Biomedical subjects
B A Stone
Publications and source records attributed to B A Stone.
A (1-->3,1-->4)-beta-glucan-specific monoclonal antibody and its use in the quantitation and immunocytochemical location of (1-->3,1-->4)-beta-glucans.
Monoclonal antibodies were raised against a (1-->3,1-->4)-beta-glucan-bovine serum albumin (BSA) conjugate. One antibody (BG1) selected for further characterization, was specific for (1-->3,1-->4)-beta-glucan, displaying no binding activity against a (1-->3)-beta-glucan-BSA conjugate and minimal binding against a cellopentaose-BSA conjugate. A range of oligosaccharides was prepared by enzymatic digestion of (1-->3,1-->4)-beta-glucan, purified by size exclusion chromatography and characterized by 1H-NMR and anion exchange chromatography. These (1-->3,1-->4)-beta-oligoglucosides, together with (1-->3)-beta- and (1-->4)-beta-oligoglucosides were used to characterize the binding site of the monoclonal antibody (BG1) by competitive inhibition. The monoclonal antibody showed maximal binding to a heptasaccharide with the structure Glc(1-->3) Glc(1-->4) Glc(1-->4) Glc(1-->3) Glc(1-->4) Glc(1-->4) Glc and was determined to have an affinity constant of 3.8 x 10(4) M-1 for this oligoglucoside. The monoclonal antibody (BG1) has been used to develop a sensitive sandwich ELISA for the specific quantitation of (1-->3,1-->4)-beta-glucans. The assay operates in the range 1-10 ng ml-1 and shows no significant cross-reaction with tamarind xyloglucan, wheat endosperm arabinoxylan or carboxymethylpachyman ((1-->3)-beta-glucan). When used with a second-stage, rabbit anti-mouse gold conjugate and viewed under the electron microscope, the monoclonal antibody probe was found to bind strongly to the walls of the aleurone in thin sections of immature wheat (Triticum aestivum) cv. Millewa grains but not to the middle lamella region.(ABSTRACT TRUNCATED AT 250 WORDS)
Compositional Changes in Compost during Composting and Growth of Agaricus bisporus.
Samples from conventional compost taken at various stages of composting and mushroom (Agaricus bisporus) growth were analyzed for changes in 80% ethanol and water extracts, monosaccharides in acid hydrolysates of polysaccharides, lignin concentration, and lignin structural features. Variable amounts of extraneous inorganic solids in the form of fine sandy particles were removed by sedimentation of the samples in a carbon tetrachloride-dibromomethane mixture. During composting, about two-thirds of the initial wall polysaccharides were consumed by compost microorganisms, and only 17% of the total polysaccharides were used during mushroom production. The relative lignin content of composts as measured by the acetyl bromide procedure increased, both during composting and mushroom growth, and the chemical structure of lignin was altered by condensation and oxidation reactions.
The use of surrogate gestational carriers for assisted reproductive technologies.
OBJECTIVES: We determined the effect of embryo transfer, zygote intrafallopian transfer, and frozen embryo transfer on clinical outcomes after surrogate gestational transfers. STUDY DESIGN: Prospective randomization was carried out. RESULTS: Forty-five infertile couples were matched with a gestational surrogate carrier and underwent 81 cycles of embryo transfer with various assisted reproductive technologic procedures. Nineteen cycles produced a clinical pregnancy, with delivery in 15 of 81 cycles (18.5% live-birth rate). Fifteen of the 45 couples (33%) had a child from the surrogate gestational carrier program. CONCLUSION: No significant differences in clinical outcome were observed on the basis of the type of procedure performed or the age of the patient.
Numbers and quality of oocytes after induction of multiple folliculogenesis in women and in mice with different lots of human menopausal gonadotropins.
OBJECTIVE: To develop a murine in vivo bioassay that is sensitive to inherent variability of hMG. DESIGN: Female F1 hybrid mice were mated after injection with fixed dosages of each of four lots of hMG. Oocytes/embryos were collected and monitored during 5 days of culture. Outcome measures were compared with retrospective outcomes of ovarian stimulation of women with the same hMG lots. The bioassay was then used to compare biopotencies of six different lots of hMG. SETTING: Center for assisted reproductive technology. INTERVENTIONS: None. PATIENTS: Outcomes of controlled ovarian stimulation and IVF were retrospectively analyzed from 43 patients. MAIN OUTCOME MEASURES: Numbers of oocytes/embryos recovered, numbers fertilized, and numbers progressing beyond fertilization. RESULTS: Differences in biopotencies of hMG lots in the murine in vivo bioassay were consistent with differences in outcomes of patients treated with the same hMG lots during controlled ovarian stimulation. Bioassay outcomes also differed between different lots of hMG. CONCLUSION: The bioassay described in this study is sensitive to between-lot differences in hMG that affect follicle recruitment and oocyte quality in women (and in mice). In this respect, the bioassay could be of value in screening hMG lots, before its exploitation in a patient population. One such application is explored.
