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Biomedical subjects

T T Yip

Publications and source records attributed to T T Yip.

At least 55 records · Page 3Linked to original sources
PubMed1990

The role of caffeinated beverages in dental fluorosis.

Recent studies have demonstrated that the incidence of dental fluorosis has increased during the past decade. Greater availability and use of fluoride-containing gels, mouth rinses, dentifrices, etc., improper prescribing of fluoride supplements and ingestion of fluoride dentifrice by some children are some of the suggested determinants of dental fluorosis. However, based on the increase in consumption of tea, coffee, and other caffeine-containing beverages by the children, and the augmentative effect of caffeine on fluoride bioavailability, we theorize that the rise in incidence of dental fluorosis in North America is mainly due to the replacement of water intake by caffeine-containing beverages among the young population.

Adolescent↗
PubMed1990

Structural homology of human, bovine, and porcine milk lactoferrins: evidence for shared antigenic determinants.

Although some degree of structural homology has been demonstrated among lactoferrins of different species, other reports suggest that these proteins are immunologically distinct. Human, bovine, and porcine lactoferrins were purified to homogeneity from colostral whey by affinity chromatography on immobilized single-stranded DNA. Evidence for shared antigenic determinants among human, bovine, and porcine lactoferrins was demonstrated by Ouchterlony immunodiffusion and immuno "dot" blots using whole antisera and purified Ig directed against each species of lactoferrin. There was no evidence that human transferrin was recognized by any of the lactoferrin-specific antisera evaluated. The degree of cross-reactivity between the lactoferrins and their trypsin digestion products was also evaluated after SDS-PAGE by immunoblotting. These data demonstrate that human, porcine, and bovine lactoferrins share common antigenic determinants and are likely to be more homologous in tertiary structures than suggested previously. Thus, investigations of human or bovine lactoferrin metabolism in infants that are based upon immunologic methods alone should be conducted cautiously in those cases where the presence of both human and bovine lactoferrin is suspected.

Animals↗
PubMed1990

Unique molecular properties of a urea- and salt-stable DNA-binding estrogen receptor dimer covalently labeled with the antiestrogen [3H]desmethylnafoxidine aziridine. A comparison with the estrogen-receptor complex.

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.

Animals↗
PubMed1989

Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence.

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.

Amino Acid Sequence↗
PubMed1989

Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography.

High-performance immobilized metal ion affinity chromatography was utilized to evaluate the adsorption properties of 67 synthetic, biologically active, peptides ranging in size from 5 to 42 residues. The metal ions, Cu(II), Ni(II) and Zn(II), were immobilized by iminodiacetic acid (IDA) coupled to TSK gel 5PW (10 microns). Two types of gradient elution (imidazole and pH) were used to evaluate peptide retention by the metal ions. A decreasing pH gradient and an increasing imidazole gradient eluted the peptides in similar order. IDA-Cu(II) and IDA-Zn(II) showed very similar selectivities for the peptides analyzed; however, IDA-Zn(II) displayed a weaker affinity for the peptides. IDA-Ni(II) showed a slightly different pattern of selectivity. Peptide adsorption effects contributed by the metal-free gel matrix were found to be relatively minor. The concentration and type of salt included in the mobile phase could affect the relative affinities of the peptides for the immobilized metal ions. Retention coefficients were assigned to individual amino acid residues by multiple linear regression analysis. Histidine showed the largest positive correlation with retention, followed by aromatic amino acid residues. Modified N-terminal residues resulted in negative contributions to retention. Analyses of peptide amino acid composition alone allowed prediction of peptide retention behavior on immobilized metal ion affinity columns.

Amino Acid Sequence↗
PubMed1989

Multiple DNA-binding estrogen receptor forms resolved by interaction with immobilized metal ions. Identification of a metal-binding domain.

