Correlation of 137Cs-induced micronuclei with survival fraction in human HT29 cancer cells.
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Cell Survival↗
Biomedical subjects
T K Lee
Publications and source records attributed to T K Lee.
Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.
Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.
Amino Acid Sequence↗
Thermal stability and domain-domain interactions in natural and recombinant protein C.
Scanning microcalorimetry and spectrofluorimetry were applied to a study of the thermal stability and interaction of the modules within natural human protein C (PC) and recombinant protein C (rPC), a potential therapeutic anticoagulant expressed in transgenic pigs. Upon heating in the presence of 2 mM EDTA, pH 8.5, each protein exhibited a similar heat absorption peak with a Tm of approximately 62 degrees C corresponding to the melting of the serine protease (SP) module. Deconvolution of this peak indicated that the SP module consists of two domains that unfold independently. At pH below 3.8, a second peak appeared at extremely high temperature corresponding to the unfolding of the two interacting epidermal growth factor-like (EGF) domains. This second peak occurred at a temperature about 20 degrees C lower in rPC than in PC indicating that the EGF domains in the recombinant protein are less stable. The isolated 6-kDa gamma-carboxyglutamic acid-rich (Gla) fragment as well as a 25-kDa Gla-(EGF)2 fragment both exhibited a sigmoidal fluorescence-detected denaturation transition in the same temperature region as the SP domains, but only in the presence of Ca2+. In 2 mM Ca2+, the first heat absorption peak in both intact proteins became biphasic, indicating Ca(2+)-induced structural changes. By contrast, Ca2+ had very little effect on the melting of Gla-domain-less protein C. This indicates that not Ca2+ itself but the Ca(2+)-loaded Gla domain is responsible for conformational changes in the SP domain of the parent protein. Detailed analysis of the shape of the endotherms obtained in Ca2+ and EDTA suggests that Ca2+ induces compact structure in the Gla domain which appears to interact strongly with the SP domain(s) of protein C.
Calcium↗
Postoperative selective bowel decontamination prevents gram-negative bacterial translocation in small-bowel graft recipients.
Gram-negative septic episodes are a potential risk of small-bowel transplantation; bacterial translocation through the graft is considered the mechanism. As a measure to prevent this complication, we evaluated postoperative selective bowel decontamination (SBD) in the rat model of orthotopic small-bowel transplantation [Lewis (LEW) and Brown-Norway (BN) rats as donors and recipients]. For 4 days after transplantation we gave FK 506, 2 mg/kg, which prevents rejection and results in indefinite recipient survival. For SBD, 24 mg/kg/day polymyxin E and 20 mg/kg/day tobramycin were administered via orogastric gavage to allograft recipients, both with and without FK 506 therapy. On Day 9, all rats were sacrificed, the peritoneal cavity was swabbed, and mesenteric lymph nodes (MLN), spleen, liver, and ileum were harvested for microbial qualitative and quantitative analysis. Animals with positive peritoneal swab cultures were excluded. SBD resulted in a significant reduction of the quantitative gram-negative bacterial flora in the ileum and cecum and of bacterial translocation to the MLN [0% versus 50% (no FK 506 therapy) and 8% versus 50% (FK 506 treated)]. In the allograft groups not treated with FK 506, SBD failed to significantly prolong survival, suggesting that acute rejection is not hastened by infection (bacterial translocation). We conclude that SBD in small-bowel-graft recipients prevents bacterial translocation by reducing intestinal gram-negative bacterial flora; this may reduce local and systemic infections by gut-derived organisms.
Animals↗
Production of engineered IgM-binding single-chain antibodies in Escherichia coli.
Two single-chain antibodies were engineered and tested as novel binding proteins with specificity for immunoglobulin M. Genes for the two single-chain Fv proteins were assembled from the variable light chain cDNA and variable heavy chain cDNA of monoclonal antibodies DA4.4 and Bet 2, which specifically bind human IgM and mouse IgM, respectively. Both single-chain Fv proteins were designed with a 14-amino acid linker which bridged the variable light chain and variable heavy chain domains. The two proteins were expressed in Escherichia coli, purified and assayed for IgM-binding activity. Both proteins demonstrate a binding specificity for their corresponding IgM which is similar to the monoclonal antibodies from which they were derived. These small IgM-binding proteins may have applications in the investigation of the immune response and in the detection and purification of monoclonal antibodies, cell-associated antibodies, and IgM from serum.
Amino Acid Sequence↗
Noninvasive blood pressure monitoring from the supraorbital artery using an artificial neural network oscillometric algorithm.