Identification of glutamic acid 105 at the active site of Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase using epoxide-based inhibitors.
Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) was modified by the mechanism-based, affinity-labeling reagent [14C](3,4)-epoxybutyl beta-D-cellobioside. Following partial inactivation a completely inactivated enzyme preparation containing 1.1 mol of covalently bound inhibitor/mol of protein was obtained by chromatography on a cellulosic matrix. The inactivated enzyme was digested with endoproteinase Glu-C and radioactive peptides purified by reversed-phase high performance liquid chromatography (HPLC). The affinity label was esterified exclusively to the gamma-carboxylate of Glu105 in the sequence Gly-Thr-Pro-Trp-Asp-Glu-Ile-Asp-Ile-Glu109. The sequence motif Glu-(Ile/Leu)-Asp-Ile is found in many glucanases and xylanases and may therefore serve to identify the catalytic nucleophile in beta-glycanases, which otherwise exhibit a low degree of sequence identity. The esterification of Glu105 by the affinity label abolished endoproteinase Glu-C-mediated hydrolysis of the Glu-Ile106 peptide bond. Identification of phenylthiohydantoin-Glu105 during automated sequence analysis was not possible unless the affinity label was liberated by prior base hydrolysis. These observations formed the basis for the development of a highly sensitive approach for the identification of catalytic carboxylates in polysaccharide hydrolases employing non-radioactive inhibitors, comparative HPLC mapping, electrospray mass spectrometry, and Edman degradation.
Ovarian responses to menopausal gonadotropins in groups of patients with differing basal growth hormone levels.
OBJECTIVE: To investigate the relationship between endogenous serum levels of human growth hormone (hGH) and ovarian response to human menopausal gonadotropins (hMG). DESIGN: Retrospective analysis of patient response to hMG. SETTING: Center for assisted reproductive technology. PATIENTS: Eighty women who had undergone controlled ovarian hyperstimulation with hMG. Basal levels of hGH in sera from 40 of these patients were less than 5.0 microIU/mL (low hGH), values for the remaining 40 were greater than 5.0 microIU/mL (high hGH). Levels of hGH in day 2 sera were analyzed against numbers of oocytes recovered in an additional 182 patients. MAIN OUTCOME MEASURES: Serum estradiol (E2) levels and numbers of oocytes recovered at oocyte pick-up. RESULTS: Average (+/- SE) levels of hGH in sera of high-hGH and low-hGH patients were 10.2 +/- 0.6 and 2.47 +/- 0.3 microIU/mL, respectively (P less than 0.05). Respective peripheral levels of insulin-like growth factor-I were 105.3 +/- 2.9 and 97.2 +/- 2.8 ng/mL. Levels of E2 in serum of high-hGH patients exceeded respective (P less than 0.05) low-hGH values throughout folliculogenesis, and more oocytes were recovered from high-hGH patients (8.1 +/- 0.9 versus 4.7 +/- 0.5 for low-hGH patients; P less than 0.05). Serum progesterone values did not differ. Higher day 2 hGH levels were associated with higher numbers of oocytes recovered after controlled ovarian hyperstimulation. CONCLUSIONS: The present findings indicate that endogenous hGH may augment gonadotropins during follicle recruitment and during multiple folliculogenesis in women. The phase of maximum ovarian sensitivity to hGH/gonadotropin synergism and the nature of synergism remain unclear.
Kinetic characteristics of alpha 1-adrenergic contractions in human corpus cavernosum smooth muscle.
Kinetic studies were conducted on the contractile response elicited by phenylephrine (PE) activation of the alpha 1-adrenergic receptor subtype in vascular smooth muscle isolated from the corpus cavernosum of impotent men. PE-induced contractions were separated into distinct phasic and tonic components, and the tonic portion was analyzed using a first-order rate equation to determine the maximal rate constant for onset of contraction (kobs max) and the maximum amplitude of the steady-state contractile response (Req max). The kobs max value in tissues from insulin-dependent diabetic patients was significantly greater than that in tissues from either noninsulin-dependent diabetics or nondiabetics. Additionally, the mean kobs max value in older patients (60-70 yr) was significantly greater than the mean kobs max value in younger patients (32-59 yr). Significant diabetes-related, but not age-related, alterations were also found in Req max. The observed changes in contractility resulted in dramatic age- and pathology-dependent alterations in the initial rate and/or magnitude of PE-induced response generation. These kinetic studies extend our previous observations at steady state and provide further evidence for heightened corporal tissue tone in the etiology of erectile dysfunction.