Immobilized metal ions have been used to characterize and locate metal ion-specific binding domains on the surface of the DNA-binding form of the estrogen receptor protein. Soluble estrogen receptors in calf uterine cytosol were labeled with [3H]estradiol and transformed to the DNA-binding configuration by brief exposure (30 min) to 3 M urea at 0-4 degrees C. The transformed receptors were purified in the presence of 3 M urea using single-stranded calf thymus DNA-agarose and characterized by high-performance size-exclusion chromatography (Stokes radius of 7.0-7.5 nm) and sucrose density gradient centrifugation (4.25 S) as dimers of 130,000 Da. Such receptor preparations subsequently labeled with [3H]desmethylnafoxidine aziridine by ligand exchange revealed one major peak of radioactivity (67 kDa) by sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis. When analyzed by immobilized metal ion affinity chromatography on iminodiacetate (IDA)-agarose loaded with Cu(II), Ni(II), or Zn(II) ions, the receptor was bound with various degrees of affinity and metal interaction heterogeneity even in the presence of 0.5 M NaCl to neutralize electrostatic interactions. The intact DNA-binding receptor dimers were most tightly bound to IDA-Cu(II) and IDA-Ni(II), but were eluted with 100-200 nM imidazole. The receptors were bound less tightly to IDA-Zn(II), and four separate peaks of receptor activity were resolved by elution with 10, 15, 30, and 100 mM imidazole (n = 27). Limited trypsin digestion of the DNA-binding receptor forms resulted in the generation of a 2.8-nm fragment with both the DNA-binding and metal-binding domains removed or destroyed. These results demonstrate that DNA-binding estrogen receptor dimers have high affinity metal ion-binding sites which are located at the DNA-binding domain. We have found (Zn(II) interaction chromatography to be unique thus far in its ability to resolve separate DNA-binding receptor forms.

Affinity Labels↗
PubMed1989

Interaction of human lactoferrin with DNA: one-step purification by affinity chromatography on single-stranded DNA-agarose.

Human lactoferrin has been purified to apparent homogeneity with high recovery (greater than 95%) in one chromatographic step using immobilized single-stranded DNA-agarose. Human colostral whey, at neutral pH, was loaded onto columns of DNA-agarose; a single purified protein, lactoferrin, was eluted using elevated ionic strength buffers. The lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, and reverse-phase (C18) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels and silver staining showed the purified lactoferrin preparation to contain a single protein of 78 kD with intact immunologic determinants. Similar results were obtained before and after iron saturation of the colostral whey proteins. Apolactoferrin purified in this manner was shown to bind iron with high affinity. These results demonstrate the effectiveness of immobilized DNA as one of the most rapid and complete lactoferrin purification procedures described thus far.

Chromatography, Affinity↗
PubMed1989

Selective removal, recovery, and characterization of immunoglobulins from human colostrum.

Investigations into the mechanisms by which Ig in human colostrum influence the development and maturation of both the gastrointestinal and the immune systems of human milk-fed term and preterm infants have been restricted by the paucity of purified human milk Ig. We have developed a simple adsorption procedure for the selective removal and quantitative recovery (95-100%) of intact Ig (secretory IgA, IgG, and IgM) present in human colostral whey. The procedure exploits the rapid, ionic-strength dependent, thiophilic adsorption of Ig during a single pass of colostral whey through a column of beaded agarose with immobilized thioether-sulfone ligands (Anal Biochem 1986;159:217-226). The purity and composition of the adsorbed Ig were verified by SDS-PAGE and sensitive silver-staining protein detection procedures; proteins of approximately 78-80 kD (secretory component), 50-60 kD (heavy chain), and 25 kD (light chain) were observed. The identity, structural integrity, and relative concentrations of the recovered Ig were confirmed by high-performance size-exclusion chromatography, Ouchterlony immunodiffusion, rocket immunoelectrophoresis and ELISA. These results were analyzed and compared with reported values for the concentration of human milk Ig. Thus, the use of thiophilic adsorption appears to facilitate 1) selective removal of Ig from colostrum, enabling the evaluation of remaining components for growth- and immune-potentiating properties, and 2) selective immobilization and recovery of Ig from colostrum under conditions consistent with preserved biologic activity.

Colostrum↗
PubMed1989

Purification and characterization of intact lactoferrin found in the urine of human milk-fed preterm infants.

Little is known about the metabolic fate of ingested lactoferrin in human-milk-fed term or preterm infants. Enzyme-linked immunosorbent assays and sodium dodecyl sulfate/polyacrylamide gradient gel electrophoresis (SDS-PAGGE) with immunoblots have demonstrated that the urinary excretion of lactoferrin by preterm infants fed exclusively human milk exceeded that by formula-fed infants. The origin and molecular integrity of the excreted lactoferrin, however, are unclear. We have developed extraction and separation procedures involving a stationary phase of immobilized single-stranded DNA (ssDNA) that allows the efficient (greater than 80%) and rapid isolation of pure, intact lactoferrin from infants' urine. We purified lactoferrin to apparent homogeneity from infants' urine in one step, using the immobilized ssDNA with mobile phases containing up to 6 mol of urea per liter. The purified lactoferrin was evaluated by SDS-PAGGE; silver-staining revealed one protein band at 78 kDa; immunoblots confirmed the presence of intact lactoferrin. High-performance affinity chromatography with use of immobilized metal ion (Cu2+) suggested the presence of intact, iron-saturated lactoferrin. Subsequent chromatography on high-performance reversed-phase (C18) columns independently verified sample identity and purity.

