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Biomedical subjects

T D Lee

Publications and source records attributed to T D Lee.

At least 73 records · Page 4Linked to original sources
PubMed1996

Prolongation of rat kidney allograft survival by nematodes.

Nippostrongylus infection strongly stimulates TH2 activity in vivo. Given the evidence of cross regulation between TH2 and TH1 cells, and the link between TH1 activity and graft rejection, we examined the effects of Nippostrongylus infection on the fate of kidney allografts in rats. Both prior Nippostrongylus infection and prior treatment with a soluble worm product significantly delayed kidney allograft rejection. Control graft rejection occurred at 9.7 +/- 1.2 days whereas grafts in Nippostrongylus- or worm extract-treated recipients lasted 32.7 +/- 11.3 days and 21.5 +/- 4.6 days, respectively. At day 5 posttransplant mononuclear cell infiltration was much reduced in the Nippostrongylus-treated recipients. Flow cytometry of isolated graft-infiltrating leukocytes showed a marked decrease in infiltrating T cells (82.8% reduction) with both CD4+ cells (81.0% reduction) and CD8+ cells (84.6% reduction) being reduced. CD8+ T cells, in particular, made up a much smaller proportion of the graft-infiltrating cells (22% rather than 49%) in the Nippostrongylus-treated animals as compared with untreated controls. Immunohistochemical assay of the graft tissue confirmed the flow cytometric results. Interleukin 4 expression was clearly demonstrated by RT-PCR of the isolated graft-infiltrating leukocytes from the Nippostrongylus-treated recipients but not from the control recipients. These data are consistent with our current hypothesis that Nippostrongylus delays graft rejection by inducing a cross-regulatory suppression of TH1 activity.

Animals↗
PubMed1996

Importance of minor histocompatibility antigens in the development of allograft arteriosclerosis.

Although acute rejection has been mostly ameliorated with the use of powerful immunosuppressive drugs, kidney and heart transplants continue to succumb to a more chronic response characterized by intimal lesions in the graft vasculature. This late-stage response is referred to as allograft arteriosclerosis. Allorecognition is clearly involved in the initiation of this response but the relative importance of major histocompatibility (MHC) and minor histocompatibility (mH) antigens remains unclear. By taking advantage of the B10 congenic set of mice and our newly described mouse aortic interposition graft model we have been able to assess the contribution of these antigens to the development of the concentric intimal lesions characteristic of allograft arteriosclerosis. We performed transplants between syngeneic animals, animals which were disparate at both MHC and multiple mH, animals which were disparate at MHC only, and animals which were disparate at multiple mH antigens only. H-Y antigen variation was controlled for by performing all transplants between female mice. In all cases the recipients were C57BL/10 (H-2b) mice. Both cellular infiltration into the intima and resulting intimal thickness were measured at 2, 4, 8, and 13 weeks posttransplant. At all time points, the grafts from MHC disparate only donors showed less severe intimal lesions than the grafts from fully disparate or mH disparate donors. This difference reached statistical significance at 4 and 13 weeks. This suggests that mH antigens are as immunogenic as MHC antigens with respect to the generation of allograft arteriosclerosis. These findings are not unique to vascular grafts and may relate to the importance of indirect antigen presentation in the development of chronic rejection.

Animals↗
PubMed1996

Effects of task instructions and oscillation frequency on bimanual coordination.

Increases in the oscillation frequency of bimanual movements produce a switch from an antiphase (180 degrees relative phase) to an in-phase (0 degrees relative phase) coordination pattern. This finding is observed when subjects are instructed not to intervene when they feel themselves slipping out of the anti-phase pattern. The question addressed in this study concerned how performance would be affected if subjects were instructed to try to maintain the pattern at all times. This issue was addressed using two separate groups of subjects: one group was given the "do not intervene" instructions, the other group was told to try to stay with the pattern at all times. Forearm rotations were tested in 15 s trials, paced by an auditory metronome set at 1.0, 1.5, 2.0, 2.5, and 3.0 Hz. Frequency distributions of the point estimates of relative phase were analyzed. The Do not Intervene group replicated previous findings, as indicated by the development of a bimodal histogram of relative phase distributions with increases in oscillation frequency. However, a very different pattern of findings emerged with increases in oscillation frequency for the group told to stay with the anti-phase pattern. Rather than a bimodal distribution being developed, the data maintained 180 degrees as its central tendency--no secondary distribution developed around 0 degrees relative phase. These data suggest that volitional control can override the inherent dynamical tendencies of the motor system.