The beta-glucan synthase from Lolium multiflorum. Detergent solubilization, purification using monoclonal antibodies, and photoaffinity labeling with a novel photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose.
The membrane-bound beta-glucan synthase from Italian ryegrass (Lolium multiflorum L.) endosperm cells has been solubilized by both non-ionic and zwitterionic detergents. A complex relationship exists between the ratio of (1----3)-, (1----4)-, and (1----3, 1----4)-beta-glucan products of the solubilized enzyme, the cations present, and the concentration of the uridine 5'-diphosphoglucose substrate. Monoclonal antibodies directed against the beta-glucan synthase complex were generated by immunization of mice with an unfractionated microsomal reparation. Hybridoma cell lines were screened using a combination of indirect enzyme-linked immunosorbent assay followed by an enzyme-capture assay. The purified monoclonal antibodies were used with Pan-sorbin (stablized protein A-bearing staphylococcal cells) to immunoprecipitate an active beta-glucan synthase complex which had been solubilized from a microsomal preparation with 0.6% CHAPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated synthase complex revealed four major polypeptides of apparent molecular mass 30, 31, 54, and 58 kDa together with several minor components. The immunoprecipitated beta-glucan synthase complex was capable of synthesizing both (1----3)- and (1----4)-beta-glucans. A new photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose, 5-[3-(p-azidosalicylamide]allyl-uridine 5'-diphosphoglucose was synthesized in a three-step reaction sequence involving mercuration of UDP-Glc, alkylation of 5-Hg-UDP-Glc, and acylation of 5-(3-amino)allyl-UDP-Glc and characterized by chemical and spectroscopic analysis. The analogue inhibits (Kiapp 16 microM) and, upon UV irradiation, irreversibly inactivates the beta-glucan synthase. The analogue was iodinated with Na125I to give a radiolabeled, photoreactive compound, and was used in photoaffinity labeling of UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and several putative UDP-Glc-binding proteins from L. multiforum. The radiolabeled analogue specifically labeled the 31-kDa polypeptide in the immunoprecipitated synthase complex. The photolabeling of this polypeptide is strictly dependent on UV irradiation, is blocked by uridine 5'-diphosphoglucose and uridine 5'-diphosphate, and reaches saturation at analogue concentrations above 300 microM. These results indicate that the 31-kDa polypeptide in the beta-glucan synthase complex bears a uridine 5'-diphosphoglucose-binding site and is involved in the catalysis of beta-glucan synthesis.
Active site-directed inhibition by optically pure epoxyalkyl cellobiosides reveals differences in active site geometry of two 1,3-1,4-beta-D-glucan 4-glucanohydrolases. The importance of epoxide stereochemistry for enzyme inactivation.
1,3-1,4-beta-D-Glucan 4-glucanohydrolases (EC 3.2.-1.73) from Bacillus subtilis and barley (Hordeum vulgare) with identical substrate specificities but unrelated primary structures have been probed with (R,S)-epoxyalkyl (-propyl, -butyl, -pentyl) beta-cellobiosides and with optically pure (3S)- and (3R)-3,4-cellobiosides as active site-directed inhibitors. The optimal aglycon length for inactivation differs for the two enzymes, and they are differentially inhibited by the pure epoxybutyl beta-cellobioside diastereoisomers. The (3S)-epoxybutyl beta-cellobioside inactivates the B. subtilis enzyme much more efficiently than does the (3R)-isomer, whereas the reverse is true for the barley enzyme. Both enzymes are inactivated by a mixture of the stereoisomers at a rate intermediate of that observed with the individual isomers. The two beta-glucan endohydrolases may therefore employ sterically different mechanisms to achieve glycoside bond hydrolysis in their common substrate. The efficiency and specificity of epoxide-based "suicide" inhibitors may be enhanced significantly by the use of inhibitors bearing only one stereoisomeric form of the epoxide group.
Growth hormone in serum of women during the menstrual cycle and during controlled ovarian hyperstimulation.
