Enzymatic and chemical digestion of proteins for mass spectrometry.
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Amino Acid Sequence↗
Biomedical subjects
T D Lee
Publications and source records attributed to T D Lee.
Identification of the major Ascaris allergen and its purification to homogeneity by high-performance liquid chromatography.
The major allergen from the body fluid of adult Ascaris suum (ABF) has been identified and purified to homogeneity using gel permeation high-performance liquid chromatography (HPLC). The purity of the Ascaris body fluid allergen (ABA-1) was confirmed by HPLC and SDS-PAGE. ABA-1 appears to be a 25-kDa dimer in its native form, which runs as a 10-kDa monomer on SDS-PAGE under reducing conditions. The allergenicity of the HPLC-purified protein was confirmed using isolated in vivo-sensitised mast cells from infected rats in an in vitro histamine release assay. ABA-1 is responsible for less than 80% of the allergenicity of ABF in this sensitive and specific system. Amino acid composition analysis and N-terminal amino acid sequencing were performed on ABA-1. Comparisons are made between ABA-1 and some of the heterogeneous Ascaris allergen preparations previously published. It is suggested that Asc-1 and allergen A both contain ABA-1 in large quantities and that discrepancies in the literature result from contaminating proteins in these preparations and technical differences in characterization of the predominant molecules present in the preparations. Compositional data suggests that the ABA-1 monomer is a molecule of 94 amino acids (based on a molecular weight estimate of 10 kDa) with a composition resembling that previously published for allergen A. The first 10 amino acids are identical to those of a protein affinity purified from the body fluid of Ascaris suum at the Wellcome Laboratories for Experimental Parasitology (WLEP-14K). The similarity between ABA-1 and WLEP-14k is also apparent on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)
Allergens↗
Development of a novel series of (2-quinolinylmethoxy)phenyl-containing compounds as high-affinity leukotriene receptor antagonists. 1. Initial structure-activity relationships.
This series of reports describes the development of orally active, highly potent, specific antagonists of the peptidoleukotrienes containing a (2-quinolinylmethoxy)phenyl moiety. Described in this first report are the structure-activity relationships that led to a more than a 20-fold improvement of the potency and selectivity of the initial chemical lead (RG 5901). From this series of compounds, RG 7152 (16) was identified and selected for further evaluation in the clinic as an antiasthmatic agent. Compound 16 competitively inhibits [3H]LTD4 binding to membranes from guinea pig lung (Ki = 38 +/- 6 nM) and the spasmogenic activity of LTC4, LTD4, and LTE4 in parenchymal lung strips from guinea pigs. Unlike the original lead (RG 5901), compound 16 does not inhibit 5-lipoxygenase from guinea pig PMNs. Following oral administration to guinea pigs, 16 blocks LTD4-induced dermal permeability (ED50 = 6.9 mg/kg), LTD4-induced bronchoconstriction (ED50 = 1.1 mg/kg), antigen-induced bronchoconstriction (ED50 = 2.5 mg/kg), and anaphylactic-induced mortality (ED50 = 16 mg/kg). These studies on structure-activity relationships indicate that there is a requirement for an acidic function and the presence of the (2-quinolinylmethoxy)phenyl moiety in a specific geometric arrangement.
Animals↗
The development of a novel series of (quinolin-2-ylmethoxy)phenyl-containing compounds as high-affinity leukotriene receptor antagonists. 3. Structural variation of the acidic side chain to give antagonists of enhanced potency.
This paper is the third in a series outlining the development of orally active sulfido peptide leukotriene antagonists containing a (quinolin-2-ylmethoxy)phenyl moiety. In this work the systematic variation of the acid side chain substituents led to dramatic and reproducible changes in the oral activity of these compounds, presumably due to alterations in their pharmacokinetic properties. The most potent compound identified, 5-[4-[4-(quinolin-2-yl-methoxy)phenyl]-3-methylbutyl]tetrazole (32), represents a convergence of good in vitro antagonist activity and a 3-10-fold improvement in oral potency over the current clinical candidate 2. The new findings from these optimization studies are as follows: oxygen substitution in the acid side chain was not necessary for antagonist activity, in vitro and in vivo activity was enhanced by alkyl or phenyl substitution on the gamma-carbon of the acid side chain of para-substituted (quinolin-2-ylmethoxy)phenyl derivatives, and free rotation about the side chain carbon atom adjacent to the (quinolin-2-ylmethoxy)phenyl ring was required for activity. The lead compound of this report (32) is a competitive inhibitor of [3H]LTD4 binding to receptor membrane purified from guinea pig lung (Ki = 12 +/- 3 nM) and of the spasmogenic activity of LTC4, LTD4, and LTE4 in guinea pig lung strip. Dosed orally in guinea pigs, this compound blocks LTD4-induced bronchoconstriction (ED50 0.8 mg/kg) and antigen-induced systemic anaphylaxis (ED50 = 1.2 mg/kg).
