assay standard for quantitation. In control plasmas, circulating A alpha FDPs accounted for less than 2% of their respective intact fibrinogen A alpha chain concentration, suggesting a physiologic low level of proteolysis occurring at the extreme COOH-terminal portion of the molecule. Plasma A alpha FDPs were elevated (2.3% to 7.8% of their respective intact fibrinogen A alpha chain concentration) in a group of plasma from patients with documented, high serum FDPs (21 to 41 micrograms/mL). Application of the two ELISAs to characterize the course of A alpha chain proteolysis during thrombolytic therapy (TIMI phase 1) indicated that A alpha FDPs were a very early marker of the lytic state (detectable 15 minutes after treatment had been initiated), and that streptokinase and recombinant tissue plasminogen activator appeared to produce significantly different A alpha chain degradation profiles.

Chromatography, Affinity↗
PubMed1994

Structure of human chorionic gonadotropin at 2.6 A resolution from MAD analysis of the selenomethionyl protein.

BACKGROUND: Human chorionic gonadotropin (hCG) is a placental hormone that stimulates secretion of the pregnancy-sustaining steroid progesterone. It is a member of a family of glycoprotein hormones that are disulfide-rich heterodimers, with a common alpha-chain and distinctive beta-chains specific to their particular G-protein linked receptors. RESULTS: We have produced recombinant hCG in mammalian cells as the selenomethionyl protein, and have determined its structure (after partial deglycosylation) at 2.6 A resolution from multiwavelength anomalous diffraction (MAD) measurements. Despite only limited sequence similarity (10% identity), the alpha- and beta-subunits of hCG have similar tertiary folds. Each subunit has a cystine-knot motif at its core of extended hairpin loops. There is a very extensive subunit interface featuring two inter-chain beta-sheets and a unique, disulfide-tethered 'arm' from the beta-subunit which 'embraces' the alpha-subunit. The carboxy-terminal peptide of the beta-subunit, which is rich in O-linked sugars, is disordered. CONCLUSIONS: Structural and sequence comparisons indicate an evolutionary homology, albeit remote, between the glycoprotein hormone chains and other cystine-knot proteins, notably platelet-derived growth factor. Segments of the alpha- and beta-chains that have been convincingly implicated in receptor binding by hCG are juxtaposed on one side of the molecule. A glycosylation site implicated in signal transduction but not in binding is also close to the presumed binding site suggesting a possible coupling between ligand binding and signaling. This study with selenomethionyl protein produced in mammalian cells extends the realm of MAD phasing.

Amino Acid Sequence↗
PubMed1994

Crystal structure of human chorionic gonadotropin.

The three-dimensional structure of human chorionic gonadotropin shows that each of its two different subunits has a similar topology, with three disulphide bonds forming a cystine knot. This same folding motif is found in some protein growth factors. The heterodimer is stabilized by a segment of the beta-subunit which wraps around the alpha-subunit and is covalently linked like a seat belt by the disulphide Cys 26-Cys 110. This extraordinary feature appears to be essential not only for the association of these heterodimers but also for receptor binding by the glycoprotein hormones.

Amino Acid Sequence↗
PubMed1994

Development, characterization, and application of monoclonal antibodies to the native and synthetic beta COOH-terminal portion of human chorionic gonadotropin (hCG) that distinguish between the native and desialylated forms of hCG.