STUDY OBJECTIVE: To investigate the interrelationships between circulating levels of endogenous human growth hormone (hGH), gonadotropins, estradiol (E2), and progesterone (P). DESIGN: Retrospective. SETTING: Assisted reproductive technology center. PATIENTS: Nine normally cycling women from whom blood samples were drawn daily; 18 patients undergoing controlled ovarian hyperstimulation. RESULTS: During the menstrual cycle, average (+/-SE) hGH levels in serum ranged between 7.97 +/- 2.71 microIU/mL on day 11 and 2.11 +/- 0.38 microIU/mL on day 22. Serum hGH levels did not correlate with peripheral levels of follicle-stimulating hormone (FSH), luteinizing hormone, P, or E2 and were related to the serum E2:FSH ratio (adhibited as an index of ovarian response to FSH) on cycle day 10 only. Levels of hGH in sera of patients undergoing controlled ovarian hyperstimulation were near 4 microIU/mL and were directly related to peripheral E2 levels at the time of induction of oocyte maturity. When corrected for differences in follicle number between stimulated patients, changes in serum E2 content were independent of respective hGH levels. CONCLUSION: The findings challenge speculation that endogenous peripheral hGH content might relate to ovarian responsiveness to FSH.
Effect of human follicular fluids from pregnant and nonpregnant patients on the development of mouse zygotes in vitro.
Mouse zygotes were cultured in medium containing follicular fluid from patients who had follicles containing oocytes which fertilized, did not fertilize, or were atretic and who did or did not become pregnant after in vitro fertilization and embryo replacement. The inhibitory effect was least with the follicular fluid from follicles in which the oocytes subsequently fertilized, greater when the oocytes did not fertilize, and most inhibitory when the follicle contained an atretic oocyte. More mouse zygotes developed to blastocysts when culture medium was supplemented with follicular fluid from patients who became pregnant compared to those who did not become pregnant. There was no difference in pregnancy outcome when an oocyte which subsequently fertilized was obtained from the follicle. These results indicate that follicles contain a substance(s) which inhibits mouse zygote development in vitro and that the inhibitory activity is related to the developmental potential of the oocyte in the follicle.
Suboptimal laboratory conditions can affect pregnancy outcome after embryo transfer on day 1 or 2 after insemination in vitro.
We have demonstrated that the replacement of pronuclear stage human embryos on the day after insemination in IVF-ET results in a pregnancy rate as good as, if not better than, the rate after ET on day 2. The pregnancy rate appears to be influenced by the culture environment used for the gametes. We would recommend that ET be undertaken as soon as possible after insemination on day 1 so that if suboptimal culture conditions are present, they do not have sufficient time to affect the viability of the embryos and reduce the subsequent chance of pregnancy after replacement. If culture conditions have been optimized there is no difference in pregnancy rate after embryo transfer on day 1 or day 2 and one can then replace embryos on either day and expect the pregnancy rate obtained in either case to be similar.
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Between-lot variability in chromatographic and biochemical properties of hMG.
Ten lots of dissociated hMG were characterized by reverse-phase gradient high-performance liquid chromatography. Areas of 12 discrete peaks were directly related to dosages of hMG injected. The lots were further analysed for immunoactive-FSH (41.6-106.2 IU/ampule), immunoactive-LH (11.0-20.4 IU/ampule), bioactive-LH (2.7-17.1 IU/ampule) and bioactive-hMG (149-298 pg E2/mIU immunoactive-FSH/ml). Relationships between integrated areas of the HPLC peaks and biochemical properties of the hMG lots were analysed by stepwise multiple linear regression. Between-lot differences in immunoactive-LH and immunoactive-FSH were related to HPLC peak areas (p less than 0.05); differences in bioactive-LH were not. Areas of 8 peaks were related to differences in bioactive-hMG activity, facilitating close approximation of bioactive-hMG from the derived multi-linear model (p less than 0.001). Rapid characterization of hMG by HPLC is of relevance as recent reports have shown that ovarian responses and pregnancy outcomes of patients are related to the immunoactive and bioactive gonadotropin content of hMG preparations used to induce multiple folliculogenesis before oocyte aspiration, in vitro fertilization, and embryo replacement.
Concentrations of follicle regulatory protein, steroids and gonadotrophins in antral fluids from women stimulated with metrodin and hCG.
