VACTERL association with high prenatal lead exposure: similarities to animal models of lead teratogenicity.
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Abnormalities, Multiple↗
Biomedical subjects
F Levine
Publications and source records attributed to F Levine.
The electron-transfer site of spinach plastocyanin.
Two sites for electron transfer have been proposed for plastocyanin: one near the copper ion and the other close to the acid patch formed by residues 42-45. Calculations of electrostatic properties of spinach plastocyanin and ionic strength dependences of electron-transfer reactions of this protein have been used to distinguish between these two sites. Calculations show that the electric potential field of spinach plastocyanin is highly asymmetric and that the protein has a dipole moment of 360 D. The negative end of the dipole axis emerges between the negative patches formed by residues 42-45, which is though to be the cation binding site, and residues 59-61. The angles between the dipole vector and vectors from the center of mass to the copper ion and to the acid patch are 90 degrees and 30 degrees, respectively. The angle between the dipole vector and a line from the center of mass to the site of electron transfer is evaluated from the ionic strength dependence of electron-transfer rates at pH 7.8 with the help of equations developed by Van Leeuwen et al. [van Leeuwen, J.W., Mofers, F.J.M., & Veerman, E.C.I. (1981) Biochim. Biophys. Acta 635, 434] and Van Leeuwen [van Leeuwen, J.W. (1983) Biochim. Biophys. Acta 743, 408]. The angles found are 85 degrees, 110 degrees, and 75 +/- 15 degrees for reactions with tris(1,10-phenanthroline)cobalt(III), hexacyanoferrate(III), and ferrocytochrome c, respectively. The electric potential field calculations suggest that the hexacyanoferrate(III) interaction angle corresponds to a unique site of minimum repulsion at the hydrophobic region of the protein surface, close to the copper ion.(ABSTRACT TRUNCATED AT 250 WORDS)
Binding Sites↗
Catalysis of superoxide dismutation by manganese aminopolycarboxylate complexes.
Complexes of manganese, copper, cobalt, and iron with a variety of aminopolycarboxylates at concentrations from 2 X 10(-7) to 3 X 10(-6) M were tested for superoxide dismutase activity with horse ferricytochrome c as the competing reagent for superoxide. In the presence of excess ligand only manganous nitrilotriacetate and manganous ethylenediaminediacetate showed activity with catalytic rate constants of 2.2 X 10(7) and 1.8 X 10(7) M-1 S-1, respectively, at pH 6, 22 +/- 1 degree C, and 10 mM ionic strength. These rate constants decrease considerably at higher pH. Manganous N-hydroxyethylethylenediaminetriacetate is oxidized by superoxide, but does not appear to have catalytic activity. From the experimental conditions under which the two complexes mentioned above exhibit catalysis, and the inactivity of other metal chelates, it is concluded that an open coordination site is essential but not sufficient to catalyze the dismutation reaction.
Binding Sites↗
Surgical treatment of medically refractory variant angina pectoris: segmental coronary resection with aortocoronary bypass and plexectomy.
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Angina Pectoris, Variant↗
HLA class II regulation and structure. Analysis with HLA-DR3 and HLA-DP point mutants.
Point mutations that affect HLA-DR structure or expression have not previously been described. In the present study, we isolated such mutants by immunoselection of an ethyl methanesulfonate-mutagenized HLA-DR3 cell line with an anti-HLA-DR3 monoclonal antibody, 16.23. To facilitate analysis, we used a parent cell line with a preexisting deletion of one haplotype encompassing DR and DQ alpha and beta. The selection yielded two sets of mutants, one with defects in DR3 structure, the other with defects in different steps leading to DR expression. Of the expression-defective mutants, one had undergone a second deletion removing the remaining DR alpha gene but no other class II genes. It had a normal abundance of DR beta mRNA but had lost binding of DR monomorphic antibodies, indicating that DR beta chains do not form noncognate dimers. A second mutant had an abnormally large DR alpha mRNA, probably resulting from a splice site mutation. Several mutants had marked reductions in DR beta mRNA levels; in two of these, the lesion appeared to be transcriptional because the reduction in DR beta mRNA was paralleled by an altered methylation pattern of one of the DR beta genes. Other expression-defective mutants had different posttranscriptional defects. Some of the mutations were similar to those that have been found in mouse strains defective in I-E expression, whereas others have no known natural counterpart. The matrix of reactivities of anti-HLA class II monomorphic antibodies with these and similar mutants allowed us to define the gene products recognized by these antibodies. A set of seven mutants were "epitope defective," that is, they expressed normal or near normal levels of HLA-DR3 but no longer bound 16.23. Unexpectedly, each of the epitope mutants had decreased DR dimer stability. These mutants should be useful in localizing the DR3 alloepitope and in elucidating its contribution as a restriction element in the presentation of soluble antigen to immune T cells.