Although the pregnancy hormone hCG has been extensively mapped immunochemically, few monoclonal antibodies have been produced to the unique COOH-terminal region of its beta-subunit (beta CTP). We now report the development and characterization of five such monoclonal antibodies. Three of these antibodies were developed to the synthetic peptide analog of the hCG beta-(109-145) region coupled to diphtheria toxoid, and two antibodies to a conjugate of bovine thyroglobulin and the peptide hCG beta-(115-145) prepared from hCG with its carbohydrate moieties intact. The monoclonal antibodies raised against the synthetic peptide bound hCG, desialylated hCG, and synthetic peptide to a similar extent, whereas antibodies generated to the natural hCG peptide did not bind to the synthetic peptide analog of the COOH-terminal peptide (beta CTP) region or to desialyated hCG. These new monoclonal antibodies could distinguish between native and desialyated hCG in liquid phase immunoassays as well as by Western blots. They are highly specific reagents for such Western blotting and were used for studies of a crude human pituitary gonadotropin preparation to demonstrate that it contained intact hCG beta without the internal peptide bond cleavages found in the subunit present in human blood and urine. Competition experiments using combinations of monoclonal antibodies and rabbit anti-beta CTP antiserum demonstrated that two epitopes exist within the beta-(115-145) region of hCG, one of which depends on the presence of carbohydrate. In summary, the new monoclonal hCG beta CTP antibodies reported here can 1) discriminate between native and desialylated hCG, 2) identify hCG and nicked hCG on Western blots, 3) provide an immunoaffinity purification tool for hCG, and 4) bind to two distinct epitopes on the beta CTP.

Animals↗
PubMed1993

Structure and significance of human luteinizing hormone-beta core fragment purified from human pituitary extracts.

A fragment of the hCG beta-subunit is present in high concentrations in the urine of pregnant women and the urine from individuals with ovarian or other cancers. The utility of immunoreactive measurement of this fragment to monitor therapy of such cancers is compromised, however, because high concentrations of the molecule are also detected in the urine of healthy postmenopausal women. It has been suggested that the latter observations may be due to a cross-reacting human (h) LH beta core fragment, presumably of pituitary origin, but no such fragment had ever been isolated. We have now isolated a hLH beta core fragment from a pituitary tissue extract. Its structure is exactly analogous to that of the hCG beta core fragment. The finding of a discrete hLH beta core fragment in a tissue extract suggests that it may be produced within pituitary tissue, rather than by a peripheral degradation process. We have also found that the same immunoaffinity method used to extract the hLH beta core fragment from the pituitary extract purified several protein fragments from postmenopausal urine, none of which was related to hLH or hCG. The availability of the pituitary hLH beta core fragment may allow development of assays that distinguish it from its hCG analog.

Amino Acid Sequence↗
PubMed1992

Prospective assessment of early fetal loss using an immunoenzymometric screening assay for detection of urinary human chorionic gonadotropin.

OBJECTIVE: To develop an economical, nonradiometric immunoenzymometric assay (IEMA) for the detection of urinary human chorionic gonadotropin (hCG) in studies of early fetal loss. To be effective, the IEMA must have a sensitivity equal to the standard immunoradiometric assay (IRMA) and sufficient specificity to eliminate the need for screening most nonconceptive cycles with the expensive and labor-intensive IRMA. DESIGN: Two different assays were used to measure hCG in daily early morning urine samples from potential conceptive cycles. SETTING: Women undergoing donor artificial insemination (AI) were evaluated in a prospective study. PATIENTS, PARTICIPANTS: Ninety-two women volunteers were selected on the basis of apparent normal reproductive health. INTERVENTIONS: Artificial insemination with nonfrozen donor semen was performed by cervical cup twice each menstrual cycle at 48-hour intervals, and daily urine samples were self-collected throughout the menstrual cycle. MAIN OUTCOME MEASURES: An IEMA was developed to detect urinary hCG using the same antibodies as in the standard IRMA; a study was designed to determine whether this nonradiometric assay could successfully detect the early fetal loss that was detected by the IRMA. RESULTS: Of 224 menstrual cycles analyzed by both assays, a total of six early fetal losses were detected by the IRMA. When the tentative screening rule was set to allow all six of these losses and 95% of future losses to be detected by the IEMA, an additional 34 false-positive results were detected by the IEMA. The specificity of the IEMA with this rule was calculated to be 84%. CONCLUSION: An IEMA based on the same antibodies used for the standard IRMA can serve as an efficient screening assay for the detection of early fetal loss. When the IEMA is used in this manner, nearly 80% of screened menstrual cycles can be eliminated without further testing by the IRMA.

Abortion, Spontaneous↗
PubMed1991

Biological markers on human pregnancy.

