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Biomedical subjects

T D Lee

Publications and source records attributed to T D Lee.

At least 127 records · Page 7Linked to original sources
PubMed1992

Serological identification of HLA-B13 subtypes.

A serological approach is used to confirm subtypes of HLA-B13 originally observed by one-dimensional isoelectric focusing (1D-IEF). Sixty anti-B13 alloantibody sera were screened against Chinese panel cells. Two clusters of sera showing distinct reactive patterns were identified. One is a shorter reactive pattern than the other. Using these serological reaction patterns, the B13 antigen can be divided into two subtypes, B13.1 and B13.2, in the Chinese population. These serological subtypes appear to correlate well with the 1D-IEF patterns of B13 subtypes. The serological subtyping is also in agreement with the differences in nucleotide sequence previously determined to exist in B13 antigen subtypes. Family studies show that both B13.1 and B13.2 segregate as HLA-B locus alleles. Gene frequencies for B13.1 and B13.2 were 0.0676 and 0.0612, respectively, in our study population of 337 southern Han Chinese.

Alleles↗
PubMed1992

Neonatal alloimmune thrombocytopenia due to HLA-A2 antibody.

A male, full-term baby with thrombocytopenia was born by a G3P2A1 mother who was not associated with autoimmune disease. Platelet antibody screening was positive by using lymphocytotoxicity test, platelet suspension immunofluorescence test and solid-phase red cell adherence test. The identified HLA antibody was of A2 specificity. It was confirmed by testing the mother's and the baby's sera against the lymphocytes and platelets of 10 HLA-A2-positive donors. The possibility of platelet-specific antibody as the cause of neonatal alloimmune thrombocytopenia was ruled out by testing against platelets of 10 HLA-A2-negative donors and the known platelet-specific antigens utilizing immobilized, purified platelet glycoprotein as targets. The mother's serum reacted strongly with both the father's and the baby's platelets and lymphocytes. This neonatal thrombocytopenia was most likely due to the maternal HLA antibody, which was induced by her antecedent gestations.

Antigens, Human Platelet↗
PubMed1991

Leucine aminopeptidase-like activity in Aplysia hemolymph rapidly degrades biologically active alpha-bag cell peptide fragments.

We have investigated the role that proteolytic enzymes in Aplysia hemolymph play in the inactivation of the neurotransmitter alpha-bag cell peptide (alpha-BCP(1-9), Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu). alpha-BCP fragments containing Pro in positions 1 or 2, or Tyr in position 1, were degraded relatively slowly (half-life, t1/2 = 10-64 min), whereas fragments lacking these residues were degraded relatively rapidly (t1/2 = 0.5-2.7 min). Of 12 peptidase inhibitors tested, only bestatin, amastatin, and phenanthroline significantly inhibited alpha-BCP(3-9) degradation. alpha-BCP(3-9) yielded only four observable cleavage products (in order of decreasing abundance at early time points): alpha-BCP(4-9), alpha-BCP(5-9), alpha-BCP(6-9), and alpha-BCP(7-9). Degradation of alpha-BCP(3-9), alpha-BCP(4-9), alpha-BCP(5-9), alpha-BCP(6-9), or alpha-BCP(7-9) was strongly inhibited by bestatin, moderately inhibited by amastatin, and not inhibited by arphramenine B. The rates of degradation of eight alpha-BCP fragments and three other peptides in plasma were well correlated with their rates of degradation in mammalian leucine aminopeptidase (LAP, EC 3.4.11.1). Collectively our data support the following ideas. 1) In hemolymph one or more LAP-like enzymes rapidly and sequentially cleave alpha-BCP(3-9) or other small peptides lacking Pro at positions 1 or 2 or Tyr at position 1. 2) LAP-like peptidases in hemolymph may act in concert with previously described ganglionic peptidases to degrade neurally released alpha-BCP(1-9) and alpha-BCP(1-8) into inactive fragments.

Amino Acid Sequence↗
PubMed1991

Characterization of canine intestinal cholecystokinin-58 lacking its carboxyl-terminal nonapeptide. Evidence for similar post-translational processing in brain and gut.

