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Biomedical subjects

T D Lee

Publications and source records attributed to T D Lee.

At least 19 recordsLinked to original sources
PubMed1992

Aplysia peptide neurotransmitters beta-bag cell peptide, Phe-Met-Arg-Phe-amide, and small cardioexcitatory peptide B are rapidly degraded by a leucine aminopeptidase-like activity in hemolymph.

We have been investigating the role of proteolytic enzymes in the inactivation of peptide neurotransmitters in the marine snail Aplysia. Previous studies (Squire, C. R., Talebian, M., Menon, J. G., Dekruyff, S. D., Lee, T. D., Shively, J. E., and Rothman, B. S. (1991) J. Biol. Chem. 266, 22355-22363) showed that neuroactive fragments of the neurotransmitter alpha-bag cell peptide (alpha-BCP) were rapidly degraded (t1/2 = 0.5-2.7 min) in plasma, hemolymph that had been cleared by centrifugation. Degradation was caused by one or more enzymes resembling mammalian leucine amino-peptidase (LAP, EC 3.4.11.1). In this report we show that three other Aplysia peptide neurotransmitters, beta-BCP(1-5) (Arg-Leu-Arg-Phe-His), FMRFa (Phe-Met-Arg-Phe-amide), and SCPB(1-9) (Met-Asn-Tyr-Leu-Ala-Phe-Pro-Arg-Met-amide) are rapidly degraded (t1/2 = 0.3-2.4 min) in plasma by apparently the same LAP-like enzyme(s). Our findings strongly suggest that the LAP-like enzyme(s), by means of its broad substrate specificity and access to the extracellular spaces of the nervous system in vivo, plays a significant role in the inactivation of many Aplysia peptide neurotransmitters, and they raise the possibility that proteolytic enzymes in the extracellular fluid contribute significantly to the inactivation of peptide neurotransmitters in other animal species.

Amino Acid Sequence↗
PubMed1992

Primary structure of a cardioactive neuropeptide from the tobacco hawkmoth, Manduca sexta.

The amino acid sequence of the first of a family of insect cardioregulatory peptides from the tobacco hawkmoth, Manduca sexta, has been determined using a combination of Edman degradation microsequencing and mass spectroscopy. This peptide contains 9 amino acid residues and an observed mass for the monoisotopic protonated molecule of 956.4 Da. There are two cysteines at positions 3 and 9 forming a disulfide bridge and the carboxyl-terminus is amidated. The structure of this peptide, Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, is identical to a peptide recently isolated from crabs called crustacean cardioactive peptide (CCAP) and we propose that this peptide be named Manduca CCAP.

Amino Acid Sequence↗
PubMed1992

Smoking and decreased fertilisation rates in vitro.

To examine possible mechanisms for the association between cigarette smoking and reduced fertility, we have measured the concentration of the nicotine metabolite cotinine in ovarian follicular fluid collected at the time of oocyte recovery during treatment for in-vitro fertilisation. In a group of women in whom follicular fluid cotinine could not be detected (limit of accurate measurement 20 ng/ml) 116 oocytes were collected, of which 84 became fertilised (72%), whereas among women with cotinine concentration greater than 20 ng/ml 20/45 (44%) oocytes did so (p < 0.01). The median fertilisation rates for individuals (range 1-8 eggs each) in the high and low cotinine groups were 57% and 75%, respectively (p < 0.05). These findings suggest that infertile women should be advised to stop or reduce smoking generally, and especially before treatment by in-vitro fertilisation.

Adult↗
PubMed1992

Calmodulin is a subunit of nitric oxide synthase from macrophages.

A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist-induced elevation of Ca2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino acid sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca2+. This may explain why NOS that is independent of Ca2+ and elevated CaM appears to be activated simply by being synthesized.

Amino Acid Oxidoreductases↗
PubMed1992

Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177.

A prokaryotic expression plasmid, pKK-DT2, containing the cDNA of rat liver NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase) was constructed and used to transform Escherichia coli strain JM109. The rat liver quinone reductase was expressed in strain in JM109 and was inducible with isopropyl beta-D-thiogalactopyranoside (IPTG). The expressed rat protein was purified by affinity chromatography and had kinetic and physical properties identical with the protein purified from rat liver in that it could utilize either NADH or NADPH as the electron donor and its activity was inhibited by dicoumarol. In addition, we have generated four mutants, Arg-177----His (R177H), Arg-177----Ala (R177A), Arg-177----Cys (R177C) and Arg-177----Leu (R177L), using this expression system. Several of the mutants behaved anomalously on SDS/PAGE, but all of the mutant proteins had the expected M(r) as determined by electrospray m.s. These results and those obtained from enzyme kinetic analysis, u.v./visible absorption spectral analysis, and flavin and tryptophan fluorescence analysis of the wild-type enzyme and four mutants indicated that mutations at Arg-177 changed the conformation of the enzyme, resulting in a decrease in enzyme activity. Replacing Arg-177 with leucine altered the protein conformation and decreased FAD incorporation.

