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N Lerner

Publications and source records attributed to N Lerner.

At least 19 recordsLinked to original sources
PubMed2001

Structure of c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus at 2.5 A: structural implications for thermal stability in phycobilisome assembly.

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechochoccus vulcanus has been determined by molecular replacement to 2.5 A resolution. The crystal belongs to space group R32 with cell parameters a=b=188.43 A, c=61.28 A, alpha=beta=90 degrees, gamma=120 degrees, with one (alphabeta) monomer in the asymmetric unit. The structure has been refined to a crystallographic R factor of 20.2 % (R-free factor is 24.4 %), for all data to 2.5 A. The crystals were grown from phycocyanin (alphabeta)(3) trimers that form (alphabeta)(6) hexamers in the crystals, in a fashion similar to other phycocyanins. Comparison of the primary, tertiary and quaternary structures of the S. vulcanus phycocyanin structure with phycocyanins from both the mesophilic Fremyella diplsiphon and the thermophilic Mastigocladus laminosus were performed. We show that each level of assembly of oligomeric phycocyanin, which leads to the formation of the phycobilisome structure, can be stabilized in thermophilic organisms by amino acid residue substitutions. Each substitution can form additional ionic interactions at critical positions of each association interface. In addition, a significant shift in the position of ring D of the B155 phycocyanobilin cofactor in the S. vulcanus phycocyanin, enables the formation of important polar interactions at both the (alphabeta) monomer and (alphabeta)(6) hexamer association interfaces.

Algal Proteins↗
PubMed2001

Regulation of ubiquitination and degradation of p53 in unstressed cells through C-terminal phosphorylation.

Previously, we found that the protein kinase C (PKC) inhibitor H7 stimulates p53 to accumulate in a form incapable of inducing transcription from p53-dependent promoters. We concluded that H7 inhibits constitutive C-terminal phosphorylation of p53, which regulates its turnover in unstressed cells. We now show that p53 and its inhibitor MDM2 (HDM2 in human cells) are together in the nuclei of H7-treated cells and can be co-immunoprecipitated. Despite this association of p53 with the ubiquitin ligase MDM2, ubiquitinated p53 was not detected in H7-treated cells. Furthermore, co-treatment with H7 and the proteosome inhibitor LLnL prevented the accumulation of ubiquitinated p53 that was observed in cells treated solely with LLnL. In addition, treatment of cells with the PKC activator phorbol ester stimulated the ubiquitination of p53 and reduced its ability to accumulate after stress. H7 did not induce the phosphorylation of human p53 on Ser-15 (Ser-18 in mouse protein), a modification that occurs in response to DNA damage and leads to the release of MDM2 and to transactivation by p53. We conclude that phosphorylation of the C-terminal domain of p53 by PKC increases its ubiquitination and degradation in unstressed cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Analysis of the role of detergent mixtures on the crystallization of the reaction center of Photosystem II.

We have recently reported the crystallization of the reaction center of Photosystem II in the presence of detergent mixtures [Adir N (1999) Acta Crystallogr D Biol Crystallogr D55: 891-894]. We have used high performance liquid chromatography, dynamic light scattering, native gel electrophoresis and thermoluminescence measurements to characterize the interaction between these detergent mixtures and RC II, to try and understand their role in the crystallization process. Size exclusion HPLC and dynamic light scattering confirmed that the isolated RC II used for crystallization was exclusively monomeric. Dynamic light scattering measurements show that the detergent mixtures formed single micelles within a limited range of hydrodynamic radii. Both size exclusion HPLC and dynamic light scattering were used to follow the interaction between the detergent mixtures and monomeric RC II. These techniques revealed a decrease in the detergent mixture treated RC II particle size (with respect with the untreated RC II), and that RC II from solubilized crystals contained particles of the same size. Native gel electrophoresis showed that this change in apparent size is not due to the disintegration of the internal structure of the RC II complex. Thermoluminescence measurements of solubilized RC II crystals showed charge recombination from the S(2,3)Q(A) (-) state, indicating that RC II remains functionally viable following detergent mixture treatment and crystallization. The role of the detergent mixtures in the crystallization of RC II is discussed.

Journal Article↗
PubMed1998

Ammonia from iron(II) reduction of nitrite and the Strecker synthesis: do iron(II) and cyanide interfere with each other?

The question of whether the production of ammonia, from the reduction of nitrite by iron(II), is compatible with its use in the Strecker synthesis of amino acids, or whether the iron and the cyanide needed for the Strecker synthesis interfere with each other, is addressed. Results show that the presence of iron(II) appears to have little, or no, effect on the Strecker synthesis. The presence of cyanide does interfere with reduction of nitrite, but the reduction proceeds at cyanide/iron ratios of less than 4:1. At ratios of about 2:1 and less there is only a small effect. The reduction of nitrite and the Strecker can be combined to proceed in each other's presence, to yield glycine from a mixture of nitrite, Fe+2, formaldehyde, and cyanide.