OBJECTIVE: Our objective was to overcome the limitations of linear models of oscillometric blood pressure determination by using a nonlinear technique to model the relationship between the oscillometric envelope and systolic and diastolic blood pressures, and then to use that technique for near-continuous arterial pressure monitoring at the supraorbital artery. METHODS: An adhesive pressure pad and transducer were used to collect oscillometric data from the supraorbital artery of 85 subjects. These data were then used to train an artificial neural network (ANN) to report diastolic or systolic pressure. Arterial pressure measurements defined by brachial artery auscultation were used as a reference. ANN results were compared with those obtained using a standard oscillometric algorithm that determined pressures based on fixed percentages of the maximum oscillometric amplitude. RESULTS: The ANN produced better estimates of reference blood pressures than the standard oscillometric algorithm. Mean difference between target and actual output for the ANN was 0.50 +/- 5.73 mm Hg for systolic pressures, compared to the mean difference of the standard algorithm of 2.78 +/- 19.38 mm Hg. For diastolic pressures, the ANN had a mean difference of 0.04 +/- 4.70 mm Hg, while the mean difference of the standard algorithm was -0.34 +/- 9.75 mm Hg. CONCLUSIONS: The ANN produced a better model of the relationship between the oscillometric envelope and reference systolic and diastolic pressures than did the standard oscillometric algorithm. Noninvasive blood pressure measured from the supraorbital artery agreed with pressure measured by auscultation in the brachial artery, and may sometimes be more clinically useful than an arm cuff device.
Adult↗
Optimal sites for forehead oscillometric blood pressure monitoring.
OBJECTIVE: Blood pressure is usually measured noninvasively with a cuff on the arm of the leg. Circumstances exist, however, when an alternative site for blood pressure measurement is desirable. This study is designed to identify a location on the forehead where blood pressure can be reliably measured noninvasively. METHODS: We mapped the superficial temporal artery and/or the supraorbital artery in 65 volunteers and found a rectangular area where an adhesive pressure pad could be placed over each artery. Oscillometric signals were recorded from four different locations over the forehead in 19 of the 65 volunteers to compare the amplitude of the signal and mean blood pressure between locations. RESULTS: The course of the supraorbital artery is quite consistent. It passed through a 2.5- x 1-cm rectangular area on the forehead in all volunteers in which it was mapped. The medial border of the rectangle is 0.5-cm medial and 1-cm above the medial corner of the left eyebrow. The course of the superficial temporal artery differed remarkably from person to person. We could not find an area of reasonable size to cover the artery in all wounds. Mean blood pressures were the same in all forehead locations. The signal was the weakest on the center of the forehead and strongest directly over the superficial temporal artery. CONCLUSIONS: Our results show that the supraorbital artery, an end-artery of the internal carotid artery, which emerges through the supraorbital foramen and cross the forehead near the center, is the preferred site to monitor blood pressure noninvasively on the forehead with an adhesive pressure bladder.
Adult↗
Assessment of two protocols for the human lymphocyte cytokinesis-blocked micronucleus assay.
This study indicated that by adding cytochalasin B (6 micrograms/ml) at 24 h, the lymphocyte culture time for micronucleus (MN) assay could be shortened to 64 h. In both unirradiated and ex-vivo irradiated (2 Gy) lymphocytes from three populations, we found that the differences in MN yield obtained by our modified cytokinesis-blocked time frame and that recommended by Fenech and Morley (1985) were insignificant (P = 0.66-0.87). We believe that the shorter assay time may enhance the applicability of MN assay for the rapid assessment of ionizing radiation overexposures.
Analysis of Variance↗
Proteolytic processing of human protein C in swine mammary gland.
Maturation of human Protein C (HPC) precursor to a zymogen in the liver requires endoproteolytic cleavages after a basic dipeptide, Lys-2-Arg-1 in the propeptide and Lys156-Arg157 connecting the light and heavy chain. Recombinant human Protein C (rHPC) was expressed in the mammary gland of transgenic swine and its proteolytic processing was monitored. We found that about 10-20% of rHPC purified from the milk still retained the propeptide and 30-40% was in the single-chain form, indicating inefficient proteolytic cleavage. This demonstrates that endoprotease(s) of the swine mammary epithelial cells do not process fully the precursor of heterologous protein. rHPC was fractionated by anion exchange chromatography and polypeptides with novel N-termini at positions -1,152 and 157 were detected in addition to the known N-termini at residues -24, +1, and 158. Since rHPC was found to be stable both in milk and after purification, it is possible that these new cleavages on the amino side of arginine at dipeptide sites Lys-2-Arg-1, Lys151-Arg152, and Lys156-Arg157 could have occurred in the mammary gland. Thus, our results suggest that a portion of HPC precursor was proteolytically processed in swine mammary gland differently than those in other expression systems.