Animals↗
HLA, GM, KM, and Diego blood group typing of Chippewa Indians.
This is the first report characterizing HLA antigen distribution in North American Indians of the Chippewa tribe. One hundred seventy-four Chippewa from Minnesota underwent HLA-A,B,C,DR, and DQ typing in a search for a single unrelated bone marrow donor. The high matching rate of this successful search is attributed to homozygosity and the extreme frequency of certain antigens in this small ethnic community. It is emphasized that smaller donor pools are required in searches within a minority population. GM and KM allotype typing as well as blood group Diego typing show patterns similar to those reported in other North American Indian groups.
Blood Group Antigens↗
On the role of knowledge of results in motor learning: exploring the guidance hypothesis.
Recent work on the role of knowledge of results (KR) in motor learning has challenged some traditional assumptions. In particular, the guidance hypothesis suggests that there is a detriment to learning when KR guides the learner toward correct performance. Three experiments that explored this hypothesis are reported here. These experiments contrasted the effects of relative frequency of guidance versus the relative frequency of KR. According to the guidance hypothesis, it was predicted that parallel effects of the relative frequency manipulation on motor learning and performance would result under guided and KR conditions. The movement task was a reciprocal timing task that was either paced by a metronome (Experiment 1) or augmented by auditory KR (Experiments 2 and 3). The results of Experiments 1 and 3 revealed a number of parallel effects, thus providing support for the guidance hypothesis. The contrast of Experiments 1 and 2 resulted in a number of dissociable effects, however. These findings are discussed in relation to the potential guiding properties of KR and their impact on motor performance and learning.
Journal Article↗
Histocompatibility antigen patterns in haemophilic patients with factor VIII antibodies.
A number of studies suggest that there is a genetic basis for the formation by some haemophilia A patients of antibodies that inactivate factor VIII. In our study, human leucocyte antigen (HLA)-A, B, C, DR and DQ typing was carried out for 44 haemophilia A patients, including 16 who had developed an antibody to factor VIII. In contrast to previous reports, we found no association between HLA-DR antigens and haemophilia A per se or the formation of a factor VIII inhibitor. However, there was an absence of HLA-Cw5 in the 16 haemophilic patients who had formed an antibody to factor VIII. This finding, consistent with a previous report, identified a statistically significant difference in HLA-Cw5 frequency when the inhibitor patient group was compared to multi-transfused haemophilic patients who had no inhibitor (11/28).
Adolescent↗
HLA-DR, DQ antigens in North American Caucasians.
HLA-DR, DQ specificities are determined by serological methods in 2,586 North American Caucasians. Antigen frequency, gene frequency and haplotype frequency are computed for each phenotype observed. The DR and DQ loci antigen distributions are well-fitted to a Hardy-Weinberg equilibrium (p greater than 0.25 for DR locus, p greater than 0.10 for DQ locus). All World Health Organization (WHO) recognized HLA-DR,DQ specificities were found except HLA-DRw18, which has been identified only in the black population. DR and DQ linkage disequilibria among recently defined splits is observed. The following DR and DQ associations are found: DR1 and DQw5; DR4 and DQw7, DQw8; DR7 and DQw2, DQw9; DR8 and DQw4, DQw6, DQw7; DR9 and DQw2, DQw9; DRw10 and DQw5; DRw11 and DQw6, DQw7; DRw12 and DQw5, DQw7; DRw13 and DQw6, DQw7; DRw14 and DQw5, DQW7; DRw15 and DQw6, DQw7; DRw16 and DQw5; DRw17 and DQw2. In this large study population, the following unusual DR and DQ associations are found: DR4, DQw2; DR4, DQw1; DR1, DQw7; DR7, DQw5; DRw17, DQw6; and other unusual haplotype phenotypes containing DRX, DQX.
Gene Frequency↗
Seven FMRFamide-related and two SCP-related cardioactive peptides from Helix.