Amino Acid Sequence↗
PubMed1992

Cloning and characterization of inducible nitric oxide synthase from mouse macrophages.

Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.

Amino Acid Oxidoreductases↗
PubMed1992

P-type calcium channels blocked by the spider toxin omega-Aga-IVA.

Voltage-dependent calcium channels mediate calcium entry into neurons, which is crucial for many processes in the brain including synaptic transmission, dendritic spiking, gene expression and cell death. Many types of calcium channels exist in mammalian brains, but high-affinity blockers are available for only two types, L-type channels (targeted by nimodipine and other dihydropyridine channel blockers) and N-type channels (targeted by omega-conotoxin). In a search for new channel blockers, we have identified a peptide toxin from funnel web spider venom, omega-Aga-IVA, which is a potent inhibitor of both calcium entry into rat brain synaptosomes and of 'P-type' calcium channels in rat Purkinje neurons. omega-Aga-IVA will facilitate characterization of brain calcium channels resistant to existing channel blockers and may assist in the design of neuroprotective drugs.

Amino Acid Sequence↗
PubMed1992

Antagonism of synaptosomal calcium channels by subtypes of omega-agatoxins.

Venom of the funnel web spider Agelenopsis aperta inhibits the binding of 125I-omega-conotoxin GVIA (omega-CgTx) to calcium channels in chick brain synaptosomal membranes. Fractionation of the venom by liquid chromatography shows that this inhibitory activity is associated primarily with a diverse class of peptide toxins called omega-agatoxins (omega-Aga). Using binding inhibition as an assay, we purified and identified the novel, 76-amino acid toxin, omega-Aga-IIIA. Inhibition of 125I-omega-CgTx binding to chick synaptosomal membranes by omega-Aga-IIIA and omega-Aga-IIA is correlated with block of potassium-stimulated 45Ca entry into synaptosomes; omega-Aga-IA neither inhibits 125I-omega-CgTx binding nor 45Ca entry under identical conditions. omega-Aga-IIA and omega-Aga-IIIA are 20-30-fold more potent than omega-CgTx as antagonists of synaptosomal calcium channels. However, whereas omega-CgTx completely blocks 45Ca entry into synaptosomes at saturating concentrations, the omega-agatoxins maximally block only 60-70% of 45Ca entry. Pretreatment of synaptosomes with omega-Aga-IIIA occludes block of 45Ca entry by omega-CgTx. The results indicate that, while the omega-agatoxins bind to the entire population of omega-CgTx-sensitive calcium channels in chick synaptosomal membranes, they exert only a partial block of 45Ca flux. Such block could occur via two distinct mechanisms. Toxin binding may alter the kinetics of a homogeneous population of channels, resulting in lower overall conductance upon depolarization. Alternatively, the omega-agatoxins may bind to two distinct channel subtypes, only one of which is blocked as a result of toxin occupation.

Agatoxins↗
PubMed1992

Patterns of prohormone processing. Order revealed by a new procholecystokinin-derived peptide.

An 83-amino acid cholecystokinin peptide with a sulfated tyrosine and an amidated carboxyl terminus (CCK-83) was purified from human intestinal mucosa. The purified peptide was chemically characterized, and its bioactivity was compared to CCK-8. Several post-translational processing steps such as cleavage at basic residues, sulfation, and amidation are necessary to form biologically active cholecystokinin from its nascent prepropeptide. The discovery of CCK-83 gives new insight into the order of preprohormone processing. The processing of prepro-CCK appears to be in the order of: 1) signal peptidase cleavage, 2) tyrosine sulfation, 3) cleavage after a carboxyl-terminal pair of basic residues, 4) carboxypeptidase B-like cleavage of these basic residues, 5) amidation (which results in the formation of CCK-83), and 6) cleavage at monobasic residues by endopeptidases (which results in the smaller molecular forms of cholecystokinin). The characterization of biologically active CCK-83 with a sulfated tyrosine and an amidated carboxyl terminus establishes the site of signal peptidase action and suggests an order of post-translational modifications that give rise to the various molecular forms of cholecystokinin.

Amino Acid Sequence↗
PubMed1992

Analysis of peptide mixtures by capillary high performance liquid chromatography: a practical guide to small-scale separations.

Capillary HPLC is a very effective means of separating small amounts of peptides and proteins. Capillary columns ranging from 0.01 mm to 0.5 mm in diameter can be constructed using recycled supports and inexpensive fused silica capillary tubing. Commercial pumping systems and UV detectors can be readily converted for operation in the flow rate range of 0.5-50 microL/min. Detailed procedures are given for the construction of columns and UV detector flow cells. A mixture of peptides derived from the endo Lys C digest of horse heart cytochrome c was used to illustrate various aspects of capillary chromatography of peptides and compares the performance of various-sized capillary columns and UV detector flow cell types.

Amino Acid Sequence↗
PubMed1992

A hypothalamic activator of calmodulin-dependent enzymes is thymosin beta 4 (1-39).