Amino Acid Sequence↗
Impact of alcohol consumption and cigarette smoking on stroke among the elderly in Taiwan.
BACKGROUND AND PURPOSE: We investigated the influence of alcohol consumption and cigarette smoking on all types of stroke and cerebral infarction, in particular among a representative sample of elderly residents in Taiwan. METHODS: This study was a component of a nationwide survey of health and living status of residents aged 65 years or older in Taiwan in which subjects received detailed physical, neurological, and laboratory examinations. Inquiries were made about medical history, and information on the amount and duration of drinking and smoking was obtained. Diagnoses of stroke were made according to the results of brain computed tomography at the onset of disease or were based on criteria established by the World Health Organization. RESULTS: Of the 2600 subjects, there were 155 elderly persons with stroke (prevalence, 6%). Excessive drinking of more than 367.6 g/wk of alcohol was associated with a high prevalence of cerebral infarction. Consumption of < or = 367.5 g/wk of alcohol did not have an influence on stroke prevalence. The relationship between duration of alcohol drinking and stroke was equivocal. More than 30 pack-years of cigarette smoking was a significant risk factor for all types of stroke and cerebral infarction in particular. Using multiple logistic regression to control for possible confounders, it was found that smoking was an independent risk factors for all stroke and was of borderline significance for cerebral infarction. Although excessive drinking was a significant risk factor for cerebral infarction in univariate analysis, this effect was lost after adjustment for other confounders. CONCLUSIONS: Cigarette smoking was a more important risk factor for stroke and cerebral infarction than excessive drinking of alcohol.
Aged↗
The anatomy of the basal ganglia and Parkinson's disease: a review.
The present understanding of the anatomy of the basal ganglia has been updated. Recent work has produced a primate model of Parkinson's disease for study of its pathogenesis and treatment. In the last two decades, administration of dopamine agonist has been the mainstay of treatment of Parkinson's disease in the humans. However, recent use of dopamine-rich tissue such as adrenal gland or human foetal cells is opening up a new frontier for the treatment of more severe Parkinsonism. Nevertheless, there is still much to be learned at the basic neuroscience level before such procedure could be used widely in clinical practice.
Animals↗
A method for simultaneous identification of human active and active-site alkylated O6-methylguanine-DNA methyltransferase and its possible application for monitoring human exposure to alkylating carcinogens.
Cells resist the major mutagenic effects of alkylating agents by the action of O6-methylguanine-DNA methyltransferase (MGMT), which transfers the alkyl (R) group of O6-alkylguanine, produced in DNA by these chemicals, to a cysteine residue in its active site (formation of R-MGMT). We demonstrate that cellular R-MGMT (which represents a record or memory within the cells exposed to these chemicals) can be assayed by its sensitivity toward proteolysis by protease V8. The possible use of this assay for monitoring exposure to alkylating carcinogens was investigated by using cultured cells and a preliminary study with the use of human blood from normal subjects and patients undergoing chemotherapy. Cultured cell experiments show that R-MGMT is sufficiently stable for the monitoring purpose and its level bears a dose-response relationship to the concentrations of the alkylating agent used. Interestingly, experiments with blood from patients undergoing chemotherapy show a gradual formation of R-MGMT in 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and an induced MGMT deficiency in cyclophosphamide-treated patients. The use of this methodology, which allows for the possible quantification of active MGMT (cellular DNA repair capacity) and R-MGMT (recent exposure) simultaneously, in monitoring human exposure to alkylating carcinogens is discussed.
Adolescent↗
Radiation dose-dependent variations of micronuclei production in cytochalasin B-blocked human lymphocytes.
Using the cytokinesis-block technique, lymphocytes from healthy volunteers (n = 9) were evaluated for 1) the radiation dose-response curve for micronuclei (MN) expression; 2) technique variables on the yield of MN; and 3) the shortest lymphocyte incubation time required for the MN assay. We found that the best fitting of relationships between increasing MN production and increasing irradiation dose (0-4.0 Gy) was the linear-quadratic model as expressed by the yield equation Y = C+alpha D+beta D2 (P = 0.0003). When lymphocytes were irradiated in vitro with 2.0 Gy and harvested at various time intervals, MN increased during the entire 84 hr culture time. The radiation caused a division delay in lymphocyte as indicated by an increased frequency of mononucleated cells and a decreased number of mitotic indices. The data showed that a shortened culture time (60 hr) for the MN assay is possible and that binucleated cells with > or = 3 MN were found only in cells irradiated at > or = 2.0 Gy. These findings suggest that scoring of MN in lymphocytes may be a practical biological dosimeter for the rapid screening of accidental radiation exposure victims, especially when their clinical manifestations are not obvious.