Antibodies, Monoclonal↗
Different roles for cytosine methylation in HLA class II gene expression.
We studied the role of cytosine methylation in the control of HLA class II gene expression in isogenic sets of cells whose members differ in their expression of HLA class II genes. These included: T5-1, 6.1.6, and P30, which are a class II expressing B-cell line, a class II nonexpressing mutant derived from T5-1, and an HLA-DR expressing partial revertant derived from 6.1.6, respectively; the class II expressing B-cell line, SB, and the class II non-expressing T-cell line, HSB, from the same individual. The use of sets of cells that differ in the way their class II genes are regulated allows us to study how that difference is reflected in the methylation state of their class II genes. At least five out of six class II genes in nonexpressing cells have a CpG site that is demethylated, when compared with the same class II gene in the respective expressing cells. The results presented in this paper indicate that most methylation changes in and around class II genes have a correlation with their state of expression. Some of these changes reflect rather than determine the state of expression. Other methylation changes appear to directly affect expression, whereas some methylation differences neither correlate with nor influence gene expression. Although 5-azacytidine does not affect class II expression in T5-1 or 6.1.6, it does induce expression in HSB. This indicates that the basis for nonexpression of class II genes is different in 6.1.6 and HSB.
B-Lymphocytes↗
Deletion mapping of HLA and chromosome 6p genes.
We have analyzed a set of heterozygous mutants with deletions that encompass parts of HLA and surrounding regions of chromosome 6p. By a combination of Southern blotting, serologic, enzymatic, and cytogenetic analyses, we have ordered eight independent deletion break points into a sequence that divides chromosome 6p into six regions. The deletion mutants have been used in conjunction with the Southern blot technique to map HLA and other 6p gene probes into those regions. On the basis of these and other data we propose a genetic and physical map of HLA and surrounding regions of chromosome 6p. We find that for HLA probes, most of which hybridize with more than one gene, the multiple copies recognized by single probes map to single regions. Any chromosome 6p gene can now be regionally mapped by using these mutants.
Chromosome Deletion↗
Differential Ia antigen expression by autologous human erythroid and B lymphoblastoid cell lines.
Human erythroid precursors express Ia antigens that have serology, function, molecular nature, and genetic regulation that are largely unknown. To approach these issues, Ia+ and Ia- subclones of the HEL human erythroleukemia cell line (HEL-DR+ and HEL-DR-, respectively) and an autologous B lymphoblastoid line (B line) were isolated. These erythroid and lymphoid lines were compared with respect to their binding of monoclonal HLA-D subregion-specific antibodies, the ability to trigger in vitro alloproliferation, expression of class II molecules, and transcription of class II-related genes. Unlike the DP+/DQ+/DR+ B lines, HEL-DR+ differentially expressed DP and DR, but not DQ specificities. Also unlike the autologous B line, HEL-DR+ appeared to be unable to trigger primary or secondary allogeneic T cell proliferation, despite the presence of responder monocytes in these cultures and irrespective of lymphokine addition. HEL-DR+ expression of bona fide class II molecules similar to B line DR heterodimers was verified by two-dimensional gel electrophoresis of material immunoprecipitated from 125I-labeled cells. Northern blot analysis of cytoplasmic RNA from these lines indicated that differential class II gene transcription could readily explain the distinct, lineage-related Ia phenotypes of HEL-DR+ and B line. In addition, the lack of invariant chain and class II transcripts in HEL-DR- implied that expression of these unlinked genes in HEL cells is co-regulated.
Antigen-Antibody Reactions↗
Evidence for two trans-acting genes regulating HLA class II antigen expression.
Previous studies have shown that a trans-acting regulatory factor is required for expression of HLA class II molecules in B-LCL, and that a trans-active factor derived from B-LCL induces de novo synthesis of T-LCL-encoded class II antigens in hybrids of T-LCL and B-LCL. To further examine the genetics of class II antigen regulation, we have now fused two different T-LCL with the class II antigen negative B-LCL 6.1.6, a regulatory mutant that does not synthesize a trans-acting factor necessary for transcription of class II genes. Northern blot analysis and cell surface immunofluorescence studies indicate that the hybrids express class II antigens encoded by both 6.1.6 and T-LCL parent cell lines. Thus, expression of class II antigens in these hybrids is controlled by a minimum of two trans-acting regulatory genes. One of these genes is inactivated in the 6.1.6 mutant and is provided by the T-LCL fusion partner. The second regulatory gene may still be active in 6.1.6, but it alone is insufficient for maintenance of class II antigen expression.