This article reviews a selected set of recently described pregnancy-associated proteins which possess potential for both signaling pregnancy onset and monitoring its course. These molecules are compared and contrasted with human chorionic gonadotropin, the first pregnancy-associated protein to be discovered, and the standard biomarker of pregnancy to which all others must still be referenced. Recent advances in hCG research have focused on the structural determination and diagnostic significance of the subunits and fragments of the hCG molecule, particularly in urine. An outline of the potential utility of this approach is also presented.

Chorionic Gonadotropin↗
PubMed1990

Isolation and partial structural characterization of an equine fibrinogen CNBr fragment that exhibits immunologic cross-reactivity with an A alpha-chain cross-linking region of human fibrinogen.

Immunochemical studies of equine fibrinogen were conducted to characterize the structural basis for the immunologic cross-reactivity observed between human and equine A alpha chains when employing an antiserum to the 26K, human cyanogen bromide (CNBr) fragment, A alpha 241-476 (CNBr VIII). A 38K, equine CNBr fragment that reacts with this antiserum was isolated from CNBr-digested equine fibrinogen by Sephadex G-100 gel filtration. It was further purified by sequential hydrophobic chromatography on phenyl-Sepharose CL-4B, followed by reversed-phased (C-8) high-performance liquid chromatography (HPLC). NH2-Terminal analysis of the purified fragment, designated EqA alpha CNBr, identified one major sequence whose first three residues, E-L-E, were identical with those of human CNBr VIII. Tryptic and staphylococcal protease digests of the equine fragment were resolved by reversed-phase HPLC (C-4, C-18), and the separated components were characterized by amino acid analysis and automated Edman degradation. A total of 34 tryptic and 20 staph protease peptides yielded sequence information that permitted the alignment of 271 equine residues with residues A alpha 241-517 from the COOH-terminal two-thirds of the human A alpha chain so that 63% of the possible matches were identical. Other features of interest included (1) an amino acid substitution in which the methionine residue at A alpha 476 in the human A alpha chain was replaced by a valine residue, thus accounting, in part, for the larger EqA alpha CNBr fragment obtained from the equine molecule, and (2) a region of striking homology in which 36 successive residues, corresponding to A alpha 428-464 in the human A alpha chain, were identical in both species. These findings, together with available structural data for the COOH-terminal portion of the rat and bovine A alpha chains, indicate that the region corresponding to (human) A alpha 240-517 represents a conserved portion of the fibrinogen molecule. This may, in turn, explain the difficulties encountered when trying to raise monoclonal antibodies to cross-linking regions that are contained within the COOH-terminal two-thirds of the human A alpha chain.

Amino Acid Sequence↗
PubMed1989

Crystallization and characterization of human chorionic gonadotropin in chemically deglycosylated and enzymatically desialylated states.

Crystals suitable for X-ray diffraction studies at moderate resolution have been grown from two forms of human chorionic gonadotropin (hCG): HF-treated hCG and neuraminidase-treated hCG. The enzymatically desialylated form of hCG produced crystals that diffract to 2.8 A as compared to the HF-treated hCG crystals that diffract to 3.0 A. Although it was assumed that the high and heterogeneous carbohydrate content of the glycoprotein hormones inhibited their crystallization, this report suggests that it is the negatively charged surface sugars and neither the total carbohydrate content nor its heterogeneity which interferes with crystal formation. Chemical deglycosylation resulted in significantly increased protein degradation during crystal growth. Such peptide bond cleavages were observed to a much lesser extent in the crystals grown from neuraminidase-digested hCG. Sequence analysis of the HF-treated hCG crystals suggested that up to 45% of the molecules within the crystal had an acid-labile peptide bond cleaved. In contrast, the neuraminidase-treated hCG exhibited less than 9% of this type of cleavage. The increase in heterogeneity of the polypeptide chains within both crystals over that existent in the starting proteins was apparently due to changes occurring during crystal growth. The manner in which hCG was treated prior to crystallization was found to be a very important factor in the extent of peptide bond cleavages occurring during crystal growth. HF treatment of glycoproteins may render glycoproteins more susceptible to peptide bond cleavages during crystal growth.

Amino Acid Sequence↗