Adult↗
PubMed1996

Prevention of experimental postoperative peritoneal adhesions by N,O-carboxymethyl chitosan.

BACKGROUND: Postsurgical adhesion formation can result in significant morbidity and, to a lesser extent, death. The purpose of this experiment was to assess the ability of N,O-carboxymethyl chitosan (NOCC) to prevent postsurgical adhesion formation in vivo. METHODS: Randomized groups of Sprague-Dawley rats were studied under two abdominal surgery models, the uterine horn model and the small bowel laceration model, for the ability of NOCC to reduce the incidence and severity of adhesion formation. Adhesions in animals were assessed after death by a blinded observer 10 to 14 days after surgical manipulation. RESULTS: NOCC consistently reduced the size, strength, and number of adhesions in both rat models. NOCC was also found to be more effective than hyaluronic acid at inhibiting adhesion formation. CONCLUSIONS: NOCC is a more effective antiadhesion agent than is the more expensive hyaluronic acid. Although the exact mechanism of NOCC's antiadhesion activity is as yet unclear, the novel chemical is of particular interest for clinical use.

Animals↗
PubMed1996

Delayed visual feedback while learning to track a moving target.

Two studies investigated the effects of delayed visual feedback on manual tracking. In Experiment 1, individuals practiced with visual feedback provided either immediately (0 delay) or with a 333-ms delay. During acquisition, the 0 delay group performed with less error than the 333-ms delay group. A retention test with 0 delay feedback was performed with the least error by the 0 delay group. A transfer test using a different 0 delay tracking pattern, was performed with the least error by the 333-ms delay group. In Experiment 2, individuals practiced at six different delays. Error increased as training feedback delay increased. For retention there were no differences between the delay groups during the 0 delay retention. At a 417-ms retention, test error decreased as training feedback delay increased. Results indicate that error during acquisition does not necessarily impair learning and that feedback delays can be beneficial for learning.

Analysis of Variance↗
PubMed1996

Large-scale DRB and DQB1 oligonucleotide typing for the NMDP registry: progress report from year 2.

DNA typing of HLA class II alleles of the DRB1/3/4/5 and DQB1 loci using sequence-specific oligonucleotide probes and polymerase chain reaction amplified DNA has been used for the large-scale typing of donors for the National Marrow Donor Program unrelated donor registry. The results of quality control analysis for the second year of the project (10/1/939/30/94) show the typing continues to be highly accurate, specific, and reliable. The average percent of correctly classified HLA oligotypes (groups of alleles defined by a hybridization pattern with a panel of sequence-specific oligonucleotide probes) based on 9,244 DRB1 and 7,244 DQB1 assignments was 99.8% (range 99.4%100.0%) for DRB1/DRB3/DRB4/DRB5 and 99.8% (range 99.6%100.0%) for DQB1. This level of accuracy is particularly remarkable because the 4,636 DRB quality control samples were tested blindly and could not be distinguished from 57,580 donor samples tested at the same time by the laboratories.

Alleles↗
PubMed1996

An apparent functional correlation between variations in amino acid residues in HLA-DR4.1 and 4.2 serological subtypes and oligonucleotide characterization.