Pulmonate snails have a more complex array of cardioexcitatory peptides than other molluscs, and Helix has a more complex array than most other pulmonates. Since a full characterisation of the cardioexcitatory peptides is necessary for an understanding of physiology, we sought to identify the members of two families of such peptides - the small cardioactive peptides (SCPs) and the FMRFamide-related peptides (FaRPs) - from Helix aspersa. We characterised the peaks of immunoreactivity from HPLC both by their elution times and by their molecular weights as determined by fast atom bombardment mass spectrometry (FABms). These two criteria, used in parallel, facilitated our identification of several known peptides: MNYLAFPRMamide, identical to SCPB of Aplysia; two tetrapeptide FaRPs, FRMRamide and FLRFamide; and three hepatapeptide FaRPs, NDPFLRFamide, SDPFLRFamide and pQDPFLRFamide. Of these peptides, only FMRFamide and pQDPFLRFamide have previously been reported from Helix. We also discovered an additional SCP and two novel FaRPs and sequenced them. The SCP is Ser-Gly-Tyr-Leu-Ala-Phe-Pro-Arg-Met-amide (SGYLAFPRMamide), and the heptapeptide FaRPs are Asn-Asp-Pro-Tyr-Leu-Arg-Phe-amide (NDPYLRFamide) and Ser-Glu-Pro-Tyr-Leu-Arg-Phe-amide (SEPYLRFamide). When these nine peptides were tested on isolated Helix ventricles, the SCPs were the most potent cardioexcitors, the heptapeptide FaRPs were next, and the tetrapeptides had the least activity.
Amino Acid Sequence↗
Immunoprecipitation of in vitro-translated products of Nippostrongylus brasiliensis RNA.
RNA has been isolated from adult Nippostrongylus brasiliensis (NB) homogenized in guanidium isothiocyanate. The mRNA has been translated in vitro utilizing a rabbit reticulocyte system and translated products immunoprecipitated with rabbit anti-adult NB antiserum and rat infection serum.
Animals↗
Inhibition of rat liver NAD(P)H:quinone acceptor oxidoreductase (DT-diaphorase) by flavonoids isolated from the Chinese herb scutellariae radix (Huang Qin).
The glucuronide conjugates of oroxylin A and two other flavones, baicalein, and wogonin, were isolated from the methanol extract of the herb scutellariae radix (Huang Qin) and were found to be inhibitors of rat liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2). Baicalin (baicalein 7-O-glucuronide) and oroxylin-A 7-O-glucuronide are approximately 50-fold more potent than wogonin 7-O-glucuronide. The enzyme kinetic analysis revealed that oroxylin-A 7-O-glucuronide is a competitive inhibitor with respect to NADH (the electron donor), with a Ki value of 63 nM. Considering the similarities of their structures and inhibition kinetics to those of dicoumarol, it is thought that oroxylin-A 7-O-glucuronide and the other two flavonoids bind to an identical site and inhibit this quinone reductase in the same fashion as dicoumarol. The results also suggest that the inhibition of NAD(P)H:quinone acceptor oxidoreductase or another vitamin K reductase by oroxylin-A 7-O-glucuronide and the related flavonoids may be one of the steps associated with the anticoagulation action of the herb. These compounds are potentially useful anticoagulant drugs.
Animals↗
HLA haplotype frequencies of Taiwan-Chinese families.
HLA haplotype frequency was studied by typing 201 members of 32 unrelated families. Linkage disequilibrium was determined by observed and expected haplotype frequencies. The two-locus haplotype frequencies with most significant linkage disequilibrium were A30-B13, Aw33-B17, Bw46-Cw11, B12-Cw8, A1-Cw6 and A33-Cw3. No locus recombination was noted among 137 children.
Asian People↗
Structural and functional analysis of NADPH-cytochrome P-450 reductase from human liver: complete sequence of human enzyme and NADPH-binding sites.
The complete amino acid sequence of human liver NADPH-cytochrome P-450 reductase has been determined by microsequence analysis and mass spectrometry. The total sequence consists of 676 amino acids initiated by an amino-terminal acetyl group. There is no evidence for posttranslational modifications, including Asn-linked glycosylation. The human enzyme exhibits sequence homology in the range of 92-95% with other mammalian enzymes. Sequence differences were mainly confined to several hydrophilic regions in the NH2-terminal and COOH-terminal domains. Since the human enzyme is immunochemically distinct from the rabbit enzyme despite similar enzymatic properties, it is likely that these variable hydrophilic regions are potential antigenic determinants. The NADPH-depleted enzyme is inactivated by either fluorescein isothiocyanate, a lysine-specific reagent, or 5-(iodoacetamido)fluorescein, a cysteine-specific reagent. In both cases, protection by NADP(H) prevents enzyme inactivation by the reagents. Isolation of fluorescent peptide from 5-(iodoacetamido)fluorescein-inactivated enzyme identified Cys 565 as the specifically NADPH-protected residue.
Adult↗
Characterization of the carboxyl terminal flanking peptide of rat progastrin.
