Biomedical subjects

E Lee

Publications and source records attributed to E Lee.

At least 19 recordsLinked to original sources

PubMed1995

Allelotype analysis of childhood acute lymphoblastic leukemia.

To identify the genetic events that may play an important role in leukemogenesis of childhood ALL, we report for the first time the allelotyping of childhood ALL. Twenty-four cases of childhood ALL were screened for loss of heterozygosity (LOH) using 101 highly polymorphic microsatellite markers, which are distributed among all autosomal chromosomes. For LOH analysis on both chromosomes 9 and 12, 54 childhood ALL samples were examined. The most frequent allelic loss was found on chromosomal arm 9p, where 20 of 50 (40%) informative samples showed LOH. Moreover, nearly 30% of samples that did not have either homozygous deletions or point mutations of the putative tumor suppressor genes CDKN2/INK4A/p16 and INK4B/p15 on chromosomal arm 9p had LOH at D9S171. Loss of chromosomal arm 12p was also frequent (26%). Mutational analysis suggested that the altered gene on 12p is not the cyclin-dependent kinase inhibitor p27/Kip1, which is also on 12p. Several other regions that had LOH included 1p, 4q, 5p, 6q, 7p, 8p, 9q, 10q, 13q, 17p, 17q, 18q, 19q, and 22q. Of 24 patients, 19 (79%) showed allelic loss on at least one chromosomal arm. Samples of two patients (8%) showed LOH on almost all chromosomes. Fractional allelic loss, calculated for each sample as the total number of chromosomal arms lost/total number of arms with information, showed a median value of 0.04 and a mean of 0.123 (range, 0 to 0.95). This fractional allelic loss is lower than those reported for many solid tumors. This analysis shows the extreme power of LOH analysis using microsatellite markers in childhood ALL.

Adolescent↗

PubMed1995

Tertiary and quaternary structural changes in Gi alpha 1 induced by GTP hydrolysis.

Crystallographic analysis of 2.2 angstrom resolution shows that guanosine triphosphate (GTP) hydrolysis triggers conformational changes in the heterotrimeric G-protein alpha subunit, Gi alpha 1. The switch II and switch III segments become disordered, and linker II connecting the Ras and alpha helical domains moves, thus altering the structures of potential effector and beta gamma binding regions. Contacts between the alpha-helical and Ras domains are weakened, possibly facilitating the release of guanosine diphosphate (GDP). The amino and carboxyl termini, which contain receptor and beta gamma binding determinants, are disordered in the complex with GTP, but are organized into a compact microdomain on GDP hydrolysis. The amino terminus also forms extensive quaternary contacts with neighboring alpha subunits in the lattice, suggesting that multimers of alpha subunits or heterotrimers may play a role in signal transduction.

Binding Sites↗

PubMed1995

Murray valley encephalitis virus envelope protein antigenic variants with altered hemagglutination properties and reduced neuroinvasiveness in mice.

Neutralization escape variants of Murray Valley encephalitis virus were selected using a type-specific, neutralizing, and passively protective anti-envelope protein (E) monoclonal antibody (4B6C-2) which defines epitope E-1c. Nucleotide sequence analysis revealed single nucleotide changes in the E genes of 15 variants resulting in nonconservative amino acid substitutions in all cases. One variant had a three-nucleotide deletion in the E gene which resulted in loss of serine at residue 277. Changes were clustered into two separate regions of the E polypeptide (residues 126-128 and 274-277), indicating that E-1c is a discontinuous epitope. One variant (BHv1), altered at residue 277 (Ser-->Ile), failed to hemagglutinate across the pH range 5.5-7.5, in contrast to parental virus and the other escape variants which hemagglutinated at an optimal pH of 6.6. BHv1 was also of reduced neuroinvasiveness in 21-day-old mice following intraperitoneal inoculation compared to the other viruses. Parental virus and the neutralization escape variants grew equally well in both vertebrate and invertebrate cell cultures, indicating that the reduced neuroinvasiveness of BHv1 was not due to a major abnormality of replication.

Amino Acid Sequence↗

PubMed1995

The modulation of phosphatidylinositol biosynthesis in hamster hearts by methyl lidocaine.