HLA-DR4 can be subdivided serologically into two specificities, DR4.1 and DR4.2, using well-defined monospecific alloantisera used in the 11th International Histocompatibility Workshop. In this study, a total of 1095 random DR4-positive individuals from several ethnic groups were tested first for serotype DR4.1/4.2 and then for DRB1*04 alleles using polymerase chain reaction (PCR) followed by sequence-specific oligonucleotide probe hybridization (SSOPH). An almost 100% correlation between samples testing positive for DR4.1 and the presence of alanine at position 74 was observed, while samples testing positive for DR4.2 correlated with the presence of glutamic acid at position 74. DRB1*04 alleles 0401, 0402, 0404, 0405, 0408, 0409 and 0410 are aligned in functional groups which coincide with the serological subtype of DR4.1. DRB1*04 alleles 0403, 0406, 0407 and 0411 coincide with subtype DR4.2. Amino acid substitutions at positions 57, 71 and 86 indicate other significant variations between alleles within the serological subgroup of DR4.1 and define five minor subgroups. The serologic and oligonucleotide allelic subgroups are in turn correlated with recognized cellular Dw antigens. While sequence data provide evidence of structural differences, data on cellular antigens support a functional association between these designated groups and their significance in transplantation and GVHD. Testing results are categorized by ethnic group in order to establish frequency data for donor selection criteria.

Amino Acid Sequence↗
PubMed1996

Evidence that CCK-58 has structure that influences its biological activity.

Many biologically active peptides exist in multiple molecular forms, but the functional significance of regions outside the region of bioactivity is unknown. The biological and immunological data presented in this study indicate that cholecystokinin-58 (CCK-58), unlike other forms of cholecystokinin, has structure that influences its bioactivity. CCK-58 was purified from acid extracts of canine intestinal mucosa until a single absorbance peak was obtained during reverse-phase chromatography. Amino acid analysis precisely determined the peptide concentrations of purified CCK-58 and synthetic CCK-8. Our hypothesis was that if the amino terminus of CCK-58 influences its bioactivity then its activity would be modified when this region was removed from the peptide. To evaluate the importance of the amino terminus of CCK-58 to influence its biological activity, the abilities of CCK-58 and CCK-8 to release amylase from pancreatic acini were compared before and after tryptic digestion. Tryptic digestion of CCK-58 decreased the half-maximal stimulation (EC50) for amylase release from 96 to 28 pM. The EC50 for digested CCK-58 was similar to that for CCK-8 (17 pM). These results suggest that CCK-58 has a structure that shields its bioactive carboxyl terminus. This is further supported by the finding that carboxyl fragments generated from CCK-58 by trypsin or by partial acid hydrolysis were greater than twofold more immunoreactive than the intact CCK-58. The diminished activity of CCK-58 SK shields the carboxyl terminus, which is important to its biological and immunological activities.

Acids↗
PubMed1996

The peptide pQFYRFamide is encoded on the FMRFamide precursor of the snail Helix aspersa but does not activate the FMRFamide-gated sodium current.

The complete sequence of the FMRFamide precursor cDNA from Helix aspersa is reported here. Since the 5' end of this cDNA is identical to that of the precursor that encodes the heptapeptide analogs of FLRFamide, the two transcripts are probably derived by alternative RNA splicing. A novel pentapeptide, Glp-Phe-Tyr-Arg-Phe-NH2 (pQFYRFamide), predicted from the cDNA sequence, was purified from extracts of H. aspersa ganglia and identified by mass spectroscopy. Partial gene sequences for the FMRFamide precursors of two closely related pulmonate species-Cepaea nemoralis and Polydontes acutangula-were also determined and compared with the cDNA sequence of H. aspersa and a partial gene sequence previously determined from H. pomatia. Not only are the FMRFamide-related sequences and proteolytic processing sites conserved, but the linear arrangement of these landmarks is also retained. Synthetic pQFYRFamide has some effects on the isolated heart and on neuronal potassium currents in H. aspersa that are similar to those of FMRFamide, but it does not activate the neuronal FMRFamide-gated sodium channel.

Amino Acid Sequence↗
PubMed1995

A microscale electrospray interface for on-line, capillary liquid chromatography/tandem mass spectrometry of complex peptide mixtures.