Adult↗
Mucosal glutaminase activity and histology as parameters of small bowel preservation injury.
The present study in Lewis rats was designed to assess the predictive value of the mucosal enzyme activities of glutaminase, maltase, and xanthine oxidase and of histology as parameters to delineate the degree of small bowel preservation injury. Small bowel grafts were flushed with saline or a modified phosphate-buffered sucrose (PBS) solution, stored at 8 degrees C for 1, 6, or 12 hr, and transplanted heterotopically. Tissue samples for determination of mucosal enzyme activities were taken after the cold storage period, 20 min after reperfusion and 2 and 7 days postoperatively. Biopsies for light microscopic evaluations were obtained at the same time points, but not after cold storage. Glutaminase activity was well maintained after cold storage, regardless of the duration of preservation. Enzyme activities measured 20 min after reperfusion decreased with increasing duration of preservation (saline: R2 = 32.8%; P < 0.01; PBS: R2 = 52.3%; P < or = 0.001) and with increasing histologic preservation injury. Glutaminase activities were predictive for survival of grafts preserved with the PBS solution (R2 = 49.6%; P < or = 0.001; sensitivity 92%; specificity 100%), while the activities of maltase and of xanthine oxidase failed to do so. The degree of histologic preservation injury seen in graft specimens obtained 20 min after reperfusion was a good predictor of graft survival with a sensitivity of 90% for saline-preserved grafts and 92% for PBS-preserved grafts and a specificity of 88 and 67%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Mucosal recipient-type mononuclear repopulation and low-grade chronic rejection occur simultaneously in indefinitely surviving recipients of small bowel allografts.
Lewis rat recipients of long-term, surviving, orthotopic Brown-Norway rat intestinal allografts, initially treated with cyclosporin A (CyA) or FK 506, were evaluated for their functional capacity and morphology over 1 year after the immunosuppressive therapy had been discontinued. Functional parameters such as nitrogen and fat balances, maltose absorption, blood chemistry, hematologic studies, and the weight gained by the allografted animals did not differ from those of syngeneically grafted or age-matched normal animals. Immunohistochemical studies showed that the lamina propria of the allografts was repopulated with recipient MHC class II+ mononuclear cells and that a normal distribution of T helper, T suppressor/killer, and IgA+ plasma cells had occurred. However, fibrous replacement of the mesenteric lymph nodes and Peyer's patches were detected in all, and an inflammatory obliterative arteriolopathy developed in the mesenteric vasculature of half of the allografted animals. No such findings were observed in recipients of syngeneic grafts. These results demonstrate that the limited use of potent immunosuppressive agents immediately after transplantation averts rejection and is followed by recipient-type mucosal lymphocytic repopulation. Simultaneously, a clinically not recognizable chronic rejection evolves. This suggests that the timely diagnosis of chronic rejection may not be possible with the use of standard tests of gut function and random mucosal biopsies alone.
Animals↗
Preservation of cytoplasm in the human lymphocyte micronucleus assay.
A major limitation in the quantitative accuracy of the human lymphocyte micronucleus (MN) assay is preservation of the cytoplasm during the cell harvesting. In this short communication, an improved method for cytoplasm preservation in a cytokinesis-blocked, whole-blood microculture (0.3 ml) technique is described. We believe that the timing of the hypotonic treatment, speed of centrifugation, handling of the cell suspension and proper Giemsa staining are important variables in the human peripheral lymphocyte MN assay.
Azure Stains↗
Role of transesophageal echocardiography in the diagnostic assessment of cardiac sources of embolism in patients with acute ischemic stroke.
To evaluate the role of transesophageal echocardiography (TEE) in the diagnostic assessment of cardiac sources of embolism in acute ischemic stroke, 94 consecutive patients (44 men and 50 women, aged 22-82 years) with acute ischemic stroke were prospectively studied. Of these, 34 patients had clinical evidence of heart disease. Both transthoracic and transesophageal echocardiograms were recorded on the same day for each patient. Transthoracic echocardiography (TTE) identified a possible cardiac source of embolism in 2 patients (6%) with and in 3 (5%) without clinical heart disease. TEE identified a possible cardiac source of embolism in 21 patients (62%) with and in 18 (30%) without clinical heart disease. TEE was superior to TTE for the detection of a cardiac source of embolism in patients with acute ischemic stroke (41 vs. 5%, p < 0.001). Factors significantly associated with a greater likelihood of such cardiac sources of embolism included left atrial enlargement, atrial fibrillation and a younger age. The yield of TEE in identifying a possible cardiac source of embolism was higher in patients with clinical evidence of heart disease than in those without.
Acute Disease↗
Bacterial translocation and the role of postoperative selective bowel decontamination in small intestinal transplantation.
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Animals↗