Methyl lidocaine is an experimental anti-arrhythmic drug which has been shown to enhance the biosynthesis of phosphatidyl-inositol (PI) in the hamster heart. In this study, the effect of methyl lidocaine on enzymes involved in the biosynthesis of PI in the heart was examined. When the hamster heart was perfused with labelled methyl lidocaine, the majority of the compound was not metabolized after perfusion. The direct action of methyl lidocaine on an enzyme was studied by the presence of the drug in enzyme assays, whereas its indirect action was studied by assaying the enzyme activity in the heart after methyl lidocaine perfusion. CTP:phosphatidic acid cytidylyl-transferase, a rate-limiting enzyme in PI biosynthesis, was stimulated by methyl lidocaine in a direct manner. Kinetic studies revealed that methyl lidocaine caused a change in the affinity between the enzyme and phosphatidic acid and resulted in the enhancement of the reaction. Alternatively, acyl-CoA:lysophosphatidic acid acyltransferase, another key enzyme for PI biosynthesis, was not activated by the presence of methyl lidocaine. However, the enzyme activity was stimulated in hearts perfused with methyl lidocaine. The enhancement of the acyl-transferase by methyl lidocaine perfusion was found to be mediated via the adenylate cyclase cascade with the elevation of the cyclic AMP level. The stimulation of protein kinase A activity by cyclic AMP resulted in the phosphorylation and activation of the acyltransferase. Interestingly, the activity of protein kinase C was not stimulated by methyl lidocaine perfusion. We conclude that the enhancement of PI biosynthesis by methyl lidocaine in the hamster heart resulted from the direct activation of the cytidylyltransferase, as well as the phosphorylation and subsequent activation of the acyltransferase.

Acyltransferases↗

PubMed1995

Site-specific cleavage of chromosomes in vitro through Cre-lox recombination.

Site-specific recombination systems are useful tools for chromosome engineering in vivo and site-specific DNA cleavage methods have applications in genome analysis and gene isolation. Here, we report a new method to fragment chromosomes in vitro using the Cre-lox site-specific recombination system. Two lox sites were targeted into the 5.7 Mb chromosomes I of Schizosaccharomyces pombe. In vitro recombination between chromosomal lox sites and exogenously provided lox oligonucleotides 'cleaved' the chromosome at the defined lox sequences. Site-specific cleavage of lox sites in the tobacco genome was also demonstrated. This recombination-based cleavage method provides a novel approach for structural and functional analyses of eukaryotic chromosomes as it allows direct isolation of chromosome regions that correspond to phenotypes revealed through Cre-lox mediated chromosome rearrangements in vivo. Moreover, recombination with end-labeled lox oligonucleotides would permit the specific end-labeling of chromosome segments to facilitate the long range mapping of chromosomes.

Base Sequence↗

PubMed1995

Function of the upstream hypersensitive sites of the chicken beta-globin gene cluster in mice.

We have shown previously that the chicken beta A-globin gene, with its 3' enhancer, is expressed in a copy number-dependent manner in transgenic mice. The expression level was low but increased approximately 6-fold upon inclusion of 11 kb of upstream DNA containing four DNase I hypersensitive sites. To study the effect of the individual upstream hypersensitive sites on transgene expression, we produced lines of mice in which the individual upstream sites were linked to the beta A gene and enhancer. RNA levels were measured in blood from adult animals. With each of these four constructs, the level of transgene RNA per DNA copy varied over a > 20-fold range. These data suggest that addition of a hypersensitive site to the beta A-globin/enhancer region abrogates its position independent expression. The average beta A-globin expression per copy in the lines carrying an upstream site was comparable with that in lines without an upstream site. Thus, no single upstream hypersensitive site accounts for the higher level of beta A-globin expression seen in mice containing the complete upstream region. We had shown previously that control of the chicken beta-globin cluster is distributed between at least two regions, the beta A/epsilon enhancer and the upstream region. Our current results suggest that the control mediated by the upstream DNA is itself distributed and is not due to a single hypersensitive site.

Animals↗

PubMed1995

Lead-induced nephropathy: relationship between various biological exposure indices and early markers of nephrotoxicity.