A peptide identical in structure to the carboxyl-terminal flanking nonapeptide of rat progastrin, predicted by cDNA sequence, was synthesized. The synthetic peptide was used for production of a rabbit antiserum. This antiserum was used to develop a radioimmunoassay specific for rat carboxyl terminal flanking peptide. This assay was used to monitor the purification of immunoreactivity from rat antral extracts. Gel permeation, anion exchange and reverse phase chromatography steps resulted in a single absorbance peak associated with the carboxyl terminal flanking peptide immunoreactivity. The purified peptide eluted in the same position as the synthetic peptide during all three types of chromatography. This material was shown to be identical in mass to Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn, the predicted sequence of the carboxyl terminal nonapeptide of rat progastrin.
Amino Acid Sequence↗
Multiple gastrin-releasing peptide gene-associated peptides are produced by a human small cell lung cancer line.
Products of the gastrin-releasing peptide gene were isolated from culture medium supernatant of a small cell lung cancer line, NCI-H345, by several (high performance liquid chromatography) HPLC steps. The column eluates were monitored by immunoassay and absorbance profiles. Gastrin-releasing peptide was identified in HPLC eluates by a specific radioimmunoassay. Two carboxyl-terminal gastrin-releasing peptide gene-associated peptides were identified by a radioimmunoassay specific for their predicted carboxyl terminus. The amino termini of these two peptides were determined by microsequence analysis. The shorter peptide was revealed to be a fragment of the larger peptide. Expression of an alternate mRNA was shown by isolation and characterization of a novel tetradecapeptide. Amino acid analysis, microsequence analysis, and mass spectral analysis confirmed that the structure was Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser. This peptide represents the carboxyl terminus of a peptide resulting from alternate processing of gastrin releasing peptide mRNA. This mRNA contains a 19-base deletion, creating a frame shift. A radioiodinated synthetic analog of this peptide (Tyr-Leu-Val-Asp-Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser ) bound specifically to a small cell cancer line with high affinity, suggesting possible biological activity of the isolated peptide.
Amino Acid Sequence↗
Gm and Km allotypes in 74 Chinese populations: a hypothesis of the origin of the Chinese nation.
This paper reports the distribution of immunoglobulin Gm and Km allotypes in 74 Chinese geographical populations. These populations are derived from 24 nationalities comprising 96.6% of the total population of China. A total of 9,560 individuals were phenotyped for Gm(1,2,3,5,21) factors, and 9,611 were phenotyped for Km(1). Phylogenetic trees were constructed on the basis of Gm haplotype frequencies and genetic distances. The results of cluster analysis show the heterogeneity of the Chinese nation, and confirm the hypothesis that the modern Chinese nation originated from two distinct populations, one population originating in the Yellow River valley and the other originating in the Yangtze River valley during early neolithic times (3,000-7,000 years ago). Frequencies of the Gm haplotype of 74 Chinese populations were compared with those of 33 populations from major racial groups. The results suggest that during human evolution, the Negroid group and Caucasoid-Mongoloid group diverged first, followed by a divergence between the Caucasoid and Mongoloid. Interrace divergence is high in comparison with intrarace divergence. There appear to be two distinct subgroups of Mongoloid, northern and southern Mongoloid. The northern and southern Mongoloid have Gm1;21 and Gm1,3;5 haplotypes as race-associate markers, respectively. Furthermore, the Caucasian-associated haplotype Gm3;5 was found in several of the minorities living in the northwest part of China. The presence of the Gm3;5 haplotype is attributed to the Caucasians living in Central Asia throughout the Silk Road. The amount of Caucasian admixture has been estimated. In contrast to the Gm haplotype distribution, Km1 gene frequencies showed a random distribution in the populations studied.
Asian People↗
The identification, localization, and pharmacology of FMRFamide-related peptides and SCPB in the penis and crop of the terrestrial slug, Limax maximus.
1. The molluscan neuropeptides FMRFamide, pQDPFLRFamide, and SCPB were tested on the isolated crop and penis of the terrestraial slug, Limax maximus. FMRFamide and pQDPFLRFamide stimulated the penis and inhibited contractions of the crop. In contrast, SCPB either stimulated or relaxed the penis and increased the tone of the crop. 2. Fibers and varicosities containing immunoreactive (ir-) FMRFamide and ir-SCPB were located in the penis and crop. 3. Extracts of penes, crops, ganglia, and whole animals all contained FMRFamide, FLRFamide, SDPFLRFamide, NDPFLRFamide, and pQDPFLRFamide. 4. These results suggest that the FMRFamide-related peptides and SCPB are involved in the regulation of the reproductive and digestive activities of Limax.
Animals↗
Highlights of protein structural analysis.
This article describes highlights of the state of the art in protein structural analysis, and comments on the current trends toward increased sensitivity and integrated isolation-structure methodologies.
Amino Acids↗