Antigens, Surface↗
Transcriptional regulation of HLA class II and invariant chain genes.
Class II (Ia) antigens are coded for by a family of genes located in the human MHC (HLA). These genes are regulated in a complex manner, being constitutively expressed, inducibly expressed, or not expressed, depending on the cell type examined. 6.1.6 is a variant of a normal B lymphoblastoid line that has lost expression of all class II molecules and has previously been shown to have a defect in the regulation of class II genes. In this report, we have examined those genes by Southern and Northern blotting and have found that 6.1.6 is severely deficient in mRNA for all class II genes examined, although the genes are structurally intact. P30, a partial revertant of 6.1.6, re-expresses mRNA for a subset of class II genes. mRNA for the class II-associated invariant chain is substantially reduced but not absent in 6.1.6.
Genes↗
Revertants from the HLA class II regulatory mutant 6.1.6: implications for the regulation of Ia gene expression.
Class II (Ia) genes of the human MHC code for developmentally regulated, highly polymorphic cell surface glycoproteins. The control of expression of this highly complex genetic region has not been extensively investigated. We used 6.1.6, a variant of a normal B lymphoblastoid cell that has lost expression of all class II molecules, to study this question. In previous studies, we have shown that the structural genes for class II molecules are intact but are not expressed in 6.1.6 (6), and that the defect is at the transcriptional level (7). In this report, we describe the isolation and characterization of revertants of 6.1.6 that reexpress class II molecules. These revertants have four unusual properties: 1) they are found at a higher frequency (0.7%) than would be expected for a mutational event; 2) they are unstable; 3) they reexpress only a subset of class II molecules; and 4) they have only been found in tetraploid cells. On the basis of previous work with 6.1.6 and the properties of reversion in 6.1.6, we propose a hypothesis for the nature of the defect in 6.1.6 and a model for the regulation of class II molecules.
Antibodies, Monoclonal↗
Two-dimensional echocardiographic detection and diagnostic features of tricuspid papillary fibroelastoma.
Cardiac papillary fibroelastomas are rare and benign primary tumors in the cardiac valves or occasionally the mural endocardium. Before 1977, these tumors were diagnosed exclusively at postmortem examination. Over the last few years, a handful of cases have been diagnosed in vivo by echocardiography. In this report, we describe the first tricuspid valve papillary fibroelastoma detected by echocardiography in an adult. Clinical and echocardiographic features are discussed.
Echocardiography↗
Results of coronary artery surgery in patients with poor left ventricular function (CASS).
We identified 420 medically treated and 231 surgically treated patients (coronary graft plus myocardial surgery in 30%) who had severe left ventricular dysfunction manifest by an ejection fraction below 0.36 and markedly abnormal wall motion. Compared with medically treated patients, those treated surgically had more severe angina (56.7% vs 29.0% class III or IV; p less than .001), less heart failure as predominant symptom (11.1% vs 18.8%; p less than .003), more severe coronary disease (66.7% vs 50.2% three-vessel disease; p less than .001), a greater concentration of left main coronary artery lesions greater than 70% (12.6% vs 3.8%: p less than .001), and a greater estimated extent of jeopardized myocardium (p less than .001). Multivariate regression analysis of survival, which adjusts for the above covariates, showed that surgical treatment prolonged survival (p less than .05), although it ranked below severity of heart failure symptoms, age, ejection fraction, and left main stenosis greater than 70% in determining prognosis. Surgical benefit was most apparent for patients with ejection fractions below 0.26 who had a 43% 5 year survival with medical treatment vs 63% with surgery. Surgically treated patients experienced substantial symptomatic benefit compared with medically treated patients if their presenting symptoms were predominantly angina; however, there was no relief of symptoms caused primarily by heart failure. We conclude that patients with predominantly ischemic pain symptoms, despite poor left ventricular function, benefit from surgery; however, operative mortality in this high-risk subset must equal or better the 6.9% obtained in this study.
Actuarial Analysis↗
Preservation of platelet function and number by prostacyclin during cardiopulmonary bypass.
Thirty-four dogs underwent total cardiopulmonary bypass for 1 or 2 hours with a bubble oxygenator and cardiotomy suction. The dogs were divided into three groups: control dogs which received heparin alone, dogs which received prostacyclin (PGI2) and heparin, and dogs which received prostacyclin alone. PGI2 was given as a bolus (6 to 60 microgram) and then as a constant infusion (0.2 to 0.8 microgram/kg/min) in the venous outflow line. The pump flows were equal in the three groups. PGI2 as a bolus led to transient hypotension, and 60 microgram reduced cardiac output temporarily. During cardiopulmonary bypass, dogs treated with prostacyclin and heparin had low mean arterial perfusion pressures which responded to fluid infusion or phenylephrine. After 1 hour of cardiopulmonary bypass and reversal with protamine, dogs treated with prostacyclin and heparin had shorter bleeding times (p < 0.02) and better platelet function than control animals. After 2 hours, they had normal platelet number, but only half showed preservation of platelet function. When heparin was omitted, PGI2 preserved platelet number, but consumption coagulopathy developed, with prolonged prothrombin, partial thromboplastin, and bleeding times and decreased fibrinogen levels. PGI 2 preserves platelet number and function but may cause hypotension, and it cannot replace heparin in cardiopulmonary bypass.