An antibody raised against a synthetic cholecystokinin (CCK) analog, (1-27)-(CCK)-33, corresponding to the midregion of CCK-58, detected immunoreactivity in intestinal extracts which eluted between the positions of CCK-33/39 and CCK-58 on high performance liquid chromatography. This peak, lacking carboxyl-terminal cholecystokinin immunoreactivity, was purified by reverse phase and cation-exchange chromatographies. Amino acid, mass spectral, and microsequence analysis established that it was the amino-terminal desnonapeptide fragment of cholecystokinin-58, (1-49)-CCK-58. It was demonstrated further that CCK-58 has less biological activity than CCK-8, suggesting that the amino terminus either sterically hindered the ability of CCK-58 to exert its biological activity or that its amino terminus acted at another site to inhibit release of amylase from rat pancreatic acini. The desnonapeptide of CCK-58 by itself had no biological activity, nor did it affect CCK-8-stimulated amylase release from isolated rat pancreatic acini, suggesting that the amino terminus shields the carboxyl terminus from expressing its biological activity. Its presence in intestine suggests that it is released into the circulation where it could be detected by midregion antibodies. The presence of high proportions of (1-49)-CCK-58 indicates that most CCK-8 is directly derived from CCK-58. Its occurrence in brain and intestine indicates similar processing for procholecystokinin in both tissues.

Amino Acid Sequence↗
PubMed1991

Purification and structural characterization of progastrin-derived peptides from a human gastrinoma.

Several peptides derived from the gastrin-predicted preprohormone sequence were isolated from a human gastrinoma by gel permeation, anion exchange, and reverse phase chromatography. The peptides were identified and characterized structurally by a combination of radioimmunoassays, mass spectral analysis, and microsequence analysis. The largest peptide, progastrin-(1-35) (cryptagastrin), extends from the putative processing site for the signal peptidase to the double basic residues adjacent to the amino terminus of gastrin 34. A shorter form of this peptide, progastrin-(6-35) (cryptagastrin-(6-35), was also isolated in smaller amounts. In addition, sulfated and nonsulfated gastrin 17 amides (progastrin-(55-71)) and the glycine-extended nonsulfated gastrin 17 (progastrin-(55-72)) were identified by radioimmunoassay, and their structures were confirmed by mass spectral analysis. Isolation of cryptagastrin indicates that the signal peptide of human preprogastrin contains 21 amino acid residues, and progastrin, therefore, contains 80 amino acids. There is minimal processing of the cryptic peptide preceding the sequence of gastrin 34. An amidated gastrin form larger than gastrin 34 could contain 71 amino acids. No evidence was obtained for processing that would produce gastrins containing more than 34 but less than 71 amino acid residues.

Amino Acid Sequence↗
PubMed1991

Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus alpha-glucosyltransferases.

An active-site peptide containing an aspartic acid implicated in catalysis has been isolated and sequenced from two Streptococcus sobrinus extracellular glucosyltransferases: sucrose:1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase (GTase-I) and sucrose:1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase (GTase-S). The sequenced peptides, tagged with radiolabeled glucose, were isolated from a pepsin digest of a stabilized glucosylenzyme complex prepared by rapidly denaturing a reaction of enzyme and radiolabeled sucrose. The glucosyl linkage had previously been characterized as a beta-anomer bound to an active-site carboxyl group. Purified GTase-I and GTase-S glucosyl-peptides had the following similar but not identical sequences: GTase-I, Asp-Ser-Ile-Arg-Val-Asp-Ala-Val-Asp; and GTase-S, Asp-Gly-Val-Arg-Val-Asp-Ala-Val-Asp. Each has 3 aspartic acids as potential sites of glucose conjugation, but the relevant residue was not identified in sequence analysis because the highly base-labile glucosyl bond was cleaved in the first sequence cycle. As an alternative, the GTase-I glucosyl-peptide was partially digested at the N terminus with cathepsin C and at the C terminus with carboxypeptidase P. Analysis of the truncated products by fast atom bombardment mass spectrometry localized the glucosyl group to Asp-6 i the GTase-I peptide. In the native enzyme, this sequence is found near the N terminus, well-removed from the glucan-binding site located on a 60-kDa domain at the C terminus. The catalysis-dependent method of incorporating a glucosyl label implicates the aspartic acid as the residue involved in stabilizing an oxocarbonium ion transition state. The peptide segment is highly conserved and homologous to a peptide from sucrase-isomaltase labeled by site-directed irreversible inhibition and peptide segments common to a broad array of alpha-glucosidases and related transferases.