A new class of stimulators of basal activity of a number of calmodulin-dependent enzymes have been previously isolated from bovine hypothalamus. One of these stimulators, denoted as C3, has been purified to homogeneity by reverse phase HPLC and tentatively identified as thymosin beta 4 (1-39) by mass spectrometry and Edman microsequence analysis. The stimulating effect of C3 on rabbit skeletal muscle MLCK basal activity was compared with that of thymosin alpha 1 and thymosin beta 4 (16-38). Evidence is presented that all the indicated compounds are Ca(2+)-independent high-affinity MLCK stimulators. The potency of the stimulators in activating the enzyme was: C3 greater than beta 4 greater than (CaM+Ca2+) greater than alpha 1.

Amino Acid Sequence↗
PubMed1992

Post-translational processing of membrane-associated recombinant human stem cell factor expressed in Chinese hamster ovary cells.

This report describes the structure of soluble human stem cell factor isolated from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with stem cell factor (SCF) cDNA, which encodes a leader sequence plus 248 additional amino acids. The 248 amino acids include a hydrophobic transmembrane region at positions 190-212. The isolated material is glycosylated and three bands (apparent M(r) 28,000, M(r) 35,000, and M(r) 40,000) are evident by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After complete deglycosylation, the molecular weight by SDS-polyacrylamide gel electrophoresis is 18,000-19,000. Structural analyses of the intact SCF, the deglycosylated SCF, and a deglycosylated C-terminal peptide were performed by laser desorption, fast atom bombardment, or electrospray mass spectrometry. Pulse-labeling of cells with 35S-labeled Met and Cys resulted in cell-associated glycosylated SCF of M(r) 33,000-45,000 which was converted to M(r) 33,000 by in vitro treatment with glycosidases. During a chase with unlabeled Met and Cys, labeled SCF of M(r) 28,000, M(r) 35,000, and M(r) 40,000 appeared in the medium; it was converted to M(r) 18,000-19,000 by glycosidase treatment. SCF at the surface of the transfected CHO cells could be demonstrated by immunofluorescence. The data obtained indicate that the recombinant human stem cell factor, as isolated, represents proteolytically processed forms containing amino acids 1-165, derived from the initially synthesized membrane-bound form of 248 amino acids. Further characterization indicated that the M(r) 28,000 form is glycosylated at Asn120, the M(r) 35,000 form at Asn120 and Asn65, and the M(r) 40,000 form at Asn120, Asn93, and Asn65. Each form also contains O-linked carbohydrate. The N-linked glycosylation, particularly that at Asn93 and at Asn65, adversely affects in vitro biological activity and receptor binding.

Amino Acid Sequence↗
PubMed1992

Effects of the model's skill level on observational motor learning.

Observation of a model prior to physical practice often facilitates the acquisition of motor skills. The majority of research studies on observational motor learning has used a skilled model for the demonstration. Recent research, however, suggests that observing an unskilled (learning) model may also be effective. The experiment reported here compared motor skill acquisition following observation of a learning model or a skilled model to the performance of subjects who lacked the benefit of observation. The task was to play a computer tracking game. Subjects were tested in pairs. Observers watched either a skilled or a learning model perform 3 trials. The observers then practiced the game for 3 trials. Observation of another 12 trials was followed by 12 more practice trials. Substantial observational learning was found, as both groups of observers performed better than the learning models after both the 3 initial trials of observation and after 12 more observation trials. However, there were no differences due to observing the skilled or the learning model. These findings are discussed in relation to theoretical issues of observational learning.

Analysis of Variance↗
PubMed1992

Effects of aging and schedules of knowledge of results on motor learning.

Little research has been conducted on aging and the learning of motor skills. In this study, we examined the effects of different schedules of knowledge of results (KR) on the acquisition and retention of a movement timing task by young adults (20-23 years) and older adults (60-82 years). The results indicated that there were differences between young and older adults in the accuracy and consistency of motor performance when KR was provided. Accuracy effects persisted during a retention interval when KR was no longer provided, although there were no differences in variability. There were no interactions of age with any of the KR-related variables. These findings suggest that the ability to process KR, and the effects of KR on motor learning, are similar in young and older adults. These findings are discussed in terms of age-related issues in movement control and learning processes.

Adult↗
PubMed1992

Antigen expression of frozen platelets.

Platelet antigens of platelet samples from 36 donors, frozen for different intervals, were evaluated by the platelet suspension immunofluorescence test (PSIFT). A, B, PLA1(HPA-1a) and various HLA antigens were tested by their corresponding antisera. The antigen could be detected in almost all the samples after one month of freezing. After 3 and 6 months, the platelet antigens could only be detected in 29.2% and 3.7% of the samples, respectively. There was no difference in decay of antigen expression among A, B, PLA1 and HLA antigens. When compared with the freshly prepared platelets, frozen platelets presented stronger antigen expression after 2 to 4 weeks of storage. This may suggest that the frozen platelets could be used for platelet crossmatching procedures without loss of their antigenicity within one month.

Antigens↗
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↗