Lead nephropathy in adults is silent and insidious, characterized by the absence of proteinuria in its early phase. Of the early markers of nephrotoxicity, urinary N-acetyl-beta-D-glucosaminidase (NAG) appears to be the only one that is elevated in early lead nephropathy. However, the elevation in urinary NAG activity may be a response to a sharp increase in renal burden of lead. Its usefulness as a marker of chronic lead nephropathy is thus in doubt. There is a need, then, to identify a reliable early biological indicator of lead-induced kidney damage. Furthermore, there is also a need to identify suitable markers of chronic exposure to describe meaningful dose-response and dose-effect relationships. Traditionally, blood lead (PbB) was used, but the current blood lead level (PbBrec) is more an indicator of recent exposure. Time-integrated blood lead indices (PbBint) derived from repeated serial PbB measurements can be used as indices of chronic exposure. In 128 lead-exposed workers, the PbBint was the most important exposure variable in describing the variability in urinary alpha 1-microglobulin (U alpha 1 m), urinary beta 2-microglobulin (U beta 2m), and urinary retinol binding protein (URBP). U alpha 1m was the only marker that was significantly higher in the exposed group, with a good dose-response and dose-effect relationship with PbBint. The lack of dose-response and dose-effect relationships in other studies may be due to inappropriate exposure markers as well as less sensitive response markers. PbBint has a better correlation than PbBrec. Furthermore, U alpha 1m may be the most sensitive of the markers because of its higher molecular weight.

Adolescent↗

PubMed1995

Glomerular function of lead-exposed workers.

Among lead-exposed workers, there is evidence of increased mortality from chronic renal diseases (nephritis and nephrosis). Epidemiological studies using early markers of nephropathy among lead-exposed workers failed to demonstrate early renal changes. This study is aimed at assessing the glomerular function of 137 lead-exposed subjects and at evaluating whether changes in markers of glomerular function are related to exposure indices derived from longitudinal blood lead data. A control group of 153 postal workers was also investigated. Several exposure indices were derived for the exposed workers, including a time-integrated index Pb in blood (PbB)int and the number of times the PbB was above critical values (PbB400, PbB500, PbB600). Through multiple linear regression analysis, PbBint was the best predictor of variation in serum beta 2-microglobulin (S beta 2m) and alpha 1-microglobulin (S alpha 1m) and urinary albumin (UA1b). A small but statistically significant difference in the mean beta 2m was found. S beta 2m was also the only marker showing a significantly higher prevalence rate ratio (PRR) of abnormalities among lead-exposed workers. Though there was no clear dose-response relationship with PbBint as the index of dose, all the 15 subjects with abnormal S beta 2m in the older age group were found in the highest PbBint group. Furthermore, of the 8 subjects with low 4-h creatinine clearance (CrCl4h), 6 had abnormal levels of beta 2m. Two subjects with CrCl4h of less than 75 ml/min/1.74 m2 had high PbBint values, thus suggesting that high blood lead levels over a prolonged time may be associated with decreased CrCl4h. Though the long-term significance of elevated S beta 2m and UA1b is unclear, their association with high PbBint and decreasing CrCl4h indicate a potentially adverse effect. Their relationship with PbB400 and PbB600 suggests that the threshold of 700 micrograms/l for PbB may not prevent the occurrence of lead nephropathy.

Adolescent↗

PubMed1995

Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome.

The bacteriophage P1 Cre-lox site-specific recombination system has been used to integrate DNA specifically at lox sites previously placed in the tobacco genome. As integrated molecules flanked by wild-type lox sites can readily excise in the presence of Cre recombinase, screening for mutant lox sites that can resist excisional recombination was performed. In gene integration experiments, wild-type and mutant lox sites were used in conjunction with two strategies for abolishing post-integration Cre activity: (i) promoter displacement of a cre-expression construct present in the target genome; and (ii) transient expression of cre. When the promoter displacement strategy was used, integrant plants were recovered after transformation with constructs containing mutant lox sequences, but not with constructs containing wild-type lox sites. When cre was transiently expressed, integrant plants were obtained after transformation with either mutant or wild-type lox sites. DNA rearrangements at the target locus were less frequent when mutant lox sites were used. DNA integration at the genomic lox site was usually without additional insertions in the genome. Thus, the Cre-lox site-specific recombination system is useful for the single-copy integration of DNA into a chromosomal lox site.