Ammonia↗
PubMed1990

Chelation therapy and cardiac status in older patients with thalassemia major.

Cardiac dysfunction is the most common cause of death in patients with homozygous beta-thalassemia. We studied a group of 10 older patients (mean age 17.5 years) with and without preexisting cardiac dysfunction who had begun chelation therapy on the average of 10 years after regular transfusions were initiated. Over the 4-year study period, two patients were noncompliant with deferoxamine therapy. Their clinical status and cardiac function deteriorated, and both died with evidence of arrhythmia and congestive heart failure. The remaining eight patients were compliant. Despite a drop in mean serum ferritin from 3,814 +/- 577 (SE) ng/ml to 1,056 +/- 146 ng/ml (p less than 0.01), two patients with preexisting cardiac problems and one patient without preexisting heart disease developed further abnormalities. Of the three patients whose status declined, one ultimately improved with alternative chelation therapy. These data suggest that for a few older patients, improvement or stabilization of cardiac status may not be achieved with improved compliance and reduced serum ferritin levels. For these patients, new approaches appear to be warranted. On the other hand, we have demonstrated that in most cases, older patients who began chelation therapy years after transfusions began have benefited from compliance with standard subcutaneous deferoxamine regimens.

Adolescent↗
PubMed1989

Gene transfer. A potential approach to gene therapy for sickle cell disease.

The current status of gene transfer experiments indicates that it is possible to provide (1) safe and efficient retroviral packaging lines for gene transfer; and (2) vectors containing the human beta-globin genes and selectable marker genes which can be transmitted into erythroid cells and are appropriately expressed. In animal autologous bone marrow transplantation experiments, stable and high-level expression of retroviral vectors containing human beta-globin genes has not yet been achieved. New retroviral vectors are being tested that contain different components of the retrovirus as well as the newly described enhancer elements 5' to the epsilon gene and surrounding the beta-globin gene. The long-term goal of human gene therapy for sickle cell disease then consists of constructing optimally safe and efficient retroviral packaging lines as well as retroviral vectors containing the human beta-globin gene and selectable markers such as the neoR gene. One would then remove bone marrow cells from patients with sickle cell disease, transfer the retroviral vectors into the bone marrow cells, and subject the cells to G418 selection in vitro. Next, one would ablate the host bone marrow and autotransplant the manipulated bone marrow bearing the retroviral vector. Finally, one would analyze the reconstituted bone marrow for human beta-globin gene expression. These experiments must await the demonstration of safe and efficient gene transfer in animals, and particularly experiments must be done in monkeys prior to the use of these approaches in humans. Alternative approaches to gene therapy include direct correction of the defect in the beta-globin gene by site-specific recombination of the defective gene with incoming normal gene sequences. This technology is not yet achievable, although preliminary experiments have been performed in which gene correction at a low frequency has been obtained. The obvious advantage of this approach is that the native human beta gene would be reconstituted. Site-specific recombination and addition of normal beta genes to the human genome represent exciting and feasible approaches to human gene therapy using the extraordinary resources of modern molecular and cellular biology. Success in treating disorders of human hemoglobin only awaits additional technical advances for increasing the efficiency of gene transfer and the level of gene expression.

Anemia, Sickle Cell↗
PubMed1987

Human beta-globin gene expression after gene transfer using retroviral vectors.

A retroviral vector containing a 4.4-kb Pst I human beta S-globin gene and a neomycin resistance gene was used to infect NIH-3T3 and mouse erythroleukemia cells (MELC). In MELC, human beta-globin mRNA transcripts are transcribed and properly initiated and spliced. In some cases, there is an appropriate increase in beta-globin mRNA on addition of dimethylsulfoxide (DMSO), an inducer of hemoglobin synthesis and erythroid differentiation in these cells. When NIH-3T3 cells are infected with the same retroviral vector, there is less globin mRNA accumulation and no evidence for appropriate regulation. Human beta-globin gene expression in MELC clones induced with DMSO is 2-3% that of endogenous mouse beta-globin gene expression. These results indicate that retroviral vectors can be used to transfer and appropriately express human beta-globin genes in erythroid cells.

Cells, Cultured↗
PubMed1987

Prenatal diagnosis of sickle hemoglobinopathies: the experience of the Columbia University Comprehensive Center for Sickle Cell Disease.