A microcapillary liquid chromatography (HPLC) system designed for the gradient elution of peptide and protein samples at flow rates < 1 microL/min has been coupled to a triple-sector quadrupole mass spectrometer via a simple sheathless electrospray interface (microspray). The microspray interface used a flame-drawn, uncoated, fused silica needle with tip outer diameters in the range of 15-20 microm and an opening less than 5 microm in diameter. Online sample filtration to prevent clogging of the drawn needle was accomplished by using a hydrophilic PVDF membrane filter integrated into the needle assembly. The spray potential (0.5-1 kV) was applied directly to the sample stream through the capillary union. Stable electrospray conditions were obtained over the full range of the gradient (0-90% acetonitrile in water) and was generally independent of flow rate. Both off-line and online analyses of proteins and peptide digest mixtures were performed at sample levels less than 10 fmol. HPLC parameters could be optimized for either rapid LC/MS analysis or enhanced performance in LC/MS/MS experiments by modulation of the eluting peak widths. Additionally, flow could be greatly reduced as selected components pass through the interface to prolong the time available to collect mass spectral data. The reduced spectral background and peak width manipulation facilitated the acquisition of peptide production spectra (MS/MS) using real-time, automated instrument control procedures.

Chromatography, High Pressure Liquid↗
PubMed1995

Amino acid sequence of CAP2b, an insect cardioacceleratory peptide from the tobacco hawkmoth Manduca sexta.

The primary structure of a novel insect neuropeptide, Cardioacceleratory Peptide 2b (CAP2b), from the tobacco hawkmoth Manduca sexta has been established using a combination of mass spectroscopy, Edman degradation microsequencing, amino acid analysis, and biological assays. The sequence of CAP2b, pyroGlu-Leu-Tyr-Ala-Phe-Pro-Arg-Val-amide, has a molecular weight of 974.6 and is blocked at both the amino and carboxyl ends. Examination of several national computer protein data bases failed to reveal other peptides or proteins with any sequence homology to CAP2b indicating that this is likely to be a novel insect neuropeptide. This peptide may be a general activator of insect viscera since it causes an increase in heart rate in Manduca and in Drosophila, and has also been implicated in the regulation of fluid secretion by the Malphigian tubules of Drosophila.

Amino Acid Sequence↗
PubMed1995

Microscale immobilized protease reactor columns for peptide mapping by liquid chromatography/mass spectral analyses.

Methods and procedures for the construction and operation of a microscale immobilized protease (trypsin) reactor are described. Optimization of reactor efficiency with regard to reactor flowrate, length, and temperature as well as sample concentration has been examined, and a novel system for sample preconcentration is presented. The structural analysis of a standard protein (cytochrome c) by a combination of trypsin microreactor digestion followed by off-line electrospray ionization MS analysis is demonstrated at the 10-pmol level. The application of these techniques for the rapid confirmation (1-2 h) of a genetic variant (hemoglobin E) including MS/MS analysis of the variant peptide is included.

Amino Acid Sequence↗
PubMed1995

Development of a mouse aortic transplant model of chronic rejection.

Chronic rejection is the most common cause of late graft failure after solid organ transplantation. A model of chronic rejection, the rat aortic allograft, has histologic features that parallel those in the vessels of human transplanted organs. However, the molecular tools required to dissect the immunology of chronic rejection are unavailable in the rat. We developed aortic transplantation in the mouse as a new model of chronic rejection. This will allow the use of the diversity of recombinant cytokines and monoclonal antibodies available for the mouse and its well-defined genetics to investigate chronic rejection in greater detail. We describe the perioperative care and surgical technique for the model in which a 1 cm segment of donor thoracic aorta was used to replace a section of recipient abdominal aorta below the renal arteries and above the aortic bifurcation. Mortality rates were initially high (70%) due to thrombosis and shock. Changes in technique and operator facility resulted in a high rate of success (75%). After 192 operations, the current success rate is > 80%. Mice free from complications at 12 hrs postop had indefinite survival, and after 2 months the typical vascular lesion of chronic rejection was present. This new model of chronic rejection will be a valuable tool to study the molecular immunology and genetics of chronic rejection.

Anastomosis, Surgical↗
PubMed1995

IgE regulation by nematodes: the body fluid of Ascaris contains a B-cell mitogen.