Bacteriophage P1↗

PubMed1995

Proteinuria and mortality in diabetes: the WHO Multinational Study of Vascular Disease in Diabetes.

The relation between proteinuria and mortality was investigated in 1188 patients with Type 1 diabetes and 3234 patients with Type 2 diabetes, aged 35-55 at baseline and followed up for a mean of 9.4 +/- 3.1 years in the WHO Multinational Study of Vascular Disease in Diabetes. Baseline prevalence of light or heavy proteinuria was the same (25%) in both types of diabetes after adjustment for differences in diabetes duration. Compared with patients with no proteinuria, all cause mortality ratios were 1.5 (95% confidence interval 1.1-2.0) and 2.9 (2.2-3.8) for Type 1 patients with light and heavy proteinuria, respectively, and 1.5 (1.2-1.8) and 2.8 (2.3-3.4) for Type 2 patients, after adjustment for age, duration of diabetes, blood pressure, cholesterol, and smoking. Proteinuria was associated with significantly increased mortality from renal failure, cardiovascular disease, and all other causes of death. In both types of diabetes, the association was strongest for renal deaths, and of similar magnitude for cardiovascular and all other causes of death. In conclusion, proteinuria is a common, important, and rather non-specific risk factor for increased morbidity and mortality in diabetes. The relation of proteinuria to mortality is similar for both types of diabetes. The benefits and risks of proteinuria reduction should be examined in large randomized trials with clinical endpoints.

Adult↗

PubMed1995

Expression of the chicken beta-globin gene cluster in mice: correct developmental expression and distributed control.

To investigate the regulation of gene clusters, we introduced the entire chicken beta-globin cluster into mice. This 35-kb region includes the four globin genes (rho-beta H-beta A-epsilon), the four upstream hypersensitive sites, and the intergenic beta A/epsilon enhancer. The chicken globins are not arranged in order of developmental expression, which is unlike the case for the human beta-globin cluster, in which gene order plays a role in the regulation of globin expression. Mice carrying the chicken cluster expressed the transgenes with the same developmental patterns as seen in the chicken. Therefore, stage-specific erythroid transcriptional milieus existed before the divergence of birds and mammals and have been conserved since then. Mice bearing the complete cluster except for a deletion removing the beta A/epsilon enhancer displayed markedly reduced expression of the beta H, beta A, and epsilon genes with efficient (but variable) rho expression. Mice carrying the four genes and beta A/epsilon enhancer but without the upstream hypersensitive sites showed reduced expression of rho, beta H, and beta A, with variable expression of epsilon. We conclude that (i) all of the genes (except possibly rho) are under the control of both the upstream hypersensitive sites and the enhancer, (ii) the influence of the control elements can extend beyond the nearest active gene, (iii) a single element (the enhancer) can influence more than one gene in a single developmental stage, (iv) the enhancer can work bidirectionally, and (v) neither the upstream sites (as a group) nor the enhancer showed developmental stage specificity. Thus, the regulation of this cluster is achieved by interaction of two distinct control regions with each of the globin genes.

Age Factors↗

PubMed1995

Human vascular smooth muscle cell-monocyte interactions and metalloproteinase secretion in culture.

Degradation of the atherosclerotic plaque extracellular matrix could destabilize the lesion, rendering it more prone to rupture. Both macrophages and vascular smooth muscle cells (SMCs) are potential sources of matrix metalloproteinases (MMPs), secreted enzymes that can digest vascular matrix. We explored interactions between human vascular SMCs and human monocytes that result in the secretion of interstitial collagenase (MMP-1) and stromelysin (MMP-3). Monocytes alone or those treated with SMC-conditioned media did not secrete these metalloproteinases as detectable by Western blot analysis. SMCs increased secretion of both MMP-1 and MMP-3 greater than 20-fold when cocultured with monocytes or when treated with monocyte-conditioned media. Addition of macrophage colony stimulating factor (< or = 1000 U/mL) to cocultures of monocytes and SMCs did not affect metalloproteinase secretion. Recombinant interleukin (IL)-1 receptor antagonist inhibited MMP-1 and MMP-3 induction in SMC cultures treated with monocyte-conditioned media (94% and 96% reduction, respectively), while a neutralizing antibody to tumor necrosis factor-alpha had no significant effect on metalloproteinase secretion. In contrast to the induction by monocyte-conditioned media of MMP-1 and MMP-3 secretion by SMCs, monocyte-conditioned media did not increase secretion of 72-kD gelatinase (MMP-2). Thus, monocytes induce MMP-1 and MMP-3 secretion by vascular SMCs through an IL-1-dependent mechanism. This response of SMCs to a defined macrophage product may contribute to plaque destabilization by mononuclear phagocytes in the lesion.