Animals↗
Effect of hemoperfusion of clearance of gentamicin, cephalothin, and clindamycon from plasma of normal dogs.
Nine normal dogs were divided into three groups of three. Group 1 was given an overdose of gentamicin; group 2, cephalothin; and group 3, clindamycin. Group 1 had hemoperfusion with Amberlite XE-336, and groups 2 and 3 with Amberlite XAD-4 resin adsorbents, for 6 hr with a blood flow rate of 300 ml/min. The plasma clearance and removal rates of antibiotics by the hemoperfusion columns were high. The clearance rate of gentamicin from plasma (mean +/- standard deviation) ranged from 59 +/- 30 to 199 +/- 6 ml/min, of cephalothin from 66 +/- 14 to 157 +/- 8 ml/min, and of clindamycin from 55 +/- 9 to 125 +/- 16 ml/min. Of the total dose of antibiotic administered, the hemoperfusion columns removed 67% from theplasma in group 1, 41% in group 2, and 18% in group 3. The fact that antibiotics may be rapidly removed from the blood during hemoperfusion should be considered in calculation of the therapeutic dose of antibiotic required for patients who receive this preocedure. Also, hemoperfusion can effectively and rapidly remove certain antibiotics from the blood of patients who have had a potentially toxic overdose.
Animals↗
Protection from cell death in cultured human fetal pancreatic cells.
Endocrine cells from the human fetal pancreas will proliferate in vitro on extracellular matrix but lose hormone expression, and redifferentiation requires removal of the expanded cells from the matrix and reaggregation into cell aggregates. However, extensive cell death occurs during manipulation and culture. The mechanism of cell death was examined at each stage throughout the process under different experimental conditions to determine optimal protocols to increase cell viability. During shipment, the addition of trehalose to the media to prevent necrosis increased yield 17-fold, while during culture as islet-like cell clusters the apoptosis inhibitor Z-VAD increased yield 1.8-fold. Following disruption of cell matrix interactions and reaggregation, there was marked evidence of apoptotic bodies by the TUNEL assay. Addition of nicotinamide or Z-VAD, or removal of arginine from the media during reaggregation, reduced the number of apoptotic bodies and the effect was additive. However, a combination of treatments was necessary to significantly increase the yield of viable cells. We conclude that cell death of human fetal pancreatic tissue in culture results from both necrosis and apoptosis and that understanding the mechanisms at the cellular level will lead to protocols that will improve cell viability and promote beta-cell growth.
Amino Acid Chloromethyl Ketones↗
Differential integrin expression facilitates isolation of human fetal pancreatic epithelial cells.
We have studied the expression of the beta 1 family of integrins in fetal and adult human pancreas. Immunohistochemical staining with a monoclonal anti-beta 1 antibody revealed that the epithelial cells of the human fetal pancreas express high amounts of beta 1 integrin, while the pancreatic stromal cells express substantially lower amounts. Islets of Langerhans from human adult pancreas also expressed high amounts of beta 1 integrin. Taking advantage of the extremely high affinity binding between the invasin protein of Yersinia pseudotuberculosis and many beta 1 integrins, we have been able to isolate highly enriched populations of fetal pancreatic epithelial cells. Epithelial-enriched cell populations retain the ability to differentiate into mature endocrine cells following transplantation into nude mice.
Adhesins, Bacterial↗
Isolation and characterization of a cell line from the epithelial cells of the human fetal pancreas.
Pancreatic cell lines are useful for basic studies of pancreatic biology and for possible application to cell transplantation therapies for diabetes. A retroviral vector expressing simian virus 40 (SV40) T antigen and H-rasval12 was used to infect a monolayer culture of epithelial cells from an 18-wk human fetal pancreas. Infected cells gave rise to a clonal epithelial cell line, designated TRM-1. This cell line expresses epithelial markers as well as gult2 and small amounts of insulin and glucagon. TRM-1 is the first cell line to be generated from the human fetal pancreas and also the first cell line derived directly from the fetal pancreas of any species. The approach that we have used to develop TRM-1 should be applicable to isolating cell lines from other stages of human pancreatic development.
Animals↗