We report here an evaluation of 55 pregnancies at risk for a sickle hemoglobinopathy prenatally diagnosed by restriction-endonuclease analysis, with the endonucleases MstII and HpaI, of amniocyte DNA. The diagnosis was completed in all cases. Eleven fetuses were predicted to be affected, of which six were terminated. Forty-one of the 55 cases were confirmed. One false-negative was reported in a case predicted to be hemoglobin AS but that was determined to be hemoglobin SS at birth. We estimate that the 55 cases represent only 5% of the pregnancies at risk for a sickle hemoglobinopathy in the New York metropolitan area during the study period. We conclude that the prenatal diagnosis of sickle hemoglobinopathies by molecular methods is reliable. However, the efficiency of utilization and effectiveness of prenatal testing is dependent on the early prospective identification of couples at risk and on the education of communities concerning the significant morbidity of the sickle hemoglobinopathies and the reproductive choices now available to them.

Anemia, Sickle Cell↗
PubMed1987

Human globin gene expression after gene transfer.

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.

Anemia, Sickle Cell↗
PubMed1986

T-cell chronic lymphocytic leukemia with pure red cell aplasia: laboratory demonstration of persistent leukemia in spite of apparent complete clinical remission.

A 40-year-old woman presented with splenomegaly, macrocytic anemia, and red cell aplasia. Although lymphocytosis was absent in the peripheral blood, large atypical lymphoid aggregates were present in the bone marrow. Splenectomy resulted in partial remission of red cell aplasia, but a gradual increase in the number of peripheral blood lymphocytes followed during the next 36 months. Flow cytometric analysis demonstrated that the majority of these peripheral blood lymphocytes had suppressor, natural killer T-cell phenotype. No other treatment was given until red cell hypoplasia worsened 42 months after initial presentation. Repeat bone marrow evaluation again demonstrated severe erythroid hypoplasia and large abnormal lymphocytic infiltrates. Cyclophosphamide given for 8 months resulted in complete resolution of the red cell aplasia and complete clinical remission of CLL. However, flow cytometric analysis revealed persistent increase in bone marrow T-cells, and bone marrow co-culture studies demonstrated residual ability of peripheral blood mononuclear cells to inhibit erythropoiesis in vitro, suggesting that residual, clinically undetectable leukemia persists in spite of complete clinical remission.

Adult↗
PubMed1981

Positive cooperativity of the estrogen receptor.

The equilibrium [3H]estradiol binding by the partially purified estrogen receptor from calf uteri was measured at 25 degrees C. The Scatchard plot of the binding data showed a convex curve characteristic of positive cooperativity and a Hill coefficient of 1.58 +/- 0.21, at receptor concentrations of 1 to 10 nM. Below a receptor concentration of approximately 0.3 nM the Scatchard plot approached linearity, suggesting that the cooperative interactions are dependent upon a monomer--dimer equilibrium. Trypsin pretreatment of the receptor resulted in a loss of dimer formation and of the cooperative interactions. The positive cooperative characteristics of the estrogen receptor were shown not to be produced by receptor inactivation, failure to complete the [3H]estradiol--receptor equilibrium reaction, or radioimpurity of the [3H]estradiol. These findings indicate that the activated 5S estrogen receptor is a homodimer and that its formation is associated with a positive cooperative estradiol-binding reaction.

Allosteric Regulation↗
PubMed1979

The role of ligand-binding as a determinant of the structure and activation of the estrogen receptor.

The dissociation of estradiol from the estrogen receptor occurs in two kinetic phases: a fast component having a half-time of approximately 3 min and a slower, or second, dissociating component having a half-time of approximately 95 min at 28 degrees. The fast component is produced by the dissociation of estradiol from the nonactivated 4 S receptor, a monomer. Thus, the magnitude of the fast component of the [3H] estradiol biphasic dissociation curve is proportional to the fraction of the receptor in the nonactivated state. The slow component is the estradiol dissociating from the activated 5 S receptor, a dimer. The salt-extracted estrogen receptor isolated from uterine nuclei shows a single, slow dissociating component equal to the slower component of the cytoplasmic biphasic dissociation curve. Estradiol binding shifts the receptor equilibrium from the low affinity nonactivated 4 S receptor toward the high affinity activated 5 S receptor. The kinetics of estriol dissociation from the receptor shows a larger fractional magnitude for the fast component and a faster second dissociating component than estradiol. This suggests that estriol transforms a smaller fraction of the receptor to the activated state and that the activated estriol receptor has a shorter half-time than estradiol. The biphasic dissociation kinetics of an estrogen from the receptor provides a new and sensitive criterion for measuring receptor activation.

Animals↗