Cells, Cultured↗

PubMed1995

Receptors for atrial natriuretic peptide are decreased in the kidney of rats with streptozotocin-induced diabetes mellitus.

To determine whether decreased renal responsiveness to atrial natriuretic peptide (ANP) in diabetes is mediated by alterations in the renal ANP receptor, ANP receptor density and affinity were measured 17-20 d after streptozotocin injection and compared with values in vehicle-treated controls and streptozotocin-treated rats made euglycemic with insulin. Plasma ANP concentration was significantly greater in hyperglycemic diabetic rats than in control or euglycemic diabetic rats. Both in glomeruli and inner medulla, ANP receptor dissociation constant did not differ among the three study groups, whereas the maximum binding capacity was decreased significantly in hyperglycemic diabetics in comparison with controls and euglycemic diabetics. Glomerular clearance receptors were also decreased significantly in hyperglycemic diabetic rats in comparison with control and euglycemic diabetic rats. To determine whether the decreased number of renal ANP receptors in diabetic rats was associated with a decreased biological response, we measured ANP-dependent cyclic GMP (cGMP) accumulation by isolated glomeruli and inner medullary collecting duct cells in vitro. cGMP accumulation was significantly less in hyperglycemic diabetic rats than in controls or euglycemic diabetic rats both in the presence or absence of the phosphodiesterase inhibitor zaprinast. cGMP phosphodiesterase activity in inner medullary collecting duct cells obtained from control and hyperglycemic diabetic rats did not differ. Thus, the decreased number of biologically active ANP receptors in the kidneys of diabetic rats is accompanied by decreased biological responsiveness in vitro and provides a potential explanation for the reduction in renal sensitivity to ANP in this condition.

Animals↗

PubMed1995

Alterations of the p16 (MTS1) gene in testicular, ovarian, and endometrial malignancies.

PURPOSE: The p16 (MTS1) gene codes for a cyclin-dependent kinase inhibitor and may be a new tumor suppressor gene. It is frequently mutated in a variety of cell lines established from tumors. This is the first report of screening for alteration of the p16 gene in testicular, ovarian and endometrial malignancies. MATERIALS AND METHODS: We examined alterations of p16 in 78 primary genital tumors (42 testicular, 21 ovarian and 15 endometrial cancers) and mononuclear cells from 2 patients with Lynch syndrome II as well as 5 testicular tumor cell lines by single-strand conformation polymorphism (SSCP) and Southern blot hybridization. RESULTS: The DNA from the p16 gene of 2 testicular tumors (5%), an ovarian cancer (4%) and a testicular tumor cell line (20%) had altered migration in gel electrophoresis as shown by SSCP. Analysis of DNA sequence of these samples revealed a polymorphism at codon 140. Southern blot hybridization detected neither deletions nor rearrangements of the p16 gene in any of the samples. CONCLUSIONS: Taken together, these results suggest that p16 alterations probably are not important for tumorigenesis of testicular, ovarian and endometrial tumors.

Base Sequence↗

PubMed1994

Role of TCR zeta chain in T cell development and selection.

Signals mediated by the T cell receptor (TCR) are required for thymocyte maturation and selection. To examine the role of TCR zeta chain signals in development, TCR expression was restored in zeta-deficient mice with transgenic zeta chains that partially or completely lacked sequences required for signal transduction. The zeta chain played a role in thymic development by promoting TCR surface expression, but zeta-mediated signals were not essential because TCRs that contained signaling-deficient zeta chains promoted T cell maturation and transduced signals associated with thymic selection.

Amino Acid Sequence↗

PubMed1994

Structure of the tomato Adh2 gene and Adh2 pseudogenes, and a study of Adh2 gene expression in fruit.