Blotting, Western↗
PubMed1988

High-performance immobilized metal ion affinity chromatography of peptides: analytical separation of biologically active synthetic peptides.

The separation of more than 30 biologically active synthetic peptides and their analogs on a high-performance immobilized metal ion affinity chromatography column is described. The metal chelating gel (TSK gel chelate-5PW) contains iminodiacetic acid (IDA) covalently coupled to a hydrophilic, resin-based matrix with a bead diameter of 10 micron. The retention of the peptides on Cu(II), Ni(II), and Zn(II) ions immobilized on the chelating gel showed that some of them can be separated by isocratic elution while the majority of them are retained and are separated into distinct fractions by elution with a linear imidazole gradient or with a continuously decreasing pH gradient. Of the three immobilized metal ions investigated here, the IDA-Cu(II) chelate column gave the best resolution irrespective of the type of gradient used. This is amply illustrated by the resolution of angiotensins I and II and their seven synthetic analogs. The results obtained here serve as guidelines for the future exploitation of this separation method for the efficient fractionation of a wide variety of peptides on an analytical or preparative scale.

Buffers↗
PubMed1988

Protein interaction with immobilized ligands: quantitative analyses of equilibrium partition data and comparison with analytical chromatographic approaches using immobilized metal affinity adsorbents.

Quantitative or analytical affinity chromatography has been successful primarily for the analysis of biologically determined macromolecular affinity relationships. Quantitative approaches are also needed to better characterize simpler, chemically defined immobilized ligands with potential for selective interaction with specific, predetermined protein surface groups. Protein interaction with immobilized metal is a rather selective and versatile, high-affinity adsorption technique for which there is little quantitative information. Using model protein interactions with immobilized Cu2+ ions, we have compared analytical frontal affinity chromatographic methods to a simple, nonchromatographic protocol for the rapid determination of quantitative affinity relationships. Values obtained for the equilibrium dissociation constant (Kd) and binding capacity (Lt) characterizing the interaction of lysozyme with immobilized Cu2+ were quite similar by frontal analysis (Kd = 37-42 X 10(-6) M; Lt = 6.8-7.4 X 10(-6) mol protein/ml gel) and by equilibrium binding analyses (Kd = 33 +/- 4.7 X 10(-6) M; Lt = 5.8-6.1 X 10(-6) mol protein/ml gel; 14 determinations). The interaction of ovalbumin with immobilized Cu2+ was characterized by an affinity (Kd = 4.2-4.8 X 10(-6) M) and capacity (Lt = 1.5-2.1 X 10(-6) mol protein/ml gel) which were also the same regardless of the method for affinity analysis. These values indicate that the total protein bound at saturation corresponds to as much as 17% of the total immobilized Cu2+ ions (approximately 40 X 10(-6) mol/ml gel). Thus, depending on the fraction of total immobilized Cu2+ available for interaction with a given protein (e.g., lysozyme), the number of individual immobilized ligands actively participating as well as those rendered unavailable upon individual protein binding events may be greater than 1. Linear Scatchard plots obtained for both lysozyme and ovalbumin (purified) suggest the presence of only a single type of immobilized Cu2+-protein interaction operative under the experimental conditions employed. However, Scatchard analyses of data obtained by the nonchromatographic equilibrium binding method also demonstrated the ability to simultaneously resolve the contribution of two components whose presence was predicted by frontal chromatography. Our results support the validity and utility of equilibrium binding data analyzed according to the equations outlined by Scatchard and others as an alternative to analytical chromatographic methods.

Chromatography, Affinity↗
PubMed1987

Interaction of proteins with immobilized Cu2+. Quantitation of adsorption capacity, adsorption isotherms and equilibrium constants by frontal analysis.