Amino Acid Sequence↗
PubMed1991

Human plasma fibronectin. Demonstration of structural differences between the A- and B-chains in the III CS region.

Fibronectins are a class of cell-adhesion proteins produced from a single gene. The soluble plasma form is synthesized by hepatocytes and the insoluble cellular form by fibroblasts and other cell types. The proteins possess multiple binding domains for macromolecules including collagen, fibrin and heparin along with at least one cell-binding domain. Cellular as well as plasma fibronectins are dimers of similar but not identical polypeptides. Their differences are the result of internal amino acid sequence variability due to alternative RNA splicing in at least three regions (ED-A, ED-B and III CS). We have been studying this polymorphism at the protein level in plasma fibronectin (pFn). Cathepsin D-digested pFn applied to a heparin-agarose column and eluted with an NaCl stepwise gradient (0.1 M, 0.25 M and 0.5 M) released two polypeptides (75 kDa and 65 kDa) in the 0.5 M-NaCl peak. Immunoblots with monoclonal antibodies IST-2 (specific for the C-terminal heparin-binding domain) and AHB-3 (specific for the III CS domain) suggest that both peptides contain the C-terminal heparin-binding (Hep-2) domain, but that only the larger fragment possesses the III CS region. These two polypeptides (75 kDa and 65 kDa) were digested with trypsin, and the resulting peptides were analyzed by fast-atom-bombardment mass spectrometry and compared with the known cDNA-derived peptide sequence. Peptides that were unique to the III CS region were further characterized by micro sequence analysis. The 75 kDa fragment is derived from the A-chain and contains the III CS region (89 amino acid residues) along with the C-terminal heparin-binding (Hep-2) domain and the fibrin-binding (Fib-2) domain. A single galactosamine-based carbohydrate group was detected at Thr-73/74 of the III CS region present in the 75 kDa fragment. The 65 kDa fragment is derived from the B-chain and lacks the entire III CS region but does contain the Hep-2 and Fib-2 domains.

Alkylation↗
PubMed1991

The SALMFamides: a new family of neuropeptides isolated from an echinoderm.

We have isolated two novel related neuropeptides from the radial nerve cords of the starfishes Asterias rubens and Asterias forbesi. One is an octapeptide with the amino acid sequence Gly-Phe-Asn-Ser-Ala-Leu-Met-Phe-NH2 and the other is a dodecapeptide with the amino acid sequence Ser-Gly-Pro-Tyr-Ser-Phe-Asn-Ser-Gly-Leu-Thr-Phe-NH2. The peptides were purified using high performance liquid chromatography (HPLC) and a radioimmunoassay for the molluscan FMRFamide-related neuropeptide, pQDPFLRFamide. Both peptides share minimal sequence identity with members of the family of FMRFamide-like peptides so we have designated them as founder members of a new family, the SALMFamides. We refer to the octapeptide as SALMFamide 1 (S1) and the dodecapeptide as SALMFamide 2 (S2). S1 and S2 are the first neuropeptides identified in species belonging to the phylum Echinodermata.

Amino Acid Sequence↗
PubMed1991

Characterization of the aqueous decomposition products of (+)1,2-bis(3,5-dioxopiperazinyl-1-yl)-propane (ICRF-187) by liquid chromatographic and mass spectral analysis.