Nematode infection of human beings or laboratory animals leads to markedly increased levels of circulating IgE, most of which is not specific to worm antigens. This phenomenon is known to be interleukin-4-dependent, but little is known about the mechanism of activation of the response. In an attempt to elucidate this mechanism, we have used a reductive approach with worm products rather than infections. In a previous article we showed that injection of the body fluid of the nematode Ascaris yields a marked increase in circulating IgE. In this study we demonstrate that the body fluid contains a B-cell mitogen. Incubation with purified splenic B cells with 50 micrograms/ml body fluid yields marked proliferation of B cells, as measured by tritiated thymidine uptake. Similarly, Ascaris body fluid stimulates G0 B cells to enter the cell cycle. T cells are unaffected by the mitogen, and the response is dependent on viable accessory cells. Contamination of Ascaris body fluid or reagents by bacterial lipopolysaccharide has been ruled out as a source of artefactual data. A model is proposed, which suggests that the B-cell mitogen in Ascaris body fluid stimulates polyclonal B-cell activity and that other nematode factors either stimulate the release of interleukin-4 or act in an interleukin-4-like manner to cause class switch to IgE.

Animals↗
PubMed1995

Structural characteristics of the Ascaris allergen, ABA-1.

The structure of the Ascaris allergen, ABA-1 was characterized at several levels. Purified allergen monomers eluted from reducing PAGE were found to reassociate into dimers in phosphate buffered saline containing 0.9 mM Ca2+. This association may involve the formation of disulfide bonds between monomers. The primary amino acid sequence was used to predict secondary structure and compare the allergen to other known proteins sequences. ABA-1 appears to be highly helical protein of two domains. Sequence analysis reveals short regions (25 amino acids) of high homology (76%) between ABA-1 and the major body wall myosin of Onchocerca volvulus. In addition, ABA-1 has sequence similarity to a family of EF-hand containing calcium binding proteins called S100 proteins. The dimerization and two-domain structure of ABA-1 is consistent with the possibility that ABA-1 is a member of the S100 family of calcium binding proteins.

Allergens↗
PubMed1994

Purification and sequence of rat oxyntomodulin.

Structural information about rat enteroglucagon, intestinal peptides containing the pancreatic glucagon sequence, has been based previously on cDNA, immunologic, and chromatographic data. Our interests in testing the physiological actions of synthetic enteroglucagon peptides in rats required that we identify precisely the forms present in vivo. From knowledge of the proglucagon gene sequence, we synthesized an enteroglucagon C-terminal octapeptide common to both proposed enteroglucagon forms, glicentin and oxyntomodulin, but sharing no sequence overlap with glucagon. We then developed a radioimmunoassay using antibodies raised against the octapeptide that was specific for enteroglucagon peptides without cross-reacting with glucagon. Rat intestine was extracted, and one presumptive enteroglucagon form was purified by following the enteroglucagon C-terminal octapeptide-like immunoreactivity through several HPLC purification steps. Structural characterization of the material by amino acid composition, microsequence, and mass spectral analyses identified the peptide as rat oxyntomodulin. The 37-residue peptide consists of pancreatic glucagon plus the C-terminal extension, Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala. This now permits synthesis of an unambiguous duplicate of endogenous rat oxyntomodulin for physiological studies.

Amino Acid Sequence↗
PubMed1994

Gallinacins: cysteine-rich antimicrobial peptides of chicken leukocytes.

We purified three homologous antimicrobial peptides ('gallinacins') from chicken leukocytes, examined their antimicrobial activity in vitro, and established their primary sequences by a combination of gas phase microsequencing and on-line LC-ESI-MS analysis of endo- and exoprotease peptide digests. The peptides contained 36-39 amino acid residues, were relatively cationic due to their numerous lysine and arginine residues, and each contained 3 intramolecular cystine disulfide bonds. Gallinacins showed primary sequence homology to the recently delineated beta-defensin family, heretofore found only in the respiratory epithelial cells and neutrophils of cattle, suggesting that beta-defensins originated at least 250 million years ago, before avian and mammalian lineages diverged. The 9 invariant residues (6 cysteines, 2 glycines and 1 proline) common to avian gallinacins and bovine beta-defensins are likely to constitute the essential primary structural motif of this ancient family of host-defense peptides.

Amino Acid Sequence↗