Concentrations of immunoreactive follicle regulatory protein (FRP) were determined in 184 follicular fluid samples recovered from 30 patients in whom ovarian stimulation before oocyte recovery and in-vitro fertilization was induced with FSH (150 i.u./day). Ovum recovery was scheduled when the diameters of greater than or equal to 2 follicles reached 15-17 mm and serum oestradiol values were 740 pM/follicle. The mean level of FRP in fluid from follicles yielding oocytes which fertilized and cleaved within 48 h of recovery (24.4 +/- 3.08 immunoreactive units [IRU]/ml; 1 IRU = approximately 1 ng pig FRP) was higher than that in fluid from follicles yielding oocytes which did not fertilize (10.5 +/- 1.67 IRU/ml, P less than 0.05). FRP in fluid from follicles yielding oocytes which fertilized but did not cleave within 48 h of recovery was 17.2 +/- 2.89 IRU/ml. Overall, concentrations of FRP did not correlate with follicular fluid volume or with FSH or LH concentrations, but were positively related to prolactin, oestradiol and total protein levels (P less than 0.04). The results indicate that the FRP content of follicular fluid may be predictive of follicle/oocyte maturity. A dose-dependent increase in release of FRP by pig granulosa cells cultured in medium supplemented with 10-100 ng prolactin/ml was demonstrated. Prolactin may, therefore, be an important determinant of FRP production by granulosa cells.
Characterization of the hydroxyproline-rich protein core of an arabinogalactan-protein secreted from suspension-cultured Lolium multiflorum (Italian ryegrass) endosperm cells.
An arabinogalactan-protein (AGP) purified from the filtrate of liquid-suspension-cultured Italian-ryegrass (Lolium multiflorum) endosperm cells by affinity chromatography on myeloma protein J539-Sepharose was deglycosylated with trifluoromethanesulphonic acid to remove polysaccharide chains that are covalently associated with hydroxyproline residues in the peptide component of the proteoglycan. The protein core, which accounts for less than 10% (w/w) of the intact proteoglycan, was purified by h.p.l.c. It has an apparent Mr of 35,000, but reacts very poorly with both Coomassie Brilliant Blue R and silver stains. Amino-acid-sequence analysis of the N-terminus of the h.p.l.c.-purified protein core and of tryptic peptides generated from the unpurified protein reveals a high content of hydroxyproline and alanine. These are sometimes arranged in short (Ala-Hyp) repeat sequences of up to six residues. Polyclonal antibodies raised against the protein core do not cross-react with native AGP, the synthetic peptide (Ala-Hyp)4, poly-L-hydroxyproline or poly-L-proline. The results suggest that the polysaccharide chains in the native AGP render the protein core of the proteoglycan inaccessible to the antibodies and that the immunodominant epitopes include domains of the protein other than those rich in Ala-Hyp repeating units.
Differences in active site structure in a family of beta-glucan endohydrolases deduced from the kinetics of inactivation by epoxyalkyl beta-oligoglucosides.
The active sites of a spectrum of beta-glucan endohydrolases with distinct, but related substrate specificities have been probed using a series of epoxyalkyl beta-glycosides of glucose, cellobiose, cellotriose, laminaribiose, laminaritriose, 3O-beta-D-glucosyl-cellobiose and 4O-beta-D-glucosyl-laminaribiose with different aglycon chain lengths. The inactivation of each of the endohydrolases by these compounds results from active site-directed inhibitor action, as indicated by the dependence of the inactivation rate on pH, glycosyl chain length and linkage position, aglycon length, and the protective effect of disaccharides derived from the natural substrates. Comparisons of inhibitor specificity between a Bacillus subtilis 1,3;1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73), a Streptomyces cellulase (EC 3.2.1.4), a Schizophyllum commune cellulase (EC 3.2.1.4), a Rhizopus arrhizus 1,3-(1,3;1,4)-beta-D-glucan 3(4)-glucanohydrolase (EC 3.2.1.6), and a Nicotiana glutinosa 1,3-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) demonstrated different tolerances for glycosyl linkage positions in the inactivation process and a critical role of aglycon length reflecting differences in the active site geometry of the enzymes. For the B. subtilis endohydrolase it was concluded that the aglycon residue of the inhibitor spans the glycosyl binding subsite occupied by the 3-substituted glucosyl residue involved in the glucosidic linkage cleaved in the natural substrate. Appropriate positioning of the inhibitor epoxide group with respect to the catalytic amino acids in the active site is crucial to the inactivation step and the number of glucosyl residues in the inhibitor affects aglycon chain length specificity. The importance of this effect differs between the glucanases tested and may be related to the number of glycosyl binding subsites in the active site.