High-performance liquid chromatography (HPLC) and fast atom bombardment mass spectrometry (FAB-MS) were employed to separate and identify the aqueous decomposition products of (+)1,2-bis(3,5-dioxopiperazinyl-1-yl)-propane (ICRF-187; 1), a drug active against several forms of human cancer and which also has recently been shown to display potent cardioprotective activity in patients treated with the antitumor antibiotic doxorubicin. Two reversed-phase HPLC columns were used to separate the hydrolysis products of 1, a Waters muBondapak phenyl column and an LKB Spherisorb ODS2 column. Incubation of 20 microM 1 in phosphate-buffered saline (PBS) at 37 degrees C for 21 h resulted in 47% decomposition, with three hydrolysis products detected (compound 2, Waters column retention time (RT) = 3.7 min, observed monoisotopic protonated molecular ion (MH+) m/z value of 305.1; compound 3, RT = 4.1 min, MH+ m/z value of 287.1; compound 4, RT = 4.8 min, MH+ m/z value of 287.1). The RT and MH+ m/z values for 1 were 17.1 min and 269.1, respectively. Based on the FAB-MS data, 2 corresponds to ICRF-198, the polar diacid diamide derivative of 1, while peaks 3 and 4 represent the monoacid monoamide derivatives of 1. Using B/E linked scan daughter FAB-MS analysis, 3 displayed a prominent fragment with a m/z value of 160, indicating that it corresponds to the monoacid monoamide derivative of 1, with the methyl group adjacent to the hydrolyzed ring. Compound 4, displaying a fragment with a m/z value of 142 in its B/E linked scan daughter ion spectrum, corresponds to the monoacid monoamide derivative of 1, with the methyl group adjacent to the closed ring.

Chromatography, High Pressure Liquid↗
PubMed1991

Massive cluster impact mass spectrometry: a new desorption method for the analysis of large biomolecules.

A new ion desorption method is described that utilizes a primary beam of massive, multiply charged cluster ions to generate secondary ions of peptides in a glycerol matrix. The massive cluster ion beam is generated via electrohydrodynamic emission using a 1.5 M solution of ammonium acetate in 30% aqueous glycerol. Negative ion spectra of peptides obtained using this technique show greatly decreased relative intensities for fragment ions and 'chemical noise' background when compared to spectra obtained using a xenon atom primary beam. The near absence of fragments derived from radiation damage to the sample solution is attributed to the impact of primary particles with energies less than 1 eV/nucleon.

Amino Acid Sequence↗
PubMed1991

Structural analysis of proteins by laser desorption and electrospray mass spectrometry.

The development of electrospray and matrix-assisted laser desorption mass spectrometry has provided protein chemists with tools for peptide and protein structure analysis with unprecedented sensitivity and molecular weight range. The two technologies can be viewed as competitive with respect to their molecular mass determinations, but complementary with respect to their differences in instrumentation, sample preparation methods, and nature of spectra produced.

Animals↗
PubMed1991

What is repeated in a repetition? Effects of practice conditions on motor skill acquisition.

The nature of repetition and its contribution to the acquisition of motor skills in neurologically healthy subjects are examined in this article. We argue that cognitive processing is a key component of practice, which is undermined by repetitive performances. The effects on motor learning of contextual interference, knowledge-of-results delivery schedules, and observation of models are examined, with particular reference to the nature of practice. The role of repetition in learning with respect to physical therapy is also discussed.

Cognition↗
PubMed1991

HLA-A, -B, -DR, -DQ antigens in black North Americans.

HLA antigens of black North Americans are not as well-characterized as those of other ethnic groups. We present results from HLA typing of black Americans using the 10th International Histocompatibility Workshop (IHW) sera. 1502 black Americans are typed for HLA-A, -B antigens. 128 black Americans are typed for HLA-DR, -DQ antigens. New specificities and splits established by the 10th IHW are identified in our data. Antigen frequency, gene frequency and haplotype frequency are computed for each phenotype observed. Haplotype A30--Bw42--DRw18--DQw4 and haplotype Aw36--Bw53 in strong linkage disequilibrium is characteristic of this black population. DR and DQ association patterns are similar to those of white North Americans. A major difference is the association of DRw8 with DQw7 in blacks versus an association of DRw8 with DQw4 in whites.

Black People↗
PubMed1991

Helminthotoxic responses of intestinal eosinophils to Trichinella spiralis newborn larvae.