A cDNA library was constructed from RNA from the pericarp of ripe tomato fruit and four cDNAs encoding ADH2 were isolated and characterized. The cDNAs encode a peptide 379 amino acids in length. They hybridized strongly with a 1.8 kb RNA species well represented in RNA from ripe, but not from mature, unripe fruit, and strongly to a similar RNA species present in hypoxic, but not in aerobic roots. Northern analysis showed that the mRNA for ADH2 in fruit increased in abundance through ripening, particularly during late ripening. In pericarp tissue of fruit, the Adh2 mRNA level increased to a maximum within 8-16 h of exposure to atmospheres with 3% (v/v) oxygen, and returned to the basal level within 16 h of a return to air. The mRNA level was sensitive to the oxygen level in the atmosphere, increasing 20-fold in 12% (v/v) oxygen and 100-fold in 3% oxygen. The homologous tomato Adh2 gene was isolated from a genomic library. The gene has an overall length of 2334 bp from transcription start site to poly(A) addition site and includes eight introns. Southern blot analysis of tomato genomic DNA identified multiple Adh2-related sequences. Two of these, PSA1 and PSA2, were cloned and found to have 94% similarity with each other and 77% similarity with the tomato Adh2 gene over a 1000 bp region. The homologous regions include introns and exons but the equivalent exons contain frame shifts, deletions and stop codons. The two regions are therefore presumptive pseudogenes.

Alcohol Dehydrogenase↗

PubMed1994

Metaanalysis of prophylactic drug treatment in the prevention of postoperative bleeding.

Prophylactic drug treatment is one of several strategies to reduce postoperative blood loss and potentially limit homologous blood use in open heart surgery. A computerized MEDLINE search supplemented with manual bibliography reviews was performed for randomized clinical trials published in peer-reviewed English-language journals from January 1980 to June 1993. A metaanalysis was conducted of trials evaluating desmopressin (group DD, n = 13), epsilon-aminocaproic acid or tranexamic acid (group EA, n = 4), and aprotinin (group AP, n = 16). Eligible studies used placebo controls and administered the drug in a prophylactic manner. The primary study end point was postoperative chest tube loss (mL, mean +/- standard deviation). There was a significant reduction in postoperative chest tube loss detected for each of the active treatments versus the placebo (DD versus controls: percent reduction 0.11, p = 0.0021; EA versus controls: percent reduction 0.30, p < 0.0001; and AP versus controls: percent reduction 0.36, p < 0.0001). Therapy with EA or AP was associated with a greater reduction in chest tube loss than DD (EA versus DD, p = 0.0033, and AP versus DD, p < 0.0001). Secondary study end points were transfusion requirements, chest reexploration, and perioperative mortality. The volume of postoperative red cell transfusion (mean +/- standard deviation) was reduced with EA (p < 0.0001) or AP treatment (p < 0.0001) compared with a placebo or DD, whereas the proportion of patients given transfusions was limited only in the AP-treated patients (odds ratio 0.23; 95% confidence interval, 0.16 to 0.33; p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Aminocaproic Acid↗

PubMed1993

Hydrolysis of myelin basic protein in myelin membranes by granzymes of large granular lymphocytes.

Immune-mediated demyelination initially manifests as a separation of myelin lamellae followed by loss of myelin proteins and eventual loss of myelin membranes. Myelin basic protein, one of the major structural proteins of myelin, is highly vulnerable to various proteases derived from diverse cell types. Killer lymphocytes such as CTL, in addition to other immune effectors, have been implicated in inflammatory demyelination. In addition to a pore forming peptide, the granules of CTL and large granular lymphocytes (LGL) contain a number of serine esterases collectively called as granzymes. We studied the effect of these granule enzymes on myelin and found that LGL granule-extracts (LGL-g) hydrolyzed MBP in myelin membranes. LGL-g also cleaved purified MBP at neutral pH in a Ca2+ independent manner and this hydrolysis was inhibited by serine esterase inhibitors. In addition, absorption of LGL-g with antigranzyme A significantly reduced MBP hydrolysis. These findings implicated the granzymes in LGL-g, especially granzyme A, as the agent causing MBP degradation. Inasmuch as activation of CTL in the vicinity of myelinated axons can lead to granule exocytosis, hydrolysis of MBP by granzymes may be a significant event in myelin destruction.

Cell Membrane↗