The interaction of lysozyme, ovalbumin, bovine and pig serum albumins with Cu2+ immobilized on Chelating Sepharose Fast Flow or TSK gel chelate-5PW was studied by frontal analysis at various initial concentrations of these solutes. The chromatographic data so obtained served as a basis for evaluating some relevant affinity chromatography parameters by adapting previously reported equations to this system. The TSK-based adsorbent had lower adsorption capacity for all the model proteins compared to the agarose-based adsorbent, due primarily to its lower porosity which has a marked influence on the accessibility of the immobilized ligand to the proteins. On the other hand, the TSK-based adsorbent offers almost ideal conditions for studying adsorption equilibria under column chromatographic conditions. The adsorption capacity of these adsorbents for the model proteins ranges from about 0.6 to 7 mumol/ml, equivalent to 40-100 mg/ml, of adsorbent. The following equilibrium constants for the interaction of the proteins with immobilized Cu2+ were obtained: lysozyme, 1.8.10(4); ovalbumin, 1.5.0(5); BSA, 1.7.10(5); PSA, 3.7.10(5) and imidazole, 8.10(3) M-1. Despite the comparatively low affinity of imidazole for the adsorbent, it is an effective competing ligand, at comparatively high concentrations, for adsorbed proteins primarily because all adsorption sites are available to it. The results obtained suggest that about 1/3 to 1/2 of the potential adsorption sites on the model proteins are involved in forming coordination complexes with Cu2+ immobilized to covalently bound iminodiacetate groups on insoluble gel matrices.

Adsorption↗
PubMed1987

High-performance analytical applications of immobilized metal ion affinity chromatography.

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.

Antibodies, Monoclonal↗
PubMed1987

Evidence that epidermal growth factor is present in swiftlet's (Collocalia) nest.

1. An epidermal growth factor (EGF)-like activity was detected and partially purified from swiftlet's nest extract. 2. The partially purified EGF-like activity was able to (a) generate competitive binding curves parallel to the standard curves in radioreceptor assay and (b) stimulate thymidine incorporation in quiescent culture of 3T3 fibroblasts and the latter activity can be suppressed by mouse EGF antibody. 3. Partial characterization of the EGF-like activity in terms of pI, molecular weight and its behavior on gel filtration column suggest that it bears similar physical properties to the EGFs isolated from the mouse and the shrew.

Animals↗
PubMed1986

Epidermal growth factor from deer (Cervus elaphus) submaxillary gland and velvet antler.

Epidermal growth factor (EGF)-like activity was isolated for the first time from the submaxillary gland (SMG) and the velvet antler of red deer (Cervus elaphus) by a combination of Sephadex gel or DEAE-Sephacel and IMAC columns in succession. The semipurified cervine EGF-like activity (cEGF), with specific activity of 4.7 ng/micrograms protein from the velvet tissues, can generate a completely parallel competitive binding curve against mouse EGF in both radioreceptor assay (RRA) and radioimmunoassay (RIA). Mitogenic activity of EGF from both tissues was demonstrated by stimulating the incorporation of [3H]thymidine in two different cell lines of fibroblast culture in a dose-dependent manner. The velvet layer may be the site of EGF synthesis outside the SMG.

Animals↗
PubMed1986

Studies on shrew (Suncus murinus) epidermal growth factor.

The shrew SMG contains a very high level of EGF. By heterologous radioreceptor assay, the EGF content was determined to be about 300 pmol/mg wet weight in the male. The content is sex dependent; in the female gland, it is only 4% of the male gland level. EGF level and EGF receptor content in other tissues showed a reciprocal distribution pattern with the parotid gland containing the second highest level of EGF and the liver containing the largest amount of EGF receptor. Shrew EGF is heat stable. It could induce early eyelid opening and incisor eruption in neonatal rat pups at a very low dose level. There is apparently no high molecular weight form of EGF in shrew tissues and body fluids. However, gel chromatography revealed that the foetus contained multiple forms of EGF. The contribution of a high EGF level to the active life in adult shrews and the exceptionally high growth rate in shrew pups are discussed.

Animals↗
PubMed1985

Isolation of the epidermal growth factor from the shrew submaxillary gland.

The epidermal growth factor (EGF) from the submaxillary glands of the Chinese Shrew (Suncus murinus) is purified to apparent homogeneity by using a sequence of four chromatographic steps, viz. gel filtration on Sephacryl S-200, affinity chromatography on immobilized Ni, hydrophobic interaction on phenyl-Sepharose CL-4B and reverse-phase HPLC. An 800-fold increase in specific activity and an overall recovery of 46% were achieved. The most effective step in its purification is the successful use of immobilized metal ion affinity chromatography (IMAC). This method was very selective, reproducible and requires a minimum of sample pre-treatment prior to chromatography.

Animals↗