Because the gastrointestinal lamina propria is the first line of defense against invasion with Trichinella spiralis newborn larvae, we investigated the helminthotoxic characteristics of isolated lamina propria eosinophils. Eosinophils were isolated from the intestinal lamina propria of rats and purified to nearly 90% purity by a combination of velocity sedimentation through Percoll and unit gravity sedimentation through a continuous gradient of bovine serum albumin. Isolated eosinophils were of high viability and responded to surface receptor stimulation. Freshly isolated intestinal eosinophils lacked cytotoxic capacity when incubated with newborn larvae in the presence of specific antiserum. Peritoneal eosinophils from the same rats exhibited 100% helminthotoxicity after 24 h. Cytotoxicity could be stimulated in the intestinal eosinophils by the addition of recombinant murine interleukin-5.

Animals↗
PubMed1990

Domain structure in yeast tRNA ligase.

Yeast tRNA ligase is one of two proteins required for the splicing of precursor tRNA molecules containing introns. The 95-kDa tRNA ligase has been purified to homogeneity from a strain of Escherichia coli which overexpresses the protein. The ligation reaction requires three enzymatic activities: phosphodiesterase, polynucleotide kinase, and ligase. By partial proteolytic digestion, we have produced fragments of tRNA ligase which contain the constituent activities. These results provide evidence for a model in which the three constituent activities of ligase are located in three distinct domains separated by protease-sensitive regions. We have also located the active adenylylated site in the ligase domains. It is lysine-114. The tRNA ligase sequence in this region has limited homology to the active-site region of T4 RNA ligase.

Adenosine Triphosphate↗
PubMed1990

MacProMass: a computer program to correlate mass spectral data to peptide and protein structures.

A program known as MacProMass has been written for Macintosh computers to assist in the analysis of mass spectral data of peptides and proteins. The program employs a user friendly, graphical interface and accommodates a variety of protein structures including cyclic peptides and multiple chain proteins. In addition to molecular mass calculations for positive and negative molecular ions, MacProMass also calculates elemental composition, amino acid composition, isoelectric point, surface free energy, and high-performance liquid chromatography index values for whole structures and peptide fragments resulting from enzymatic or chemical degradation. Users can program their own amino acid residues and terminal groups. In addition to search routines for both mass and sequence, theoretical fragment ions for peptide mass spectra can be calculated. Analysis of variant proteins is facilitated with a subroutine that systematically catalogs single amino acid substitutions that correspond to mass differences between observed and expected molecular ions. Interchain and intrachain disulfide bonds and other types of linkages are maintained throughout the chemical and enzymatic degradation operations.

Amino Acid Sequence↗
PubMed1990

Structural analysis of a 64-kDa major structural protein of human cytomegalovirus (Towne): identification of a phosphorylation site and comparison to pp65 of HCMV (AD169).

The major 64-kDa structural protein of human cytomegalovirus (pp64) was isolated from a guanidinium chloride extract of the virions and dense bodies of HCMV (Towne) by reverse-phase HPLC. Purified pp64 was reduced and alkylated followed by digestion with trypsin. The molecular mass of each of the tryptic peptides was determined by fast atom bombardment/mass spectrometry and compared with the predicted molecular mass of the fragments deduced from the corresponding DNA-derived peptide sequence of pp65 from HCMV (AD169). Microsequence analysis was employed to confirm selected peptides. Results of protein sequence analysis of pp64 from HCMV (Towne) are in complete agreement with the DNA-derived protein sequence of pp65 predicted for HCMV (AD169) with the following exceptions and modifications. The protein isolated from HCMV (Towne) was found to contain an Ala at position 448 instead of Ser448 reported for the protein from HCMV (AD169). We also identified Ser472 as a site of phosphorylation in pp64 from HCMV (Towne). Finally, on the basis of the sequence of HCMV (AD169) DNA fragment encoding the matrix protein and on S1 nuclease protection analysis, it has been predicted that one version of the matrix protein (possibly the lower matrix protein, Mr 65K) is encoded by an mRNA that is formed through splicing of a short intron. However, we have obtained peptides that contain sequences spanning through the splice-junction region, suggesting that in HCMV (Towne), the matrix protein is encoded by an unspliced message.

Amino Acid Sequence↗